Datasets:

pszemraj

/

summary-souffle

like 0

Background LSC relies heavily on its Office of Compliance and Enforcement (OCE) and its Office of Program Performance (OPP) to carry out activities related to grant awards, grantee program effectiveness, and grantee compliance responsibilities. According to LSC officials, LSC established OCE in 1997 and OPP in 1999 to (1) help ensure compliance with requirements of the LSC Act, and (2) evaluate, fund, monitor and oversee grantee programs, including quality of services provided. Figure 1 shows staffing levels for OPP and OCE and LSC overall between 1999 and 2009. As shown in figure 2, the Directors of OPP and OCE report to the Vice President for Programs and Compliance, who reports to the LSC President. LSC’s President reports to an LSC board composed of 11 members. In April 2010, the 11 member board was undergoing transition, with: 1 board member continuing, 6 of the remaining 10 being sworn in during April, 2 board members awaiting to be named, and 2 others awaiting Senate confirmation. (OPP) According to the LSC Vice President of Programs and Compliance’s goals and objectives document (LSC workplan), the Vice President for Programs and Compliance is responsible for coordinating OPP and OCE; implementing efforts to improve LSC’s oversight of grantees; assessing LSC component directors’ staffing allocations and assignments; conducting quarterly joint staff meetings and training sessions; and overseeing LSC’s internal quality agenda, including providing staff training. In accordance with the LSC Workplan, the Vice President for Programs and Compliance also oversees LSC’s grantee compliance and program functions, with emphasis on intra-office coordination, improved grantee guidance, and improved grantee follow-up activities by OCE and OPP. According to LSC’s policy and the 2009 OPP Procedures Manual, OPP’s responsibilities include designing and administering LSC’s process for awarding competitive grants, and developing and implementing strategies to improve grantee program quality. In carrying out its responsibilities, OPP is to issue requests for proposals, guide grant applicants through the application process, and evaluate applications against performance criteria. According to the 2008 Roles and Responsibilities of LSC Offices Responsible for Grantee Oversight, OCE is charged with reviewing grantees’ compliance with the LSC Act and implementing regulations, responding to inquiries and written complaints concerning grantees received from members of the public or Congress, and providing follow up on the referrals of findings from LSC’s Office of Inspector General. In carrying out its responsibilities, OCE is to conduct grantee case service reports and case management system site visits; review grantee compliance with the LSC accounting manual and fiscal-related regulations; review the audited financial statements of grantees; and initiate questioned-cost proceedings as necessary. To increase compliance, OCE is also responsible for issuing corrective action notices to grantees and for following up on corrective action plans through conducting interviews, reviewing grantee corrective action plans, and performing follow-up reviews. Figure 3 presents an overview of LSC grant award process responsibilities as prescribed by LSC’s policies and procedures. In addition, the Office of Legal Affairs (OLA) has some responsibilities with respect to LSC’s grantee oversight. Specifically, according to the Roles and Responsibilities of LSC Offices Responsible for Grantee Oversight, OLA, headed by a Vice President of Legal Affairs who reports to LSC’s President, is responsible for providing legal services for LSC, such as interpreting statutory and regulatory authorities applicable to LSC grantees and approving contracts prior to award. OPP and OCE and other operating units seek legal counsel and information from OLA on application of relevant laws and regulations, as well as legal issues arising from oversight and enforcement activities. Controls over Grant Application Review and Award Process Need Improvement LSC controls over reviewing and awarding grants are intended to help ensure the fair and equitable consideration of applicants. Recently LSC has taken action intended to improve controls in this area. For example, LSC enhanced documentation of its grant application evaluation process through its 2010 Reader Guide. In addition, the LSC grants system contains detailed application evaluation questions based on the LSC Performance Criteria, and LSC has developed training materials and provided training to OPP personnel on the application evaluation process. However, at the time of our review, we found LSC’s controls over reviewing grantee applications and awarding grants were deficient in the following areas: documenting grant award decisions, carrying out and documenting management review of grant applications, and using automated grantee data available in the LSC Grants system. These deficiencies increase the risk that LSC may not be considering all relevant information in a consistent manner, limit LSC’s ability to explain the results of award decisions, and have resulted in incomplete and inaccurate information in the LSC grants grantee application evaluations. Grant Application Evaluations and Decisions Lacked Supporting Documentation LSC’s grant application evaluation process and basis for the resulting decisions were not clearly documented, including key management discussions in the evaluation-making process. According to the Standards for Internal Control in the Federal Government, all significant events should be clearly documented, and readily available for examination. We found LSC procedures did not require, nor did the staff maintain, a comprehensive record documenting (1) the extent to which management held discussions and considered all available, relevant information in the grant funding decision-making process for each applicant, and (2) that a complete record of the deliberative process (i.e., inputs, discussions, decisions made) was used, leading up to a grant application being funded or denied by LSC. Instead, LSC uses presentation notebooks, including multiple data sources, including grant applicant information, which are prepared for OPP staff funding recommendation presentations to OPP management and later for presentations to LSC management and the LSC President. Final grant award decisions are summarized in a chart initialed by responsible staff, LSC management and the President and individual grant award letters are certified by the LSC President. LSC’s procedures provided for documenting summaries of grantee application data. Specifically, LSC procedures required a one-page applicant overview and a two-page program summary for each applicant. OPP staff prepare the one-page applicant overview to document (1) information (such as poverty levels) about the applicant’s service area, (2) an overall score based on the reviewer’s evaluation, and (3) whether there are any special grant conditions, such as those due to prior grantee problems, including noncompliance with LSC regulations. OPP staff also prepare a two-page program summary that is to document their assessment of the grantee considering past performance as well as information in the application related to the following four performance areas: (1) effectiveness in identifying the most pressing civil legal needs of low- income people in the service area and targeting resources to address those needs, (2) effectiveness in engaging and serving the low-income population throughout the service area, (3) effectiveness of legal representation and other program activities intended to benefit the low-income population in the service area, and (4) effectiveness of governance, leadership, and administration. According to the Vice President for Programs and Compliance, while not explicitly required to do so by current LSC procedures, LSC officials also develop and use other data and analyses in addition to these two summary documents. Specifically, LSC staff prepare other relevant information and record the information in notebooks, such as the results of prior site visits. LSC staff use these notebooks to facilitate discussions with management about prospective grantee awards. However, the extent to which this other relevant information influenced award decisions was not documented. During a part of our review, we were not able to determine the extent to which the information in any of the notebooks we obtained was used or how it was considered in the funding decisions. LSC managers held a series of meetings where funding and award decisions were discussed. Following these meetings, LSC staff prepared a funding decision chart that was initialed by the Director of OPP, Vice President for Programs and Compliance, and the LSC President to document the final funding decisions. This chart, however, does not document how the managers’ consideration of various elements or relative risks contributed to the final decisions. Therefore, this lack of documentation of the factors considered in making these decisions increases the risks that grantee application evaluation and funding decisions may not consider all key, relevant information and makes it difficult to describe the basis for decisions later. Grant Application Evaluation Process Lacked Requirements for Managerial Review LSC has no requirement for carrying out and documenting OPP Director managerial review and approval of competitive grant evaluations or renewals by the OPP primary staff reviewers. According to the Standards for Internal Control in the Federal Government, control activities, such as conducting and documenting reviews, are an integral part of an entity’s stewardship of government resources and achieving effective results. Existing LSC guidance, such as the 2010 Reader Guide, provides that each application be reviewed against specific elements (derived from the LSC Performance Criteria and the ABA Standards for the Provision of Civil Legal Aid). The Guide is used in conjunction with an automated evaluation form in LSC Grants that reviewers use to record their assessments of each grant application. However, the guidance does not provide specific steps to carry out or document management review of the application evaluation in the LSC grants system. Consequently, the OPP grant application evaluations we reviewed lacked any evidence in LSC Grants that the OPP Director had reviewed them. The OPP Director did not sign any of the evaluation forms we reviewed in the LSC grants system, a key internal control activity. Specifically we selected a probability sample of 80 grantees from a population of 140, which encompassed 57 renewal applications and 23 competitive grant applications. We found that none of the 80 (100 percent) grant files contained any documentation demonstrating that managers had reviewed and approved the OPP staffs’ evaluation of the application. This lack of documented management review impairs LSC’s ability to identify gaps or incompatible data in the applications or evaluations prior to making the grant award. We found instances where an effective OPP manager’s review should have identified and corrected evaluation errors. For example, we identified 14 grant applications where the reviewer incorrectly identified projected expenses for the grant as matching the projected expenditures in another section of the application. Full LSC Grants System Capabilities Not Utilized LSC Grants is a computer-based application intended to assist LSC in data collection and review of applications submitted in response to an LSC Request for Proposal. However, because LSC’s Grants system lacked basic automated controls to ensure integrity over information in the system related to its grants application evaluation process, the system’s full capabilities were not utilized. The Standards for Internal Control in the Federal Government provide that entities should have application controls designed to help ensure the completeness and accuracy of transactions. Specifically, we found the data in LSC Grants was erroneous and inconsistent because the system did not have edit checks preventing the OPP staff reader from entering incomplete or incompatible data. Lacking complete and reliable grantee applicant evaluation data in LSC Grants, required LSC management to instead rely on inefficient, manual compilation and review of grantee application evaluation data in making decisions about whether to approve and fund a grantee. Our review found 7 of the 57 (12 percent) renewal grantees’ files had input fields that were blank and required information was not included. Similarly, we found 3 of the 23 competitive grantees (13 percent) where essential grantee evaluation data were not filled out. We also found numerous instances in both the renewal grantees, 15 out of 57 (26 percent) and competitive grantees, 6 out of 23 (26 percent), where grantees entered data in different parts of the grant application and the data were inconsistent. In addition, we found one grantee where the grant was to be funded with restrictions on the length of the grant term. However, the space where the reason for this restriction was required was left blank by the OPP staff. According to LSC, the evaluation process relies on both a qualitative and substantive analysis of an applicant’s proposal narrative to assess its capacity to provide high quality legal services. OPP staff’s judgment inherent in the substantive evaluation cannot be flagged or assessed by information validation fields. Nonetheless, LSC acknowledged the consistency and accuracy of information within the application can be addressed. LSC management also informed us that it is reviewing the LSC grants system for improvements. LSC’s external auditor’s 2008 report identified similar issues concerning inconsistent documentation of grantee evaluations. The auditor noted incomplete data in the grants system, used prior to LSC Grants, for 12 out of 32 grantee evaluations. The auditor recommended that the Office of Program Performance establish procedures to ensure that evaluation forms are properly completed before grant awards are made. Control Weaknesses Hinder LSC’s Ability to Oversee Grantees and Ensure Compliance While LSC recognized the importance of grantee site visits and had established overall policies and reasonable risk-based criteria to be used for such visits, it had not yet established detailed procedures on (1) conducting and documenting site visit selection, (2) timely completion of site visit reports, and (3) timely resolution of site visit recommendations and corrective actions. Control weaknesses hampered effective grantee site visits. These control weaknesses hinder LSC’s ability to effectively oversee its grantees’ compliance with LSC regulations and limits its ability to ensure grantees are visited according to their relative risk levels and that any compliance issues are identified and resolved in a timely manner. Risk Assessment Process for Program Visits Not Documented or Consistently Applied We observed good site visit planning techniques and interview execution in Philadelphia, Pennsylvania, and Indianapolis, Indiana. We also noted that LSC has an overall goal that provides for grantee site visits at least once every 3 years; however, LSC did not have procedures detailing how identified risks factors are to be used in a risk-based determination of which grantees should receive site visits by either OPP or OCE personnel. According to the Standards for Internal Control in the Federal Government, management’s internal control assessment should consider identified risks and their possible effect. By not formally documenting specific procedures on how risk assessment criteria are to be used in decisions about which sites to visit, LSC does not have adequate assurance that grantees with the greatest risk of noncompliance receive priority attention and oversight. In a prior GAO report, we recommended that LSC develop and implement an approach for selecting grantees for internal control and compliance reviews that is founded on risk-based criteria, uses information and results from oversight and audit activities, and is consistently applied. Although LSC has identified risk factors to consider,as of April 2010 it did not yet have procedures for how each risk factor is to be applied or considered when determining which grantee sites to visit. OPP officials told us that their program liaisons make recommendations for visits, which are reviewed by the three OPP regional teams (North, South and West). Then OPP meets as a group to discuss the teams’ recommendations and make preliminary recommendations for the next year’s visits. The OPP director and deputy director meet with the OPP regional teams when those recommendations are made and with all of OPP program staff to make final recommendations. After consultations with OCE, OPP’s recommendations are sent to the Vice President for Program Performance and Compliance. The deputy director and director approve the final list when they send it to the Vice President for Program Performance and Compliance for approval. However, we found no documentation demonstrating whether regional teams appropriately applied the risk factors, nor whether risk assessment results were summarized consistently in making the final recommendations for site visits. OCE Site Visit Reports Have Not Been Timely As shown in table 1, our review of all OCE site visit reports on grantee compliance, completed between October 2007 and July 2009, showed that 15 of 22 exceeded the 120 day goal set for reporting on grantee compliance. OCE’s Procedures Manual provides that OCE’s grantee compliance site visit final reports are to be issued within 120 days of each site visit trip’s completion. According to LSC, the OCE Procedures Manual was updated in April 2008 to establish a time frame of 120 days for completing site visits. Overall, our analysis showed that the average length of time required to complete the OCE site visit reports was about 150 days. Delays in formally communicating grantee site visit findings to grantees can delay grantees’ resolution of any internal control weaknesses (for example, if the grantees did not inquire about prospective income during client intake) and legal noncompliance issues identified during the site visits. Information on any continuing or serious internal control or compliance issues can be critical in making grantee funding decisions. According to LSC, there are informal means by which LSC informs grantees of preliminary findings. For example, OCE staff generally provides grantees with technical assistance in correcting compliance matters during site visits to facilitate immediate correction. LSC officials further stated that at the end of the visit staff hold an exit conference to advise the grantee of the preliminary findings and discuss how to make the necessary corrections. The LSC financial statement auditor also reported in 2010 that its review of OCE site visit reports found that 2009 grantee site visit reports were not issued on time, based on OCE’s 120 day goal. For example, the auditor reported that one out of the nine reports they sampled was issued 289 calendar days after the completion of fieldwork. One of the keys to completing timely OCE site visit reports within prescribed time frames is obtaining timely OLA opinions on LSC regulations. An LSC Director told us that site visit reports are held pending receipt of any requests to OLA for a legal opinion related to a possible noncompliance issue. However, LSC did not have specific procedures defining expected time frames and for overseeing OCE receipt of OLA opinions within such time frames. As of January 28, 2010, OLA had issued 47 opinions since January 2004. The average time elapsed from the date of the request for an OLA opinion and the issuance of the opinion was approximately 200 days. Of those 47 opinions, over 50 percent (25) took longer than 120 days to issue, with an average delivery time of approximately 334 days. As of January 28, 2010, two opinions had been outstanding for 721 and 603 days, respectively, and two other reports were not complete due to a pending legal opinion on prospective income, which was issued 465 days after being requested. LSC Did Not Track Resolution of Site Visit Recommendations for Improvement While our review found indications that cognizant LSC components share visit reports, LSC did not require and document its process for tracking and assessing actions in response to site visit recommendations and corrective actions. According to the Standards for Internal Control in the Federal Government, an entity’s internal control activities should include monitoring control improvement efforts. It further provides that such controls should assess the quality of performance over time and ensure the findings of audits and other reviews are promptly resolved. Over time, the trend of the number and types of findings, recommendations, and corrective actions, if analyzed and used appropriately, should provide information that could assist LSC management in determining and addressing any issues concerning the quality of grantee program performance and compliance. Consequently, the absence of required documented procedures for tracking OPP and OCE recommendations and corrective actions reduces LSC’s assurance that site visit results information is monitored for necessary corrective action and appropriately shared among cognizant LSC component organizations. According to an OIG manager and the OPP and OCE Directors, OPP and OCE share information on site visit recommendations through the LSC intranet—where site visit reports are posted. Although not required by LSC procedures, according to an LSC Director, OCE submitted site visit reports on grantee compliance—including recommendations and needed corrective actions—to OPP staff responsible for grant awards and monitoring of grantee program performance. According to LSC’s President, OPP staff are in regular contact with grantee executive directors and other program management and program engagement visits are often used as a vehicle for following up on recommendations. The OCE Director told us that OPP staff provided program quality information obtained through its review of site visit reports to OCE for consideration in grantee compliance reviews. Although staff may share information about site visits, an LSC official who is responsible for monitoring program performance told us that LSC does not consider or track whether recommendations are open or closed, but rather provides the recommendations as possible best practices for grantees to consider implementing as their programs develop. Therefore, an LSC Director told us that the site visit report recommendations are not tracked for remediation purposes or for trending and analysis by LSC because these recommendations are considered best practices which may or may not be implemented. The Vice President for Program Performance and Compliance said that OPP prioritizes the recommendations included in its reports and only includes what OPP believes to be the most important recommendations. By undertaking the effort to make recommendations and prioritizing them to highlight important areas, but not tracking their completion and analyzing the results, LSC is missing an opportunity to assess the extent of progress made and leverage the value of these recommendations. Performance Measures Could Be Better Aligned with Core Activities and Organizational Responsibilities LSC performance measures were not aligned with LSC’s core activities nor were they linked to specific offices responsible for making grant awards and monitoring grantee program performance and grantee compliance functions. Further, LSC did not have procedures in place to periodically reassess measures to ensure they are current. According to GAO’s Executive Guide: Effectively Implementing the Government Performance and Results Act, as a best practice, entities should assess performance to ensure that programs meet intended goals, assess the efficiency of processes, and promote continuous improvement. It further provides that performance measures should be linked directly to organizational components that have responsibility for making programs work and that routinely revisiting and updating an entity’s performance measures would help ensure they are relevant in providing feedback about whether the entity is achieving its current objectives. Performance measures that are not linked to the responsible office hinder accountability for program results, including the extent to which the LSC organizational components contribute toward LSC’s mission and where improvements are needed, and limit transparency and accountability to LSC’s Board on any organizational performance issues. LSC issued a Strategic Directions plan in 2006 laying out LSC’s performance measures. However, the plan’s performance measures did not account for the full range of LSC’s key grant awards and monitoring of grantee program performance and organizational grantee compliance responsibilities. For example, LSC’s plan did not include metrics to measure performance in the following core LSC activities related to its key grant awards or monitor grantee program performance and grantee compliance with respect to: identifying and targeting LSC’s own resources to address the most pressing civil legal needs of low-income individuals in the nation, and ensuring that grantees use the funding they receive to serve the low- income population throughout the nation. In addition, not all measures in LSC’s strategic plan were linked to specific LSC components. For example, LSC did not link scores on competitive grant evaluations with either OPP’s or OCE’s performance, even though these offices have responsibility for grantee program quality and compliance oversight. In addition, LSC did not link the performance measure number of technical assistance and training sessions conducted by LSC to the OPP organization even though OPP has organizational responsibility for such technical assistance. Further, we found LSC did not have procedures providing for periodic reassessment of key metrics to ensure they reflect up-to-date LSC mission priorities and objectives. According to the Chief Administrative Officer, LSC has recognized that its existing performance measures should be revised and periodically reassessed to ensure they are up-to-date and have begun actions in this regard. For example, since 2006, management has been developing a performance measure to obtain current information on “timeliness and degree of resolution of OCE corrective action notices.” LSC reviewed the results of a number of follow-up visits to confirm grantee resolution of OCE corrective action notices. The review found that the existing measure based on using the corrective action notices as an indicator of timeliness of resolution was insufficient. Instead, it was determined that without site visit verification of the resolution of original site visit findings the performance measure could not be reported on. LSC Management Has Not Consistently Focused on Key Human Capital Issues LSC’s employee handbook provides overall policy direction over its human capital practices. However, we found existing procedures were flawed in several key respects concerning staffing needs assessments, evaluating performance, and providing appropriate internal control training. Specifically, LSC did not (1) systematically assess short- and long-term workload and staffing needs in relation to the corporation’s strategic goals and objectives, (2) provide required performance reviews for OPP staff in 3 of the 6 years we reviewed and for OCE staff in 2 of the 6 years we evaluated, or (3) provide formal training for current and incoming staff on internal controls. Standards for Internal Control in the Federal Government provides that all personnel are to possess and maintain a level of competence enabling them to effectively accomplish their assigned duties. In addition, Human Capital Principles for Effective Strategic Workforce Planning provides that effective staffing assessments should provide short- and long-term strategies for acquiring, developing, and retaining staff to achieve programmatic goals. Strategic human capital practices are key to ensuring that an entity (1) has the staff capabilities needed to meet short- and long-term goals, (2) can effectively address performance problems, and (3) has staff who are trained in internal controls and related sound management practices. Our review found that LSC did not have procedures for assessing staffing needs. According to the Chief Administrative Officer and Director of Human Resources, LSC does not use mission priorities to establish staffing needs. Instead, the Vice President for Programs and Compliance said OPP and OCE consider workload needs and required staffing levels when preparing their budgets. According to the LSC employee handbook, LSC’s policy is that employee performance is to be evaluated annually at the beginning of the calendar year by the supervisor of record, based on job performance in the prior year. OPP staff stated that it is through the annual employee performance evaluation process that training needs are identified. However, LSC did not have procedures for ensuring review of employee performance and training. For calendar years (CY) 2003 and 2005, OPP and OCE personnel did not receive annual performance evaluations, and for CY 2008 OPP personnel did not receive performance evaluations. For 2003 and 2005, the Director of OHR stated that LSC did not follow its employee performance evaluation policy for conducting the required staff evaluations in 2003 and 2005 because of concerns about the appraisal process. As a result, LSC’s President suspended the appraisal process for these years. In 2008, according to the OPP director, OPP personnel did not receive appraisals because of a concern that evaluations would have to be done by a combination of people, none of whom had complete responsibility for overseeing the work throughout the year. Without the employee performance appraisals for all of its staff, LSC has limited its opportunities to encourage high performance, identify training needs, and communicate with staff. Controls over Contract Approval and Budgetary Tracking Were Not Always Effective Although LSC had policies requiring approval and funds availability determination before issuing contracts for its grant activities and programs, it had not established specific funds tracking procedures to ensure that necessary approvals were obtained and funds were available before awarding contracts. Lacking effective contract approval and fund availability controls, LSC is at increased risk of improper contract awards and undetected budget shortfalls. LSC’s Administrative Manual’s policy requires approvals from OLA, the Comptroller, and, if the contract is over $10,500, the President, before contract award. However, our review found that LSC did not obtain contract approvals by OLA, the Comptroller, and LSC President—a critical accountability control—for any of the nine contracts over $10,500 issued in fiscal years 2008 and 2009. Our review of the nine contracts that exceeded the $10,500 presidential approval threshold revealed that LSC lacked any documentation showing that the required Contract Approval Form was completed before the contracts were awarded. The LSC Chief Administrative Officer (CAO) told us that verbal approvals were given by the President for five of the contracts. Of the remaining four contracts, one had the LSC President’s approval on the contract itself (but not the contract approval form), while the remaining three LSC contracts did not have any evidence of approvals. The LSC Administrative Manual, issued in February 2005, requires review and approval of all contracts before award by (1) office directors to ensure that they are within budgetary limitations; (2) OLA for legal assurance; (3) the Comptroller to ensure the requirements of the Administrative Manual were followed and to start a purchase order; and (4) if over $10,500, the LSC President. In accordance with the LSC Administrative Manual, a Contract Approval Form, which shows all approvals by designee signature, must be used to meet documentation requirements and be retained for all contracts awarded. Two contracts that did not follow LSC’s approval process resulted in an unplanned budgetary adjustment for fiscal year 2009. Specifically, we found two Office of Information Technology (OIT) contracts supporting grants management and administration that were not properly authorized and for which fund availability was not determined prior to contract award, which resulted in a LSC budget shortfall of over $70,000 in fiscal year 2009. According to the Director of OIT, after verbal approval by the LSC CAO, these contracts were executed by the Director of OIT without taking any action to determine that sufficient monies were available to fund the contracts, and without obtaining the required prior approval of OLA, the Comptroller, and the LSC President. LSC’s Comptroller informed the Board of Directors, President, and Inspector General of OIT’s overspending and asked for and received a $70,000 internal budgetary adjustment on August 31, 2009, to transfer budgeted funds from LSC’s capital expenditures account to the consulting budget. Consistent with our findings, the LSC financial statement auditor reported in its January 2010 Report of Deficiencies in Internal Control Over Financial Reporting and Other Matters for 2009 that the Contract Approval Forms were not used as required by the LSC Administrative Manual, and there was no evidence of approval by OLA. The auditors recommended in January 2010 that LSC implement procedures to ensure policies for contract awards are followed. LSC recently revised its Administrative Manual, effective October 1, 2009, to include a Contract Approval Form, with a provision that the LSC President approve all contracts over $10,500. Further, the LSC CAO stated that training was provided for all administrative staff on the proper procedures to follow for processing contracts. Such training should help ensure that a Contract Approval Form accompanies all LSC contracts, and that OLA and the Comptroller both review and document approval of all contracts and sign off on the Contract Approval Form before contract execution. However, the training may be of limited value unless LSC also establishes specific, detailed procedures on the steps required to ensure that all necessary approvals and fund availability certification is carried out and documented. Conclusions Effective governance, accountability, and internal control are key to maintaining public trust and credibility. As such, identifying and implementing effective internal controls will assist LSC in ensuring that the federal funds LSC receives are being used efficiently and effectively. LSC has taken actions to improve its governance and accountability practices by implementing or partially implementing all 17 of the recommendations from our August 2007 and December 2007 reports. Progress continues since our prior testimony in October 2009 as LSC has implemented two additional recommendations and continues to take actions on the remaining recommendations. However, several key recommendations related to LSC’s grantee oversight responsibilities remain to be fully implemented. The control deficiencies we identified, along with the continuing nature of several related deficiencies first identified nearly 3 years ago, are indicative of weaknesses in LSC’s overall control environment. A weak control environment limits LSC’s ability to effectively manage its grant award and grantee performance oversight responsibilities. As such, it will be important for the LSC President and Board of Directors to continue to set a “tone at the top” supportive of establishing and maintaining effective internal control not only by managers but also by personnel throughout the entity’s program operations. In this regard, LSC would benefit from an entitywide internal control assessment, including whether the risks associated with grantee selection are effectively considered, past recommendations and corrective actions are properly tracked, and whether effective controls are in place over performance measurement, performance evaluation, and contract awards. LSC could also strengthen its overall control environment by providing training to staff throughout the entity on how internal controls, when functioning as intended, are integral to the achievement of the entity’s mission objectives. In the near term, it will be important for LSC leadership to direct immediate action to address the continuing weaknesses, as well as those identified in our current review. For the long term, LSC will need to focus on monitoring the sustained commitment to an effective overall system of internal controls necessary to achieve a solid basis for effectively accomplishing its core mission of enabling the grantees to provide legal services to individuals who otherwise could not afford such services. Recommendations for Executive Action In order to improve key control processes over grant awards and monitoring of grantee program performance and grantee compliance, we recommend the President of LSC, and the Vice President for Programs and Compliance, take the following 17 actions: Grant Application Processing and Award Develop and implement procedures to provide a complete record of all data used, discussions held, and decisions made on grant applications. Develop and implement procedures to carry out and document management’s review and approval of the grant evaluation and award decisions. Conduct and document a risk-based assessment of the adequacy of internal control of the grant evaluation and award and monitoring process from the point that the Request for Proposal is created through award, and grantee selection. Conduct and document a cost benefit assessment of improving the effectiveness of application controls in LSC Grants such that the system’s information capabilities could be utilized to a greater extent in the grantee application evaluation and decision-making process. Develop and implement procedures to ensure that grantee site visit selection risk criteria are consistently used and to provide for summarizing results by grantee. Establish and implement procedures to monitor OCE grantee site visit report completion against the 120 day time frame provided in the OCE Procedures Manual. Execute a study to determine an appropriate standard timeframe for OLA opinions to be developed and issued. Develop and implement procedures to monitor completion of OLA opinions related to OCE site visits against the target time frame for issuing opinions. Develop and implement procedures to provide a centralized tracking system for LSC’s recommendations to grantees identified during grantee site visits and the status of grantees’ corrective actions. Develop and implement procedures to link performance measures (1) to specific offices and their core functions and activities, and (2) to LSC’s strategic goals and objectives. Develop and implement procedures for periodically assessing performance measures to ensure they are up-to-date. Develop and implement procedures to provide for assessing all LSC component staffing needs in relation to LSC’s strategic and strategic human capital plans. Develop and implement a mechanism to ensure that all LSC staff receive annual performance assessments. Develop and implement a process to monitor contract approvals to ensure that all proposed contracts are properly approved before award. Develop and implement procedures for contracts at or above established policy thresholds, to ensure the LSC President provides written approval in accordance with policy before contract award. Develop and implement procedures to ensure budget funds are available for all contract proposals before contracts are awarded. Develop and implement procedures for providing and periodically updating training for LSC management and staff on applicable internal controls necessary to effectively carry out LSC’s grant award and grantee performance oversight responsibilities. Establish a mechanism to monitor progress in taking corrective actions to address recommendations related to improving LSC grants award, evaluation, and monitoring. Agency Comments and Our Evaluation We provided copies of the draft report to LSC’s management for comment prior to finalizing the report. We received a written comment letter from LSC’s President on behalf of LSC’s management (see appendix III). In its written comments, LSC agreed with our findings and recommendations and identified specific actions it has taken and plans to take to implement these recommendations. LSC also provided technical comments which we considered and incorporated as appropriate. As agreed with your office, unless you publicly announce the contents of this report earlier, we will not distribute it until 30 days from its date. At that time, we will send copies of the report to other appropriate congressional committees and the president of LSC. This report will also be available at no charge on the GAO Web site at http://www.gao.gov. If you have any questions or would like to discuss this report, please contact me at (202) 512-9095 or by e-mail at raglands@gao.gov. Major contributors to this report are listed in appendix IV. Appendix I: Objectives, Scope, and Methodology Our reporting objectives were to determine the extent to which the Legal Services Corporation (LSC) properly implemented key internal controls in awarding grants and overseeing grantee program performance; measured its performance in awarding grants and overseeing grantees; evaluated staffing needs for grant awards management and grantee performance oversight; and followed appropriate budget execution processes for awarding contracts related to grants award and grantee performance and oversight. To address the first two objectives, we interviewed current members of LSC’s management and staff, staff in LSC’s Office of Inspector General (OIG), and the audit firm employed by the OIG to obtain information on the functions and processes of LSC’s grant awards and monitoring of grantee program performance and grantee compliance. We also reviewed LSC documentation on internal control activities related to the awarding of grants and oversight of grantee programs, including policy manuals, audit reports, and management reports. In addition, we selected a probability sample of 80 out of 140 grantees and reviewed related grant applications and application evaluations (for the 2009 funding year), and compared evaluation results with instructions in LSC Grants, a computer-based grants application system. Results based on probability samples are subject to sampling error. The sample we drew for our review is only one of a large number of samples we might have drawn. Because different samples could have provided different estimates, we express our confidence in the precision of our particular sample results as a 95 percent confidence interval. This is the interval that would contain the actual population values for 95 percent of the samples we could have drawn. All survey estimates in this report are presented along with their margins of error. We analyzed the document setting out LSC-wide and component-specific goals and performance measures and compared this to federal guidance on performance measurement. We also observed LSC site visits at two grantees in Philadelphia and Indianapolis. To obtain information on LSC controls for assessing staffing needs for its grants functions, we interviewed LSC management and reviewed policies and procedures for evaluating staffing needs, training, and professional development, and reviewed relevant literature. We compared LSC’s staffing needs assessment processes to federal best practices in workforce planning principles. To obtain information on controls over contract approval and budget execution, we reviewed LSC’s administrative policy and procedure manual and consolidated operating budget guidance, documented budget execution requirements, and tested contracts for proper approval. For each of our objectives, we compared the information obtained with federal best practices in internal control in GAO’s Standards for Internal Control in the Federal Government. We conducted our work in Washington, D.C.; Indianapolis, Indiana; and Philadelphia, Pennsylvania, from March 2009 to May 2010 in accordance with generally accepted government auditing standards. Those standards require that we plan and perform the audit to obtain sufficient appropriate evidence to provide a reasonable basis for our findings and conclusions. We believe the evidence obtained provided a reasonable basis for our audit findings and conclusions. Appendix II: Prior GAO Reports Recommendation Status Our August 2007 report recommendations to improve and modernize the governance processes and structure of LSC, along with our views on the status of LSC’s efforts to implement these recommendations (as of March 2010), are summarized in table 2. LSC data, which we obtained and analyzed as part of our follow-up work conducted between May 2009 and March 2010, showed that the board had fully implemented five of the eight recommendations, and had taken some action on the remaining three recommendations. Our August 2007 report recommendations to improve and modernize key management processes at LSC, along with the status of LSC’s efforts to implement those recommendations (as of March 2010), are summarized in table 3. Our December 2007 report recommendations to improve LSC’s internal control and oversight of grantees, along with our views on the status of LSC’s efforts to implement those recommendations (as of March 2010), are summarized in table 4. Appendix III: Comments from Legal Services Corporation Appendix IV: GAO Contact and Staff Acknowledgments Acknowledgments In addition to the contact named above, Kimberley A. McGatlin, Assistant Director; Lisa Crye; Patrick Frey; Cole Haase; Bernice M. Lemaire; Mitch Owings; Melanie Swift; and Carrie Wehrly made key contributions to this report. F. Abe Dymond, Lauren S. Fassler, and Justin Fisher provided technical assistance.

The Legal Services Corporation (LSC) was created as a private, nonprofit corporation to support legal assistance for low-income individuals on civil legal matters, primarily through federal grants and is primarily funded through federal appropriations. Effective internal controls over grant awards and oversight of grantees' performance are critical to LSC's mission. GAO and the LSC Inspector General have previously reported weaknesses and made recommendations. GAO's objectives for this report were to determine the extent to which LSC (1) implemented key internal controls in awarding and overseeing grantees, (2) measured its performance, (3) evaluated staffing needs, and (4) adhered to its budget execution processes. GAO analyzed key records and prior recommendations as well as interviewed LSC officials regarding LSC's internal control and performance frameworks, staffing, and contract processes. Although LSC's controls over reviewing and awarding grants are intended to help ensure fair and equitable consideration, they need improvement. Final award and fund decisions are documented and approved; however, LSC's grant application evaluation process and associated decisions were not documented, including key management discussions in the evaluation process. This lack of documentation of factors considered in making these decisions increases the risk that grantee application evaluation and funding decisions may not consider all key relevant information and makes it difficult to describe the basis for decisions later. In addition, LSC has no requirement for carrying out and documenting managerial review and approval of competitive grant evaluations or renewals, limiting its ability to identify gaps or incompatible data in applications. Although LSC has efforts underway to ensure it visits all grantee sites at least once every 3 years, LSC did not consistently or explicitly document the application of risk criteria when selecting which grantees to visit, complete timely site visit reports, or track the recommendations from the site visits. These weaknesses hindered LSC's ability to effectively oversee grantees. LSC is not required to follow the Government Performance and Results Act but has developed a Strategic Directions document with some performance measures. However, these measures do not reflect all of LSC's core activities and are not linked to its two primary offices for awarding and overseeing grants. Therefore, LSC cannot effectively measure its performance in several key dimensions, such as identifying and targeting resources in addressing the most pressing civil legal needs of low-income individuals across the nation. LSC has not systematically assessed its long-term staffing needs to achieve strategic goals and objectives, which could help ensure it has the staff capabilities needed to meet its short- and long-term goals. LSC has not consistently provided performance reviews for all of its staff, limiting opportunities to encourage high performance, identify training needs, and communicate with staff. At times, LSC did not adhere to its budget execution process in awarding contracts supporting its key grant-making responsibilities. Because officials did not follow LSC's approval controls for two contracts and there was a breakdown in tracking funds, LSC had a budget shortfall of $70,000 in 2009. Missing or flawed internal controls limit LSC's ability to effectively manage its grant award and grantee performance oversight responsibilities. Although LSC has taken steps to address all 17 GAO recommendations identified in prior work, several have yet to be fully addressed. In the near term, it will be important for LSC leadership to address both current and continuing weaknesses. For the long term, LSC will need to focus on strengthening its overall system of internal controls in order to establish a solid basis for effectively accomplishing its core mission.

gov_report

Phleboviruses transmitted by sandflies are endemic in the Mediterranean area. The last decade has witnessed the description of an accumulating number of novel viruses. Although, the risk of exposure of vertebrates is globally assessed, detailed geographic knowledge is poor even in Greece and Cyprus where sandfly fever has been recognized for a long time and repeatedly. A total of 1,250 dogs from mainland Greece and Greek archipelago on one hand and 422 dogs from Cyprus on the other hand have been sampled and tested for neutralising antibodies against Toscana virus (TOSV), Sandfly fever Sicilian virus (SFSV), Arbia virus, and Adana virus i. e. four viruses belonging to the 3 sandfly-borne serocomplexes known to circulate actively in the Mediterranean area. Our results showed that (i) SFSV is highly prevalent with 71. 9% (50. 7–84. 9% depending on the region) in Greece and 60. 2% (40. 0–72. 6%) in Cyprus; (ii) TOSV ranked second with 4. 4% (0–15. 4%) in Greece and 8. 4% (0–11. 4%) in Cyprus; (iii) Salehabad viruses (Arbia and Adana) displayed also substantial prevalence rates in both countries with values ranging from 0–22. 6% depending on the region and on the virus strain used in the test. These results demonstrate that circulation of viruses transmitted by sand flies can be estimated qualitatively using dog sera. As reported in other regions of the Mediterranean, these results indicate that it is time to shift these viruses from the" neglected" status to the" priority" status in order to stimulate studies aiming at defining and quantifying their medical and veterinary importance and possible public health impact. Specifically, viruses belonging to the Sandfly fever Sicilian complex should be given careful consideration. This calls for implementation of direct and indirect diagnosis in National reference centers and in hospital microbiology laboratories and systematic testing of unelucidated febrile illness and central and peripheral nervous system febrile manifestations. In the Old world, phleboviruses (Bunyaviridae family, Phlebovirus genus) transmitted by phlebotomines consist of three species or antigenic groups, namely Sandfly fever Naples, Salehabad, and Sandfly fever Sicilian serocomplexes. Each species contains several viruses among which Naples, Sicilian and Toscana virus cause 3-day fever, commonly called sandfly fever in humans; Toscana virus (TOSV) causes of neuroinvasive human infections such as meningitis and encephalitis [1]. In Greece, outbreaks of sandfly fever were reported in Athens among the local population, and among American, British and German troops during World War II [2]. Sandfly fever has been described in Cyprus and Greece with both sporadic cases and epidemics [3–7]. In both countries, the high rates of antibodies observed in seroprevalence studies indicate that viruses belonging to Sandfly fever Naples and Sandfly fever Sicilian serocomplexes are transmitted by local sand flies to human populations [7–10]. Sandfly fever Cyprus virus (SFCV), closely related to Sandfly fever Sicilian virus (SFSV), was isolated during a large outbreak of sandfly fever in Swedish United Nations troops stationed in Cyprus; few cases were also caused by TOSV [5]. In Greece, in recent sporadic cases of meningitis, (i) TOSV RNA was detected in the CSF of a patient [11], and (ii) viral RNA corresponding to Adria virus, a novel virus belonging to the Salehabad species, was also identified in the CSF [12,13]. To date, SFSV or another SFS-like virus have been neither isolated nor detected by molecular techniques in Greece. During the last decade, field-to-laboratory integrated studies associating virologists, parasitologists and entomologists have discovered several new phlebotomine-borne phleboviruses; thus there is an increased diversity in each of the three aforementioned species or serocomplexes [14]. Although the pathogenicity of most of these newly discovered viruses remains unknown, they are sympatric with recognized pathogenic phleboviruses [15–17]. Because several viruses of the same serocomplex co-circulate in various regions, interpretation of seroprevalence studies requires using techniques that hold the capacity to discriminate between these antigenically-related viruses. To the best of our knowledge, all studies performed in Greece and Cyprus used either ELISA or IFA tests, which are notoriously prone to cross-reactivity between viruses belonging to the same serocomplex [7–10]. To conduct our nation-wide (mainland Greece, Greek islands, Cyprus) seroprevalence study in dogs, we selected neutralisation tests which is the most discriminant assay as previously reported in Algeria, Tunisia, Turkey and Portugal [15,18–20]. Although virus exposure to viruses may be quantitatively different in humans and dogs, because of different feeding preferences of phlebotomines, recent studies suggest that virus circulation can be estimated using either human or dog sera since dogs live in close proximity to humans and are readily infected by these viruses [15,18,20,21]. In our study, dog sera were tested for the presence of neutralising antibodies against TOSV, SFSV, and two viruses belonging to the Salehabad complex (Arbia virus isolated in Italy and Adana virus isolated in Turkey). From 2005 to 2010, a total of 422 and 1,250 dog sera were collected in Cyprus and Greece, respectively. These sera originated from the five districts of Cyprus and 32 prefectures belonging to 12 regions of Greece (Table 1). Veterinarians were asked to provide dog samples from animals visiting their clinic for any reason: vaccination, hair cut, nail cut, deworming, general check up, treatments, and other purposes, without discrimination. The animals were examined clinically and peripheral blood samples (without EDTA) was collected, after the written consent of the owner, and questionnaires with personal, epidemiological, and clinical data for each dog were completed. Only domestic dogs that were raised in the area were considered for the study. The domestic dogs were included after owners’ informed consent. Information regarding age, sex, was obtained after interviewing dog owners (Table 1). Each dog was examined clinically by the veterinarian and blood samples were collected. Whole blood samples were collected (1–2 mL) by cephalic or jugular venipuncture and serum was separated by centrifugation and stored at −20°C. Data on the region, gender, and age (distributed according to 3 classes: young 6–11 months, adult 12–83 months, senior ≥ 84 months) were recorded. This study was ethically approved by the Institutional Animal Care and Use Committee of the University of Crete Medical School and conform with the European Union Directive 2010/63/EU regarding use of animals and biological specimens in research, as well as the relevant Hellenic legislation (Presidential Decree 160/91, under the Code Numbers 31 EE 05,31 EPR 04 and 31EP 020). Written informed consent was obtained from the dog owners, according to the aforementioned national legislations. Sera were tested by the virus microneutralisation assay (MN), described for phleboviruses [19] in parallel for 3 distinct sandfly-borne phleboviruses: (i) TOSV strain MRS2010-4319501 (TOSV belongs to the Sandfly fever Naples virus species or complex) [22], (ii) SFSV strain Sabin [23], (iii) Arbia-like virus strain T131 (Salehabad species or complex), and (iv) Adana virus strain 195 (Salehabad species or complex) [15]. Briefly, two-fold serial dilutions from 1: 10 to 1: 80 were prepared for each serum and a volume of 50μL was pipeted into 96-well plate. Viruses were titrated in Vero cells (ATCC CCL81). A volume of 50 μL containing 1000 TCID50 was added into each well except for the controls that consisted of PBS. A volume of 50 μL of EMEM medium enriched with 5% fetal bovine serum, 1% Penicilin Streptomycin, 1% L-Glutamine 200 mM, 1% Kanamycin, 3% Fungizone, was added to each well of the controls. The plates were incubated at 37C° for one hour. Then, a 100μL suspension of Vero cells containing approximately 2 x105 cells/mL of EMEM medium (as previously described) was added to each well, and incubated at 37C° in presence of 5% CO2. The first row of each plate contained control sera diluted 1: 10 and Vero cells without virus. After 5 days (Toscana and Arbia virus) and 7 day (Sicilian and Adana virus), the microplates were read under an inverted microscope, and the presence (neutralization titer at 20,40,80 and 160) or absence (no neutralization) of cytopathic effect was noted. Cut-off value for positivity was set at titre ≥ 20 [15,18,20,21]. Due to insufficient volume in Greek samples, ADAV was used for testing Cyprus specimens, only. Dog seroprevalence for each virus was estimated for each prefecture and mapped using the geographical information system software (GIS, Redlands, CA; ArcGIS 10). The chi-square or Fisher’s exact tests were used to compare percentages of positivity among categories of the same independent variables and also the total prevalence of each virus. A p value < 0. 05 was considered as statistically significant. Analyses were performed with StatLib and SPSS® 21 software for Windows. In Greece, a total of 1,250 sera (540 male and 710 female, sex ratio 0. 76) were collected. The median age was 36 months (range: 3–216). The sera were collected from 32 prefectures, but owing to the variability in the number of collected sera from each prefecture (range: 1–410), the sera were grouped into 12 regions. Of these 12 regions, Thessaly was not included in the analyses because it consisted of 1 serum only. For the other 11 regions, the number of sera ranged from 14 to 410. In Cyprus, a total of 442 sera (202 male and 240 female, sex ratio 0. 84) were collected. The median age was 36 (range: 3–144). They consisted of 67,27,97,74, and 177 sera collected from the districts of Ammochostos, Larnaca, Limassol, Nicosia, and Paphos, respectively. The two dog populations had the same median age (36 months) and a similar sex ratio (0. 76 vs 0. 84). Characteristics of the dogs and their geographic origin are presented in Table 1. Results for domesticated dogs living in Greece and in Cyprus are presented in Table 2 and Table 3, respectively. Toxic activity in the serum was detected in 65 and 73 sera from Greece and Cyprus, respectively; therefore calculations were done on the basis of 1,185 and 369 sera of Greece and Cyprus, respectively. As previously shown [26,27], a cut-off titre ≥ 20-when used for 1000TCID50 inoculum, is equivalent to a cut-off titre ≥ 40 when a 100 TCID50 is used [28]. In Greece, a much higher rate of SFSV-NT-Ab was observed (71. 9%) compared with 4. 4% and 2. 6% for TOSV and ARBV, respectively (Table 1 and Fig 1). Similar results were observed in Cyprus where SFSV-NT-Ab was present in 60. 2% of the dog sera, whereas 16. 3%, 8. 4% and 5. 4% of sera were positive for ADAV, TOSV and ARBV, respectively (Table 2 and Fig 2). The distribution of TOSV positive sera is quite homogenous within the studied regions (p = 0. 248, p = 0. 094). There is no significant difference according to the sex of the dogs. In contrast, it appears that the prevalence increases with the age, although it is not statistically significant even when the results of dogs from Greece and Cyprus are merged (3. 7% / 5. 3% / 7. 7%, p = 0. 3). In both countries, a statistic association was found between SFSV prevalence and geographic area. In Cyprus, none of the sera containing ARBV-NT-Abs were also positive for ADAV-NT-Abs and vice versa; this demonstrated that there is no cross-reactivity through MN assay between these two viruses despite the fact that they belong to the same serocomplex. Exposure to ARBV and ADAV is significantly associated with the age with higher rates observed in older dogs. In contrast, the high rates of SFSV-NT-Abs were observed in the 6-11-month age class in Cyprus and Greece. At the outset of our study, the following data were available for Greece: (i) there was no serological data in domestic animals (cattle, goats, sheep, dogs or cats) for any phlebovirus transmitted by sand flies, (ii) in the 1970' s, 13. 1% of 38 adults living in Crete had NT-Abs against Naples virus, and 24. 7% and 8. 5% of 632 human sera from Athens inhabitants had NT-Abs against Naples virus and Sicilian virus, respectively [29]; (iii) more recent studies reported various rates of TOSV IgG using ELISA and/or IIF tests in continental Greece as well as in the Ionian and Aegean islands [8–10]; (iv) TOSV RNA (belonging to the lineage C) was detected in the CSF of a patient with meningitis [11], and (v) viral RNA corresponding to Adria virus, a novel virus belonging to the Salehabad species, was also identified in the CSF of a patient with meningitis [12,13]. To date, SFSV or another SFS-like virus have been neither isolated nor detected by molecular techniques in Greece. At the outset of our study, the following data were available for Cyprus: (i) there was no serological data in domestic animals (cattle, goats, sheep, dogs or cats) for any phlebovirus transmitted by sand flies; (ii) first evidence of the presence of TOSV, Naples and Sicilian viruses were observed in Swedish soldiers of the United Nations force [3] through detection of NT-Abs and isolation of strains of Naples and Sicilian viruses [30]; (iii) NT-based seroprevalence results showed that Naples, Sicilian, and TOSV were endemic with respective rates of 57%, 32% and 20% [7]; (iv) investigation of a second outbreak in Greek troops stationed in Nicosia of which almost 50% developed febrile syndrome had resulted in isolation of Cyprus virus (SFCV), closely related but distinct from Sicilian virus although belonging to the SFSV serocomplex [4,5]. The recent discovery of several new viruses belonging to the three species associated with phlebotomines in the Old World has raised questions about the viral strain currently circulating in the two regions. Since broadly cross-reactive techniques such as ELISA and IIF are not capable to distinguish between viruses belonging to the same serocomplex, we decided to use microneutralisation assay using viral strains or surrogates which presence had been assessed in Greece and Cyprus. Indeed, we consider it valid to use SFSV as a surrogate for SFCV (isolated in Cyprus) and other SFSV-related viruses because amino acid distances observed between the proteins that elicit neutralizing antibodies (Gn and Gc) are well within the acceptable range, ie <5% different for SFSV and SFSV-related viruses[25,31]. Thus, neutralising antibodies are unlikely to discriminate between closely-related SFSV isolates. Since collecting human sera displaying a large geographic distribution was challenging, we decided to use dog sera; indeed, dog sera can be good surrogates for the following reasons: (i) dogs are readily infected with phlebotomine-borne phleboviruses which are human pathogens [18,20,31,32]; (ii) domestic dogs live in close contact with humans and therefore are exposed to sandfly bites, although different feeding preferences of sand fly species have to be considered [31]. The largest amount of data available on dogs, at the outset of this study, concerned TOSV, which observed rates (4. 4% in Greece and 8. 4% in Cyprus) are in the same order of magnitude as those recently reported in dogs in Tunisia (6. 8%, [20]), in Algeria (4. 3%, [18]), in France (3. 9%, [21]) and in Portugal (6. 8% [31]. Because all these studies measured neutralising antibodies against TOSV, their results are comparable and they reflect local circulation of TOSV only, not other viruses belonging to the SFNV complex. Together these results demonstrate that TOSV can readily infect dogs. Exposure level of dogs and humans may be drastically different in the same area as previously shown in Tunisia where MN-based seroprevalence rates were respectively at 6. 8% in dogs compared with 41% in humans [20]. In the present study, dogs living in the Ionian island of Corfu showed a much lower seroprevalence compared to the human population living in the same island (3. 9% vs 51. 7%) [8]; however, in this case the techniques used were different; in the human study, ELISA/IIF detected not only TOSV IgG but also IgG raised after infection with other viruses belonging to the Sandfly fever Naples serocomplex in which 6 new viruses were described during the last decade (Arrabida, Fermo, Granada, Massilia, Punique, Zerdali) [16,33–37] in addition to Naples virus (a proven human pathogen) and Tehran virus. Although none of these viruses were detected or isolated in Greece or Cyprus, the presence of one of these 6 recently discovered viruses or of a yet to be discovered virus may account for these apparently discrepant results. Last, these techniques do not hold the same sensitivity [38]. The same explanation applies for discrepancies observed between high rates of ELISA/IFA TOSV IgG reported in Aegean islands (17. 6%, 11. 5%, 20%, 22% and 34. 7% for Lesbos, Rodos, Siros, Crete and Evia, respectively) [9] compared with our findings: 5. 3% in north Aegean islands (Chios and Lesbos), 0% in south Aegean islands (Rodos, Siros and Santorini), 1% in Crete and 4. 4% in Evia (Stere Hellas) (Table 2). In Central Macedonia, 7. 3% of dog sera contained TOSV-NT-Abs, which is in agreement with reported cases of human infections [38,39] and a recent study showing that TOSV and/or antigenically related viruses are circulating extensively in the area [10]. It is worth underlining that, despite using the same technique, discrepant prevalence rates were also described between dogs and humans in Tunisia [19,20]. Therefore, it is difficult to compare results of serological studies performed with different techniques. When using the same technique, results observed in humans and in dogs consistently detected TOSV although they varied considerably quantitatively; therefore dogs can serve as sentinel for humans and vice versa for assessing the presence of TOSV although quantitative results must be interpreted carefully. The absence of cross-protection between ARBV-NT-Abs and ADAV-NT-Abs confirm previous data from Turkey [15]. Accordingly, cumulative percentage of viruses belonging to the Salehabad species is 21. 7%. This is congruent with the results observed in Adana, southern Anatolia, Turkey where domestic animals were presenting high rates of NT-Ab against viruses belonging to the Salehabad serocomplex [15]. Tesh et al [29] did not detect NT-Abs against Salehabad virus (SALV) in human populations suggesting that SALV was not infecting humans. In contrast, NT-Abs against Medjerda Valley virus were described in 1. 35% (14/1260) of human sera collected from the general population living in Northern Tunisia [24]. This suggests that at least some viruses belonging to the Salehabad complex can infect humans and other vertebrates. Interestingly, Adria virus RNA has been detected in the CSF of a Greek patient presenting with meningitis [13] but was never isolated precluding serological studies aiming at defining the possible impact of this virus in the region and beyond. However, molecular detection of Adria virus in Albania (in sand flies) and in Greece (in human) suggests that its distribution might cover a large geographic area. This constituted the first direct evidence supporting human pathogenicity of a virus belonging to the Salehabad virus complex. Isolation of Adria virus is now a priority in order to pursue the studies using neutralization-based serological studies in humans and animals. Very high rates of SFSV-NT-Abs were observed in Cyprus and Greece. In the latter, rates were consistently above 50% (range 50. 7–84. 9%); in Cyprus, rates were above 40% (range 40. 0–72. 6%) except in Ammochostos (26. 3%). The extremely high prevalence rates observed with SFSV in young dogs show that this virus continues to circulate very actively in these regions, and beyond as recently described in dogs from Tunisia (50. 8%, [20]) and in Portugal (38. 1%, [31]). In both countries, a statistic association was found between SFSV prevalence and geographic area. The differences of prevalence depending upon the region may be due to the geographic and climatic characteristics of these regions which affect the distribution, proliferation and abundance of phlebotomine vectors of SFSV. Analysis of the questionnaires did not identify any clinical manifestations such a fever and/or neurological signs during the past weeks and months in the SFSV-positive dogs. This tends to suggest that SFSV is not or mildly affecting dogs during the viremic period. Whether or not dogs can play a role as reservoir in the natural cycle remains to be studied. To do so, experimental studies to understand the virus kinetics are necessary. Also, studies aiming at the identification of viremic domestic dogs should be planned in high prevalence areas. The massive prevalence of SFSV-NT-Ab observed in our study is not unexpected and is congruent with entomological and human data in the literature: (i) isolation of Corfu virus on the eponymous island from Phlebotomus neglectus [17]; (ii) SFSV IgG detected by IFA in human sera in Northern Greece (Macedonia), Central Greece (Evritania and Larisa), North–Western Greece (Epirus), and Corfu Island; (iii) detection of Chios virus, SFSV-like, in Chios island; (iv) sandfly fever epidemics were reported in Swedish UN soldiers and Greek soldiers in 1984 and 2002, respectively [3–5]; (v) a high attack rate (63%) in tourists hosted in Cyprus for a short period [6]; (vi) a 32% prevalence rate of SFSV IgG in Cyprus native population [7]. In contrast with the two other serocomplexes which display an important range of genetic distance between their respective members, Sicilian virus strains are genetically and antigenically much more closely related [14,16]; therefore, exposure to different SFSV strains (Italy, Turkey, Cyprus, Greece, Ethiopia) can be measured by using the prototypic Italian strain. Despite high rates of antibodies in humans and other vertebrates and successive outbreaks in Italy, Cyprus, Greece and Ethiopia [3,5, 23,40], SFSV remains a neglected pathogen, almost never included in diagnostic algorithms despite repeated and accumulating evidence of its involvement in febrile syndromes and in neuroinvasive infections. In conclusion, this study indicates that (i) sandfly-borne phleboviruses belonging to 3 distinct genetic and antigenic groups are widely spread and co-circulate; (ii) dogs represent excellent qualitative sentinels for virus transmitted by sandflies and further studies must be done to estimate the role of dogs in the dynamics of transmission, and whether they play a role as reservoir hosts in the natural cycle of these viruses. Since several of these viruses are proven human pathogens, our results plead for performing similar studies using human sera to identify geographic hot spots. The increasing number of sequence data for these phlebotomine-borne phleboviruses now enables to design and develop real-time molecular assays. The improved diagnostic toolbox will allow to investgate the medical impact of these viruses in patients presenting unexplained febrile illness and neuroinvasive infections.

Phleboviruses transmitted by sandflies are endemic in the Mediterranean basin. An increased number of new viruses was described during the last decade. However, levels of exposure of human and animal populations are poorly known. A total of 1,250 dogs from Greece and 422 dogs from Cyprus were tested for the presence of neutralising antibodies signing previous infection with selected phleboviruses representing the 3 serological complexes known to be present in the Old World: Toscana virus (TOSV), Sandfly fever Sicilian virus (SFSV) and Salehabad viruses (Arbia and Adana viruses). Our data showed that (i) SFSV is largely predominant with infection rates higher than 50%, (ii) TOSV is widely distributed with 4. 4% and 8. 4% in Greece and Cyprus, respectively, and (iii) that viruses belonging to the Salehabad serocomplex should be further studied for their capacity to cause human disease in view of prevalence rates in dogs up to 22. 6%. These findings confirm that dogs can be considered as excellent sentinels for sandfly-borne phleboviruses. The results also underline the importance to study the role of SFSV in humans and may lead to the set-up diagnostic tests for patients presenting unexplained febrile illness and neuroinvasive infections. Further studies are also needed to define whether these viruses cause diseases in dogs.

lay_plos

ROME (Reuters) - On January 9, 1324, the Venetian traveler, merchant and writer Marco Polo was preparing for his final journey - to the afterlife that he as a God-fearing Christian was certain existed. Aged 70, Polo summoned a priest-notary to his home in Venice to record his words in Latin on a sheepskin measuring about 67 x 27 cm (26 x 10 inches). Now, a three-year study of the will by scholars and historians offers a fresh glimpse into Polo as well as more support for the commonly-held view that he visited China, which some historians had questioned. Venice’s National Marciana Library, custodian of the will, has co-published a large-format book, which contains a parchment reproduction of the nearly 700-year-old will, including marks left when the sheep was sheared and ink stains on the writing side. The work, called “Ego Marcus Paulo Volo et Ordino” (“I, Marco Polo, Desire and Order”), is intended mostly for collectors, history buffs and libraries. It was produced with Scrinium, a Venetian publishing house. “The last ‘diplomatic transcription’ of the will is 150 years old,” said Stefano Della Zana, Scrinium’s cultural director, referring to the term used by experts who study ancient letters and handwriting to produce modern interpretations. “This was done with the latest scientific techniques and scholarly standards of philology, so previous errors have been corrected,” he said. In one case, a scribbled Latin word that had previously been believed to refer to fabric was corrected to mean a residence. An exact replica of Marco Polo's 700-year-old last will and testament, is seen in Rome, Italy, April 10, 2018. Picture taken April 10, 2018. REUTERS/Alessandro Bianchi One of the causes of the misunderstanding, according to Professor Attilio Bartoli Langeli, was because the notary’s handwriting was “decidedly bad cursive script, awkward and careless”. Polo left money to Church institutions in Venice, forgave outstanding debts, and freed his indentured servant, a Tatar he had named Peter, “so that God may absolve my soul from all guilt and sin”. In the late 20th century a few historians argued that Polo never made it to China but picked up stories of the Mongol Empire from Persian merchants he met on the Black Sea. Most historians have rejected this view and Della Zana said the fact that Polo’s servant was a Tatar, who were a Mongol people, supports evidence that he did get to China. Further evidence lies in an inventory of Polo’s possessions that surfaced after his death and listed items from the far east, such as expensive musk. Apart from the money bequeathed to the Church, Polo left nearly everything else to his wife and three daughters. Della Zana said this was uncommon because it was then customary for men without sons to leave their assets to male members of the extended family. Slideshow (4 Images) “In that sense, he was well ahead of his times,” Della Zana said. Pointe-à-Callière, Montréal’s Archaeology and History Complex, is bringing its visitors an exhibition that will fire their imaginations and take them on an incredible voyage: Marco Polo — An Epic Journey transports visitors to the 13th century, taking them from Venice to China, along a route so astonishing and exceptional that it is still being discussed over seven centuries later. This event demonstrates the immense impact of the journey described by Marco Polo in the Description of the World, as his accounts allowed for the development of map-making, which in turn led to major exploration of the globe beginning in the 15th century. In fact, the book had a hand in influencing Christopher Columbus to set sail towards the west to open a new route to India and finally discover America in 1492. Marco Polo, before the big adventure! Marco Polo was born in Venice in 1254, into a family of merchants. The Polo family was not part of the well-off elite of the day. It was, however, well-versed in matters of trade, having for generations travelled seas controlled by Venice, going as far as Constantinople and beyond, on the shores of the Black Sea. Niccolo and Matteo Polo, Marco’s father and uncle, had made an initial trip to the Orient, coming home in 1269, after having met Emperor Kublai Khan in China. **The ongolian ruler had entrusted them with the mission of getting him some sacred oil from the lamp of the Holy Sepulchre, bringing back 100 priests capable of convincing people of the superiority of the Christian faith, and a letter from the Pope. They therefore left Venice again to fulfil this mission, this time accompanied by young Marco who had no idea, when he began his journey at the age of 17 in 1271, that he would be 41 years old upon his return in 1295. His journey would take 24 years. The magnificent voyage described in the Description of the World Marco Polo travelled over 20,000 kilometres on his journey, going mostly by land and returning by sea. The exhibition gives visitors a chance to experience several stops along the route. And what better guide is there than the explorer’s travelogue, The Travels of Marco Polo or Description of the World, in which Marco Polo tells the story of his voyage, as described to writer Rustichello da Pisa who was imprisoned with Marco Polo in Genoa from 1296 to 1298. The book is no doubt the most famous travel guide ever written! Landscapes; climates; distances travelled on foot, horse, or camel; dangers; rites and customs; clothing; fauna; flora; festivities in the court of Emperor Kublai Khan… Marco Polo described everything he saw along the way, as well as everything he was told and all that he heard during his many experiences. These memories serve as our guide through the exhibition. The Travels of Marco Polo or Description of the World, in which Marco Polo tells the story of his voyage, Marco Polo, the risk-taker The exhibition illustrates the extent to which young Marco Polo was no doubt a risk-taker—courageous, adventurous, and equipped with an iron constitution… all requirements for undertaking such an expedition. Desert crossings, thieves, precarious means of communication, insecurity, harsh climates, and the possibility of falling sick are but a few of the obstacles he had to face on his way to the kingdom of Kublai Khan. The long journey was an extremely difficult and dangerous attempt in the 13th century, with some of the countries along the way still difficult to access in the present day. A delight for the eyes and the soul—magnificent objects! Among the over 200 artefacts and objects featured in the exhibition are some that deserve special mention. The first of these—in fact, the most valuable—comes to us from St. Mark’s Basilica in Venice: a perfume burner or lamp of Byzantine influence, transformed into a reliquary of the Holy Blood, dating from the 12th century, reminiscent—given its magnificent cupolas—of St. Mark’s Basilica. A perfume burner or lamp of Byzantine influence, reminiscent—given its magnificent cupolas—of St. Mark’s Basilica. A splendid bronze—very popular in the court of China—from the 13th-15th centuries, depicting a dancing barbarian, is on loan from the Musée Cernuschi in Paris. Silk, a luxurious product from the Orient, is well represented in the exhibition with magnificent pieces from the 12th, 13th, and 14th centuries, lent by the Musée des tissus et des arts décoratifs in Lyon. An enamelled copper shrine or case, which contained a relic of St. Thomas Becket dating to the 13th century, on loan from the Musée de Cluny in Paris. An impressive six-foot tall high relief showing the crucifixion of Christ with Mary, Saint John, and angels, from the 13th-14th centuries, comes to us from the Museo Correr in Venice. Statuettes of women, from 1279, representing people of the court in the era of Mongolian emperor Kublai Khan, are on loan from the Musée d’art et d’histoire in Saint-Denis. From the Cité de la Céramique in Sèvres come several ceramics in the shape of crosses or stars that illustrate the Persian decorative genius. There are also treasures of rock crystal, gold, and jade—true wonders worthy of princely families — including a pair of winged chimeras dating back to the Liao Dynasty, in the 10-11th centuries, on loan from the Samuel and Myrna Myers collection in Paris. Also on display are saddles from the Émile Hermès collection, each more magnificent than the last. Two were made during the Qing Dynasty (1644-1911), and all demonstrate the importance of the horse—and the wealth of certain horsemen—in Marco Polo’s time. A central object in the ongoing fascination with Marco Polo’s journey, a facsimile of The Travels of Marco Polo, commissioned by John the Fearless in the years 1410-1412 and lent to us by the Bibliothèque nationale de France, is featured in the exhibition. Illuminations from the book are on display throughout the exhibition, allowing visitors to closely follow Marco Polo’s path. Among these is a very evocative image showing Marco at age 17, astride his horse, between his father and his uncle, as they begin their epic journey. The illustrator quite accurately depicted the seriousness of the goodbyes. One can easily understand what must be going through their minds, wondering if they are ever to return, given the many dangers that await them on the road ahead… Also of note: Christopher Columbus himself annotated a version of The Travels of Marco Polo, and this precious document is today preserved in Seville. Saddle from the Émile Hermès collection A magnificent Thangka (something that is unrolled) depicting Dalha, one of the Five Spirits of Destiny. A thangka is a typically Tibetan type of painting on canvas. Thangkas are used to guide the meditation of Gelugpa monks who practice Tibetan Lamaist Buddhism, Kublai Khan’s religion. Mongolia, 18th-19th century. A fascinating journey along the Silk Road Upon entering the exhibition room, visitors find themselves in 13th century Venice, with a little help from a projection created by Ubisoft. There they learn about the importance of the prosperous city-state of Venice, its maritime and commercial power, and its role in trade between the East and the West. The Earth, or what was known of it at the time, consisted of just three continents: Europe, Africa, and Asia. Along the route, visitors are taken from one place to another, exploring such sublime landscapes as those found along the Silk Road. Marco Polo was, in fact, one of the first to explore this route that has transcended time and has always held great interest. He brought back precious stones like lapis lazuli from Afghanistan, spices and cotton from India, and jade and silks from China. During his long stay in China, he took note of its people’s many inventions, and brought back a few in his luggage: who today is aware that the Chinese invented paper currency and charcoal? From Venice to China, by way of Persia, the Mongolian steppe, and the shores of India, visitors explore—as Marco Polo did—the know-how of Europe and Asia’s master craftsmen, and take part in a multi-sensory examination of furs, silks, and woollen fabrics they are invited to touch! The scent of rare spices from exotic places also serves to awaken visitors’ senses. A space representing a magnificent mosque and its evocative objects welcomes visitors to the oasis of Yazd in Persia. Farther along in the exhibition is the recreation of a Mongolian yurt—a model of adaptability and hospitality—, offering visitors a short break, as well as an opportunity to examine its layout. Other exotic locations await visitors along the way: the Holy Land, where the sacred oil requested by Kublai Khan was found; Armenia and its herds; Georgia and its mountains; Persia and its treasures; Afghanistan, the crossroads of Asia; the Pamir Mountains, which Marco Polo was the first European to cross; and the dreadful Taklamakan Desert. Winged chimera dating back to the Liao Dynasty. In the Footsteps of Marco Polo Visitors get a chance to appreciate the challenges the explorer faced in a screening of excerpts from the film In the Footsteps of Marco Polo, produced in 2008 by present-day explorers who followed in Marco Polo’s footsteps over a two-year period. A fascinating experience! In the early 1990s, Denis Belliveau and Francis O’Donnell, two long-time friends from Queens, New York, decided to follow Marco Polo’s path with the explorer’s travelogue as their only guide. They made the long journey on foot, on horseback, and by sea. These two regular guys undertook an extraordinary project that no one else had yet attempted. A valuable contributor In producing this exhibition, Pointe-à-Callière would like to thank Jean-Paul Desroches, the exhibition curator. Mr. Desroches worked as the General Conservator at the Musée Guimet in Paris for 35 years, in addition to having been a professor in the Far East department at the École du Louvre for 20 years. For this exhibition, Mr. Desroches lent Pointe-à-Callière a variety of objects from the Mongolian civilization. Explore the exhibition with Jean-Pierre Desroches (in french) Marco Polo (1254-1324) was a Venetian merchant believed to have journeyed across Asia at the height of the Mongol Empire. He first set out at age 17 with his father and uncle, traveling overland along what later became known as the Silk Road. Upon reaching China, Marco Polo entered the court of powerful Mongol ruler Kublai Khan, who dispatched him on trips to help administer the realm. Marco Polo remained abroad for 24 years. Though not the first European to explore China—his father and uncle, among others, had already been there—he became famous for his travels thanks to a popular book he co-authored while languishing in a Genoese prison. Marco Polo: The Early Years Marco Polo was born around 1254 into a prosperous merchant family in the Italian city-state of Venice. His father, Niccolò, and his uncle Maffeo had left the year before on a long-term trading expedition. As a result, he was raised by extended relatives following his mother’s death at a young age. Niccolò and Maffeo first spent about six years in Constantinople (now Istanbul, Turkey), which had been under Latin control since the Fourth Crusade of 1204. The two brothers then went to the port city of Soldaia (now Sudak, Ukraine), where they owned a house. Did you know? Christopher Columbus purportedly sailed to the New World with a copy of Marco Polo’s “Travels” in tow. Thinking he would reach Asia and having no idea about the Mongol Empire’s collapse, Columbus marked up the book with notes in preparation for a meeting with Kublai Khan’s descendent. ADVERTISEMENT Thanks for watching! Visit Website ADVERTISEMENT Thanks for watching! Visit Website The Byzantine re-conquest of Constantinople in 1261, along with upheavals in the Mongol Empire, may have blocked their way home. Niccolò and Maffeo therefore turned east in order to trade in such things as silk, gems, furs and spices. After spending three years in Bukhara in present-day Uzbekistan, they were encouraged by a Mongolian embassy to visit Kublai Khan, grandson of Genghis Khan, who controlled a huge swath of Asia. Kublai quizzed them on European affairs and decided to send them on a goodwill mission to the pope. In 1269, the two brothers finally made it back to Venice, where Niccolò and Marco Polo met each other for the first time. ADVERTISEMENT Thanks for watching! Visit Website Marco Polo’s Travels Along the Silk Road Two years later, Niccolò and Maffeo sailed to Acre in present-day Israel, this time with Marco at their side. At the request of Kublai Khan, they secured some holy oil from the Church of the Holy Sepulchre in Jerusalem and then backtracked to Acre to pick up gifts, papal documents and two friars from newly elected Pope Gregory X. The friars quickly abandoned the expedition, but the Polos continued on, possibly by camel, to the Persian port city of Hormuz. Failing to find any boats to their liking, they instead took take a series of overland traders’ routes that, in the 19th century, would become known as the Silk Road. Over the next three years they slowly trekked through deserts, high mountain passes and other rough terrain, meeting people of various religions and cultures along the way. Finally, around 1275, they arrived at Kublai Khan’s opulent summer palace at Shangdu, or Xanadu, located about 200 miles northwest of his winter quarters in modern Beijing. Kublai, who generally relied on foreigners to administer his empire, took Marco Polo into his court, possibly as a tax collector. At one point, the Venetian was sent on official business to the port city of Hangzhou (then called Quinsai), which, like Venice, was built around a series of canals. Marco Polo also purportedly journeyed across inland China and into present-day Myanmar. After many years of seeking a release from service, the Polos finally secured permission from Kublai to escort a young princess to her intended husband Arghun, the Mongol ruler of Persia. In 1292 the Polos joined a flotilla of 14 boats that set out from Zaitun (now Quanzhou, China), stopped briefly in Sumatra and then landed in Persia 18 months later, only to find out that Arghun was dead. The princess was made to marry Arghun’s son. The Polos, meanwhile, stayed on with Arghun’s brother for nine months before heading to Venice via Trebizond (now Trabzon, Turkey), Constantinople and Negrepont (now Euboea, Greece). They arrived home in 1295, the year after Kublai’s death sent the Mongol Empire into an irrevocable decline. Marco Polo in Venice Shortly thereafter, Marco Polo was captured in battle by Venice’s archrival Genoa. While in prison he met the Arthurian adventure writer Rustichello of Pisa, with whom he would collaborate on a 1298 manuscript called “Description of the World.” It has since become better known as “The Travels of Marco Polo” or simply “The Travels.” With the help of notes taken during his adventures, Marco Polo reverently described Kublai Khan and his palaces, along with paper money, coal, postal service, eyeglasses and other innovations that had not yet appeared in Europe. He also told partially erroneous self-aggrandizing tales about warfare, commerce, geography, court intrigues and the sexual practices of the people who lived under Mongol rule. A Genoese-Venetian peace treaty in 1299 allowed Marco Polo to return home. He probably never left Venetian territory again. The following year, he married Donata Badoer, with whom he would have three daughters. Not much is known about his golden years except that he continued trading and litigated against a cousin. Marco Polo died in January 1324, having helped to inspire a later generation of explorers. Everything we know about him comes from his own text and a few Venetian documents; Asian sources never mentioned him. This lack of hard evidence has caused a small number of skeptics to question whether Marco Polo actually made it to China. They back up their case by pointing to certain inaccuracies in “The Travels,” as well as his failure to report such practices as chopstick use and foot binding. Nonetheless, most scholars are convinced by the detailed nature of Marco Polo’s account, which, they say, overwhelmingly checks out against available archaeological, historical and geographical records.

In 1298, Marco Polo wrote a manuscript, now dubbed The Travels of Marco Polo, describing his adventures along the Silk Road to China, his meeting with Mongol ruler Kublai Khan, and the innovations he came across, like eyeglasses. In the centuries since, some historians have doubted whether the Venetian actually traveled as far as he claimed, based in part on his failure to mention chopsticks, per History.com. But nearly 700 years after his death, Polo is setting the record straight himself. A three-year study of Polo's sheepskin will, written in Latin by a priest-notary in 1324, offers more evidence of Polo's adventures across Asia, reports Reuters. For instance, the will secured the freedom of Polo's indentured servant, a man Polo named Peter, who was from the Tatar clan of Mongols. Researchers say this-and an inventory of possessions mentioning musk from the far east-suggests Polo did in fact reach China, which is now the accepted narrative. Christopher Columbus apparently believed it, having carefully perused his own copy of The Travels of Marco Polo, which Pointe-à-Callière Museum refers to as "the most famous travel guide ever written." The study also helped correct errors in Polo's will blamed on the notary's poor handwriting (a word believed to refer to fabric actually meant a residence) and highlighted the explorer's possible belief in gender equality: Though possessions were usually left to male relatives at the time, Polo gave almost everything to his wife and three daughters.

multi_news

FIELD OF THE INVENTION [0001] The present invention relates to wringing devices for use in the floor-maintenance industry. More particularly, the present invention relates to a mop wringer having a filter for prolonging the useful life of a floor care fluid used with mops during floor maintenance, while also reducing the labor associated with such floor maintenance. BACKGROUND OF THE INVENTION [0002] Labor is the largest expense item in cleaning. Innovations which minimize the amount of time required to complete cleaning activities reduce overall costs associated with such necessary maintenance. [0003] In the prior art, a mop is often used with a bucket/wringer combination. The bucket is filled with a cleansing fluid, which is usually a mixture of water and detergent. The mop is inserted into the bucket to rinse it and/or to absorb the cleansing fluid. The wringer is used to remove excess fluid from the mop before it is used to clean a floor or other surface. The fluid from the wringer is returned to the bucket, allowing it to be reused. However, dirt and other contaminants that are picked by the mop during mopping accumulate within the bucket. Within a relatively short period of time, the cleansing fluid can become too dirty to continue to be used effectively, and will need to be replaced. [0004] In a mopping activity, the amount of time required to complete a task is prolonged because of the need to frequently replace the cleansing fluid used with the mop. Reducing the number of times that the cleaning fluid needs to be replaced can lead to significant savings in both time and cost. [0005] Therefore, there is a need for a device that will allow for the reclamation of cleansing fluid from a mop that has been used while simultaneously removing dirt particles, waste particles and other contaminants from the cleansing fluid, thus prolonging the useful life of the cleansing fluid. SUMMARY OF THE INVENTION [0006] The present invention described herein solves the problem of removing dirt particles and other contaminants from the cleansing fluid used with mops by providing a wringer with a filter that filters dirt particles and other contaminants from the cleansing fluid that is wrung from the mop by the wringer. [0007] In a preferred embodiment of the present invention as described herein, a wringer comprises an open top and a plurality of side surfaces, with the plurality of side surfaces forming an internal volume. The wringer further comprises a first wringing surface that is movable between a first position and a second position, a second wringing surface having at least one perforation, and a filter that is disposed on the second wringing surface. The internal volume of the wringer body is decreased as the first wringing surface is moved from the first position to the second position, and the filter is configured and adapted to prevent dirt particles or other waste particles from flowing through the at least one perforation during wringing. [0008] In a second preferred embodiment, a wringer comprises a wringer body having an open top and a plurality of side surfaces, with the plurality of side surfaces forming an internal volume, a first wringing surface movable between a first position and a second position and having at least one perforation, and a filter that is disposed on the first wringing surface. The internal volume of the wringer body is decreased as the first wringing surface is moved from the first position to the second position, and the filter is configured and adapted to prevent dirt particles from flowing through the at least one perforation during wringing. [0009] In a third preferred embodiment, a wringer comprises a compartment having a variable internal volume, a perforated section forming a portion of the compartment, and a filter disposed within the compartment. Fluid within the compartment passes through the filter before flowing through the perforated section. [0010] In a fourth preferred embodiment, a wringer comprises a container having an open top and an internal volume, a movable part that is movable between a first position and a second position, a perforated section with an internal surface, and a filter. The internal volume of the container is decreased as the movable part is moved between the first position and the second position, and the filter is attached to the internal surface of the perforated section. BRIEF DESCRIPTION OF THE DRAWINGS [0011] FIG. 1 is a perspective view of one preferred embodiment of the present invention incorporated on a sidepress wringer. [0012] FIG. 2 is another perspective view of the embodiment of FIG. 1. [0013] FIGS. 3A and 3B are cross-sectional views of the embodiment of FIG. 1. FIG. 3A shows the wringer with a movable part in a first position, and FIG. 3B shows the wringer with a movable part in a second position. [0014] FIG. 4 is a partial cross sectional view of the embodiment of FIG. 1 attached to a bucket. [0015] FIG. 5 is a partial cross section view of another embodiment of the present invention also in a sidepress wringer. [0016] FIG. 6A and 6B are partial cross-sectional views of the embodiment of FIG. 5. FIG. 6A shows the wringer with the movable part in a first position, and FIG. 6B shows the wringer with a movable part in a second position. [0017] FIGS. 7A and 7B are plan views of different arrangements of perforations that may be used in accordance with the present invention. [0018] FIG. 8 is a cross sectional view of a filter in accordance with a preferred embodiment of the present invention. [0019] FIGS. 9A and 9B are partial views of a preferred embodiment of the invention showing the attachment of the filter. FIG. 9A is a partial plan view, and FIG. 9B is a partial top cross-sectional view. [0020] FIGS. 10A and 10B are perspective views of another preferred embodiment of the present invention incorporated in an industrial type wringer; [0021] FIGS. 11A and 11B are perspective views of yet another preferred embodiment of the present invention incorporated in a downpress type wringer; [0022] FIGS. 12A and 12B are perspective views of the embodiment shown in FIGS. 11A and 11B detailing the attachment of the filter to the wringer in a first preferred configuration; and [0023] FIGS. 13A and 13B are perspective views of the embodiment shown in FIGS. 11A and 11B detailing the attachment of the filter to the wringer in a second preferred configuration. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [0024] FIGS. 1-4 illustrate a first preferred embodiment of the present invention as described herewith. Wringer 1 generally comprises container 10, actuator 30, filter 40, and attachment member 60. Container 10 has open top 12, a plurality of side walls 14, movable section 20, and perforated section 18, which define an internal volume 11. Movable section 20 is movable from a first position A to a second position B (see FIGS. 3A and 3B, further described below), which reduces the size of internal volume 11. Sealing members 24 on edges 22 of movable section 20 minimize the leakage of fluid from container 10 where movable section 20 meets side walls 14. However, even if some fluid is able to leak out of container 10 through edges 22, this does not seriously hamper the effectiveness of the present invention. In a preferred embodiment, the components of container 10 are made of plastic, although other materials, such as metal, may also be used. [0025] Movable section 20 is generally rectangular and is attached to container 10 at its base by means of rod 22, about which movable section 20 is pivotable. The interior surface 21 of movable section 20 is generally smooth. Internal scaffolding 23 may be used to provide strength to movable section 20 while reducing overall weight. Movable section 20 further comprises arms 24, which project at an angle from the exterior surface of movable section 20 but transverse from rod 22. While two arms 24 are used in this preferred embodiment, it is noted that one arm or three or more arms may also be used without departing from the spirit and scope of the invention. Arms 24 connect movable section 20 to actuator 30, which is used to actuate movable section 20 from first position A to second position B. [0026] Actuator 30 further comprises handle 38, rod 34, and arms 36. Actuator 30 is rotatable about axis X, which is co-axial with rod 34. Rod 34 is parallel with rod 22. Arms 36 are fixedly attached to and orthogonally project from rod 34. Arms 36 are pivotably connected to arms 24 of movable section 20 by connecting rod 26 so that an acute angle is formed between arms 24 and arms 36. While connecting rod 26 is used in the preferred embodiment to provide additional structural support to arms 24 and 36, alternative mechanisms for providing connection between arms 24 and 36, such as rivets or bolts, may also be used. Arms 24 and 36 are generally flat and are oriented so that they are aligned along a plane that is orthogonal to axis X. This orientation provides the greatest resistance to bending of arms 24 and 36 due to forces experienced during actuation of movable section 20, while reducing weight and the amount of material needed for arms 24 and 36. Arms 24 and 36 also generally have rounded ends to eliminate sharp corners, helping to prevent injuries to users. [0027] Handle 38 projects orthogonally from rod 34 and provides leverage to allow a user to more easily rotate actuator 30 about axis X. As shown in FIGS. 1-2, handle 38 is generally angled upwards, allowing a user to grasp and press against handle 38 without having to bend down. The minimization of bending reduces the potential for user injury. Handle 38 may also be provided with a crosspiece 39, allowing the user to grip the top of handle 38 in a horizontal manner. Crosspiece 39 may be covered by a gripping material to further aid in gripping handle 38. In a preferred embodiment, handle 38 and rod 34 are formed from a single piece of bent metal tubing, but may also be formed of any other suitable material, e.g., molded plastic. However, handle 38 and rod 34 may be formed as two separate parts and subsequently connected together. [0028] Spring 32 is attached to rod 34 and arms 24. Spring 32 biases movable section 20 towards first position A. In a preferred embodiment, spring 32 is attached to rod 34 by inserting rod 34 through the coils of spring 32. It can be readily seen that spring 32 may be connected to movable section 20 and actuator 30 in different configurations. For example, hooks may be used to connect spring 32 to movable section 20 and actuator 30. Another alternative embodiment has spring 32 connecting arms 24 and 36. [0029] As shown in FIGS. 3A and 3B, pressing on handle 38 with force F causes arms 36 to pivot about axis X. This exerts a translational and rotational force on arms 24 through connecting rod 26. Arms 24 respond to this force by pivoting about connecting rod 26 so that the angle between arms 24 and arms 36 increases while exerting a pushing force upon movable section 20. Movement of handle 38 is thus translated into pivotal movement of movable section 20 about rod 22 towards second position B. As movable section 20 moves towards second position B, internal volume 11 of container 12 is reduced, and movable section 20 is brought closer to perforated section 18. When handle is released, the bias exerted by spring 32 returns movable section 20 and actuator 30 back to first position A. [0030] Perforated section 18 has a plurality of perforations 19 that allow fluid within internal volume 11 to flow out of container 10. Perforations 19 may take on any number of shapes, sizes, and arrangements. In a preferred embodiment, perforated section 18 is disposed on the side wall 14 that is opposite movable section 20, and perforations 19 are shaped as circular holes. In alternative embodiments, as shown in FIGS. 7A and 7B, perforations 19 are shaped as slots with rounded ends, and arranged either vertically or horizontally. Furthermore, perforated section 18 may be disposed on other parts of container 12. [0031] Filter 40 is attached to the outer surface of perforated section 18 exterior to container 10. Therefore, any fluid that exits container 10 through perforations 19 in perforated section 18 will pass through filter 40. In a preferred embodiment, as illustrated in FIG. 8, filter 40 comprises a plurality of coarse fibers 42 bonded together to form a non-woven mat 44. Fibers 42 may be aligned or randomly formed and fiber mat 44 may also be bonded to a second layer 46, which may comprise a nylon scrim. The construction of filter 40 allows fluid to pass through filter 40 while trapping dirt and other particles within fibers 42. Fibers 42 may be composed of polyester, polyolefin, acrylic, polyamide, aramid, felt or other similar durable and inexpensive material. The filter may also be formed of natural fibers, or a combination of natural and synthetic fibers. The porosity of filter may be selected to capture a variety of particle sizes (e.g., 100 to 400 microns) and different types/sizes of filters may be employed in different applications to target particles in a particular size range. As one skilled in the art will readily appreciate, the porosity for filter 40 should be selected so that the filter traps dirt and other waste particles typically encountered in cleaning floors while remaining sufficiently porous to allow fluid to easily pass through filter 40. [0032] Filter 40 may be attached to perforated section 18 by any number of methods. For example, in a preferred embodiment as shown in FIGS. 9A and 9B, filter 40 is attached to perforated section 18 by a plurality of rails 48 projecting from the inner surface of perforated section 18. Rails 48 are formed on three sides of filter 40, allowing filter 40 to be slid in and out of rails 48. Rails 48 are configured to form a slot into which filter 40 may be removably inserted. This allows a user to easily remove and re-insert filter 40 when filter 40 requires cleaning or replacement. Alternatively, as shown in FIG. 2, filter 40 may be attached to section 18 by a plurality of pins or buttons 41. In still another embodiment shown in FIGS. 13A and 13B, filter 40 may be attached using a plurality of clips 164. [0033] As shown in FIG. 4, attachment member 60 allows wringer 1 to be attached to bucket 70. In the preferred embodiment illustrated, attachment member 60 consists of projections 62 and 64, which allow wringer 1 to be placed over and seated upon lip 72 of bucket 70. Projections 62 and 64 are configured to extend substantially down the side of bucket 70, but are not actively clamped or attached to bucket 70. This allows wringer 1 to be adequately secured to bucket 70 during normal use while allowing wringer 1 to be quickly and easily removed simply by lifting wringer 1 away from bucket 70. Wringer 1 is oriented upon bucket 70 so that fluid that flows through perforated section 18 will be deposited into bucket 70. Bucket 70 may also have wheels 74, allowing the user to roll bucket 70 along the ground instead of having to carry bucket 70 from one location to the next. Wringer 1 is also oriented in relation to bucket 70 so that when the user presses against handle 38 with force F, force F is directed towards the center of bucket 70. This prevents an unbalancing of wringer 1 and bucket 70 that might cause the wringer 1 and bucket 70 to fall over. In an alternate embodiment, the wringer and bucket may be of a unibody construction. [0034] In use, a mop head is inserted into container 10 through open top 12. The user presses against handle 26, rotating actuator 30 to actuate movable section 20 from first position A towards second position B. This action squeezes the mop head between movable section 20 and perforated section 18, wringing dirty cleansing fluid from the mop head. At the same time, the cleansing fluid is forced through filter 40 after exiting container 10 through perforated section 18. As the cleansing fluid passes through filter 40, dirt and other contaminants and foreign objects are removed from the cleansing fluid after the cleansing fluid passes through perforated section 18, but before it reaches bucket 70. The dirt is thus trapped in filter 40 and kept separate from the cleansing fluid in bucket 70. The cleansing fluid used may include water, a commercial cleansing solvent, and/or some other type of suitable fluid. [0035] The placement of filter 40 within container 10 of wringer 1 is advantageous in several respects. Filter 40 is clearly visible to the user at all times. This allow for quick recognition of when filter 40 needs replacement after too much dirt has been trapped in filter 40. Filter 40 is also easily accessible for replacement or cleaning, either when filter 40 is dirty, or when a different type of filter 40 is required when a different cleansing fluid is being used. Also, the position of filter 40 within container 10 allows for the positive forcing of the cleansing fluid through filter 40, instead of relying upon gravity or other passive means for filtering the cleansing fluid. This increases the speed of the filtering process, thus further reducing the total time required during mopping. [0036] In another embodiment, illustrated in FIG. 5, perforated section 118 may be disposed on movable section 120 of wringer 100. In this embodiment, filter 140 is attached to the exterior surface of movable section 120. Preferably, this embodiment utilizes an actuator 130 that is not located in a manner that would impede the flow of fluid through perforated section 118. Furthermore, the force that actuates movable section 120 should not unbalance wringer 100 or the container to which it is attached. As shown in FIGS. 6A and 6B, a second preferred embodiment utilizes actuator 130 that comprises handle 138 attached to first gear 132, which is rotatably attached to wringer 100. The teeth of first gear 132 are interlocked with the teeth of second gear 134, is fixedly attached to movable section 120 and is coaxial with rod 122. Pressing upon handle 138 causes the rotation of first gear 132, which in turn causes the rotation of second gear 134 in the opposite direction, resulting in the pivoting of movable section 120 about rod 122 towards side wall 114. First gear 132 and second gear 134 may be dimensioned to provide the optimal mechanical advantage for user to actuate movable section 120 through the movement of handle 138. [0037] The present invention may readily be used with other styles of wringers and wringer/bucket combinations. For example, as shown in FIGS. 10A and 10B a filter 40 may be installed on an industrial type wringer 150 having no moving parts or metal components. As shown in FIG. 10B, filter 40 may be formed in a conical configuration to fit wringer 150. In an alternate preferred embodiment shown in FIGS. 11A and 11B, filter 40 may be installed on a downpress type wringer 160. As shown in FIGS. 12A and 12B, filter 40 may be attached to wringer 160 with a series of push pins 162 that mate with corresponding apertures on both sides of wringer 160. Alternatively, as shown in FIGS. 13A and 13B, a plurality of clips 164 may be used to secure filter 40 to wringer 160. In addition, as shown in FIGS. 9A and 9B, slots may be formed integrally with the wringer body to hold filter 40 in place. As another example, an electrical motor could be incorporated into the wringer to assist in squeezing a mop inserted into a compressor volume. As one of ordinary skill in the art can readily appreciate, for each of the wringer types disclosed herein, the wringer may be formed integrally with a bucket as a single unit. [0038] The present invention may also be used in other apparatus for use in floor maintenance. For example, filter 40 may be used with an apparatus used for the waxing of floors. Filter 40 would be configured to have an appropriate porosity and would be composed of suitable materials for the fluid being used. [0039] It can be readily seen by those skilled in the art that a wringer in accordance with the present invention may take many different configurations in addition to the ones presented here while remaining within the spirit and scope of the present invention. For example, the wringer may have more than one movable section, and the motion of the movable section may be different than the one herein described. Furthermore, other actuating mechanisms other than the actuator disclosed herein may also be used. Accordingly, it should be clearly understood that the embodiments of the invention described above are not intended as limitations on the scope of the invention, which is defined only by the following claims.

A wringer for use with wet mops is disclosed. The wringer includes a body having a plurality of side surfaces and at least one perforated section. A filter is disposed adjacent to the perforated section, so that fluid exiting the container through the perforated section will pass through the filter. The filter may be secured to the wringer by slots formed in the wringer body, or with pins, clips or any other similar attaching means. The filter removes dirt and other contaminants from cleansing fluid used with the mop, lengthening the useful lifetime of the cleansing fluid before replacement is needed. The wringer may also have an attachment member to allow the wringer to be used with a bucket or may be formed integrally with a bucket.

big_patent

Several recent studies have shown a genetic influence on gene expression variation, including variation between the two chromosomes within an individual and variation between individuals at the population level. We hypothesized that genetic inheritance may also affect variation in chromatin states. To test this hypothesis, we analyzed chromatin states in 12 lymphoblastoid cells derived from two Centre d' Etude du Polymorphisme Humain families using an allele-specific chromatin immunoprecipitation (ChIP-on-chip) assay with Affymetrix 10K SNP chip. We performed the allele-specific ChIP-on-chip assays for the 12 lymphoblastoid cells using antibodies targeting at RNA polymerase II and five post-translation modified forms of the histone H3 protein. The use of multiple cell lines from the Centre d' Etude du Polymorphisme Humain families allowed us to evaluate variation of chromatin states across pedigrees. These studies demonstrated that chromatin state clustered by family. Our results support the idea that genetic inheritance can determine the epigenetic state of the chromatin as shown previously in model organisms. To our knowledge, this is the first demonstration in humans that genetics may be an important factor that influences global chromatin state mediated by histone modification, the hallmark of the epigenetic phenomena. Polymorphisms and quantitative differences in gene expression provide the genetic basis for human variation. Studies in humans and other organisms suggest that variation at the transcript level accounts for the majority of the phenotypic variation among species and across individuals within species [1–4]. Recent studies have demonstrated that inherited factors influence gene expression variation between both copies of a gene within an individual [5] as well as between individuals [1,6–8]. In a large-scale analysis of allele-specific gene expression using Affymetrix HuSNP chip [9], we found that allelic variation in gene expression is common, affecting about half of the genes in human genome. This conclusion was supported from the studies of digital gene expression in UniGene database [10,11] and allele-specific gene expression using a custom-designed single nucleotide polymorphism (SNP) chip [12]. Analysis of allelic variation in gene expression can facilitate identification of regulatory SNPs when the regulatory SNPs are in linkage disequilibrium with an exonic SNP used in the analyses of allele-specific gene expression [13,14]. Eukaryotic genomes are organized into chromatin, formed by DNA and protein complex. The basic unit of chromatin is the nucleosome structure containing 146 bp DNA that wraps around a histone octamer. At the chromosome level, gene expression is regulated by distinct chromatin structures. This epigenetic information is often encoded in post-translational modifications of histone proteins such as acetylation, methylation, and phosphorylation [15]. Histone modifications can be maintained through mitotic cell divisions. This stable transmission of epigenetic state through mitosis provides the basis for cellular differentiation and organism development. Although there are a few examples of inheritance of epigenetic information across generations in model organisms [16,17], no investigation of the global effect of genetic inheritance on chromatin state in humans has been reported. In light of genetic influence on allelic gene expression variation in pedigree [5], we set out to analyze if genetic inheritance also affects chromatin variation in humans, as measured by variations in histone modifications using an allele-specific chromatin immunoprecipitation (haploChIP) assay [13]. The use of allele-specific variation in chromatin state in a heterozygous individual is a powerful approach to study genetic influence since other sources of variations in the cellular environment are likely affecting both alleles more or less equally. Our study demonstrates that specific chromatin states as a quantitative trait show familial aggregation. To evaluate the allele-specificity of our chromatin immunoprecipitation (ChIP) assay, we first examined protein binding at three imprinted genes (LIT1, H19, and SNRPN) and two X-linked genes (HPRT1 and PGK1) loci. We used 12 lymphoblastoid cells derived from 12 individuals from two Centre d' Etude du Polymorphisme Humain (CEPH) families. Each cell line was characterized by six antibodies targeting at chromatin proteins. The description of cell lines and experiments can be found in Tables S3, S6, S7, and S8. Three antibodies target at active chromatin proteins, which are RNA polymerase II (Pol II), histone H3 lysine 9/14 acetylation (H3Ac), and lysine 4 dimethylation (H3K4). The remaining three antibodies target at inactive chromatin proteins, which are histone H3 lysine 9 dimethylation (H3K9), lysine 27 dimethylation (H3K27di) and trimethylation (H3K27tri). The control DNAs were from whole cell extract, which were prepared as the ChIP experiments, except for the omission of antibodies. We refer to this control DNA as input. We analyzed DNAs that were co-immunoprecipitated by the antibodies using oligo ligation assay (OLA). The results for the differential methylation region in LIT1 promoter are shown in Figure 1A. The paternal allele was specifically pulled down by antibodies targeting at active chromatin (Pol, Ac, and K4 in Figure 1A; the paternal allele is C for GM10858, GM11872, and GM11875 and T for GM10859, GM10861, GM10870, and GM11982). This is consistent with the previous study, which demonstrated that the LIT1 gene was imprinted and was expressed from paternal chromosome only [18]. The maternal allele was preferentially pulled down by antibodies targeting at inactive chromatin (K9, K27di, K27tri in Figure 1A; the maternal allele is T for GM10858, GM11872, and GM11875 and C for GM10859, GM10861, GM10870, and GM11982). As a control, the input showed nearly equal intensities of both alleles. Promoter regions of H19, SNRPN, HPRT1, and PGK1 also displayed expected allele-specificity in our ChIP assays (Figure S6). After we have established allele-specificity for our ChIP assay using the imprinted genes and X-linked genes, we proceeded to analyze genome-wide allele-specific chromatin states by ChIP-on-chip method with a SNP chip. Since Affymetrix 10K SNP chip was designed for genotyping purpose, we had to modify the protocol in order to use the 10K SNP chip for doing ChIP-on-chip studies. The modified protocol is illustrated schematically in Figure 1B. We first repaired DNA fragments that were co-immunoprecipitated by antibodies or from the nonenriched control DNAs (input) by flushing the ends with a nuclease and adding adaptors to the DNA ends (Figure 1B). The DNA fragments were amplified and hybridized separately to the 10K SNP chips. We used 12 lymphoblastoid cell lines derived from 12 individuals, six of them from each of the two CEPH families (1347 and 1362, two parents and four children). Each cell line was analyzed with the six antibodies (Pol II, H3Ac, H3K4, H3K9, H3K27di, and H3K27tri) and two controls (input and genomic DNA using unmodified protocol), which gave 96 ChIP-on-chip experiments. The data from the 96 ChIP-on-chip experiments can be represented in a data matrix, with 96 rows (experiments) and 10,000 columns (SNPs). Each SNP had two measurements, one for chromatin binding from the A allele and the other from the B allele. We were interested in two derived values. The first one was the total intensity, which was the sum of chromatin-binding intensities from A allele plus B allele. The total intensity was similar to those obtained in conventional ChIP-on-chip experiments. The second one was the relative intensity, which was the ratio of A allele chromatin-binding intensity divided by the total intensity. The relative intensity was uniquely produced in this study due to the use of the SNP chip in ChIP-on-chip experiment. The input serves as an important control for two purposes. First, both input and the ChIP-on-chip experiment used our modified protocol. Comparison of genotype call between genomic DNA and input allowed us to evaluate the allelic specificity of our protocol for this experimental system. We found that the concordance of genotype call between genomic DNA and input was usually around 99% (Table S1). Thus, the result validated our protocol. Second, it allows us to define biological activity specifically due to chromatin beyond a baseline. The baseline can be assessed by input. Because the complexity in this high dimensional ChIP-on-chip data, we need to reduce the complexity in order to effectively understand the variance structure. We used principal component analysis (PCA) for this purpose. In our data, the first two principal components typically account for between 10%–50% of the total variance. Therefore, we can now focus our analyses in two dimensions instead of the original 10,000 dimensions. The result from PCA analysis using total intensity (A + B) for the ChIP-on-chip data is shown in Figure 2A. We plotted the 96 samples using the scores from the first principal component (PC1) and the second principal component (PC2). As shown in Figure 2A, the samples were clustered by the antibodies using the total chromatin-binding intensities (A + B). For example, the samples from antibodies targeting at active chromatin (Pol II, H3Ac, and H3K4) are on the left. Samples from two (H3K27di and H3K27tri) of the three antibodies targeting at inactive chromatin are on the right. Samples from the third antibody targeting inactive chromatin (H3K9) and the controls are in the middle. This is expected because chromatin states are determined by histone modifications and Pol II activity. Samples from the two families (red and blue, Figure 2A), are all intermixed. Therefore, we concluded that the major determinant of the total variance in the ChIP-on-chip experiments was due to variations in chromatin states as revealed by the antibodies targeting at different modification forms of histone H3 proteins and Pol II when using the total intensity. However, we got a totally different picture when PCA was performed with the relative intensity (A/A + B) (Figure 2B). Now the samples from the family 1 and family 2 (red and blue, Figure 2B) were separated from each other into two clusters. The separation was the largest for the antibodies targeting at active chromatin, which were represented by the open symbols at the bottom of Figure 2B. So the global chromatin states as measured by the relative intensity from the A allele differ for the individuals in family 1 versus the individuals in family 2. This observation led us to conclude that genetic inheritance can influence chromatin modifications. To validate this important finding, we carried out the same ChIP-on-chip experiments and analyses for two additional families (1331 and 1413). We analyzed twelve lymphoblastoid cell lines, six from each of the two CEPH families (1347 and 1362), with the two antibodies (H3Ac and H3K4) and the two controls (input and genomic DNA). Once again, we saw clustering of the samples by controls/antibody (H3Ac and H3K4) when PCA was performed using the total intensity (A + B) (Figure 3, left panels, three pair-wise comparisons among CEPH families 1347,1331, and 1413). More importantly, samples from the two different families were separated into two clusters when PCA was analyzed using the relative intensity (A/A + B) (Figure 3, right panels, three pair-wise comparisons among CEPH families 1347,1331, and 1413). To better understand the genetic influences on chromatin variation, we constructed pairs of genetically related individuals (siblings or parent-child), as well as pairs of genetically unrelated individuals from heterozygous individuals in the four CEPH families. We then computed the difference between the two individuals in each pair. To identify those SNPs that had similar chromatin state within genetically related individuals, we compared the variance in genetically related pairs versus the variance in genetically unrelated pairs. We identified seven SNPs (F-test, p < 0. 05). Variation in chromatin state for the seven SNPs was smaller in the genetically related pairs than the variation in the genetically unrelated pairs (Figure 4), indicating similar chromatin state between the related individuals. These differences were specifically observed in the ChIP experiment (in H3Ac but absent in input). Yan et al. previously demonstrated that allelic gene expression variation segregated as a Mendelian trait [5]. To evaluate if allelic chromatin variation also follows Mendelian inheritance, we performed inheritance analysis for the seven genes analyzed in Figure 4. All seven genes showed segregation patterns that were consistent with Mendelian inheritance (Figure 5 and Figure S8). For examples, ABB haplotype in GM10859 (mother in CEPH family 1347) in the case of rs938335 had low H3Ac binding activity (below two standard deviations from the mean intensity of B allele), whereas BAA haplotype in GM10859 had high H3Ac binding activity (above two standard deviations from the mean of A allele). The two heterozygous children are GM11871 and GM11875, both of whom received BAA from the mother. The allelic fraction values (A/A + B) are 0. 61 and 0. 73, respectively, which are higher than 0. 5. But the allelic values are not as extreme as the one in GM10859. This is because the paternal allele AAB has normal level of H3Ac binding activity. Similarly, BAA and ABB haplotypes in CEPH family 1362 have low H3Ac binding activity. Therefore, GM11982 and GM11983 had low allelic fraction values, 0. 41 and 0. 36, respectively. However, GM11984 received both alleles that had low H3Ac binding activities. Consequently, the allelic fraction was 0. 48, very close to 0. 5. Conversely, GM11987 received both alleles that had normal H3Ac binding activities, thus the allelic fraction value was also close to 0. 5. Note that this is different from conventional Mendelian inheritance analysis in that it uses the allelic fraction as a phenotypic trait, and this depends on relative quantities between the two alleles. Nevertheless, our results agree very well with inheritance of the chromatin state, in turn providing direct support for genetic influence on the chromatin state. However, we must qualify our results by noting that, in contrast to the relatively large number of informative individuals studied in Yan et al. [5] (eight and ten informative individuals per family for two different genes) (Figure 1), the maximum number of individuals informative for any SNP tested in any family in our study is five. This limits the statistical power of our inheritance analysis, despite a highly suggestive result. Taken together, these results suggest that inherited genetic components could determine the epigenetic state of the chromatin. To our knowledge, this is the first demonstration in humans that genetic inheritance may be an important factor directing the global chromatin state mediated by histone modification, the hallmark of the epigenetic phenomena. Our aim was to determine if genetic inheritance can influence chromatin state globally in humans. Our studies support the notion that inherited genetic components can determine the epigenetic state of the chromatin. Our strategy was to use samples from different pedigrees to assess the genetic effect. The use of the SNP chip to measure allele-specific chromatin-binding intensity in a heterozygous individual and the use of relative binding intensity between the two alleles enables us to detect difference in chromatin state in individuals between different families since other sources of variations in the cellular environment are likely affecting both alleles more or less equally. The use of PCA made it possible to focus analyses on a few components, which have the capacity to combine weak signals from multiple genetic loci. Otherwise, the weak signal may not be detectable when analyzed individually. We used a combination of 12 lymphoblastoid cell lines and six antibodies plus two controls in the experiment. This is two-factor experiment design. Genetic factor has two levels, one for each family; whereas chromatin factor has three levels, one for active chromatin, one for inactive chromatin, and one for control. This study design allows us to assess genetic inheritance effect as well as chromatin states targeted by the six antibodies on the total variance across the 96 experiment data. We are interested in the variance across the 96 samples. In our variance component model, we decomposed the total variance into three components, genetics, chromatin, and residual variance. Because the complexity in this high dimensional ChIP-on-chip data, we need to reduce the complexity to effectively understand the variance structure. We used PCA for this purpose. What PCA does is to transform the data matrix by rotating the coordinate system. After transformation, we have a new set of variables, denoted by principal components. Each principal component is a linear combination of the original variables. PCA has two useful mathematic properties. First, all principal components are orthogonal to each other, so the total variance is simply the sum of variances from each principal component. Second, principal components are ranked so that PC1 accounts for the largest variance in the data, followed by PC2. In our studies, the first two principal components usually account for about 10%–50% of the total variance. Therefore, we were able to focus the analyses in two dimensions instead of the original 10,000 dimensions. PCA using total ChIP signal as (A + B) (Figures 2A and 3, panels on the left) indicated that the total variance in the samples was comprised mostly by antibodies targeting at various chromatin proteins, which also demonstrated the specificity of the ChIP assay. In contrast, PCA using the relative signal (A/A + B) indicated that the total variance in the samples comprised primarily the difference between two families and secondarily antibodies targeting at various chromatin proteins (Figures 2B and 3, panels on the right). The separation between different families in controls served as the baseline, which captured the background level of difference due to genotypes. The separation between different families is much too large for the antibodies targeting at active chromatin, indicating specific chromatin state differences between different families. This result suggests that genetic inheritance can influence the global chromatin state. The relative intensity measurement (A/A + B) has better sensitivity in detecting the genetic effect than the total intensity (A + B), since other sources of variations in the cellular environment that could affect the total intensity are likely affecting both alleles more or less equally, thus not masking the genetic effect on the relative intensity in chromatin state. In the case of PCA of families 1 and 2 (Figure 2B) using the relative intensity, the largest variance, captured by PC1, was due to the difference between family 1 and family 2. In the case of PCA analysis of families 3 and 4 (Figure 3, top right), the largest variance (always captured by PC1 because of the algorithm) was due to the difference between control and antibodies targeting active chromatin states (H3Ac and H3K4). PC2 was the vector that contained the second largest variance in these data, corresponding to the difference between family 3 and family 4. The conclusion of genetic influence on chromatin state is supported by the clustering of the families when samples are projected in the 2-D space defined by PC1 and PC2. The conclusion is valid regardless of whether the separation is on PC1 or PC2, which is determined by the variance-covariance structure of the data. In other words, principal components are data driven. PCA is an unsupervised method. Furthermore, our allelic segregation analysis agrees very well with Mendelian inheritance of the chromatin state (Figures 5 and S8), thus providing direct support for genetic influence on the chromatin state. It is interesting to note that familial aggregation of allelic-specific DNA methylation variation at imprinted gene loci has been previously reported [19] as well as Mendelian inheritance of DNA methylation [20]. A total of three recent studies also indicated germline inheritance of methylation epimutation in MSH2 and MLH1 in families with hereditary nonpolyposis colorectal cancer [21–23]. DNA methylation and histone acetylation showed nearly identical patterns in young monozygotic twins but marked differences in old monozygotic twins [24]. All these observations support the notion of the influence of genetic inheritance on epigenetic processes. A genetic effect on chromatin state is well known in model organisms. Examples include position-effect variegation in Drosophila melanogaster [25]. A related observation is transgenerational epigenetic inheritance. For example, agouti viable yellow mice display inheritance of yellow fur as a result of incomplete erasure of the methylation signal associated with a retrotransposon insertion [16], and kinked-tail mice transmit phenotype through multiple generations due to the loss of the silent epigenetic state at the Axin gene [26] as well as a heritable white-tail phenotype associated with Kit-specific microRNAs [17]. Meiotic transmission of epigenetic states has also been described in several studies in plant [27–29]. Although total gene expression as expression quantitative trait loci was regulated by genetic loci [6–8], the detection of the expression quantitative trait loci usually required a much larger sample size than the 12 samples used here. Our ability to detect familial aggregation by allele-specific chromatin state, but not in total chromatin state, resulted from the increased specificity of probing chromatin state with the relative intensity (A/A + B). The use of PCA might further enhance our ability to detect the genetic effects on chromatin state, since PCA had the capacity to detect a robust signal captured in the principal components even though signals from individual SNPs might be weak. The allelic differences in chromatin provided an explanation for the observed allelic variation in gene expression [5]. ChIP was carried out using a ChIP assay kit (Upstate, (http: //www. upstate. com/img/coa/17-295-33519A. pdf). Lymphoblastoid cells of 24 individuals from CEPH/Utah pedigrees (family identification 1347,1362,1331, and 1413) were used in this study. ChIP was carried out using a ChIP assay kit (Upstate) with antibodies against histone H3 acetylated at K9 and K14 (Upstate, 06–599), dimethylated at K4 (Upstate, 07–030), dimethylated at K9 (Upstate, 07–441), dimethylated at K27 (Upstate, 07–452), trimethylated at K27 (Upstate, 07–449), and Pol II (Abcam, ab5408, http: //www. abcam. com). All cell lines are described in Table S3. Briefly, 2 × 107 cells were grown in RPMI medium 1,640 supplemented with 2 mM L-glutamine and 15% FBS. The cells were fixed by adding formaldehyde solution into the culture medium to a final concentration of 1%. After centrifugation the cell pellets were rinsed twice with an ice-cold PBS solution and then suspended in a lysis buffer (all buffers used in the ChIP experiment are described in http: //www. upstate. com/img/coa/17-295-33519A. pdf). Sonication was performed on ice using Cole Parmer economical ultrasonic processor at power 9 for 12 cycles of sonication, each cycle for 10 s followed by a 30-s break on ice. The cell pellets were centrifuged at 10,000 RCF (×g) for 10 min, and the resulting lysates in the supernatant were stored at −80 °C until use. The chromatin lysates were diluted by 10-fold in a ChIP dilution buffer. They were precleared by Salmon sperm protein A agarose and incubated with each of the six antibodies individually overnight at 4 °C. The chromatin complexes were sequentially washed in low salt, high salt, LiCl salt, and TE buffers. The protein/DNA complex was eluted in an SDS elution buffer (1% SDS, 0. 1 M NaHCO3). The crosslink between protein and DNA was reversed. The protein/DNA complex was treated with Proteinase K. DNAs were purified using Qiagen mini-elute reaction clean-up kit (http: //www1. qiagen. com). PCR was carried out using primer pairs described in Table S2. Antarctic Phosphatase (New England Biolabs, http: //www. neb. com) and Exonuclease I (New England Biolabs) was used to remove unincorporated primers and dNTPs. Allele-specific OLA was carried out in a 5-μl reaction containing 1× Ampligase buffer (Epicentre Biotechnologies, http: //www. epibio. com), 100 nM each ligation primers, 0. 5 U Ampligase, and 1 μl of phospho/exo treated PCR product (∼10 ng) for 30 cycles, with each cycle at 95 °C for 30 s, 50 °C for 30 s, and 65 °C for 2 min. All primers are described in Table S2. Ligation products were resolved by ABI3730XL genetic analyzer and analyzed using GeneMapper 3. 5 software (Applied Biosystems, http: //www. appliedbiosystems. com). We treated 500-ng input DNA or 50-ng immunoprecipitated DNAs in the ChIP experiment with mung bean nuclease to flush the ends. The DNA was phosphorylated and ligated to an Xba-linker (Table S2). Following an Xba I digestion, DNA was purified by Qiagen mini-elute reaction clean-up kit and was ligated to an Xba-adaptor. DNA was then amplified using an Xba-primer. This amplification step did not introduce biased representation of the initial ChIP DNA (Figure S7). It also retained the allelic specificity as demonstrated by the experiment described in Figures 1A and S6. Next, 10-μg PCR products from the input or 5-μg PCR products from the ChIP experiments were digested and labeled as described in the 10K SNP chip manual. We carried out the hybridization, washing, and scanning as described in the manual. All statistical analyses were developed using R and Splus packages. The missing values in PM or MM probes were replaced by the mean MM across all SNPs. For each SNP, we computed the ratio PM/MM and then applied the Robust Multi-array Average (RMA) method [30]. Probe intensity was computed by the function of max (mean (log2[PM/MM], 0) for allele A and allele B. The intensity at the probe set level was the average of the ten pairs of the probes from each allele of the SNP. The signal for each allele of an SNP was evaluated by t-test for the measurement of (PM − MM) with H0 hypothesis of (PM − MM) = 0 for the ten probes for a given SNP. We chose a p-value of 0. 01 as a threshold for the presence of a signal. We used PCA to visualize similarity and variability among the 96 samples containing the ChIP data done on 12 individuals, each characterized with six antibodies plus the controls of input and DNA, using either the total binding intensity (A + B) or the relative binding intensity (A/A + B). PCA transforms the data matrix by rotating the coordinate system. After transformation, we had a new set of variables, denoted by principal components. Each principal component was a linear combination of the original variables with different weights (loadings). The loadings reflected the degree of contribution of each SNP to the principal component. PCA has two useful mathematic properties. First, all principal components are orthogonal to each other so the total variance is the sum of variances from each principal component. Second, principal components are ranked in such a way so that PC1 accounts for the largest variance in the data followed by PC2. In our data, the first two principal components typically accounted for about 10%–50% of the total variance. Therefore, we focused our analyses in two dimensions instead of the original 10,000 dimensions. The utilities of the PCA in this study are 2-fold. First, PCA provides dimension reduction, allowing visualization of data structure in 2-D. Second, it provides a quantitative assessment of data structure and interactions among variables. In this study, the data structure refers to the clustering of samples by family or antibody type. The separation of samples by different principal components reflects the degree of difference due to either family or antibody. The separation in PC1 is always the largest, by definition, due to the PCA algorithm. The relative contribution of the components can be assessed by eigen-values, which are provided in Tables S4 and S5. The expected value of relative binding intensity (A/A + B) is 0. 5. Deviation from 0. 5 for an SNP among heterozygous individuals for genomic DNA and input suggests erroneous behavior of the SNP. We removed SNPs whose deviation from 0. 5 exceeded two standard deviations. A total of 2,365 SNPs were removed by this criterion. We used 0. 5 for homozygous individuals in the PCA for the relative binding intensity (A/A + B). All samples were projected in the space defined by the first and second principal components. The National Center for Biotechnology Information (NCBI) Entrez Gene (http: //www. ncbi. nlm. nih. gov/entrez/query. fcgi? db=gene) accession numbers for the genes discussed in this paper are ASTN2,23245; C6orf190,387357; CD19,930; CD3G, 917; GAPDH, 2597; MYOD1,4654; NES, 10763; PKHD1,5314; RPLP1,6176; SYT9,143425; TCBA1,154215; TIAM1,7074; and TMEM16D, 121601.

Human health and disease are determined by an interaction between genetic background and environmental exposures. Both normal development and disease are mediated by epigenetic regulation of gene expression. The epigenetic regulation causes heritable changes in gene expression, which is not associated with DNA sequence changes. Instead, it is mediated by chemical modification of DNA such as DNA methylation or by protein modifications such as histone acetylation and methylation. Although much has been known about epigenetic inheritance during development, little is known about the influence of the genetic background on epigenetic processes such as histone modifications. In this report the authors studied five histone modifications on a genome-wide level in cells from different families. Global epigenetic states, as measured by these histone modifications, showed a similar pattern for cells derived from the same family. This study demonstrates that genetic inheritance may be an important factor influencing global chromatin states mediated by histone modifications in humans. These observations illustrate the importance of integrating genetic and epigenetic information into studies of human health and complex diseases.

lay_plos

Many studies have proved that oncogenic viruses develop redundant mechanisms to alter the functions of the tumor suppressor p53. Here we show that Epstein-Barr virus (EBV), via the oncoprotein LMP-1, induces the expression of ΔNp73α, a strong antagonist of p53. This phenomenon is mediated by the LMP-1 dependent activation of c-Jun NH2-terminal kinase 1 (JNK-1) which in turn favours the recruitment of p73 to ΔNp73α promoter. A specific chemical inhibitor of JNK-1 or silencing JNK-1 expression strongly down-regulated ΔNp73α mRNA levels in LMP-1-containing cells. Accordingly, LMP-1 mutants deficient to activate JNK-1 did not induce ΔNp73α accumulation. The recruitment of p73 to the ΔNp73α promoter correlated with the displacement of the histone-lysine N-methyltransferase EZH2 which is part of the transcriptional repressive polycomb 2 complex. Inhibition of ΔNp73α expression in lymphoblastoid cells (LCLs) led to the stimulation of apoptosis and up-regulation of a large number of cellular genes as determined by whole transcriptome shotgun sequencing (RNA-seq). In particular, the expression of genes encoding products known to play anti-proliferative/pro-apoptotic functions, as well as genes known to be deregulated in different B cells malignancy, was altered by ΔNp73α down-regulation. Together, these findings reveal a novel EBV mechanism that appears to play an important role in the transformation of primary B cells. Epstein-Barr virus, also known as human herpesvirus 4 (HHV4), belongs to the gammaherpesvirus family and is largely spread as it can be detected in 90% of the worldwide population. EBV infects B cells and, in most cases, does not lead to any clinical manifestations. However, when EBV infection occurs during adolescence or young adulthood, it may cause infectious mononucleosis, a benign lymphoproliferative disease. A minority of EBV infections result in the development of several types of human B cell malignancies, including Burkitt' s lymphoma (BL), Hodgkin and non-Hodgkin lymphomas [1]. In addition, EBV has been clearly associated with epithelial cancers, i. e. nasopharyngeal carcinoma (NPC) and a sub-set of gastric carcinoma [1]. The risk of developing EBV-induced malignancies is significantly increased in immuno-compromised individuals, such as AIDS patients and organ-transplant recipients. In vitro EBV efficiently infects human resting B cells and transforms them into proliferating lymphoblastoid cell lines (LCLs) [2]. Similar to other herpesviruses, the EBV life cycle includes a latent and non-productive phase, as well as a lytic phase leading to the production of the virus progeny. After primary infection, EBV persists lifelong in a latent state in a sub-population of resting memory B cells [3]. Recent studies led to a model of EBV persistence whereby different viral transcription programs were used within the context of the normal biology of B lymphocytes in order to carry out its life cycle [4], [5]. Eleven genes can be expressed in the latency phases, namely the EBV nuclear antigens (EBNA) 1,2, 3A, 3B, 3C, LP, the latent membrane proteins (LMP) 1,2A, 2B, the untranslated EBER-1 and EBER-2 RNAs, as well as multiple microRNAs [2]. Based on the expression pattern of the different latency genes, four latency phases have been identified so far. Type I latency is normally present in Burkitt' s lymphoma and is associated with the expression of EBNA-1 as well as EBERs and miRNAs. Type II latency is frequently detected in Hodgkin' s lymphoma and nasopharyngeal carcinoma, and is linked to the expression of EBNA-1, LMP-1, LMP-2A, LMP-2B, EBERs and miRNAs. Type III latency is characterized by the expression of all 11 latency genes and is mainly found in lymphoproliferative diseases in immunocompromised individuals and in in vitro EBV-transformed LCLs. Finally, type IV latency is associated with the infectious mononucleosis and is less well defined, since the expression pattern of the latency genes may differ in different patients [6]. LMP-1 is the major EBV oncoprotein and displays transforming activities in in vitro and in vivo models [2]. It is an integral membrane protein composed of a short cytoplasmic amino-terminal domain, six hydrophobic transmembrane domains, and a cytoplasmic carboxy-terminal domain [2]. LMP-1 exerts its transforming properties by functioning as a member of the tumour necrosis factor receptor (TNFR) superfamily leading to the a constitutive activation of several cellular signaling pathways [7]–[9]. In particular, LMP-1 activates the nuclear factor-kappa B (NF-κB) signaling pathway, thus promoting cell growth and inhibition of apoptosis. In addition to LMP-1, other EBV latent proteins, i. e. EBNA-2, EBNA-3A and EBNA-3C, are involved in the immortalization of primary B cells. The role of EBNA-2 is mainly mediated by its ability to modulate the transcription of host and viral genes, while EBNA-3C plays a direct role in cellular transformation by inactivating the products of tumor suppressor genes, such as retinoblastoma (pRb) and p53 [10]–[14]. Interestingly, as for other oncogenic viruses, e. g. human papillomavirus type 16 (HPV16) [15], EBV developed multiple and redundant mechanisms to inactivate p53-regulated pathways. Indeed, EBNA-3C is able to alter the p53 transcription activity via direct binding as well as by inducing stabilization of p53 inhibitors, such as mdm2 and Gemin3 [10], [12], [14]. p73 is a closely p53-related transcription factor that shows functional similarity to p53 [16], [17]. The impact of EBV on p73 has been poorly investigated so far, although initial findings indicate that, similarly to p53, p73 is targeted by EBV. Indeed, p73 expression was found down-regulated in EBV-positive gastric carcinoma by heavy methylation of CpG islands within its promoter [18]. In addition, it has been recently shown that EBNA-3C attenuates p73 expression in LCLs by targeting the transcription factor E2F-1 [19]. However, it is likely that, in line with the previous findings on p53, EBV has developed multiple mechanisms to alter p73 function. In addition, due to the variability in the expression pattern of the latent genes in EBV-positive cancer cells, it is possible that more than one viral protein has the ability to target the p53/p73 pathway. We have previously shown that cutaneous HPV38 which belongs to an HPV subgroup potentially associated with the development of non-melanoma skin cancer (NMSC), is able to induce the accumulation of ΔNp73α which in turn alters the transcriptional functions of p53 and p73 [20], [21]. ΔNp73α is a p73 isoform that lacks transactivation (TA) domain and is accumulated in several tumors [22]. Most importantly, increase in ΔNp73α protein levels correlates with poor outcome of the disease and bad response to therapy [23]–[25]. Here we show that EBV LMP-1 activates ΔNp73α expression in B cells by favoring the recruitment of p73 to ΔNp73 promoter. This phenomenon appeared to be mediated by c-Jun NH2-terminal kinase 1 (JNK-1), and resulted in the inhibition of p53-regulated genes encoding key anti-proliferative regulators. Several isoforms of ΔNp73 have been identified, that can be generated by alternative splicing at the 5′ region of the p73 mRNA or by transcriptional initiation from a promoter (p2) within the p73 gene [26], [27]. We have previously shown that HPV38 induces the accumulation of ΔNp73α mRNA generated by the p2 promoter [20]. We therefore evaluated whether the levels of this specific ΔNp73 transcript were induced by EBV. ΔNp73α mRNA levels were determined in EBV-positive and EBV-negative B-cell lines by RT-PCR using specific primers. Six LCLs expressed high levels of ΔNp73α transcript, while no signal was detected in the EBV-negative B lymphoma cell line BJAB (Figure 1A). To assess that the over-expression of ΔNp73α was indeed linked to EBV infection, we infected primary B cells with recombinant EBV and analyzed ΔNp73α mRNA levels by quantitative RT-PCR. As previously shown, ΔNp73α is not expressed in primary B cells (Figure 1B) [28]. In contrast, an increase in ΔNp73 mRNA levels was observed between 12–36 hours post-EBV infection which correlated with LMP1 transcript levels (Figure 1B). ΔNp73 mRNA accumulation was also observed in cancer B-cell lines, RPMI, upon EBV infection cells (Figure 1C). Several C-terminus ΔNp73 isoforms have been characterized, that are generated by alternative splicing (α, β, γ, ε). The isoform alpha plays a key role in altering the p53/p73 functions and is over-expressed in several human cancers [29]. RT-PCR experiments with specific ΔNp73 isoform primers confirmed that alpha, and not beta, ΔNp73 is expressed in EBV-infected B-cells (Figure 1D). Accordingly, immunoblotting with a p73 antibody revealed a 65–70 kD protein band in LCLs that co-migrated with the ΔNp73α ectopically expressed in HEK 293 cells (Figure 1E). Taken together, these data showed that EBV specifically activates ΔNp73α transcription in primary and cancer B-cells. Studies on other oncogenic viruses demonstrated that alterations of p53-regulated pathways are normally induced by the viral oncoproteins [15], [30]. Figure 1B showed a correlation between ΔNp73α and LMP-1 expression levels, supporting the possible involvement of the viral oncoprotein in ΔNp73α up-regulation. Therefore, we determined later whether the major EBV transforming protein, LMP-1, was responsible for ΔNp73α accumulation. RPMI cells were transduced with empty (pLXSN) or LMP-1 expressing retrovirus (pLXSN-LMP-1) and ΔNp73α transcript and protein levels were determined by RT-PCR and immunoblotting, respectively. Both ΔNp73α mRNA and protein levels were strongly increased in RPMI/LMP-1 cells in comparison to cells infected with the empty retroviral vector (Figures 2A and B). To further demonstrate the role of LMP-1 in ΔNp73α up-regulation, we infected RPMI cells with a wild-type or mutated EBV, in which the LMP-1 gene was deleted (EBVΔLMP-1). Two RPMI/EBVΔLMP-1 cell lines were generated by two independent infections and ΔNp73α expression levels were compared with the ones of mock infected cells as well as RPMI/EBV. Deletion of LMP-1 gene from the EBV genome abolished ΔNp73α up-regulation, as shown by RT-PCR and immunoblot analyses (Figures 3 A and B). Transduction of RPMI/EBVΔLMP-1 cells with a recombinant retrovirus expressing LMP-1 (pLXSN LMP-1) restored the ability of EBV to promote ΔNp73α mRNA and protein accumulation (Figures 3C and D). In summary, these data highlight the central role of LMP-1 in EBV-mediated ΔNp73α up-regulation. The p73 p2 internal promoter contains a p53 responsive element (RE) which can be activated by both p53 and p73 [31]–[33]. Therefore, we evaluated whether p53 and/or p73 are involved in the regulation of p2 promoter in the presence or absence of LMP-1. Chromatin immune precipitation (ChIP) experiments using the p53 null SaOS-2 cells as experimental model showed that LMP-1 over-expression increased p73 binding affinity for the RE within the p2 promoter, while it did not influence p53 recruitment to the same site (Figure 4A). ChIP experiments with anti p73 antibody in RPMI cells transduced with empty (pLXSN) or pLXSN-LMP-1 retrovirus also showed an increased binding of p73 to the p2 promoter in presence of LMP1 (Figure 4B). In addition, DNA pull-down experiments, in which a biotinylated DNA probe containing a region of the p2 promoter encompassing the p53RE was incubated with cellular extracts of RPMI or RPMI LMP-1 cells, showed that LMP-1 increased p73 efficiency in binding DNA (Figure 4C). Silencing of p73 expression in LCL by shRNA correlated with down-regulation of ΔNp73α mRNA levels (Figure 4D). In contrast, targeting p53 with a siRNA in the same cells did not alter ΔNp73α levels (Figure 4D), indicating that p53 is not involved in the transcriptional regulation of ΔNp73α in LCLs. We conclude from this set of data that LMP-1-mediated ΔNp73α transcriptional activation is partly due to the recruitment of p73 to the p2 promoter. It is known that LMP-1 stimulates JNK-1, which in turn leads to p73 phosphorylation and increase in its transcriptional activity [34]–[36]. We therefore determined whether JNK-1 is involved in ΔNp73α accumulation mediated by LMP-1. Ectopic levels of JNK-1 in BJAB cells induced ΔNp73α accumulation (Figure 5A). In addition, treating LCLs with a specific inhibitor of JNK (SP600125) led to a time-dependent decrease in ΔNp73α mRNA levels (Figure 5B). JNK-1 down-regulation in LCL by siRNA also led to a decrease in ΔNp73α mRNA and protein levels (Figures 5C and D). LMP-1 transforming activities lie mostly on two distinct domains in its cytoplasmic C-terminus, namely C-terminal activation region 1 (CTAR1) (amino acids 187–231) and CTAR2 (amino acids 351–386). As CTAR2 LMP-1 mutant (LMP-1/378 stop) is unable to activate JNK-1 [37], we determined whether deletion of CTAR2 affected LMP-1 ability to promote p73 and ΔNp73α accumulation. We first transfected SaOS-2 cells with HA tagged p73 in the presence of wild-type or 378 stop-mutant LMP-1. ChIP experiments performed with an HA antibody showed that only the wild-type LMP-1 increased p73 recruitment to p2 promoter (Figure 5E). According to previous data [35], immunoblotting showed that wild-type LMP-1, but not the LMP-1 378 stop-mutant that is unable to activate JNK-1, induced p73 accumulation (Figure 5F). Finally, ΔNp73α expression was only detected in RPMI/EBVΔLMP-1 cells containing the wild-type LMP-1 and not the LMP-1 378 stop-mutant (Figure 5G). LMP-1 is also able, via the CTAR1 and CTAR2, to activate the cellular kinase p38 which in turn activates p73 [38]–[40]. However, p38 inhibition in LCLs by a chemical inhibitor did not result in a decrease of mRNA levels of ΔNp73α (data not shown), indicating that a different mechanism is involved in the event. Together, these data highlight a crucial role of JNK-1 in LMP-1-mediated accumulation of p73 and ΔNp73α. Emerging lines of evidence show that epigenetic changes play an important role in carcinogenesis [41]. Accordingly, several oncogenic viruses, including EBV, are able to hijack the epigenetic machinery in order to de-regulate cellular gene expression, to persist in the host cell and complete the viral cycle [42]. EZH2 is a component of the Polycomb 2 complex and is able to methylate the Histone H3 on Lysine 27, leading to chromatin condensation and gene silencing [43]. Interestingly, it has been recently shown that, upon interferon alpha treatment, EZH2 inhibits ΔNp73α expression in hepato-cellular carcinoma cells (HCC) by direct binding to the p2 promoter [44]. Therefore, we next determined whether in primary B cells and LCL the observed alterations in ΔNp73α expression may be ascribed to changes in the EZH2 recruitment to p2 promoter. ChIP experiments showed that EZH2 binds the p53 RE within the p2 promoter only in primary B cells, but not in LCL (Figure 6A). A similar pattern was observed in ChIP experiments performed with an antibody that specifically recognized H3K27 methylated form (Figure 6A). In contrast, acetylation on lysine 9 of the Histone H3, a marker of transcriptionally active chromatin, was increased at the p53/p73 RE in LCLs in comparison to primary B cells (Figure 6A). In addition, p73 was more efficiently recruited to p53/p73 RE in LCLs than primary B Cells (Figure 6B). Loss of EZH2 at ΔNp73α promoter appeared to be dependent on LMP-1 expression in LCLs and RPMI cells (Figure 6C and D). According to these results, histone H4 hyperacetylation, another event associated with active transcription, is strongly enhanced at ΔNp73α promoter in LCLs in comparison to primary human B cells (Figure 6E). JNK can induce H4 hyperacetylation to regulate gene expression [45], accordingly inhibition of JNK-1 by siRNA or chemical inhibitor in LCLs resulted in the decrease of histone H4 hyperacetylation at ΔNp73α promoter (Figure 6E and F). In addition, the recruitment of p73 to the ΔNp73α promoter in LCL is strongly reduced upon JNK-1 inhibition (Figure 6F). Immunoblotting showed that EZH2 is weakly detected in primary B cells, while its protein levels are considerably elevated in LCLs (Figure 6G and data not shown). Similarly, expression of LMP-1 in RPMI cells infected with EBV-ΔLMP-1 led to a substantial increase in EZH2 protein levels (Figure 6H). To determine whether the increase of EZH2 levels and decrease of its recruitment to ΔNp73 promoter in LMP-1 cells was due to changes in its localization, we performed cellular fractionation experiments followed by immunoblotting. LMP-1 expression did not alter EZH2 cellular localization, which appeared to be exclusively nuclear in primary B cells and LCLs (Figure 6I). However, immuno-fluorescence experiments with an anti-EZH2 antibody highlighted a different pattern of staining in primary B cells and LCLs, indicating that LMP-1 induced a redistribution of EZH2 in the nucleus (Figure 6J). Indeed, EZH2 staining appears to be punctuated in primary B cells, while it is more diffuse in LCLs (Figure 6J). Together the data show that ΔNp73α expression mediated by EBV LMP-1 correlates with the release of EZH2 from the ΔNp73 promoter, which results in the opening of the chromatin and in an increased access to transcription factors. To investigate the role of ΔNp73α in EBV infected cells, we down-regulated its protein levels by expressing an anti ΔNp73α anti sense oligo-nucleotide (AS). The sense oligo-nucleotide (S) was used as negative control. Figure 7A shows that ΔNp73α was efficiently down-regulated in a dose-dependent manner by AS in LCLs. Most importantly, ΔNp73α down-regulation led to PARP cleavage, indicating that ΔNp73α is involved in the inhibition of apoptosis in EBV infected cells (Figure 7A). FACS analysis confirmed that transfection with AS promoted cellular death (Figure 7B). To gain more insight into the biological significance of ΔNp73α expression in EBV infected cells, we compared the transcriptome profiling by RNA-seq of LCLs transfected by S or AS at two different concentrations. The expression levels of approximately 253 genes were found differentially expressed (214 up-regulated, and 39 down-regulated, p value<0. 01) in cells transfected with S and AS (Tables S2 and S3, supplementary material). Functional analysis of the data was conducted as explained in Materials and Methods. It has been previously shown that ΔNp73α, but not ΔNp63α, plays a role in nervous system development and in the prevention of nerve growth factor-induced p53-mediated apoptosis [46]. Accordingly, we observed that the decrease in ΔNp73α levels by AS in LCL also led to the deregulation of several genes encoding products that are involved in development, e. g. in neurogenesis and in the regulation of apoptosis in neurons (Tables S2 and S3, supplementary material), indicating the specificity of our approach and validating our RNA seq analysis. Most importantly, AS ΔNp73α up-regulated genes encoding proteins, which play crucial roles in cellular transformation processes, such as apoptosis, cell cycle, DNA-repair and signaling pathways, i. e. NF-κB, Notch, RAS, and toll-like receptors (TLRs) (Table S2 and S3, supplementary material). In agreement with the functions of ΔNp73α as an antagonist of p53, several p53-regulated pro-apoptotic genes were found up-regulated in LCLs transfected by AS (Figure 7C and Tables S2 and S3, supplementary material). Interestingly, the inhibition of ΔNp73α by AS in LCLs resulted in a strong increase of PLK2 mRNA levels. PLK2 is a p53-regulated gene and encodes a serine threonine kinase which is involved in cell cycle regulation and cellular response to stresses. Its expression has been often found silenced by promoter methylation in Burkitt' s Lymphomas [47]. ChIP experiments in SaOS-2 cells expressing HA-tagged-ΔNp73α demonstrated its binding to the promoter of PLK2 to the p53 binding site 1, which was further increased in the presence of LMP-1, while p73 recruitment to the same promoter was not influenced by the viral oncoprotein (Figure 7D). To corroborate the RNA-seq data, we have down-regulated ΔNp73α and determined PLK2 mRNA levels by quantitative PCR. Figure 7E shows that PLK2 and Pig3 are down-regulated in LCL in comparison to primary B cells. In addition down-regulation of ΔNp73α resulted in a rescue of their expression confirming the RNA seq data. Another gene that was found up-regulated in ΔNp73α AS-transfected LCLs is KLHDC8B which also appears to be associated with lymphomagenesis and is often found mutated in familiar and sporadic Hodgkin lymphomas [48]. KLHDC8B mRNA levels were increased by 3. 66 and 18. 44 folds in LCLs transfected with low and high doses of ΔNp73α AS, respectively (p value = 0. 001693). Taken together, these data show that in LCL, ΔNp73α plays a key role in regulating cellular genes, the products of which exert important functions in cellular transformation, including two genes, PLK2 and KLHDC8B, that have been previously reported to be associated with lymphomagenesis. Oncogenic viruses share the ability to target key pathways involved in preventing cellular transformation, considerably increasing the probability of an infected cell to evolve towards malignancy. One of the best characterized mechanisms of oncogenic viruses is the ability to inhibit the function of the tumor suppressor p53, a transcription factor that can trigger cell cycle arrest or apoptosis in response to stress or DNA damage [49]. Many oncogenic viruses, such as HR mucosal HPV types [15], EBV [10], [12], [14], Human T-cell Lymphotropic Virus (HTLV-1) [50], [51], Kaposi' s sarcoma-associated herpesvirus (KSHV) [52]–[54] have developed strategies to inactivate p53. We have recently described a novel mechanism of deregulation of p53 transcriptional functions by the beta cutaneous HPV38 which appears, together with other beta HPV types, to be linked to skin carcinogenesis. HPV38 E7 oncoprotein promotes accumulation of ΔNp73α increasing its transcription and protein half-life ([20], [21] and our unpublished data). In turn, ΔNp73α competes with p53 for binding to p53 RE elements, preventing the activation of p53-regulated genes. Although the involvement of HPV38 in NMSC is still under debate, the demonstration that also other well-established oncogenic viruses promote ΔNp73α accumulation will further highlight the importance of the event and corroborate the potential role of HPV38 in human carcinogenesis. In this study, we show that the oncoprotein LMP-1 from EBV activates the transcription of ΔNp73, favoring the recruitment of p73 to cis p53 element of ΔNp73 p2 promoter. We also demonstrated that LMP-1-mediated up-regulation of ΔNp73α transcription is dependent on JNK-1, a kinase strongly activated by LMP-1. JNK-1 inhibition by different means strongly decreased ΔNp73α expression in EBV-infected cells. In addition, expression of ectopic levels of JNK-1 in the EBV-negative B-lymphoma cell line, BJAB, resulted in the activation of ΔNp73α transcription. Accordingly, LMP-1 mutants lacking the JNK-1 activating domain (CTAR2) did not influence the ΔNp73α expression levels. It is well established that several amino acid residues of p73 are phosphorylated by JNK-1 [34]. Therefore, it is likely that p73 recruitment to the ΔNp73 promoter is mediated by its JNK-1-dependent phosphorylation. Additional experiments are required to confirm this hypothesis and establish whether the p73 affinity for ΔNp73 promoter is determined by phosphorylation of one or more specific amino acids. Previous studies have shown that inhibition of JNK-1 in LMP-1 expressing cells led to decrease of cdc2 levels and cell cycle arrest [55]. In our experimental model the inhibition of cdc2 in LCLs by the chemical inhibitor roscovitine slightly affected ΔNp73α levels (data not shown). JNK-1 could also induce ΔNp73 transcription by an alternative mechanism via activation of the proto-oncogene c-Jun. It has been shown that p73 acts in synergistic manner with c-Jun in promoting cellular survival [56]. This event is well explained by their cooperative ability to activate the transcription of specific subsets of cellular genes. ChIP-seq experiments have revealed the presence of AP1-binding motifs in close proximity to the p73 cis elements in promoters of genes encoding proteins with anti-apoptotic functions [57]. Brigati et al. have shown that TPA treatment of Germinal Center B cells, able to induce ΔNp73α expression, also leads to binding of c-Jun to an AP1 site which was located on the promoter of ΔNp73α, just upstream the p53/p73 RE [28]. According to these findings, we observed that in LCLs c-Jun is recruited to an AP1 cis element closely located to the p53/p73RE of ΔNp73 promoter (our unpublished data). ChIP experiments in primary and EBV-immortalized B cells showed that activation of ΔNp73 promoter by the recruitment of p73 correlated with the displacement of the polycomb 2 complex component EZH2 and epigenetic changes. The apparently paradoxical finding that EBV infected B cells, despite the increased intracellular levels of EZH2, show reduced amount of EZH2 and lower levels in H3K27 methylation on the promoter of ΔNp73, recalls the scenario observed in HPV16 E6/E7 expressing cells [58]. Hyland et al. observed increased levels of EZH2 in the presence of HPV16 E6 and E7 proteins, which correlated with a decrease of H3K27 methylation. The authors explained that this phenomenon was due to an increase in KDM6A and KDM6B levels, two demethylase enzymes, and a decrease in BMI1, a Polycomb1 protein which stabilizes Polycomb 2-mediated methylation. According to this model, EBV is able to trigger accumulation of KDM6B via LMP-1 ([59] and our unpublished data) as well as a reduction of BMI1 levels (our unpublished data). Accumulation of EZH2 in cells expressing LMP-1 could be a consequence of post-translational modifications that negatively regulate its enzymatic activity. Accordingly, it has been previously shown that phosphorylation of EZH2 by AKT on serine 21 suppresses methylation of lysine 27 in Histone 3 [60]. It has been reported that EBV LMP-1 triggers the AKT pathway [61] which is often found activated in NPC and Hodgkin' s lymphomas [62], [63]. Based on these findings, we could speculate that the loss of EZH2 recruitment to ΔNp73 promoter is due to serine 21 phosphorylation. We are currently assessing this hypothesis. High levels of p73 and ΔNp73 have been observed in B cell chronic lymphocytic Leukemia [64]. Although resting B cells do not express ΔNp73, epigenetic changes leading to ΔNp73 up-regulation were observed in the activated B cells compartment of the germinative center of the tonsil. Thus, it is likely that the mechanisms characterized in EBV-infected cells in this study may also occur in different scenarios independently of the presence of the viral oncoprotein. To evaluate the biological significance of EBV-mediated ΔNp73α over-expression in the transformation of B cell, we down-regulated ΔNp73α expression by AS in LCL, and compared the cellular expression profiling with one of the S transfected LCL. Decrease in ΔNp73α levels led to the alteration of the expression of cellular genes linked to neurogenesis as well as to the regulation of apoptosis in neurons. These results are consistent with the known in vivo functions of ΔNp73 in brain development and in the prevention of nerve growth factor induced p53-mediated apoptosis [46]. An additional cluster of genes that appeared down-regulated by ΔNp73α in LCL is a group of Homeobox genes. To our knowledge, this is the first time that ΔNp73α has been shown to be implicated in the regulation of HOX genes. Since both HOX genes and ΔNp73α are aberrantly expressed in cancer cells, these findings, if confirmed, could further contribute to the understanding of the events associated with carcinogenesis [22], [65]. Loss of PLK2 expression by promoter CpG island methylation is one of the most common epigenetic events in B-cell lymphomas [47]. It is worth noting that we found PLK2 gene strongly up-regulated upon inhibition of ΔNp73α in LCL. It is well known that PLK2 promoter is positively regulated by p53. Thus, it is highly likely that ΔNp73α induces PLK2 down-regulation by altering the p53 transcriptional function. For the first time, our data provide evidence for a link between PLK2 expression silencing and ΔNp73α in EBV-infected cells. Our findings also suggest that LMP-1 increases the affinity of ΔNp73α for PLK2 promoter without altering its protein levels (HA-ΔNp73α: Figure 7D and our unpublished data). It is possible that the viral oncoprotein, independently of its ability to positively regulate the ΔNp73α transcription, may increase ΔNp73α affinity for PLK2 promoter by promoting post-translational modifications. Similarly to PLK2, KLHDC8B has been linked to lymphomagenesis. Multiple cases of Hodgkin' s lymphoma with translocations or polymorphisms affecting KLHDC8B have been reported in the same family [66], indicating that inhibition of its expression plays a role in B cell transformation. Our data show that the expression of KLHDC8B was restored upon ΔNp73α down-regulation. According to the well characterized ΔNp73α ability to act as an inhibitor of p53, the expression levels of different p53 responsive genes (MDM2, APAF1, GADD45, BAX, CCND1, etc.) increased significantly after ΔNp73 down-regulation, leading to apoptosis. Other subgroups of genes that resulted to be regulated by ΔNp73α are the ones involved in lymphocyte migration and proliferation, cytokine production and innate immune response. As a whole, the RNA seq data indicate that in EBV infected cells ΔNp73α may contribute to the development of EBV-associated disease in several ways, e. g. by inhibiting apoptosis, promoting B cell growth, as well as by modulating host defence machinery allowing EBV persistence. In summary, our data underline the important function of ΔNp73α in EBV-induced cellular transformation, unveiling novel links between its accumulation and deregulation of the expression of many cellular genes. However, the degree to which ΔNp73α oncogenenic effects exceed tumor suppressor effects of p73 activation remains to be better determined. Cellular and viral genes were expressed using the retroviral vector pLXSN (Clontech, Palo Alto, CA) or the expression vector pcDNA-3 (Invitrogen). The pLXSN-LMP-1 construct has been previously described (68). The constructs pLXSN-LMP-1 mutants (LMP-1AxAxA, LMP-1 378 STOP, and LMP-1AxAxA/378STOP) were generated in this study using standard molecular biology techniques. The constructs pcDNA3 HA-ΔNp73α, pcDNA3 HA-p73α were previously described [20]. RPMI 8226 cells (harbouring mutated p53: Glu 285 to Lys) (RPMI) were kindly provided by Dr Christophe Caux (Centre Léon Bérard, Lyon, France). RPMI pLXSN or pLXSN-LMP-1 were generated as described in Fathallah et al. [67]. RPMI EBV and EBVΔLMP-1 were obtained as in [68]. Expression of LMP-1 wild-type or LMP-1 378 stop mutant in RPMI EBVΔLMP-1 was achieved by transduction with recombinant retroviruses [67]. The EBV-immortalized lymphoblastoid cell lines (LCLs), ATM14, ATM11,48513F, 48513M, 2095,2145 and EBV negative immortalized B cells, BJAB (with mutated p53: His 193 to Arg), were previously described [69] (or generated at IARC, Lyon France). Several LCLs were generated in this study by infecting primary B cells isolated from different donors as previously described [67]. Primary and immortalized B cells were cultured in RPMI 1640 medium (GIBCO; Invitrogen life Technologies, Cergy-Pontoise, France) supplemented with 10% FBS, 100 U/ml penicillin G, 100 mg/ml streptomycin, 2 mM L-glutamine, and 1 mM sodium pyruvate (PAA, Pasching, Austria). SaOS-2 and human embryonic kidney cells (HEK293) were cultured in foetal calf serum (FCS) and Dulbecco' s modified Eagle medium (DMEM) (Gibco) using standard culturing conditions. Cells were treated with JNK inhibitor SP600125 at the final concentration of 20 µM. DNA plasmids were transiently transfected in cells by using FuGENE6 or Xtreme gene 9 reagents (Roche) according to the purchased protocol. Immuno-fluorescence in primary and immortalized B cells was performed as described in Fathallah et al. [67]. For FACS staining, cells were collected and washed twice in PBS, then stained with Propidium iodide (PI) at the final concentration of 5 µg/ml. Subsequently, cells were analyzed for the % of dead cells by FACS CANTO (Becton Dickinson). Gene silencing of SAPK/JNK was performed using SignalSilence SAPK/JNK siRNAI (6269 cell signalling). Cells (8×105) were transfected with siRNA to the final concentration of 100 nM by oligofectamine (invitrogene) according to the manufacture protocol. p53 gene silencing was performed as in Accardi et al. [20]. ΔNp73α levels were down-regulated by electroporating 1. 5×106 cells with either 0. 5 or 2 µg of AS or S oligos (for AS and S oligos sequences please see [20]). Cells electroporation was performed by Neon Transfection System, using a pulse voltage of 1350 v and a pulse width of 30 ms. To specifically silence p73 isoform we used the target sequence: 5′-CAGACAGCACCTACTTCGA-3′ spanning from +71 to +90 bp downstream of the transcription start codon was cloned in OmicsLink shRNA Expression system containing a puromycin selection marker (HSH018180-6-HIVmH1, OS395979, GeneCopoeia). As negative control, a scrambled shRNA (sH1) was used. Lentivirus production was performed as previously described [70]. Lentiviral suspension was added to 1. 5×106 LCLs and selection with puromycin (0. 8 µg/ml) was initiated 24 hours later. One week post infection cells were collected and processed for the different experiments. Total cellular RNA was extracted from cells using the Absolutely RNA Miniprep kit (Stratagene). RNA Reverse transcription to cDNA was carried out by RevertAid H Minus M-MuLV Reverse Transcriptase (MBI Fermentas) according to manufacturer' s protocol. Quantitative PCR (Q-PCR) was performed in duplicate in each experiment as previously described [67]. The primer sequences used for RT and Q-PCR are indicated in Table S1A (supplementary material). Preparation of whole or fractioned cell lysates extracts, sodium dodecyl sulfate -polyacrylamide gel electrophoresis (PAGE), and immunoblotting (IB) were performed as previously described [20], [21]. The following antibodies were used for IB: β-actin (C4; MP Biomedicals), human p53 (NCL-CM1; Novocastra Laboratories Ltd.), p73 (anti-p73 Ab-1; Calbiochem), hemagglutinin (HA) -peroxidase-high affinity (3F10; Roche), anti-LMP-1 Ab (S12; a gift from Georges Mosialos, Alexander Fleming Institute, Varkiza, Greece), PARP (9542; Cell signalling), SAP/JNK1 (56G8; Cell signalling), anti-EZH2 (AC22; Cell signalling). A biotinylated fragment of ΔNp73 promoter was generated by PCR using the genomic DNA as template and a biotinylated primer Fw 5′-Btndt CTGGTGGGTTTAATTA-3′ and a non-biotinylated primer Rev 5′- AGGAGCCGAGGATGCTGG-3′ (Sigma). Cells were re-suspended in lysis buffer HKMG (10 mM HEPES, pH7. 9,100 mM KCl, 5 mM MgCl2,10% Glycerol, 1 mM DTT and 0. 5% NP-40) incubated in ice for 10 min, then lysed by sonication (25% Amp, 1 min). One mg of total cellular extracts was pre-cleared with 40 µl of streptavidine-agarose beads (Amersham Bioscience) for 1 hour at 4°C, and then incubated with 2 µg of purified DNA biotinylated probe for 16 hours at 4°C. Poly dI-dC (40 µg) was added to the reaction to avoid unspecific binding. DNA bound proteins were recovered by incubating with 60 µl of streptavidine-agarose beads for 1 hour at 4°C and washed several times with HKMG buffer [71]. Beads were resuspended in 1× SDS-PAGE loading buffer and analyzed by immunoblotting. Chromatin immunoprecipitation (ChIP) was performed with Diagenode Shearing ChIP and OneDay ChIP kits or with LowCell ChIP Kit according to the manufacturers' protocols, using the following antibodies: EZH2 (AC22; Cell signalling), Histone 3 Lysine 27 Trimethyl polyclonal antibody H3K27 (Epigentek), Histon 3 acetylated lysine 9 antibody H3K9Ac (9649S; cell signalling), Acetyl-Histone H4 (17–630; Millipore), HA hemagglutinin (HA) high affinity (3F10; Roche), and p73 (anti-p73 Ab-1; Calbiochem). The eluted DNA was used as template for Quantitative PCR as previously described [21]. Primers for Quantitative ChIP are listed in Table S1B (supplementary material). RNA integrity and quantification of the total cellular RNA from LCLs tranfected with ΔNp73α AS and S oligo-nucleotide were characterized by measuring the 28s/18s rRNA ratio and RIN using the Agilent 2100 bioanalyzer instrument, and the Agilent RNA 6000 Nano kit. 5 µg of total cellular RNAs was depleted from rRNA using the Invitrogen RiboMinus Eukaryote kit according to manufacturer' s standard protocol. The absence of 28s/18s rRNA was checked on the Agilent 2100 bioanalyzer instrument. Five hundred ng of each sample were enzymatically fragmented using 1 unit of RNase III provided in the SOLiD Total RNA-seq Kit, incubated at 37°C for 10 minutes and cleaned up using the Invitrogen RiboMinus Concentration Module according to manufacturer' s standard protocol. RNA yield and size distribution were assessed with the Agilent 2100 Bioanalyzer instrument and the Agilent RNA 6000 Pico kit. The amplified whole transcriptome library for each sample was constructed according to Lifetechnologies' s SOLiD Total RNA-seq Kit protocol (PN 4452437 Rev. B). To summarize, adaptors were hybridised and ligated to 100 ng of fragmented rRNA-depleted total RNAs followed by the construction and subsequent purifications of cDNAs using successively the SOLiD Total RNA-seq Kit and the Agencourt AMPure beads. cDNAs were then barcoded, amplified with 15 cycles of PCR and purified using the Invitrogen PureLink PCR Micro Kit. Yield and size distribution of the amplified DNA libraries were assessed with the Agilent 2100 Bioanalyzer instrument and the Agilent DNA 1000 kit. After minimizing the DNA in the 25–200 bp range, 0. 4 pM of each barcoded libraries were pooled at equimolar concentrations prior to template bead preparation, in which the pooled library is clonally amplified by emulsion PCR following the Lifetechnologies' s SOLiD EZ bead E80 protocols (PN 4441486 Rev. D, 4443494 Rev. D, 4443496 Rev. D). Two hundred and forty µl of emPCR beads were 3′ modified and deposited on 4 lanes FlowChip before being incubated for 60 minutes at 37°C. The forward 50 bp reads sequencing chemistry was applied. The secondary and tertiary analyses was done with LifeScope software v. 2. 5. 1 from Life Technologies (Build ID: LifeScope-v2. 5. 1-r0_102906_20120406100430) The raw data (xsq files) from each lane were grouped per sample (based on the barcodes) before launching the standard RNA seq workflow on the 3 samples (EBV_sense, EBV_antisens1, EBV_antisens2). We kept all the standard parameters as advised by Life Technologies. This workflow includes 3 modules: the “mapping analysis” for which we used hg19 as reference genome, the “coverage analysis” and the “count known genes and exons analysis”. After reads mapping, the R/Bioconductor package edgeR (empirical analysis of digital gene expression data in R) was used to study differential gene expression [72]. After fitting a negative binomial model, data obtained from antisense samples were grouped before applying the “common dispersion” function in edgeR. Next, differential gene expression was determined using the exact test. Heatmaps and gene set expression comparisons were performed with BRB-ArrayTools software Version 4. 2. 1. To this end, reads were RPKM (Reads Per Kilobase of exon model per Million mapped reads) normalized and corresponding gene lists were filtered for selected pathways (Table S3, supplementary material). Statistical significance was determined by Student T test. The p value of each experiment is indicated in the corresponding Figure legend. Error bars in the graphs represent the standard deviation.

Approximately 20% of worldwide human cancers have been associated with viral infections. Many oncogenic viruses exert their transforming properties by inactivating the products of tumour suppressor genes. One of the best characterized events induced by ongocenic viruses is the inactivation of the transcriptional factors p53. The mucosal high-risk HPV types, EBV, HTLV-1 and KSHV, via their viral proteins, are able to target p53 by distinct mechanisms. We have recently described a novel p53 inactivation mechanism of some cutaneous beta HPV types which have been suggested to be associated with skin carcinogenesis. Beta HPV38 induces accumulation of the p53 antagonist, ΔNp73α which in turn silences the expression of the p53-regulated genes. Here we report that also EBV, via the oncoprotein LMP-1, induces the expression of ΔNp73α which is dependent on the recruitment of p73 on ΔNp73 promoter and the activation of JNK-1. The recruitment of p73 to the ΔNp73 promoter correlated with the displacement of the histone-lysine N-methyltransferase EZH2 which is part of a transcriptional repressive polycomb 2 complex. We also show that ΔNp73α plays an important role in transformation of primary human B cells and regulates the expression of a large number of cellular genes that encode proteins linked to cancer development, including lymphomagenesis.

lay_plos

People with type 1 diabetes who used a bionic pancreas instead of manually monitoring glucose using fingerstick tests and delivering insulin using a pump were more likely to have blood glucose levels consistently within the normal range, with fewer dangerous lows or highs. The full report of the findings, funded by the National Institutes of Health, can be found online June 15 in the New England Journal of Medicine. The researchers - at Boston University and Massachusetts General Hospital - say the process of blood glucose control could improve dramatically with the bionic pancreas. Currently, people with type 1 diabetes walk an endless tightrope. Because their pancreas doesn't make the hormone insulin, their blood glucose levels can veer dangerously high and low. Several times a day they must use fingerstick tests to monitor their blood glucose levels and manually take insulin by injection or from a pump. In two scenarios, the researchers tested a bihormonal bionic pancreas, which uses a removable tiny sensor located in a thin needle inserted under the skin that automatically monitors real time glucose levels in tissue fluid and provides insulin and its counteracting hormone, glucagon, via two automatic pumps. In one scenario, 20 adults wore this device combination and carried a cell phone-sized wireless monitor around Boston for five days, unrestricted in their activities. In the other, 32 youth wore the device combination for five days at a camp for children with type 1 diabetes. Both groups were also monitored for five days wearing their own conventional pumps that deliver insulin. "The bionic pancreas system reduced the average blood glucose to levels that have been shown to dramatically reduce the risk of diabetic complications," said co-first author Steven Russell, M.D., Ph.D., assistant professor of medicine at Massachusetts General Hospital. "This is tremendously difficult with currently available technology, and so most people with diabetes are unable to achieve these levels." The researchers found about 37 percent fewer interventions for low blood glucose (hypoglycemia) and a more than twofold reduction in the time in hypoglycemia in adults using the bionic pancreas than with the manual pump. For adolescents using the bionic pancreas, results showed more than a twofold reduction in the need for interventions for hypoglycemia. As well, both groups had significant improvements in glucose levels with the bionic pancreas, particularly during the night. "The performance of our system in both adults and adolescents exceeded our expectations under very challenging real-world conditions," said Ed Damiano, Ph.D., the paper's senior author, an associate professor of biomedical engineering at Boston University and the parent of a son with type 1 diabetes. "A cure is always the end goal," he said. "As that goal remains elusive, a truly automated technology, which can consistently and relentlessly keep people healthy and safe from harm of hypoglycemia, would lift an enormous emotional and practical burden from the shoulders of people with type 1 diabetes, including my child and so many others." Just as a thermostat helps control a home's temperature, the normal pancreas senses blood glucose levels and adjusts the hormones that control it. People with type 1 diabetes, whose pancreas produces little or no insulin, have been using the equivalent of a manual thermostat, needing constant checking and adjustment. A bionic pancreas - like the one used in these studies - would function more like an automated thermostat, automatically monitoring blood glucose and delivering insulin or glucagon when needed to keep glucose within the normal range. As well, these bionic pancreas devices could be monitored remotely by the patient's medical provider or parent. "With promising results such as these, we plan to support larger multicenter trials of the artificial pancreas in the near future," said Guillermo Arreaza-Rubín, M.D., the project officer for artificial pancreas studies funded by the NIH's National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). "Within the next few years, we hope these technologies will go beyond experimental trials and be available to benefit more people with type 1 diabetes." "The landmark Diabetes Control and Complications study - also funded by NIDDK - has long shown that maintaining as normal a blood glucose level as possible early on can stave off complications, including heart, kidney and eye diseases, decades later," said NIDDK Director Griffin P. Rodgers, M.D. "By funding research on the artificial pancreas, we aim to help people with type 1 diabetes maintain healthy blood glucose levels, prevent painful and costly complications, and lead freer, healthier lives." ### Among other funding sources, this research was supported by NIDDK grants R01DK085633 and R01DK097657, and was made possible by the Special Statutory Funding Program for Type 1 Diabetes Research. The program was established by Congress for research to prevent and cure type 1 diabetes. The NIDDK, part of the NIH, conducts and supports basic and clinical research and research training on some of the most common, severe and disabling conditions affecting Americans. The Institute's research interests include: diabetes and other endocrine and metabolic diseases; digestive diseases, nutrition, and obesity; and kidney, urologic and hematologic diseases. For more information, visit http://www. niddk. nih. gov/. About the National Institutes of Health (NIH): NIH, the nation's medical research agency, includes 27 Institutes and Centers and is a component of the U.S. Department of Health and Human Services. NIH is the primary federal agency conducting and supporting basic, clinical, and translational medical research, and is investigating the causes, treatments, and cures for both common and rare diseases. For more information about NIH and its programs, visit http://www. nih. gov. NIH...Turning Discovery Into Health A portable artificial pancreas built with a modified iPhone successfully regulated blood sugar levels in a trial with people who have Type 1 diabetes, researchers reported Sunday. Type 1 diabetes, which usually starts in childhood or young adulthood, is a chronic condition in which the pancreas produces little or no insulin, the hormone that lowers blood sugar levels. Insulin works in conjunction with glucagon, a hormone that raises blood sugar. Together, they keep blood sugar in a healthy range. Currently about one-third of people with Type 1 diabetes rely on insulin pumps to regulate blood sugar. They eliminate the need for injections and can be programmed to mimic the natural release of insulin by dispensing small doses regularly. But these pumps do not automatically adjust to the patient’s variable insulin needs, and they do not dispense glucagon. The new device, described in a report in The New England Journal of Medicine, dispenses both hormones, and it does so with little intervention from the patient. “The data address some of the most difficult problems in diabetes management,” said Dr. Kevan Herold, director of the Yale Diabetes Center, who was not involved in the study. “I’d say that the effects are quite significant and noteworthy.” Dr. Fredric E. Wondisford, director of the diabetes institute at Johns Hopkins, also found the results encouraging. “To me, it’s a clear advance,” he said. But he cautioned that the effectiveness and practicality of the device had still not been tested in large numbers of patients over long periods of time. He also raised the issue of cost and insurance coverage. Treatment of Type 1 diabetes is complicated. Patients not using pumps need two or more insulin injections a day, and all have to monitor blood sugar several times a day by pricking their skin and testing their blood. Maintaining safe blood sugar levels requires precise adjustments, especially to prevent hypoglycemia, or extremely low blood sugar. Hypoglycemia can occur quickly, without the patient’s awareness, and can be a life-threatening emergency. For patients with adequate treatment, elevated blood sugar is usually not an emergency, but can cause vascular damage over time that can lead to eye problems and amputations. The artificial pancreas is the latest version of a device that researchers have been refining for several years. The system consists of an iPhone 4S with an attached glucose monitoring device, two pumps, and reservoirs for insulin and glucagon. A sensor implanted under the skin on one side of the patient’s abdomen measures the glucose in the fluid between the cells, which corresponds closely to blood glucose levels. The sensor delivers the reading to the smartphone, and the phone’s software calculates a dose of insulin and glucagon every five minutes. The medicine is then pumped through thin tubes to two tiny infusion points embedded just under the skin on the other side of the patient’s abdomen. The phone also has an app with which a patient can enter information immediately before eating, indicating whether the meal is breakfast, lunch or dinner, and whether the carbohydrate content will be small, large or typical. The device then calculates and dispenses the proper dosages. The device still requires a finger stick twice a day to get an accurate blood reading, which the patient enters into the phone. The developers tested the device over five days in two groups of patients, 20 adults and 32 adolescents, comparing the results with readings obtained with conventional insulin pumps that the participants were using. The adults in the trial each had the constant attention of a nurse, and they lived in a hotel for the five-day study. Most of the time they were free to travel around and pursue normal activities. The adolescents, 16 boys and 16 girls, lived under supervision in a summer camp for youths with diabetes. “We need to do a true home-use study, give people the device and send them home,” said the lead author, Dr. Steven J. Russell, an assistant professor of medicine at Massachusetts General Hospital. “Let them do whatever it is they’re going to do without supervision.” Several authors of the new report have received payments from medical device companies and hold patents on blood sugar monitoring technology. The artificial pancreas performed better than the conventional pump on several measures. Among the adolescents, the average number of interventions for hypoglycemia was 0.8 a day with the experimental pump, compared with 1.6 a day with the insulin pumps. Among adults, the device significantly reduced the amount of time that glucose levels fell too low. And the artificial pancreas worked well at calculating mealtime doses without the patient having to use (often inaccurate) estimates and correct a too high or too low reading after eating. Much more work needs to be done before the device can be marketed, Dr. Russell said. The senior author, Edward R. Damiano, an associate professor of biomechanical engineering at Boston University, has a 15-year-old son with Type 1 diabetes. He said he was determined to get the new device working and approved in time for his son to go off to college carrying one. An artificial pancreas developed by Boston researchers shows considerable promise to dramatically change the treatment of type 1 diabetes, potentially enabling 2 million Americans to eat what they want without counting carbohydrates or calculating insulin injections, researchers announced Sunday. Investigators from Massachusetts General Hospital and Boston University developed the experimental device, which consists of an automated pump that releases the hormones insulin and glucagon and a glucose monitoring system controlled by an iPhone app. Advertisement “We encouraged them to eat whatever they wanted while they wore the bionic pancreas,” said Dr. Steven Russell, an endocrinologist at Massachusetts General Hospital who led the study. “They went on a diabetes vacation, eating ice cream, candy bars, and other things they normally wouldn’t eat — like taking out a new sports car and seeing what it can do.” In a new study published online in the New England Journal of Medicine, the researchers found that 52 adults and teens who used the mobile system for five days had healthier blood sugar levels compared to when they used standard treatments that required them to check their own blood sugar levels and determine how much insulin to inject via a pump device. “This is not a cure,” said study coauthor Edward Damiano, an associate professor of biomedical engineering at Boston University who holds a patent on the software that makes the automatic dosing decisions. “It’s taking diabetes management to its ultimate potential and unburdens people with type 1 diabetes from thinking about all the things that go into managing diabetes every day of their lives.” Damiano, whose 15-year-old son developed type 1 diabetes during his first year of life, said he wakes up two to three times a night to check his son’s blood sugar to prevent it from falling to a dangerously low level, called hypoglycemia, which can cause seizures, a coma, and sometimes even death. Having an automated device, which is expected to become available by 2017 after more testing and upgrades, would save Damiano and other parents from having to wake up in the middle of the night to avoid medical emergencies, he said. Children with type 1 diabetes are eight times more likely to die from severe hypoglycemia at night — called dead in bed syndrome — than in a car accident. Advertisement Diabetes experts who were not involved in the study cautioned that automated devices need far more rigorous testing to determine if they’ll be safe enough to become widespread. “Quite a few things need to be addressed,” added Dr. David Harlan, chief of the diabetes division at the UMass Memorial Medical Center in Worcester. “The chances of the pump failing with two difference hormone infusions are great and it doesn’t remove constant diligence from lives of people with diabetes.” The rate of type 1 diabetes — in which the body’s immune cells attack and destroy a healthy insulin-producing pancreas — has, for unknown reasons, been surging over the past few decades, with nearly 16,000 children under age 18 now diagnosed with the condition every year. (Type 2 diabetes, by comparison, is a more common condition, and tends to occur well into adulthood and has far more explainable causes, such as genetics, obesity, and a lack of exercise.) In the new study, all of the study participants were closely monitored — adults were accompanied by nurses round-the-clock and the teens were in a summer camp for those with type 1 diabetes — to ensure that their blood sugar levels wouldn’t rise too high or fall too low, which could cause seizures or other complications. The research found that adult patients with type 1 diabetes who used the experimental device had lower blood sugar levels overall than the control group while also spending 67 percent less time in a state of hypoglycemia. Children who used the device experienced a 50 percent reduction in carbohydrate use to treat hypoglycemia, though they did not experience a significant difference in their blood sugar measurements. That’s likely because children who participated in the study were closely monitored even when they used their usual treatments, said Russell. He and his colleagues presented their results Sunday evening at the American Diabetes Association meeting in San Francisco. “The initial proof of concept is very strong,” said Dr. Guillermo Arreaza-Rubin, a director at the National Institute of Diabetes and Digestive and Kidney Diseases which funded the trial. “Parents of children with type 1 live in permanent fear and maybe this device can free them from that burden and increase the quality of life of those with this disease.” A few other groups of researchers presented findings on similar automated devices at the diabetes conference, he said, which, like the device developed in Boston, are also about three or four years away from widespread use. Christopher Herndon, 13, a study participant who tried the bionic pancreas last summer while at Camp Joslin in Charlton, said for the first time in his life he “felt steady the whole day” rather than shaky when his blood sugar levels dropped too low and “really talkative” when it was too high. Despite using an insulin pump and continuous glucose monitoring device, which many diabetes experts consider to be gold standard therapies, the Newburyport teen said he has never been able to achieve the kind of blood sugar control that he had while using the bionic pancreas. While his mother, Christina, often wakes him at 2 a.m. to have some juice if his blood sugar drops too low, he was never woken by his counselors at the camp during the five days he used the device. Mark Lorenz for The Globe Christopher Herndon said he has never been able to achieve the kind of blood sugar control that he had while using the bionic pancreas. Colby Clarizia, another study participant, said he consumed three blueberry muffins for breakfast while wearing the artificial pancreas and was surprised to see how well the device worked. The 21-year-old from Amesbury also found it easy to use. “Instead of measuring every carb in a glass of milk or serving of rice, I just had to input whether I was going to eat a large meal or small one,” he said. Such a device, he added, would make it far easier for him to eat in restaurants where carbohydrate counts aren’t posted on menus. The bionic pancreas automatically monitors blood sugar using a wireless glucose monitoring system that sends a signal every five minutes to an iPhone adapted with software. The iPhone app determines how much insulin to dispense — to make high blood sugar levels decrease — and how much of the hormone glucagon to dispense to make blood sugar levels rise; it also learns over time how to adjust the release of the two hormones based on input from the monitoring system. Harlan plans to test the device in conjunction with Russell’s team in a large multicenter study launching this week that will have less rigorous monitoring. Participants will need to stay within an hour’s drive of the study site but won’t need to be accompanied by a health professional wherever they go. “I think it’s an important step forward, but it’s going to be quite challenging to see whether this device can function safely and effectively without close supervision,” said Dr. Howard Wolpert, director of the institute for technology translation at the Joslin Diabetes Center in Boston, who reviewed the paper before publication. “An analogy I would present is that they climbed a flight of stairs but now the next stage will be like climbing a hill or even a mountain.”

While you were using your iPhone to browse Facebook and read Newser, a group of researchers was modifying an iPhone 4S to be used as a portable artificial pancreas-and in a recent trial, the device successfully regulated the blood sugar levels of people with Type 1 diabetes. About a third of people with Type 1 diabetes, which is typically diagnosed in childhood, use an insulin pump to regulate their blood sugar rather than giving themselves insulin injections. But unlike those pumps, the bionic pancreas adjusts both insulin and glucagon, a hormone that works with insulin to regulate blood sugar, automatically, the New York Times reports. It performed better than a regular pump for both adults and adolescents, according to the press release. The device is comprised of more than just the iPhone: The patient has a sensor implanted under the skin near the abdomen, which sends readings of blood glucose levels to the attached phone. The phone then calculates and sends the correct dosage of insulin and glucagon through attached pumps and tubes every five minutes. Patients can also enter information about meals before they eat, and the phone will calculate and deliver the correct dose. Twice-daily finger pricks are still required, with the blood sugar readings entered into the phone. Adult participants had about 37% fewer incidents during which low blood glucose levels required intervention-incidents that can be dangerous in the moment as well as cause health complications down the line-and the device could even allow patients to eat what they want, the Boston Globe notes. (During the trial, participants "went on a diabetes vacation, eating ice cream, candy bars, and other things they normally wouldn't eat," explains the lead researcher.) It could be available by 2017, but larger trials are the next step.

multi_news

FIELD OF THE INVENTION The present invention generally relates to artificial cardiac pacemakers, and the treatment of patients who have vasodepressor or cardioinhibitory disorders. More specifically, the present invention relates to the treatment of patients who experience vasovagal syncope episodes. BACKGROUND OF THE INVENTION Generally speaking, a cardiac pacemaker is an electrical device used to supplant some or all of an abnormal heart&#39;s natural pacing function, by delivering appropriately timed electrical stimulation signals designed to cause the myocardium of the heart to contract or &#34;beat&#34;. Vasovagal syncope is a condition marked by a sudden drop in blood pressure resulting in fainting. It is not only unpleasant for a patient, but potentially dangerous, as fainting may lead to injuries from falls. Therapy to counteract a vasovagal syncope episode is possible through use of an implanted cardiac pacemaker. What is needed, then, is a reliable means of detecting the onset of a vasovagal syncope episode, so that the pacemaker can deliver the therapy when it is needed. SUMMARY OF THE INVENTION In view of the foregoing, it is a first object of the present invention to provide a cardiac pacemaker capable of detecting episodes reflective of vasodepressor or cardioinhibitory disorders. It is a second object of the present invention to satisfy the first object specifically with respect to incipient vasovagal syncope. It is a third object of the present invention to satisfy the first and second objects with an emphasis on preventing &#34;false positives.&#34; In order to satisfy the above objects and others, the present invention provides a cardiac pacemaker for detecting the onset of a cardioinhibitory episode at least including: heart rate drop determination means for determining the size of a drop in heart rate; minimum rate drop storage means for storing a programmable minimum rate drop; minimum rate storage means for storing a programmable maximum drop ending rate; comparator means coupled to the heart rate drop determination means, the minimum rate drop storage means, and the minimum rate storage means for comparing the size of the drop in rate to the minimum rate drop, and for comparing an ending rate to the maximum drop ending rate, the ending rate being defined as the heart rate at the time the rate drop is determined; and cardioinhibitory indication means coupled to the comparator means for indicating the occurrence of a cardioinhibitory episode when the rate drop exceeds the minimum rate drop and the ending rate is below the maximum drop ending rate. The present invention further provides a cardioinhibitory detection method adapted for use in a cardiac pacemaker for detecting the onset of cardioinhibitory episode at least including the steps of: determining the size of a drop in heart rate; storing a programmable minimum rate drop; storing a programmable maximum drop ending rate; and comparing the size of the drop in rate to the minimum rate drop, and for comparing an ending rate to the maximum drop ending rate, the ending rate being defined as the heart rate at the time the rate drop is determined; and indicating the occurrence of a cardioinhibitory episode when the rate drop exceeds the minimum rate drop and the ending rate is below the maximum drop ending rate. The details of the present invention will be revealed in the following description, with reference to the attached drawing. BRIEF DESCRIPTION OF THE DRAWING The various figures of the drawing are briefly described as follows: FIG. 1 is a diagram showing the heart of a patient electrically connected to the pacemaker in FIG. 2; FIG. 2 is a schematic block diagram of a multi-sensor, rate-responsive, dual chamber implantable pulse generator (IPG) capable of subsuming the present invention; FIG. 3 is a graph of heart rate versus time illustrating the vasovagal syncope detection method of the present invention; and FIG. 4 is a flowchart detailing an alternate embodiment for the rate drop size determination of the present invention. DETAILED DESCRIPTION OF THE INVENTION Part I. Elementary Description FIG. 1 generally shows a pacemaker 10 implanted in a patient 12. The pacemaker leads 14 and 15 electrically couple the pacemaker 10 to the patient&#39;s heart 11 via a suitable vein 18. The leads act to both sense polarizations in the heart, and to deliver pacing stimuli the heart. Part II. General Description of the Pacemaker Device FIG. 2 is a block circuit diagram illustrating a multi-programmable, implantable, dual-chamber, bradycardia pacemaker 10 capable of carrying out the present invention. Although the present invention is described in conjunction with a microprocessor-based architecture, it will be understood that it could be implemented in other technology such as digital logic-based, custom integrated circuit (IC) architecture, if desired. It will also be understood that the present invention may be implemented in cardioverters, defibrillators and the like. Lead 14 includes an intracardiac electrode 24 located near its distal end and positioned within the right ventricle 16. Electrode 24 is coupled by a lead conductor 14 through an input capacitor 26 to the node 28, and to the input/output terminals of an input/output circuit 30. Similarly, the lead 15 has a distally located intracardiac electrode positioned within the right atrium 17. Electrode 22 is coupled by a lead conductor 15 through an input capacitor 75 to a node 76, and to the input/output terminals of the input/output circuit 30. Input/Output Circuit 30 contains the operating input and output analog circuits for digital controlling and timing circuits necessary for the detection of electrical signals derived from the heart, such as the cardiac electrogram, output from sensors (not shown) connected to the leads 14 and 15, as well as for the application of stimulating pulses to the heart to control its rate as a function thereof under the control of software-implemented algorithms in a Microcomputer Circuit 32. Microcomputer Circuit 32 comprises an On-Board Circuit 34 and an Off-Board Circuit 36. On-Board Circuit 34 includes a microprocessor 38, a system clock 40, and on-board RAM 42 and ROM 44. Off-Board Circuit 36 includes an off-board RAM/ROM Unit 46. Microcomputer Circuit 32 is coupled by Data Communication Bus 48 to a Digital Controller/Timer Circuit 50. Microcomputer Circuit 32 may be fabricated of custom IC devices augmented by standard RAM/ROM components. It will be understood by those skilled in the art that the electrical components represented in FIG. 2 are powered by an appropriate implantable-grade battery power source (not shown). An antenna 52 is connected to Input/Output Circuit 30 for purposes of uplink/downlink telemetry through a radio frequency (RF) Transmitter/Receiver Circuit (RF TX/RX) 54. Telemetering both analog and digital data between antenna 52 and an external device, such as an external programmer (not shown), is accomplished in the preferred embodiment by means of all data first being digitally encoded and then pulse position modulated on a damped RF carrier, as substantially described in U.S. Pat. No. 5,127,404, issued on Jul. 7, 1992, entitled &#34;Telemetry Format for Implantable Medical Device&#34;, which is held by the same assignee as the present invention and which is incorporated herein by reference. A reed switch 51 is connected to Input/Output Circuit 30 to enable patient follow-up via disabling the sense amplifier 146 and enabling telemetry and programming functions, as is known in the art. A Crystal Oscillator Circuit 56, typically a 32,768 Hz crystal-controlled oscillator, provides main timing clock signals to Digital Controller/Timer Circuit 50. A Vref/Bias Circuit 58 generates a stable voltage reference and bias currents for the analog circuits of Input/Output Circuit 30. An ADC/Multiplexer Circuit (ADC/MUX) 60 digitizes analog signals and voltages to provide telemetry and a replacement time-indicating or end-of-life function (EOL). A Power-on-Reset Circuit (POR) 62 functions to initialize the pacemaker 10 with programmed values during power-up, and reset the program values to default states upon the detection of a low battery condition or transiently in the presence of certain undesirable conditions such as unacceptably high electromagnetic interference (EMI), for example. The operating commands for controlling the timing of the pacemaker depicted in FIG. 2 are coupled by bus 48 to Digital Controller/Timer Circuit 50 wherein digital timers set the overall escape interval of the pacemaker, as well as various refractory, blanking and other timing windows for controlling the operation of the peripheral components within Input/Output Circuit 50. Digital Controller/Timer Circuit 50 is coupled to sense amplifiers (SENSE) 64 and 67, and to electrogram (EGM) amplifiers 66 and 73 for receiving amplified and processed signals picked up from electrode 24 through lead 14 and capacitor 26, and for receiving amplified and processed signals picked up from electrode 22 through lead 15 and capacitor 75, representative of the electrical activity of the patient&#39;s ventricle 16 and atrium 17, respectively. Similarly, SENSE amplifiers 64 and 67 produce sense event signals for re-setting the escape interval timer within Circuit 50. The electrogram signal developed by EGM amplifier 66 is used in those occasions when the implanted device is being interrogated by the external programmer/transceiver (not shown) in order to transmit by uplink telemetry a representation of the analog electrogram of the patient&#39;s electrical heart activity as described in U.S. Pat. No. 4,556,063, issued to Thompson et al., entitled &#34;Telemetry System for a Medical Device&#34;, which is held by the same assignee as the present invention, and which is incorporated herein by reference. Output pulse generators 68 and 71 provide the pacing stimuli to the patient&#39;s heart 11 through output capacitors 74 and 77 and leads 14 and 15 in response to paced trigger signals developed by Digital Controller/Timer Circuit 50 each time the escape interval times out, or an externally transmitted pacing command has been received, or in response to other stored commands as is well known in the pacing art. In a preferred embodiment of the present invention, pacemaker 10 is capable of operating in various non-rate-responsive modes which include DDD, DDI, VVI, VOO and VVT, as well as corresponding rate-responsive modes of DDDR, DDIR, VVIR, VOOR and VVTR. Further, pacemaker 10 can be programmably configured to operate such that it varies its rate only in response to one selected sensor output, or in response to both sensor outputs, if desired. Part III. Vasovagal Syncope Detection Feature Details of the vasovagal syncope detection feature of the present invention follow below, with reference to the graph 300 in FIG. 3. It should be understood that the present invention is not limited to the detection of vasovagal syncope, and it can be used to detect episodes reflective of other vasodepressor or cardioinhibitory disorders such as carotid sinus syndrome. In the graph, a lower rate 302 is shown--a rate below which the heart will not be allowed to fall (also known as the escape rate in this instance). The detection algorithm contains three parameters used to determine the presence, vel non., of a vasovagal syncope episode. The detection algorithm is triggered when the heart rate (atrial or ventricular) of the current cycle (R N ) is less than the heart rate of a predetermined previous cycle R N-M, where M is an integer. A programmable quantity, M equals two in the preferred embodiment, so that the reference cycle is the one twice removed (R N-2 ). It should be understood that M may be a number other than 2. For example, M equals 5 in an alternate embodiment so that the heart rate compared is five cycles removed. The drop (304 in FIG. 3) is compared to a programmable minimum rate drop, which is 20 beats per minute (BPM) in the preferred embodiment. If the actual rate drop is less than or equal to the minimum rate drop, the algorithm halts, and the control of the pacemaker 10 is returned to the main program. If the actual rate drop (also known as the drop ending rate) exceeds the minimum rate drop, the heart rate of the current cycle is then compared to a programmable maximum drop ending rate (306 in FIG. 3). The maximum drop ending rate is the highest heart rate that would not be considered severely bradycardic. If the drop ending rate is greater than or equal to the maximum drop ending rate, the algorithm continues searching for the above-mentioned trigger criteria. If the drop ending rate is below the maximum drop ending rate, the algorithm proceeds to count the number of consecutive cycles (308 in FIG. 3) that both the rate drop has been greater than the minimum rate drop and the drop ending rate has been below the maximum drop ending rate. This number is compared to a programmable minimum consecutive cycle number, which is 2 in the preferred embodiment. If the number of consecutive cycles is less than the minimum consecutive cycle number, the control of the pacemaker 10 is returned to the main program. If the number of consecutive cycles is greater than or equal to the minimum consecutive cycle number, the occurrence of a vasovagal syncope episode is indicated, causing the pacemaker 10 to deliver a predetermined vasovagal syncope prevention or elimination therapy. Segments 310 and 312 generally illustrate the application of pacemaker therapy used to prevent or eliminate a vasovagal syncope episode upon its detection. Part IV. Sleep Disabling Feature To further prevent false positives, the present invention disables the detection feature of the present invention while the patient is presumed to be asleep. Otherwise, a low heart rate normally associated with sleeping might be erroneously associated with bradycardia which accompanies a vasovagal syncope episode. Such a malfunction would unnecessarily lead to disturbing the patient&#39;s sleep by raising his or her heart rate to a high intervention rate. This feature is implemented in the present invention with the use of a diurnal clock (not shown) in the microcomputer circuit 32 which causes the microcomputer circuit 32 to disable the detection feature during presumed sleeping hours. The sleeping hours of the diurnal cycle are programmed prior to implant to suit the individual patient&#39;s lifestyle. Part V. Rate Drop Size Determination (Alternate Embodiment) FIG. 4 is a flowchart describing a program 400 for implementing an alternate embodiment for computing the rate drop size 304. To summarize the method, the pacemaker 10 compares the current rate to the highest of 5 stored rates sampled over the previous 2.5 minutes (each of the 5 samples is the highest rate of all the beats occurring in its corresponding 30 second interval) to determine the rate drop. During the operation of the program 400 two counters (not shown) keep track of the number of consecutive atrial paces (CAP) and consecutive ventricular paces (CVP) meeting the criteria for detection of a WS episode. At Step 402 CAP is incremented whenever an atrial pace occurs, and reset to zero whenever an atrial sense occurs. Likewise, CVP is incremented whenever a ventricular pace occurs, and reset to zero whenever a ventricular sense occurs. At Step 404 if CAP equals the number of consecutive beats required to indicate detection, the program 400 then advances to Step 406 where the pacemaker 10 can begin interventional therapy. If CAP does not equal the required number of detection beats, the program 400 advances to Step 408. At Step 408, if the current pacing mode is DDI and CVP equals the required number of detection beats, intervention (detection of a VVS episode) is indicated (Step 406). If these conditions are not met, the program advances to Step 410. If the drop detection feature of the pacemaker 10 is enabled, the program continues to Step 412; otherwise, the program returns to Step 402. The &#34;Current Top&#34; at Step 412 refers to the lowest rate observed during the previous 2.5 minutes. Conversely, the current top also refers to the largest interval between beats observed during the previous 2.5 minutes. If the current and previous intervals between beats are lower than the Current Top, the program advances to Step 414; otherwise the program jumps to Step 416. A new Current Top is stored at Step 414. At Step 416, the pacemaker determines whether the current and previous intervals are slower than the interval corresponding to the Drop Rate. If these intervals are not slower than the Drop Rate interval, the program returns to Step 402. If the intervals are slower the Drop Rate interval, the program advances to Step 418 to begin examining the size of the rate drop. At Step 418 the program determines the Drop Size interval from a look-up table based upon the fastest stored Top Rate and the programmed rate drop. If the current interval is slower than the fastest Top interval plus the Drop Size interval (Step 420), the program advances to Step 422. At Step 422, if the previous interval was also slower than the fastest Top interval plus the Drop Size interval, detection of a VVS episode is indicated by returning the program to Step 406. Steps 420 and 422 insure that at least two consecutive beats meet the conditions for VVS before indicating detection. This will prevent frequent, unnecessary triggering of intervention therapy where the presence of the identified characteristics is only transient in nature. Variations and modifications to the present invention are possible given the above disclosure. However, such variations and modifications are intended to be within the scope of the invention claimed by this letters patent. For example, the maximum drop need not be 20 BPM, but may be any other suitable number, as may be established by one (either at manufacture or just prior to implantation, or after implantation using remote programming means) skilled in the art. Similarly, the maximum drop ending rate is not limited to 65 BPM. And, the minimum consecutive cycle number may be any suitable whole number.

A rate-responsive cardiac pacemaker implements a novel scheme which detects incipient vasovagal syncope (or other episodes caused by a vasodepressive or cardioinhibitory disorder) when a) the heart rate drops below a programmable minimum size, and b) the rate after said drop is below a programmable maximum drop ending rate. The pacemaker implements a stability and intervention procedure upon the detection of an episode, in which it ignores transient drops in rate, and paces at a predetermined high rate if the drops are stable. The pacemaker then gradually reduces the pacing rate over a predetermined time to the pre-episodic level. A sleep disable feature disables the vasovagal syncope detection and therapy features during the patient&#39;s sleeping hours to reduce or eliminate false positive responses.

big_patent

SECTION 1. SHORT TITLE. This Act may be cited as the ``Fairness in Flood Insurance Act of 2015''. SEC. 2. APPEALS OF PROJECTED SPECIAL FLOOD HAZARD AREAS. (a) Burden of Proof.-- (1) Appeals to fema.--Section 1363 of the National Flood Insurance Act of 1968 (42 U.S.C. 4104) is amended by adding at the end the following new subsection: ``(h) Burden of Proof.--In any appeal to the Administrator, or any judicial review of a final administrative determination, regarding the designation of flood elevation determinations or the identification of special flood hazard areas, the Administrator shall have the burden of proving, by clear and convincing evidence, that the elevations proposed by the Administrator or the designation of an identified special flood hazard area, as the case may be, is scientifically and technically correct.''. (2) Determinations by scientific resolution panel.-- Subsection (c) of section 1363A of the National Flood Insurance Act of 1968 (42 U.S.C. 4104-1(c)) is amended-- (A) by redesignating paragraphs (3) and (4) as paragraphs (4) and (5), respectively; and (B) by inserting after paragraph (2) the following new paragraph: ``(3) Burden of proof.--The Scientific Resolution Panel may not resolve a dispute submitted under this section in favor of the Administrator unless the Panel determines, by clear and convincing evidence, that the data and determinations of the Administrator involved in the dispute are scientifically and technically correct.''. (b) Deadline for Appeals.-- (1) In general.--Section 1363 of the National Flood Insurance Act of 1968 (42 U.S.C. 4104) is amended-- (A) in subsection (b), by striking ``ninety-day'' and inserting ``6-month''; and (B) in subsections (c) and (d), by striking ``ninety days'' each place such term appears and inserting ``6 months''. (2) Applicability.--The amendment made by paragraph (1) shall apply with respect to second newspaper publications of notifications of the Administrator of the Federal Emergency Management Agency referred to in section 1363(b) of the National Flood Insurance Act of 1968 that occur after the date of the enactment of this Act. (c) Reimbursement for Costs of Appeal.-- (1) Applicability; implementation.--Subsection (f) of section 1363 of the National Flood Insurance Act of 1968 (42 U.S.C. 4104(f)) is amended-- (A) by striking the first sentence and inserting the following: ``When, incident to any appeal that is successful, in whole or part, regarding the designation of any aspect of a flood map, including elevation or designation of a special flood hazard area, the community, owner, or lessee of real property, as the case may be, incurs expense in connection with the appeal, including for legal services and services provided by surveyors, engineers, and scientific experts, the Administrator shall reimburse such individual or community for reasonable expenses to an extent measured by the ratio of the successful portion of the appeal as compared to the entire appeal. Any successful appeal shall be entitled to such reimbursement and reimbursement shall not be contingent upon filing within the 6-month periods referred to in subsections (c) and (d).'' ; and (B) by striking the last sentence and inserting the following: ``The Administrator shall issue guidance to implement this subsection, which shall not be subject to the notice and comment requirements under section 553 of title 5, United States Code.''. (2) Deadline.--The Administrator of the Federal Emergency Management Agency shall issue the guidance referred to in the last sentence of section 1363 of the National Flood Insurance Act of 1968 (42 U.S.C. 4104(f)), as amended by paragraph (1)(B) of this subsection, not later than the expiration of the 6- month period beginning on the date of the enactment of this Act. SEC. 3. REVISIONS OF EXISTING FLOOD INSURANCE MAPS; APPEALS. (a) Updating of Maps.--Subsection (f) of section 1360 of the National Flood Insurance Act of 1968 (42 U.S.C. 4101(f)) is amended to read as follows: ``(f) Updating of Flood Maps.-- ``(1) Upon necessity or request.--The Administrator shall revise and update any floodplain areas and flood-risk zones-- ``(A) upon the determination of the Administrator, according to the assessment under subsection (e) of this section, that revision and updating are necessary for the areas and zones; ``(B) upon the request from any State or local government stating that specific floodplain areas or flood-risk zones in the State or locality need revision or updating, if sufficient technical data justifying the request is submitted; or ``(C) upon the request from any owner or lessee of real property located in a floodplain area or flood- risk zone if sufficient technical data justifying the request is submitted. ``(2) Request by state or local government.--When the Administrator revises and updates any floodplain area or flood- risk zone pursuant to a request from any State or local government, the Administrator shall provide to that State or local government a Letter of Map Revision, Letter of Map Revision Based on Fill, or physical map revision, as appropriate, that includes a description of any revisions or modifications to such floodplain area or flood-risk zone. ``(3) Request by owner or lessee.--When the Administrator revises and updates any floodplain area or flood-risk zone pursuant to a request from any owner or lessee of real property, the Administrator shall provide to that owner of lessee a Letter of Map Amendment, Letter of Map Amendment Based on Fill, Letter of Map Revision, or Letter of Map Revision Based on Fill, as appropriate, that includes a description of any revisions or modifications to such floodplain area or flood-risk zone. ``(4) Revision of flood maps.--Any updates of flood maps, notifications of flood map changes, and compendia of flood map changes required by this section shall reflect any changes made pursuant to paragraphs (2) and (3) occurring since the most recent such update, notification, or compendia, respectively.''. SEC. 4. APPEALS REGARDING EXISTING FLOOD MAPS. (a) In General.--Section 1360 of the National Flood Insurance Act of 1968 (42 U.S.C. 4101) is amended by adding at the end the following new subsection: ``(k) Appeals of Existing Maps.-- ``(1) Right to appeal.--A State or local government, or the owner or lessee of real property, who has made a formal request to the Administrator to update a flood map that the Administrator has denied may at any time appeal such a denial as provided in this subsection. ``(2) Basis for appeal.--The basis for appeal under this subsection shall be the possession of knowledge or information that-- ``(A) the base flood elevation level or designation of any aspect of a flood map is scientifically or technically inaccurate; or ``(B) factors exist that mitigate the risk of flooding, including ditches, banks, walls, vegetation, levees, lakes, dams, reservoirs, basin, retention ponds, and other natural or manmade topographical features. ``(3) Appeals process.-- ``(A) Administrative adjudication.--An appeal under this subsection shall be determined by a final adjudication on the record, and after opportunity for an administrative hearing. ``(B) Rights upon adverse decision.--If an appeal pursuant to subparagraph (A) does not result in a decision in favor of the State, local government, owner, or lessee, such party may appeal the adverse decision to-- ``(i) the Scientific Resolution Panel provided for in section 1363A, which shall recommend a non-binding decision to the Administrator; or ``(ii) the Federal district court of appropriate jurisdiction. An appeal by a State or local government, or the owner or lessee of real property, pursuant to clause (i) shall not preclude such party from further appealing pursuant to clause (ii). ``(C) Burden of proof.--In any appeal under this subsection, the Administrator shall bear the burden of proving, by clear and convincing evidence, that the elevations proposed by the Administrator or the designation of any aspect of the special flood hazard area, as the case may be, is scientifically and technically correct. ``(4) Relief.-- ``(A) Wholly successful appeals.--In the case of a successful appeal resulting in a policyholder's property being removed from a special flood hazard area, such policyholder may cancel the policy at any time within the current policy year, and the Administrator shall provide such policyholder a refund in the amount of any premiums paid for such policy year, plus any premiums paid for flood insurance coverage that the policyholder was required to purchase or maintain during the 2-year period preceding such policy year. ``(B) Partially successful appeals.--In the case of any appeal in which mitigating factors were determined to have reduced, but not eliminated, the risk of flooding, the Administrator shall reduce the amount of flood insurance coverage required to be maintained for the property concerned by the ratio of the successful portion of the appeal as compared to the entire appeal. The Administrator shall refund to the policyholder any payments made in excess of the amount necessary for such new coverage amount, effective from the time when the mitigating factor was created or the beginning of the second policy year preceding the determination of the appeal, whichever occurred later. ``(C) Additional relief.--The Administrator may provide additional refunds in excess of the amounts specified in subparagraphs (A) and (B) if the Administrator determines that such additional amounts are warranted. ``(5) Recovery of costs.-- When, incident to any appeal which is successful in whole or part regarding the designation of the base flood elevation or any aspect of the flood map, including elevation or designation of a special flood hazard area, the community, or the owner or lessee of real property, as the case may be, incurs expense in connection with the appeal, including legal services and services provided by surveyors, engineers, and scientific experts, the Administrator shall reimburse such individual or community for reasonable expenses to an extent measured by the ratio of the successful portion of the appeal as compared to the entire appeal. The Administrator may use such amounts from the National Flood Insurance Fund established under section 1310 as may be necessary to carry out this paragraph. ``(6) Guidance.--The Administrator shall issue guidance to implement this subsection, which shall not be subject to the notice and comment requirements under section 553 of title 5, United States Code.''. (b) Deadline.--The Administrator of the Federal Emergency Management Agency shall issue the guidance referred to section 1361(k)(6) of the National Flood Insurance Act of 1968 (42 U.S.C. 4101(k)(6)), as added by the amendment made by subsection (a) of this section, not later than the expiration of the 6-month period beginning on the date of the enactment of this Act. SEC. 5. CONSIDERATION OF FLOOD MITIGATION FACTORS IN ESTABLISHING FLOOD HAZARD AREAS. Section 1360 of the National Flood Insurance Act of 1968 (42 U.S.C. 4101), as amended by the preceding provisions of this Act, is further amended by adding at the end the following new subsection: ``(l) Consideration of Flood Mitigation Factors.--In identifying, designating, and establishing any areas having special flood hazards, including in reviewing, revising, or updating of such areas, the Administrator and the Technical Mapping Advisory Council established under section 100215 of the Biggert-Waters Flood Insurance Reform Act of 2012 (42 U.S.C. 4101a) shall take into consideration any factors that mitigate against flood risk, including ditches, banks, walls, vegetation, levees, lakes, dams, reservoirs, basin, and retention ponds, and the extent to which such factors mitigate against flood risk.''. SEC. 6. CONSIDERATION OF COASTAL AND INLAND LOCATIONS IN PREMIUM RATES. (a) Estimates of Premium Rates.--Clause (i) of section 1307(a)(1)(A) of the National Flood Insurance Act of 1968 (42 U.S.C. 4014(a)(1)(A)(i)) is amended by inserting ``, taking into consideration differences between properties located in coastal areas and properties located inland,'' after ``the risk involved''. (b) Establishment of Chargeable Premium Rates.--Paragraph (1) of section 1308(b) of the National Flood Insurance Act of 1968 (42 U.S.C. 4015(b)(1)) is amended by inserting ``due to differences between properties located in coastal areas and properties located inland and'' after ``differences in risks''. (c) Rate Tables.--Not later than the expiration of the 180-day period beginning on the date of the enactment of this Act, the Administrator of the Federal Emergency Management Agency shall revise and expand the rate tables for premiums under the National Flood Insurance Program to implement the amendments made by this section and reflect differences between properties located in coastal areas and properties located inland. SEC. 7. STREAMLINING OF FLOOD MAP PROCESSES. The Administrator of the Federal Emergency Management Agency shall consult with the Technical Mapping Advisory Council established under section 100215 of the Biggert-Waters Flood Insurance Reform Act of 2012 (42 U.S.C. 4101a) regarding methods of or actions to-- (1) make the flood map processes of the Council more efficient; (2) minimize any cost, data, and paperwork requirements of the Council; and (3) assist communities, and in particular smaller communities, in locating the resources required to successfully appeal flood elevations and flood hazard area designations. Not later than the expiration of the 1-year period beginning on the date of the enactment of this Act, the Administrator shall submit a report to the Committee on Financial Services of the House of Representatives and the Committee on Banking, Housing, and Urban Affairs of the Senate setting forth any recommendations for methods or actions developed pursuant to the consultation required under this section. SEC. 8. SUFFICIENT STAFFING FOR OFFICE OF FLOOD INSURANCE ADVOCATE. (a) In General.--Section 24 of the Homeowner Flood Insurance Affordability Act of 2014 (42 U.S.C. 4033) is amended by adding at the end the following new subsection: ``(c) Staff.--The Administrator shall ensure that the Flood Insurance Advocate has sufficient staff to carry out all of the duties and responsibilities of the Advocate under this section, which shall include providing direction as necessary, including by direct conversations with insurance agents.''. (b) Timing.--The Administrator of the Federal Emergency Management Agency shall take such actions as may be necessary to provide for full compliance with section 24(c) of the Homeowner Flood Insurance Affordability Act of 2014, as added by the amendment made by subsection (a) of this section, not later than the expiration of the 180-day period beginning on the date of the enactment of this Act. SEC. 9. GAO STUDY ON ADEQUACY OF FLOOD MAPS. The Comptroller General of the United States shall conduct a study to determine the scientific and technical adequacy of the flood maps proposed and established pursuant to chapter III of the National Flood Insurance Act of 1968 (42 U.S.C. 4101 et seq.) by the Administrator of the Federal Emergency Management Agency. Not later than the expiration of the 6-month period beginning on the date of the enactment of this Act, the Comptroller General shall submit a report to the Committee on Financial Services of the House of Representatives and the Committee on Banking, Housing, and Urban Affairs of the Senate a report setting forth the results and conclusions of the study conducted pursuant to this section. SEC. 10. GAO STUDY OF EFFECTS OF CHANGING BASE FLOOD. The Comptroller General of the United States shall conduct a study regarding the effects on the National Flood Insurance Program and otherwise of changing the standard for designation of areas as special flood hazard areas from having a 1 percent or greater chance of experiencing flooding in any given year to having a 10 percent or greater chance of experiencing flooding in any given year. Not later than the expiration of the 180-day period beginning on the date of the enactment of this Act, the Comptroller General shall submit to the Congress a report setting forth the findings and conclusions of the study conducted pursuant to this section.

Fairness in Flood Insurance Act of 2015 This bill amends the National Flood Insurance Act of 1968 to declare that, in any appeal to the Federal Emergency Management Agency (FEMA), or any judicial review of a final administrative determination, regarding the designation of flood elevation determinations or the identification of special flood hazard areas, FEMA shall have the burden of proving, by clear and convincing evidence, that the elevations proposed or the designation of an identified special flood hazard area is scientifically and technically correct. FEMA shall: reimburse the reasonable legal and related expenses of any individual or community that succeeds on such an appeal; and revise and update any floodplain areas and flood-risk zones upon the request from any owner or lessee of real property located in a floodplain area or flood-risk zone (currently, only upon the request of FEMA or of a state or local government), if sufficient technical data justifying the request is submitted. A state or local government, or the owner or lessee of real property, who has formally requested FEMA to update a flood map that FEMA has denied may at any time appeal the denial according to a specified procedure. The basis for appeal shall be possession of knowledge or information that: the base flood elevation level or designation of any aspect of a flood map is scientifically or technically inaccurate; or specified factors exist, including natural or manmade topographical features, that mitigate the risk of flooding. These flood mitigation factors shall also be considered in the establishment and updating of areas with special flood hazards. When estimating risk premium rates for flood insurance, and prescribing chargeable premium rates, FEMA shall take into consideration the differences between properties located in coastal areas and properties located inland. FEMA shall consult with the Technical Mapping Advisory Council about methods of making or actions to make the Council's flood map processes more efficient and achieve other specified goals. The Homeowner Flood Insurance Affordability Act of 2014 is amended to require FEMA to ensure that the Flood Insurance Advocate has sufficient staff to carry out all of the Advocate's duties and responsibilities, which shall include providing direction as necessary, including by direct conversations with insurance agents. The Government Accountability Office shall study: the scientific and technical adequacy of the flood maps FEMA proposes and establishes, and the effects on the National Flood Insurance Program and otherwise of changing the standard for designating special flood hazard areas from having a 1% or greater to having a 10% or greater chance of experiencing flooding in any given year.

billsum

Convergent morphologies have arisen in plants multiple times. In non-vascular and vascular land plants, convergent morphology in the form of roots, stems, and leaves arose. The morphology of some green algae includes an anchoring holdfast, stipe, and leaf-like fronds. Such morphology occurs in the absence of multicellularity in the siphonous algae, which are single cells. Morphogenesis is separate from cellular division in the land plants, which although are multicellular, have been argued to exhibit properties similar to single celled organisms. Within the single, macroscopic cell of a siphonous alga, how are transcripts partitioned, and what can this tell us about the development of similar convergent structures in land plants? Here, we present a de novo assembled, intracellular transcriptomic atlas for the giant coenocyte Caulerpa taxifolia. Transcripts show a global, basal-apical pattern of distribution from the holdfast to the frond apex in which transcript identities roughly follow the flow of genetic information in the cell, transcription-to-translation. The analysis of the intersection of transcriptomic atlases of a land plant and Caulerpa suggests the recurrent recruitment of transcript accumulation patterns to organs over large evolutionary distances. Our results not only provide an intracellular atlas of transcript localization, but also demonstrate the contribution of transcript partitioning to morphology, independent from multicellularity, in plants. Convergent morphologies have arisen multiple times in plants (Viridiplantae). Diverse cellular architectures underlie these moprhologies, with varying relationships between the number of nuclei per cell and the number of cells within an organism. Within the Chlorophyta, Acetabularia possesses an anchoring rhizoid, supporting stalk, and photosynthetic cap, but is, during most of its life cycle, a unicellular organism reaching heights of up to 10 cm with a single nucleus [1]–[3]. Another green alga, Caulerpa, is one of the largest known single-celled organisms, with stolons (up to meters in length) producing fronds and holdfasts [4]–[9] (Fig. 1A–C). Unlike Acetabularia, which is a single-celled organism, Caulerpa is coenocytic, with numerous nuclei. Siphonocladous chlorophytes have a chambered body plan compartmentalizing variable numbers of nuclei, as in Cladophora. Land plants (Embryophyta) are multicellular organisms, in which organs are composed of tissues and distinct cell types. Developmental biology in land plants was historically influenced by cell theory and studies in animals, in which organismal level morphology is an emergent property of cell division and histogenesis [10], [11]. Animal development is a poor example for land plants, in which morphogenesis is dissociated from histogenesis because cellular lineages and division patterns are largely independent from organ morphology. For the above reasons, it has been argued [11] that cell theory is not as applicable to plants as in animals with respect to explaining how complex morphologies arise. In its place, Kaplan and Hagemann [11] argued for organismal theory, which they define as “[interpreting] the living protoplasmic mass as a whole, rather than considering its constituent cells as the basic unit. ” In other words, the morphology in plants arises at the organismal level rather than as an emergent cellular property. Kaplan and Hagemann assert that “higher plants are also siphonous, but at a subtler, microscopic level. ” Some of the siphonous features they argue land plants possess include: 1) cell division through a phragmoplast, 2) plasmodesmata, 3) the symplasm, 4) a multinucleate endosperm and megagametophytes, 5) distinct cytohistological zonations of the shoot apical meristem throughout the Embryophyta, 6) that cell lineage is often independent of morphology (e. g., in leaves), 7) and convergent morphology in multicellular red algae and land plants with different cell lineage patterns. That land plants are truly siphonous is false: cell walls are a prominent features of land plants upon which morphology is dependent and land plants are multicellular organisms. However, it is useful to think about development in land plants from this unique perspective. That organ growth and morphogenesis are separate from cell division reduces the importance of cell-type specific transcript accumulation in these organisms. Transcriptomics and phylogenetics provide a mechanism to test hypotheses of cell versus organismal theory in siphonous green algae and land plants. Do the accumulation patterns of transcripts differ between single-celled and multicellular organisms with convergent morphology? Are groups of transcripts recurrently recruited to organs across large evolutionary distances regardless of whether an organism is multicellular? Here, we provide a transcriptome of the giant coenocyte Caulerpa taxifolia. We detect a strong apical-basal gradient of transcript accumulation within the cell. Groups of transcripts with distinct functionalities accumulate in relevant pseudo-organs (morphological structures equivalent to a multicellular organ but not comprised of tissues or cells). Cell compartmentalization is partitioned in Caulerpa, despite its polynucleate condition, and transcripts are patterned according to the flow of genetic information, from transcription-to-translation in a basal-to-apical fashion. An analysis of the intersection of the transcriptomic atlases of a land plant (tomato, Solanum lycopersicum) and Caulerpa demonstrates a limited, recurrent recruitment of genes with similar functions to morphological structures. Our results provide a broad, evolutionary context for the relationship between the cell and organismal morphology at a molecular level within plants, confirming and expanding upon the organismal theory originally proposed by Kaplan and Hagemann [11]. To develop a resource to address how organismal morphology can arise in the absence of multicellularity, we sequenced transcripts from multiple pseudo-organs and de novo assembled the intracellular transcriptome of Caulerpa taxifolia (see sequence submission information and S1–S4 Datasets). Caulerpa taxifolia stolons, upwards of 1 m in length, bear fronds (typically 15–30 cm long at maturity) with pinnately-arranged, tapered pinnules. The pinnules arise from active growth at the frond apex, which superficially resembles, in form and function, the apical cells and meristems of embryophytes (Fig. 1B). Caulerpa taxifolia is anchored by holdfasts, which take up phosphorus, nitrogen, and carbon from the substrate, and harbor both ecto- and endosymbiont bacteria that aid this process [9]. We sampled five replicates each of 1) the frond apex, 2) rachis, 3) pinnules, 4) the lower third of the frond base, 5) stolon, and 6) holdfast regions (Fig. 1C). One holdfast sample was lost when thawing for library preparation, reducing holdfast sampling to four replicates. The sample we sequenced was clonal in origin, having proceeded through numerous rounds of asexual reproduction. In its vegetative phase, Caulerpa taxifolia is a haplophasic diploid. Caulerpa taxifolia has one of the smallest genome sizes in its genus (∼100 Mbp, approximately the size of the Arabidopsis thaliana genome) and unlike other Caulerpa species does not exhibit extensive endopolyploidy [12], [13]. The transcriptome of Caulerpa taxifolia is dominated by patterning along the apical-basal axis. Throughout this manuscript, we use the terms “accumulation” and “abundance” in a relative sense to describe transcript accumulation patterns. Transcript accumulation in replicates derived from basal regions (holdfast, stolon, frond base) is highly similar and distinct from apical regions (frond apex, rachis, pinnules), as shown in a Principal Component Analysis (PCA) performed on replicates (Fig. 1D; S5–S20 Datasets). The growing frond apex in particular exhibits a unique transcriptomic signature, perhaps indicative of the “meristemplasm” found in this region, as previously described [5], [14]. A Self-Organizing Map (SOM) was used to partition transcripts into six clusters (nodes), each with a distinct accumulation pattern (Figs. 2, S1; S21 Dataset). These nodes explain prominent densities of transcripts with similar accumulation patterns across organs, as visualized using a PCA (Fig. 2A–B). The nodes are roughly organized along the apical-basal axis (Fig. 2C). For example, Node 3 transcripts exhibit high frond apex accumulation, and progressing basally to Node 5 transcripts which accumulate at high levels in the holdfast, nodes with intermediate accumulation patterns along the apical-basal axis are observed. The overall patterns of transcript accumulation, visualized using the combination of SOMs and PCA, can be explored for a random subset of genes in an interactive graphic we have prepared (http: //danchitwood. github. io/CaulerpaGeneExpression/, for use with a Google Chrome web browser). Such strong apical-basal, intracellular partitioning of transcript accumulation is not surprising considering the influence of gravitropism on regeneration and anchoring [6], circadian movements of chloroplasts into and out of the apex, and cytoplasmic streaming along the frond and pinnule lengths [5]. Transcripts belonging to each node are highly enriched for associated Gene Ontology (GO) terms, often specific to cellular functions and organelles (S22–S28 Datasets). For example, Node 2 transcripts, which accumulate at high levels in the pinnules and rachis, are enriched for photosynthetic GOs, but additionally those associated with mitochondria, respiration, the electron transport chain and ATP synthesis, as well as the production of secondary metabolites. Node 3 transcripts with high abundances in the frond apex are enriched for COPI/II vesicle coat proteins and kinases. Most surprising is the overwhelming concentration of transcripts associated with nuclear functions—DNA replication and damage, chromatin, RNAi, and even the subunits of DNA polymerase II—in the frond base, stolon, and holdfast. Multicellular land plants possess an inherent constraint at the cellular level. Generally, every cell must have a nucleus, plastids, mitochondria, and cytoskeletal components to carry out basic metabolism, cell division, and differentiation, although numerous exceptions exist. But how is cellular compartmentalization distributed over similar morphology in Caulerpa? One hypothesis is that because morphogenesis is decoupled from multicellularity in Caulerpa, the distribution of different cell compartment identities might consolidate within distinct organs. That is, each cell is subcompartmentalized in a multicellular land plant, whereas the siphonous body plan of Caulerpa may maintain compartmentalization in pseudo-organs. Indeed, GO enrichment analysis reveals that transcript identity loosely follows the flow of genetic information progressing in a basal to apical direction in Caulerpa (Fig. 2, S22–S28 Datasets). Transcripts associated with transcriptional gene regulation accumulate at high levels in the holdfasts, stolon, and frond base, whereas those associated with translation are more abundant in the pinnules. Vesicular trafficking and kinase activity, associated with the cytoplasm and plasma membrane, are enriched within the frond apex. To explore the fundamental relationship between cell compartmentalization and organism morphology, we selected all transcripts belonging to significantly enriched GO terms related to transcriptional gene regulation, translation, and other important organellar and cell biological functions (Fig. 3, S29 Dataset). Transcripts encoding RNA polymerase II subunits are highly abundant in the holdfast. Those encoding numerous chromatin, epigenetic, DNA recombination, repair, and replication, and RNAi machinery components are highly abundant in the stolon. Transcripts related to translation accumulate at high levels in the photosynthetic tissues, mostly in the pinnules and somewhat in the rachis. Proteolysis transcripts are found in these regions too, but additionally in the frond apex where translational components accumulate at lower levels. COPI/II coat proteins and numerous kinases are highly abundant in the frond apex. The association of COPI/II trafficking with the apex, an active growth region containing white “meristemplasm, ” is consistent with the previously reported enrichment of rough endoplasmic reticulum and Golgi bodies in this region [4], [5], [14]. The overall transcriptomic signature in Caulerpa—a single cell—is striking. From the holdfast to the frond apex, transcript accumulation loosely follows a nuclear-to-cytoplasm and transcriptional-to-translational pattern of identity (Fig. 3). The Caulerpa body plan is compartmentalized as if a single land plant cell, and different cellular compartments in Caulerpa are associated with different types of morphogenesis. Land plant morphology, and the numerous and diverse morphologies of various chlorophytes, are derived from the monophyletic inheritance of a core gene set. In some instances, as between land plants and Caulerpa, convergent structures with related functions (for example, leaves and fronds, and roots and holdfasts) have evolved using these genes. If land plant morphology is viewed from the perspective of organismal theory proposed by Kaplan and Hagemann [11], and land plants are even considered to be siphonous and cellularization patterns arbitrary, then the accumulation of transcripts throughout the organism can be compared to detect molecular homology. To what degree have similar transcript accumulation profiles been recruited to morphological structures in land plants and Caulerpa? To answer this question, we analyzed the intersection of the Caulerpa transcriptomic atlas (Figs. 1–3) with a transcriptomic atlas from a land plant (tomato, Solanum lycopersicum cv. M82) that was derived using similar molecular and bioinformatics methods as presented here [15]–[17]. Putative homologous transcripts from tomato (see Materials and Methods) were used to assign Caulepra transcripts to a corresponding tomato self-organizing map node [17] (Fig. 4A). The distribution of genes from each Caulepra node across tomato nodes was then compared to the expected distribution using a χ2 test. A higher than expected enrichment of genes assigned to a particular tomato node indicates that a group of Caulerpa genes with similar accumulation patterns are associated with a specific accumulation profile in tomato (Fig. 4B). For example, Caulerpa Node 2 genes, which are highly abundant in the photosynthetically active pinnules and rachis (Fig. 2), are associated with tomato genes belonging to tomato Node 3, which are highly abundant in leaf, seedling, and vegetative apex samples (note: tomato and Caulerpa nodes are distinct and should not be confused with each other) (Fig. 4B, C). The intersection of Caulerpa Node 2 with tomato Node 3 is predominately photosynthetic genes (S30 Dataset). Although the molecular correspondence between photosynthetic structures is expected, other associations are less so. Caulepra Node 3 genes are highly abundant in the frond apex and are associated with tomato Node 8 genes that accumulate at high levels in the root and stem (Fig. 4D). Consistent with enriched GO terms in both Caulerpa and tomato, these genes are associated with vesicular trafficking (particularly COP cotamers) and vacuolar transporters (S30 Dataset). Interestingly, the Caulerpa Node 6 genes with high stolon abundance are associated with tomato Node 1 genes with high abundance in the inflorescence and relatively high abundance in the vegetative apex, both meristematic organs (Fig. 4E). The genes intersecting both nodes (S30 Dataset) are members of RNAi, chromatin, and DNA recombination, repair, and replication pathways, suggesting a molecular association between the stolon and meristems of land plants. The ability of the stolon to repetitively produce pseudo-organs (both fronds and holdfasts) and the enrichment of nuclear replication-associated transcripts indicates meristem-like identity at the molecular level. The association between transcript accumulation profiles in Caulerpa and a land plant suggests, to a limited extent, molecular homology underlying morphology. Kaplan and Hagemann [11] argued that land plants, like Caulerpa, are siphonous. While the statement is extreme and not technically correct, reevaluating land plant development from this perspective is insightful, with respect to the role cells play in determining morphology. Morphogenesis and key patterning events in land plants rely on non-cell autonomous, symplastic movement of transcription factors and small RNAs, that transcend cell division patterns [18]–[21]. Spatially restricted transcripts in a siphonous organism, and their correspondence with land plant morphology, demonstrate that the plant form is achievable without cells and questions the centrality of cell division patterns in determining plant morphology. RNA-seq libraries were prepared from at least four replicates of the frond apex, rachis, pinnules, the frond base, stolon, and holdfast, sampling a prolifically growing Caulerpa taxifolia strain obtained from an aquarium in St. Louis, MO. The sampled strain was entrained to a circadian cycle using aquarium lighting roughly synchronized with the outside light-dark cycle. Sampling occurred mid-afternoon, at a time when chloroplasts were enriched in the frond apex (an important consideration, given the nightly retreat of chloroplasts into the frond base and stolon) [5], [22]. Large, intact fragments consisting of fronds, stolons, and holdfasts were removed from the marine aquarium and cleaned in synthetically prepared seawater for approximately 5 seconds to help reduce levels of outside contamination. Different samples corresponded to separately growing clones in the same aquarium. Samples were immediately immersed in liquid nitrogen after cleaning. The samples were then removed from the liquid nitrogen, dissected before they thawed, and placed into microcentrifuge tubes that were immersed again in liquid nitrogen. Samples were then stored at −80°C until library preparation. During thawing before library preparation, one holdfast microcentrifuge sample exploded and was removed from analysis, reducing the holdfast sample number to four. All five samples from other pseudo-organs were successfully processed. Libraries were created using a custom high-throughput method for Illumina RNA-seq with a poly-A enrichment step [15], and sequenced in 100 bp paired-end format at the UC Berkeley Genomics Sequencing Laboratory on two lanes of HiSeq 2000 platform (Illumina Inc. San Diego, CA, USA). Library making was undertaken exactly as published in Kumar et al. [15] without modification of the protocol. We believe that the freezing step during sample preparation is important to bypass the Caulerpa wounding response for successful RNA isolation. Reads were preprocessed using custom perl scripts that involved removal of low quality reads with average Phred quality score <20, trimming of low-quality bases with Phred score <20 from the 3′ ends of the reads, and removal of adapter/primer contamination. In addition identical reads, which originated during the PCR enrichment step of the library preparation, were collapsed into a single read using a custom perl script in order to reduce the computational resources required for transcriptome assembly. However, PCR-duplicated reads were retained for downstream quantification of transcript abundances. The pre-processed reads were sorted into individual samples based on barcodes using fastx_barcode_splitter and barcodes were trimmed using fastx_trimmer from Fastx_toolkit (http: //hannonlab. cshl. edu/fastx_toolkit/). A total of 420 million reads (210 million paired-end 100×100), obtained after preprocessing, were used for transcriptome assembly. De novo transcriptome assembly was carried in a similar fashion as Ranjan et al. [23], but is described here again in detail. The Trinity software package (version r2013-02-16) was used to assemble, de novo, a Caulerpa taxifolia transcriptome using preprocessed RNA-seq reads [24]. The assembly was performed, using 24 large-memory computing nodes, at The Lonestar Linux Cluster at Texas Advance Computing Center (TACC, University of Texas). “Trinity. pl —seqType fq —JM 1000G —left reads-1. fq —right reads-2. fq —min_contig_length 200 —CPU 24 —bflyHeapSpaceMax 7G” was the command line used for assembly. Subsequently, assembly statistics and downstream analysis were performed in the iPlant atmosphere and Discovery computing atmosphere [25]. A total of 77,285 contigs with N50 (N50 is defined as the largest contig length such that using equal or longer contigs produces half the bases of the transcriptome) of 1243 bp, mean length of 807 bp and median of 433 bp, were assembled. In order to remove redundant and/or highly similar contigs, the contigs were then clustered using the CD-HIT-EST program from the CD-HIT suite at a sequence similarity threshold value (-c) of 0. 95 and word-length (n) of 8, leaving other parameters at default [26]. This resulted in the final Caulerpa transcriptome assembly with 57,118 contigs and N50 of 813 bp, mean length of 632 bp and median of 381 bp (see sequence submission information). The prediction of likely coding sequences from 57,118 clustered contigs, using TransDecoder (http: //transdecoder. sourceforge. net/), resulted in 35,827 putative open reading frames (ORFs) /coding sequences (CDS) (see sequence submission information). The contigs from the final Caulerpa transcriptome assembly were compared to the NCBI nr (nonredundant) database (ftp: //ftp. ncbi. nlm. nih. gov/blast/db/FASTA/nr. gz), Arabidopsis protein database (ftp: //ftp. arabidopsis. org/home/tair/Sequences/blast_datasets/TAIR10_blastsets/TAIR10_pep_20110103_representative_gene_model_updated) and tomato (Solanum lycopersicum) ITAG2. 3 protein database (ftp: //ftp. solgenomics. net/tomato_genome/annotation/ITAG2. 3_release/ITAG2. 3_proteins. fasta) using BLASTX with an e-value threshold of 1e-5 (S1 Dataset) [27]. BLAST searches against the nr database resulted in annotation of 24,589 contigs (43% of clustered contigs) of which 20,146 contigs had reasonably stringent e-value of less than 1−e10. 17427 (13698 with e-value <1e−10) and 17392 (13274 with e-value <1e−10) contigs were annotated against Arabidopsis and tomato protein databases. When BLASTX comparison was performed against the nr database, more than 49% of annotated C. taxifolia contigs found top BLAST hits against the sequences from members of Cholorophyta, such as Volvox carteri, Chlamydomonas reinhardtii, Chlorella variabilis, and Coccomyxa subellipsoidea. Almost half of the top BLAST hits to cholorophytes confirm high sequence similarity between C. taxifolia and chlorophytes. The Caulerpa contigs were, further, compared explicitly against protein sequences of the chlorophytes C. reinhardtii and V. carteri using BLASTx, and vice-versa using tBLASTn with an e-value threshold of 1e−5 [27]. Chlamydomonas reinhardtii and V. carteri protein sequences were downloaded from Phytozome v10 (http: //phytozome. jgi. doe. gov/pz/portal. html). BLAST searches against V. carteri and C. reinhardtii sequences found a hit for 19048 (33%) and 20377 (36%) of assembled Caulerpa contigs, respectively (S2 Dataset). Reciprocal BLAST searches of V. carteri and C. reinhardtii sequences against C. taxifolia contigs found homologs for 42% of sequences of each species. The BLASTX output generated against the NCBI nr database, with maximum twenty hits for each contig, was used for Blast2GO analysis to annotate the contigs with GO terms describing biological processes, molecular functions, and cellular components [28]. Blast2GO performs GO annotation by applying an annotation rule (AR) on the found ontology terms from the BLAST-hits, which is based on annotation score. The default e-Value hit filter (1e−6) and annotation cut-off (55) was used to calculate the annotation score. Our gene ontology annotations are, by necessity, based in part on the inclusion of hits using a relatively low e-value threshold. It will be critical in the future to validate these assignments using functional analysis. After the Blast2GO mapping process, proper GO terms were generated followed by use of ANNEX and GO Slim, which are integrated in the Blast2GO software, to enrich the annotation (S3, S4 Datasets). Sequence descriptions were also generated from Blast2GO, which are arbitrary nomenclature based upon degrees of similarities identified in the nr database according to e-value and identity to blasted genes (S1, S2 Datasets). BLASTX against the nr database resulted in annotation of 24,589 contigs (43% of clustered contigs) of which only 14,206 (25% of clustered contigs) were assigned GO-terms. Given the problems associated with the de novo transcriptome assembly algorithms and lack of functional tools in Caulerpa, BLASTX annotation of 43% of clustered contigs and GO annotation of only 25% of clustered contigs is not surprising. Similar functional annotation for only a fraction of assembled contigs has been noted for other de novo assembled plant transcriptomes [23], [29], [30]. These non-annotated contigs likely correspond to 3′ or 5′ untranslated regions, non-coding RNAs, or short sequences not containing a known protein domain, some of which might represent potential Caulerpa-specific genes. RSEM (RNAseq by expectation maximization), which allows for an assessment of transcript abundances based on the mapping of RNA-seq reads to the assembled transcriptome, was used for transcript abundance estimation of the de novo assembled transcripts [31]. Due to read mapping ambiguity among de novo assembled transcripts, it is common to have the same read mapped to multiple contigs. RSEM models the reads mapped at multiple contigs taking into account length of target contigs, number of mismatches, sequencing errors, etc., and generates an estimated read count for each contig. Single end reads, retaining the PCR-duplicated reads, from individual libraries of each Caulerpa sample were mapped to clustered contigs using the perl script run_RSEM_align_n_estimate. pl that employs RSEM, followed by joining RSEM-estimated abundance values for each sample using merge_RSEM_frag_counts_single_table. pl, generating raw estimated counts for each contig from each Caulerpa sample (S5 Dataset). Subsequently, differential expression analysis for each organ pair was carried out using run_DE_analysis. pl, which involves the Bioconductor package EdgeR in the R statistical environment [32]. Contigs that had RSEM-estimated counts ≥30 for all samples combined were used for transcript abundance estimates. Normalization factors were calculated using the trimmed mean of M-values method to obtain normalized read count per million for each contig of a sample. This normalized reads per million was then used for the pair-wise differential expression analysis for each organ pair using EdgeR. The lists of significant differentially expressed contigs (FDR<0. 05) for each organ-pair comparison are presented in S6–S20 Datasets. All the Perl scripts used for read mapping, generating read counts and differential expression analysis are documented with Trinity software suite [33]. Those transcripts differentially expressed between at least one organ pair (S6–S20 Datasets) were subsequently used to find clusters of genes with similar transcript accumulation patterns using Self Organizing Maps (SOMs) [34]. Differentially expressed transcripts were averaged across replicates for each pseudo-organ sample. Averaged transcript abundance values were then scaled across pseudo-organs to arrive at scaled transcript accumulation patterns which were used to assign cluster membership. Scaling was performed using the scale () function in the base package using default settings, such that the average transcript abundance value across pseudo-organs was 0 and the variance equal to 1. To cluster transcripts across organs, a 3×2 hexagonal SOM was implemented, using the Kohonen package in R [35], [36]. 100 training iterations were used during clustering, over which the α-learning rate decreased from 0. 05 to 0. 01. Mean distance of transcript accumulation patterns to their closest unit stabilized after approximately 15 iterations of training. A decision to use six clusters was arrived at by first analyzing the results of a Principal Component Analysis (PCA) on scaled transcript accumulation across tissues, using the prcomp () function in R with default settings. Average and scaled transcript accumulation levels across organs, as well as SOM cluster membership and PC values are provided (S21 Dataset). Four main densities in the variance attributable to accumulation patterns were discernable (arrows, Fig. 2A), and the results of a 4 cluster SOM largely overlap with the densities. Variance in accumulation among transcripts across organs was large, however, and the decision to specify 6 SOM clusters not only produced clusters with unique accumulation patterns and lower variance in abundance (Fig. 2C), but also yielded clusters with more interpretable GO enrichment categories (that is, significant GO enrichment consistent with known biology, such as photosynthetic GOs enriched in nodes with high pinnule transcript abundance). To verify that 6 GOs was indeed the maximum cluster number specifying unique transcript accumulation profiles without redundancy, we undertook partitioning of the PCA space into a variable number of SOM clusters over 100 iterations for each node number with random seeds. Linear Discriminant Analysis (LDA) was performed on genes maximizing separation of cluster identity using PCs 1–5 (PC6 explained negligible amounts of variance and could not be incorporated into the LDA) using the lda function from the MASS package [37]. The predict function (stats package) and table function (base package) were used to reallocate genes to predicted clusters (within MASS) using the linear discriminants. A high fraction of a node' s originally assigned transcripts by SOM being reassigned correctly indicates little redundancy in node transcript accumulation patterns. Starting with 6 nodes, reassignment using LDA begins to drop before reaching a plateau of low reassignment rates, indicating that choosing 6 nodes maximizes the number of unique accumulation profiles represented by clusters without redundancy (see S1 Fig. for results). Analysis of intersection between tomato and Caulerpa transcriptomic atlases was undertaken using data from Chitwood et al. [17]. Best BLASTX hits of Caulerpa transcripts to tomato (Solanum lycopersicum) (see “Functional annotation of transcriptome” above and S1, S2 Datasets) were used to assign tomato transcript accumulation patterns, across a number of organs, to Caulerpa transcript accumulation patterns. The distribution of tomato transcripts assigned to tomato SOM nodes was taken as the null distribution and compared to the number of Caulerpa transcripts assigned to each tomato node. p values, indicating the degree of significant difference between the two distributions, were obtained from χ2 values using the chisq. test function (stats package). Clusters of transcripts were analyzed for GO enrichment terms at a 0. 05 FDR cut-off using the “goseq” package in Bioconductor (S22–S28 Datasets) [38]. Unless otherwise specified, all statistical analyses on transcript accumulation were performed using R [36] and data visualization using the ggplot2 package [39]. The quality filtered, barcode-sorted and trimmed short read dataset, which was used for transcriptome assembly, was deposited to the NCBI Short Read Archive under accessions SRR1228213–SRR1228223, SRR1228225–SRR1228232, SRR1228234–SRR1228238 and. SRR1228240–SRR1228244. All assembled contigs have been deposited at DDBJ/EMBL/GenBank under the accession GBCY00000000. The version described in this paper is the first version, GBCY01000000. Sequences of all contigs of Caulerpa_final_transcriptome, obtained after clustering of transcripts, can be downloaded as a FASTA file at http: //de. iplantcollaborative. org/dl/d/80CF0D47-5A80-4CE7-B6DF-F4A7ED803493/Caulerpa_final_transcriptome. fasta. The contigs were named as Ctaxi_contig plus a serial number with the Trinity identifiers. Sequences of all predicted ORFs from the Caulerpa transcriptome assembly can be downloaded as a FASTA file at http: //de. iplantcollaborative. org/dl/d/40273882-35DD-4930-9DBD-6D60CEAA7890/Caulerpa_predicted_ORFs. fasta. The ORFs were named as Ctaxi_predicted_CDS plus a serial number.

Plants include both the green algae and land plants. Multiple times, root, stem, and leaf-like structures arose independently in plant lineages. In some instances, such as the siphonous algae, these structures arose in the absence of multicellularity. It has been argued by some that the morphology of multicellular land plant organs similarly arises independently of cell division patterns. Here, we explore the partitioning of gene transcripts within what is debatably the largest single-celled organism in the world, the siphonous alga Caulerpa taxifolia. We find that within this giant cell specific transcripts localize within pseudo-organs (morphological structures that are not comprised of cells or tissue). The overall pattern of transcript accumulation follows an apical-basal pattern within the cell. Moreover, transcripts related to different cellular processes, such as transcription and translation, localize to specific regions. Analyzing the signatures of transcript accumulation in land plant organs and the pseudo-organs of Caulerpa, we find that groups of transcripts accumulate together in morphological structures across evolution at rates higher than expected by chance. Together, our results demonstrate a relationship between transcript partitioning and organism morphology, independent from multicellularity, throughout diverse plant lineages.

lay_plos

Mammalian articular cartilage is an avascular tissue with poor capacity for spontaneous repair. Here, we show that embryonic development of cartilage in the skate (Leucoraja erinacea) mirrors that of mammals, with developing chondrocytes co-expressing genes encoding the transcription factors Sox5, Sox6 and Sox9. However, in skate, transcriptional features of developing cartilage persist into adulthood, both in peripheral chondrocytes and in cells of the fibrous perichondrium that ensheaths the skeleton. Using pulse-chase label retention experiments and multiplexed in situ hybridization, we identify a population of cycling Sox5/6/9+ perichondral progenitor cells that generate new cartilage during adult growth, and we show that persistence of chondrogenesis in adult skates correlates with ability to spontaneously repair cartilage injuries. Skates therefore offer a unique model for adult chondrogenesis and cartilage repair and may serve as inspiration for novel cell-based therapies for skeletal pathologies, such as osteoarthritis. Hyaline cartilage is a skeletal tissue that consists of a single cell type (the chondrocyte) embedded within a homogeneous, collagenous extracellular matrix (reviewed in Gillis, 2018). In mammals, hyaline cartilage is predominantly an embryonic tissue, making up the anlage of the axial (chondrocranial, vertebral and rib) and appendicular (limb) endoskeleton. The vast majority of mammalian hyaline cartilage is replaced by bone during the process of endochondral ossification, with cartilage persisting temporarily in epiphyseal growth plates, and permanently at relatively few sites within the adult skeleton (e. g. in joints, as articular cartilage – Decker, 2017). In juvenile mammals, growth of articular cartilage occurs by cellular rearrangement and increases in chondrocyte volume (Decker et al., 2017), and by appositional recruitment of new chondrocytes from a superficial population of slow-cycling chondroprogenitor cells (Hayes et al., 2001; Dowthwaite et al., 2004; Karlsson et al., 2009; Williams et al., 2010; Kozhemyakina et al., 2015). However, evidence for the presence of chondroprogenitor cells in adult articular cartilage is scant, and this – combined with the avascular nature of the tissue – may account for why mammalian articular cartilage cannot heal spontaneously following injury (Hunziker, 1999). Chondrichthyans (cartilaginous fishes – sharks, skates, rays and holocephalans), on the other hand, possess an endoskeleton that is composed largely of hyaline cartilage, and that remains cartilaginous throughout life. Though chondrichthyans reinforce their endoskeleton with a superficial layer of calcified cartilage (in the form of small mineralized plates called ‘tesserae’ – Dean and Summers, 2006), the core of their endoskeletal elements persists as hyaline cartilage and does not undergo endochondral ossification. Like many fishes (and unlike mammals), chondrichthyans also exhibit an indeterminate type of growth, with a continued (albeit slow) increase in size through adulthood (Dutta, 1994; McDowall, 1994; Frisk and Miller, 2006). It therefore stands to reason that, in chondrichthyans, skeletal tissues may possess a persistent pool of chondroprogenitor cells to facilitate continued growth of their cartilaginous endoskeleton throughout adulthood, and that such cells (if present) could also impart the endoskeleton with an ability to undergo spontaneous repair following injury. However, basic mechanisms of hyaline cartilage development, growth and repair in chondrichthyans remain largely unexplored. Here, we characterize the development and growth of the cartilaginous endoskeleton of a chondrichthyan, the little skate (Leucoraja erinacea), from embryonic development to adulthood. We demonstrate conservation of fundamental cellular and molecular characteristics of cartilage development between chondrichthyans and mammals, and we identify unique features of adult skate cartilage that contribute to its continued growth through adulthood. We further show that skates can repair surgically induced partial-thickness cartilage injuries, highlighting this system as a unique animal model for adult chondrogenesis and spontaneous hyaline cartilage repair. The pectoral fin endoskeleton of jawed vertebrates consisted ancestrally of three basal cartilages – from anterior to posterior, the propterygium, mesopterygium and metapterygium – and a series of articulating distal radials (Davis et al., 2004). Among extant jawed vertebrates, this ancestral ‘tribasal’ condition has been retained in the pectoral fins of chondrichthyans and non-teleost ray-finned fishes (e. g. in sturgeon, gar and bichir), but has been reduced in tetrapods and teleosts, to include only derivatives of the metapterygial and pro-/mesopterygial components, respectively (Davis, 2013). Our study focused on the metapterygium of the skate (Figure 1), as this element is relatively large, reliably identifiable across all embryonic and post-embryonic stages and easily accessible for surgical manipulation. In mammals, early cartilage development is marked by the accumulation of preskeletal mesenchyme into a ‘condensation’ at the site of future chondrogenesis (Hall and Miyake, 2000). Cells within this condensation begin to secrete cartilage extracellular matrix (ECM) components and undergo overt differentiation into chondrocytes. To investigate the early development of cartilage in the skate, we prepared a histological series of skate metapterygia from embryonic stage (S) 30 through to hatching and used a modified Masson’s trichrome stain to visualize condensation, differentiation and ECM secretion. At S30, the presumptive metapterygium exists as condensed mesenchyme, with pericellular Light Green staining (which appears blue) indicating onset of ECM secretion by cells within the condensation (Figure 2a). From S31-33 (Figure 2b–d), the metapterygium differentiates into cartilage and grows, with cells in the centre of the element adopting differentiated chondrocyte morphology (i. e. cells residing within ECM cavities or ‘lacunae’), but cells in the periphery maintaining a less differentiated appearance. By hatching (Figure 2e), the cartilage of the metapterygium is surrounded by a distinct fibrous perichondrium (Figure 2ei), beneath which we begin to observe modifications of the ECM at sites of superficial calcification (i. e. developing tesserae – Figure 2eii). By hatching, cells throughout the cartilage have adopted differentiated chondrocyte morphology, and are embedded within extensive collagenous ECM (Figure 2f). Mammalian hyaline cartilage ECM is composed largely of fibrils of type II collagen, which entrap aggregates of the hydrated proteoglycan aggrecan (Eyre, 2002; Kiani et al., 2002). Col2a1 and Agc (the genes encoding type II collagen and aggrecan, respectively), in turn, are directly transcriptionally regulated in chondrocytes by the SRY-box transcription factors Sox9, Sox5, and Sox6 (Bell et al., 1997; Lefebvre et al., 1998; Lefebvre et al., 2001). To test for conservation of these gene expression features in chondrocytes of the skate metapterygium, we characterized the co-expression of genes encoding cartilage ECM components and upstream transcriptional regulators in situ. We first cloned fragments of skate Cola2a1 (Figure 3—figure supplement 1) and Agc (Figure 3—figure supplement 2) and tested for their expression in the S32 metapterygium by chromogenic mRNA in situ hybridization. We found that both Col2a1 (Figure 3a) and Agc (Figure 3b) are expressed in chondrocytes throughout the skate metapterygium, reflecting shared ECM properties between skate and mammalian hyaline cartilage. To test for conservation of the regulatory relationship between Sox5, Sox6 and Sox9, we used multiplexed fluorescent in situ hybridization by chain reaction (HCR) to test for co-expression of these genes (Figure 3—figure supplements 3–4) in metapterygium chondrocytes. We observed co-expression of Col2a1 and Sox9 in chondrocytes throughout the metapterygium (Figure 3c–d), as well as co-expression of Col2a1, Sox5 and Sox6 (Figure 3e–f), indicating likely conservation of regulation of genes encoding cartilage ECM components by SoxE- and SoxD-class transcription factors in skate cartilage. To characterize patterns of cell proliferation within the growing metapterygium, we conducted a label retention experiment in skate hatchlings. Incorporation and detection of thymidine analogues, such as 5-ethynyl-2' -deoxyuridine (EdU), provides a sensitive readout of DNA synthesis and, by extension, cell proliferation (Salic and Mitchison, 2008). Briefly, hatchling skates were given a single intraperitoneal microinjection of EdU, and were then harvested at 1-, 5-, 10- and 40 days post-injection (hereafter referred to as 1-, 5-, 10- and 40 day chase, respectively), to test for label retention within the metapterygium. In animals analyzed at 1 day chase, EdU+ chondrocytes were recovered throughout the cartilage of the metapterygium, though with a concentration of EdU+ cells around the periphery of the element (Figure 4a), pointing to the continued proliferation of differentiated chondrocytes at hatching. We also observed EdU+ cells within the perichondrium of the metapterygium at 1 day chase (Figure 4b). These label-retaining cells exhibited a distinct, flattened nuclear morphology (relative to adjacent chondrocytes), and were recovered in increasingly greater numbers within the perichondrium at 5- (Figure 4c) and 10 day chase (Figure 4d). By 40 days chase (Figure 4d), we observed a marked decrease in the number of EdU+ perichondral cells. This pattern of label retention (Figure 4f) is suggestive of an expanding or self-renewing cell population within the perichondrium, with a greater number of label-retaining cells arising through proliferation of EdU+ progenitors but reduction in the number of label-retaining cells with eventual dilution of EdU to undetectable levels. In animals analyzed at 10- (Figure 4g) and 40 days chase (Figure 4h), we observed numerous clusters of EdU+ chondrocytes in cartilage immediately adjacent to EdU+ perichondral cells. As cells of the inner perichondrium are known to give rise to new chondrocytes in the cartilaginous anlage of the chick limb skeleton (Scott-Savage and Hall, 1979), we speculate that these subperichondral EdU+ chondrocytes are the progeny of label-retaining perichondral cells, and that growth of the hatchling metapterygium occurs both through proliferation of differentiated chondrocytes, and by recruitment of new chondrocytes from progenitor cells within the perichondrium. Using mRNA in situ hybridization by HCR, we investigated gene expression at the cartilage-perichondrium interface within the metapterygium of skate hatchlings. As in S32 embryos, we noted co-expression of Col2a1 and Sox9 (Figure 4i) and Col2a1, Sox5 and Sox6 (Figure 4j) in chondrocytes of the metapterygium. However, we also observed cells within the perichondrium that expressed Sox9, Sox5 and Sox6 but not Col2a1 (Figure 4i, j). These cells were invariably located within the innermost region of the perichondrium, at the cartilage-perichondral interface, and exhibited the flattened nuclear morphology of the EdU+ perichondral cells described above. Taken together, our label retention and gene expression data point to the perichondrium as a source of cartilage progenitor cells in skate hatchlings. We next examined histological features of the adult skate metapterygium, by vibrotomy (Figure 5a) and histochemical staining of paraffin sections with a modified Masson’s trichrome stain (Figure 5b). In transverse sections through the metapterygium, we noted that the core of the element has the glassy appearance of hyaline cartilage, while the surface of the cartilage is covered by a rind of calcified tesserae (Figure 5a–b). Cells in the core of the metapterygium exhibit typical hyaline chondrocyte morphology (Figure 5c), are embedded in an ECM that stains with Light Green, and express high levels of type II collagen (Figure 5d). A higher magnification view of the tesserae reveals that these sit beneath a well-developed, fibrous perichondrium, which stains variably red and blue/green with trichrome staining (Figure 5e). We intermittently see a thin layer of unmineralized cartilage between the surface of the tesserae and the perichondrium, likely corresponding with the ‘supratesseral’ cartilage that has been previously reported in the stingray, Urobatis halleri (Seidel et al., 2017). Closer examination of the intertesseral joint region (i. e. the zone of cartilage between adjacent tesserae – Figure 5f) reveals cells with typical chondrocyte morphology within ECM up to the boundary between hyaline cartilage and the perichondrium, as well as a distinct population of flattened, spindle-shaped cells at the boundary between cartilage and perichondrium (Figure 5fi), hereafter referred to as ‘inner perichondral cells’. Interestingly, the adult skate metapterygium is permeated by a series of canals, which originate at the surface of the cartilage, and extend toward the core of the element (Figure 5a–b). These cartilage canals occur throughout the metapterygium, originate within the perichondrium and enter the cartilage between tesserae (i. e. through the intertesseral joint region), and are not lined by an epithelium (Figure 5g). Cartilage canals contain an abundance of cells, including some red blood cells (Figure 5h), but predominantly cells with connective tissue/mesenchymal morphology – many of which appear to be invading from the canal into surrounding cartilage ECM (Figure 5i). Immunostaining for type II collagen reveals that cartilage canals are zones of active ECM synthesis, with high levels of type II collagen being secreted by cells at the periphery of the canals (Figure 5j). To test for cells that are actively expressing cartilage ECM products in the adult skeleton, we analyzed expression of Col2a1 by mRNA in situ hybridization on sections of adult metapterygium. High levels of Col2a1 transcription were detected around the periphery of the cartilage – that is in the intertesseral joint region and in cartilage adjacent to tesserae – and also in the thin layer of supratesseral cartilage that sits between the tesserae and the perichondrium (Figure 6a). In situ hybridization by HCR revealed that both supratesseral and peripheral chondrocytes co-expressed Col2a1 and Sox9 (Figure 6b–c), as well as Col2a1, Sox5, Sox6 (Figure 6d–e), pointing to retention of transcriptional features of developing cartilage around the periphery of the adult metapterygium. Interestingly, as in hatchling skates, we also observed co-expression of Sox5, Sox6 and Sox9 (but not Col2a1) in the flattened cells of the inner perichondrium (Figure 6b, d). Given the indeterminate growth of cartilaginous fishes, we speculated that the transcriptional signature of embryonic cartilage in the periphery of the adult skate metapterygium could reflect recently born chondrocytes contributing to ECM expansion, while the presence of this signature in the inner perichondrium could reflect progenitors of new chondrocytes. We therefore sought to test for the presence and fate of cycling cells in the adult skate metapterygium using a label retention experiment. Due to the relatively slow growth rate of cartilaginous fishes, we reasoned that a pulse-chase label retention experiment could be used not only to localize cell proliferation within the metapterygium, but also to lineage trace label-retaining cells to test for contributions to hyaline cartilage. Briefly, eight adult female skates were given three intraperitoneal injections of EdU, 48 hr apart, and two animals were then euthanized, fixed and processed for EdU detection 3 days, 1 month, 2 months and 5. 5 months following the final IP EdU injection (hereafter referred to as 3 day, 1 month, 2 month and 5. 5 month chase, respectively). EdU detection was performed on transverse paraffin sections though the metapterygium, with EdU+ cells scored according to their location in the outer perichondrium, inner perichondrium, cartilage canals or cartilage (i. e. chondrocytes). 30–40 sections were analyzed from each animal, but quantification was performed by counting the sum total and tissue localization of EdU+ cells in five adjacent sections through the metapterygium (as indicated in Figure 1i) after pulse + 3 days, 1 month, 2 month and 5. 5 month chase (Table 1). After a 3 day chase, EdU+ cells were recovered almost exclusively in the perichondrium, with most appearing as cells with rounded nuclei in the outer perichondrium (Figure 7a), and relatively few as flattened cells of the inner perichondrium (Figure 7b). No EdU+ chondrocytes were detected after a 3 day chase. After 1 month and 2 month chases, we continued to detect EdU+ cells within the outer and inner perichondrium, and we also observe EdU+ cells within the cartilage canals that originate in the perichondrium and permeate the core of the metapterygium (Figure 7c–d). After a 5. 5 month chase, we detected an abundance of EdU+ cells in both the outer and inner perichondrium (Figure 7e), as well as abundant EdU+ chondrocytes in the peripheral hyaline cartilage of the intertesseral joint region (Figure 7f) and in cartilage subjacent to the tesserae (Figure 7g), as well as relatively few EdU+ chondrocytes within the mineralized matrix of the tesserae (Table 1). EdU+ cells are present in greater abundance within cartilage canals after a 5. 5 month chase (Figure 7h) and given the relative dearth of EdU+ cells in cartilage canals after the 3 day, 1 month and 2 month chases, we speculate that these EdU+ cartilage canal cells are of perichondral origin. After the 5. 5 month chase, we also observed EdU+ cells that appeared to be invading hyaline cartilage from the blind ends of cartilage canals (Figure 7i), and in one individual we observed three instances of EdU+ chondrocytes immediately adjacent to the blind ends of cartilage canals (Figure 7j). Taken together, our label retention and gene expression data point to the morphologically distinct Sox5/Sox6/Sox9 + cells of the inner perichondrium as adult cartilage progenitor cells, with a capacity to give rise to new chondrocytes both in the periphery (i. e. appositional growth), and also in the core of the metapterygium, via cartilage canals (i. e. interstitial growth). Additionally, while the number of EdU+ cells is highly variable between individuals, the general trend of a greater number of label-retaining perichondral cells after the 2 month and 5. 5 month chases – and, more specifically, a greater number of label-retaining cells within the inner perichondrium – points to the likely self-renewal of perichondral cells, perhaps with a progressive sequence of differentiation from outer perichondral cell to inner perichondral cell, and eventually to chondrocyte – either in the periphery, or deeper in the core (via cartilage canals). Mammalian articular hyaline cartilage is unable to spontaneously heal following injury. Rather, articular cartilage injuries tend to infill with fibrocartilage – a subtype of cartilage that exhibits large bundles of collagen fibres, and with ECM composed substantially of type I collagen (Eyre and Wu, 1983; Benjamin and Ralphs, 2004). Fibrocartilage is mechanically inferior to hyaline cartilage at the articular surfaces of synovial joins, and its formation within articular cartilage lesions can result in the onset of degenerative osteoarthritis. We sought to test whether the persistence of adult chondrogenesis in the skate metapterygium – and the presence of cartilage progenitor cells in the perichondrium – correlated with an ability to spontaneously repair injured hyaline cartilage. We conducted a surgical cartilage injury experiment, in which a metapterygium cartilage biopsy was performed in 26 adult skates using a 4 mm biopsy punch (producing a cartilage void of ~1/4 to 1/3 diameter of the metapterygium – Figure 8a). Two animals were euthanized one-week post-operation, and at monthly intervals for the following year, and processed histologically to assess the extent of repair. In animals assessed at 1 and 2 months post-operation (mpo) (n = 4), cartilage injuries were in-filled with a fibrous connective tissue (Figure 8b, bi), but by 3mpo (n = 2), this connective tissue began to differentiate into cartilage (i. e. with typical hyaline chondrocytes, albeit at a much higher density than in the adjacent, uninjured cartilage – Figure 8c, ci). In animals assessed at 4-10mpo (n = 14), tissue within the injury sites showed varying degrees of progressive differentiation into cartilage, starting from the interface between the injury site and adjacent cartilage and progressing toward the surface of the metapterygium (Figure 8—figure supplement 1), and by 11-12mpo (n = 4), injury sites were completely or near-completely filled with repair cartilage (Figure 8d, Figure 8—figure supplement 2,3). Chondrocytes within the repair cartilage remained at much higher density than in the adjacent cartilage, and the surface of the injury site remained irregular, with some superficial red staining of the ECM (Figure 8di). This could reflect the re-appearance of tissue with a perichondral-like nature, or a step toward re-establishment of tesserae at the surface of the metapterygium, as these tissues stained variably red-blue and red, respectively, with modified Masson’s trichrome stain. However, the vast majority of repair tissue resembled typical hyaline cartilage, with a Light Green-stained ECM that integrates with adjacent uninjured cartilage and no evidence of ECM fibre bundles typical of fibrocartilage (Figure 8dii). We tested whether the ECM of repair cartilage was composed of type II collagen (as in typical hyaline cartilage) or a mixture of types I and II collagen (as in fibrocartilage) using immunofluorescence. We observed strong, positive staining for type II collagen throughout the ECM of repair cartilage, as well as pericellular staining for type II collagen in adjacent uninjured hyaline cartilage (Figure 8e – Figure 8—figure supplement 4). Conversely, we observed no positive staining for type I collagen in repair or uninjured cartilage (including cartilage canals), despite strong positive staining in adjacent skeletal muscle fibres (Figure 8—figure supplement 4). These findings suggest that adult skate repair cartilage produces ECM similar to that of adjacent hyaline cartilage and is unlike the fibrocartilaginous repair tissue that typically fills mammalian chondral defects. Interestingly, in two animals collected between 4-10mpo, our biopsy had been unsuccessful, with the biopsy punch perforating the surface of the metapterygium but failing to remove a wedge of cartilage. In both cases, a large mass of ectopic cartilage formed above the tesserae on the surface of the metapterygium, but beneath the fibrous perichondrium (Figure 8—figure supplement 5). This suggests that mechanical perturbation of the perichondrium may act as an inductive cue for onset of a chondrogenic injury response, and is consistent with the perichondrium as a source of new cartilage not only during normal adult growth, but also following injury. The endoskeleton of bony fishes (including tetrapods) forms largely through a process of endochondral ossification. In endochondral ossification, pre-skeletal mesenchyme aggregates to form a condensation at the site of skeletogenesis, and cells within this condensation undergo progressive differentiation, starting from the centre, into chondrocytes and eventually enlarged, hypertrophic chondrocytes (Hall, 2005; Karsenty et al., 2009; Long and Ornitz, 2013). Chondrocyte and hypertrophic chondrocyte fates are determined and characterized by the expression of genes encoding distinct sets of transcription factors and ECM components, with the transcription factors Sox5, Sox6 and Sox9 regulating the expression of Col2a1 and Agc1 in chondrocytes (Bell et al., 1997; Lefebvre et al., 1998; Lefebvre et al., 2001; Smits et al., 2001; Akiyama et al., 2002; Han and Lefebvre, 2008), and the transcription factor Runx2 regulating the expression of Col10a1 (the gene encoding non-fibrillar type X collagen) in hypertrophic chondrocytes (Linsenmayer et al., 1991; Takeda et al., 2001; Zheng et al., 2009; Simões et al., 2006; Higashikawa et al., 2009; Ding et al., 2012). Hypertrophic cartilage is ultimately invaded by vasculature, and is replaced by bone, with bone-forming cells (osteoblasts) arising both from the perichondrium/periosteum, and through transdifferentiation of hypertrophic chondrocytes (Colnot et al., 2004; Roach, 1992; Roach et al., 1995; Zhou et al., 2014; Yang et al., 2014; Park et al., 2015; Hu et al., 2017). Within growing endochondral bones, non-hypertrophic cartilage persists in the growth plate, where chondrocytes continue to proliferate and contribute to lengthening of an element, and at points of endoskeletal articulation. Upon cessation of growth, growth plate cartilage will hypertrophy and ossify, with non-hypertrophic cartilage persisting only at articular surfaces. In skate, onset of endoskeletal development is marked by the appearance of mesenchymal condensations at sites of skeletogenesis, and cells within condensations differentiate into chondrocytes progressively, from the centre of the condensation to the periphery. This differentiation of condensed mesenchyme is accompanied by co-expression in chondrocytes of Sox5, Sox6, Sox9, Col2a1 and Agc1, pointing to conservation of the regulatory interaction between SoxD- and E-class transcription factors and the genes encoding type II collagen and aggrecan in cartilaginous and bony fishes, and to the broad developmental and biochemical comparability of cartilage between these major vertebrate lineages. Unlike in bony fishes, however, chondrocytes in the skate metapterygium do not undergo hypertrophy, but rather remain terminally differentiated in a non-hypertrophic state. While cartilaginous fishes, strictly speaking, lack bone, they nevertheless possess the vast majority of transcription factors and ECM components required to make bone (Venkatesh et al., 2014), and there are instances of mineralization within the skeleton of cartilaginous fishes that share molecular properties with bone – for example expression of Col10a1, Col1a1 and SPARC in the mineralized areolar tissue of the vertebral column, and immunolocalization of types I and X collagen to tesserae and pre-mineralized supratesseral cartilage (Egerbacher et al., 2006; Enault et al., 2015; Criswell et al., 2017; Seidel et al., 2017; Debiais-Thibaud et al., 2019). These observations, combined with palaeontological evidence for the presence of bone along the gnathostome stem (Donoghue et al., 2006; Charest et al., 2018), are consistent with bone as an ancestral feature of jawed vertebrates, the loss of this tissue in extant cartilaginous fishes, and the independent re-deployment of deeply conserved mechanisms of cartilage mineralization at sites such as tesserae and the axial column. There has been relatively little work on postembryonic growth of hyaline cartilage in cartilaginous fishes, perhaps owing to the general difficulties of locating and maintaining a suitable range of subadult life stages. Growth of the tessellated calcified cartilage of cartilaginous fishes has been well documented, particularly in the stingray Urobatis halleri, where it has been shown that tesserae continue to grow throughout life, and that this growth likely occurs by accretion, with continuous mineralization of a thin layer of ‘supratesseral’ cartilage that sits between the tesserae and the perichondrium, and of ‘subtesseral’ cartilage beneath the tesserae (Dean et al., 2009; Seidel et al., 2016; Seidel et al., 2017). We have discovered a population of label-retaining cartilage progenitor cells (characterized by co-expression of Sox5, Sox6 and Sox9) in the inner perichondrium of adult skates, and we have traced their chondrocyte progeny to the peripheral unmineralized cartilage and (occasionally) mineralized tesserae of the metapterygium. Based on our observation of a similar cell type in the metapterygium perichondrium of hatchling skates, these observations point to a general mechanism of appositional post-embryonic cartilage growth, wherein progenitor cells of perichondral origin give rise to new chondrocytes in the periphery of the metapterygium – some of which will become incorporated into growing tesserae, while others (e. g. in the intertesseral joint regions) will persist as unmineralized hyaline cartilage (Figure 9). In addition to appositional growth of cartilage in the metapterygium, we also find some evidence of interstitial growth, by the addition of new chondrocytes to the unmineralized cartilaginous core of the metapterygium. In our pulse-chase label retention experiments, EdU+ cells are only recovered in the perichondrium after a pulse + 3 day chase (with the exception of a single label-retaining cell in a cartilage canal), with no label-retention in differentiated chondrocytes. However, we observe a striking increase in label-retaining cells within cartilage canals and core cartilage after pulse + 1–5. 5 month chases, including a small number of chondrocytes in the centre of the metapterygium cartilage core, immediately adjacent to cartilage canals. Given the initial distribution of label retaining cells after the 3 day chase, we speculate that these core chondrocytes derive from label-retaining progenitors in the perichondrium and were transported to the cartilaginous core of the metapterygium via cartilage canals (Figure 9). However, skate cartilage canals also contain red blood cells (Figure 4h), which suggests that these structures may serve to vascularise the adult cartilaginous endoskeleton. If this is the case, then such vasculature could serve to transport cartilage progenitor or mesenchymal stem cell-like cells, from niches elsewhere in body, to the core cartilage of the metapterygium. Cartilage canals that extend from the perichondrium into the core of endoskeletal elements have been shown to occur in the vertebrae, jaws and pectoral girdles of sharks and rays (Hoenig and Walsh, 1982; Dean et al., 2010). Similar canals have been described from the cartilage of embryonic tetrapods, though in tetrapods these are transient structures that function in mediating the replacement of cartilage by bone during endochondral ossification (Blumer et al., 2004; Blumer et al., 2008). Conversely, cartilage canals in cartilaginous fishes persist within the adult skeleton, have been described as containing blood vessels and lymph-like and other amorphous materials, as well as immature chondrocytes, and have been speculated to function in the nourishment and maintenance of cartilage in the adult endoskeleton (Hoenig and Walsh, 1982; Dean et al., 2010). The cartilage canals that we have described in the metapterygium of the skate closely resemble those described previously in other cartilaginous fishes, and findings from our label retention experiments are consistent with a function for these canals in transporting material (including pre-chondrocytes) to the core cartilage of the metapterygium. It therefore seems likely that cartilage canals do, indeed, function in the maintenance, nourishment, and, most likely, interstitial growth of cartilage in the endoskeleton of cartilaginous fishes. We have found that the persistence of cartilage progenitor cells and chondrogenesis in the adult skate skeleton correlates with an ability to spontaneously repair injured cartilage – albeit with a tissue containing a significantly higher density of chondrocytes relative to normal adult cartilage. A comparative analysis of the mechanical properties of normal and repair cartilage remains to be conducted, though the ECM of skate repair cartilage is composed of type II collagen and appears to integrate seamlessly with adjacent tissue, and cells within repair cartilage appear indistinguishable from adjacent chondrocytes. While it is not currently possible to precisely trace the cell lineage of repair cartilage within our injury paradigm, the demonstrated chondrogenic potential of the adult skate perichondrium during normal growth, as well as our observation that mechanical perturbation of the surface of the metapterygium is sufficient to induce a large mass of ectopic cartilage beneath the perichondrium, points to the perichondrium as the most likely source of repair cartilage following injury. Our findings stand in contrast with previous work in the dogfish (Scyliorhinus spp.), which determined that the cartilaginous skeleton of sharks could not heal following injury (Ashhurst, 2004). This was based on a design in which cartilaginous fin radials were bisected and monitored for repair over 26 weeks. It was observed that cut surfaces of dogfish fin rays were initially capped by a fibrous tissue, with subsequent appearance near the injury site of a disorganized, cartilage-like tissue that failed to integrate with existing ray cartilage or to unite the bisected elements. Importantly, the cartilage injury in that study (i. e. complete bisection) was severe, and may have posed an insurmountable barrier to repair, even for a tissue with local progenitors and repair potential (i. e. it is possible that the repair response that we report in skate requires some scaffold of normal cartilage as a foundation for repair). Additionally, fin rays are relatively small, and therefore may exhibit different growth properties relative to larger elements of the endoskeleton. Additional studies of repair potential across the range of skeletal elements and tissue types in cartilaginous fishes are needed to determine whether cartilage repair is a general feature of the skeleton, or a unique property of specific skeletal elements. Osteoarthritis (OA) is a debilitating deterioration of joint cartilage with symptoms ranging from stiffness and joint pain to complete immobility. OA can severely impact quality of life, and has an extremely high economic burden, and so there is great interest in identifying novel therapeutic strategies to promote joint cartilage repair. Joint cartilage repair still poses a substantial clinical challenge, owing to the avascular and aneural nature of articular cartilage, and therefore its limited capacity to initiate spontaneous repair. Recently, focus has shifted from surgical approaches (e. g. microfracture and autologous chondrocyte implantation – Rodrigo et al., 1994; Brittberg et al., 1994) to stem cell-based therapies for cartilage defects – namely the application of patient-derived mesenchymal stem cells (MSCs) or induced pluripotent stem cells (iPSCs) as sources of repair tissue for damaged cartilage (Bernhard and Vunjak-Novakovic, 2016; Murphy et al., 2017; Harrell et al., 2019). While these approaches hold great promise, some challenges nevertheless remain. Derivation of persistent cartilage from MSCs is challenging, as chondrogenically differentiated MSCs will often continue on a path toward hypertrophy and ultimately ossification (Pelttari et al., 2006; Steinert et al., 2007), while the use of incompletely differentiated iPSCs can result in heterogeneous repair tissue, and may come with a risk of teratoma formation (Heng et al., 2004; Saito et al., 2015). The unique endoskeletal growth and repair properties of cartilaginous fishes may offer a powerful model to inform novel cell-based strategies for mammalian cartilage repair. It remains to be determined whether the adult skate perichondrium is a homogeneous cell population with equivalent chondrogenic potential throughout, or a heterogeneous tissue containing a specialized subpopulation of chondroprogenitors. However, the ability of this tissue to give rise to cartilage as a terminal product – rather than cartilage as an intermediate step toward endochondral bone – is significant and could be exploited to further our understanding of the molecular basis of stable and reliable adult chondrogenesis in vitro and in vivo. Further characterization of the transcriptional fingerprint of cell types within the skate perichondrium, and the gene regulatory basis skate chondrocytes differentiation during growth and repair, could provide a rich source of information on how MSCs and/or iPSCs could be edited or manipulated to enhance their efficacy in mammalian articular cartilage repair. Leucoraja erinacea adults and embryos were maintained in large rectangular tanks or seatables, respectively, in flow-through natural seawater at a constant temperature of 15°C at the Marine Resources Center of the Marine Biological Laboratory in Woods Hole, MA, U. S. A. Adult skates were fed on a diet of squid and capelin. Skate embryos were staged according to the lesser-spotted dogfish (Scyliorhinus canicula) staging table of Ballard et al. (1993) and the winter skate (Leucoraja ocellata) staging table of Maxwell et al. (2008). Prior to fixation, all skate embryos and adults were euthanized with an overdose of ethyl 3-aminobenzoate methanesulfonate salt (MS-222 - Sigma) in seawater (1 g/L MS-222 buffered with 2 g/L sodium bicarbonate). Animals were kept in a euthanasia bath until the cessation of gill pumping and heartbeat, and for adults, decapitation was used as a secondary method of euthanasia. All skate embryos were fixed overnight in 4% paraformaldehyde (Electron Microscopy Science) in 1X phosphate buffered saline (PBS – ThermoFisher), rinsed 3 × 10 min in 1X PBS, dehydrated stepwise into 100% methanol, and stored in methanol at −20°C prior to analysis. Pieces of dissected adult skate cartilage to be used for histochemical staining, immunofluorescence or EdU detection were fixed in 4% paraformaldehyde in filtered seawater for 48 hr, rinsed into filtered seawater containing ~0. 5% paraformaldehyde and stored at 4°C. Pieces of dissected adult skate cartilage to be used for mRNA in situ hybridization were fixed for 48 hr in 4% paraformaldehyde in 1X PBS, then rinsed 3 × 10 min in 1X PBS, dehydrated stepwise into 100% methanol, and stored in methanol at −20°C. All work involving skate embryos and adults was conducted in strict accordance with protocols approved by the Marine Biological Laboratory Institutional Animal Care and Use Committee. For paraffin embedding of embryonic tissue, specimens were cleared 3 × 20 min in Histosol (National Diagnostics) at room temperature, transitioned through 2 × 30 min steps in 1: 1 Histosol: molten paraffin in a standard wax oven at 60°C, then left in molten paraffin (RA Lamb Wax – Fisher Scientific) at 60°C overnight. The following day, specimens were moved through four changes of molten paraffin (each >1 hr) before positioning and embedding in a Peel-A-Way embedding mold (Sigma). For paraffin embedding of adult skate tissue, samples were rinsed several times in water, and then demineralised in a 10% (w/v) solution of ethylenediaminetetraacetic acid (EDTA) in 0. 1M Tris pH 7. 2 for 20–25 days on a rocking platform at 4°C. Upon completion of demineralisation, samples were washed several times with water, and then infiltrated with paraffin under vacuum in a tissue processor (with stepwise dehydration into 100% ethanol, 3 × 1 hr washes in xylene and 4 × 45 min washes in molten paraffin) before embedding in a Peel-A-Way embedding mold. All blocks were left to set overnight at room temperature before sectioning at 8 μm on a Leica RM2125 rotary microtome. Sections were mounted on Superfrost plus slides (VWR) and stained with a modified Masson’s trichrome stain, according to the protocol of Witten and Hall (2003). All histochemical staining was carried out at least in triplicate (i. e. on three separate stage-matched individuals). Slides to be used for immunofluorescence were dewaxed in histosol and rehydrated through a descending ethanol series into 1X PBS + 0. 1% Triton X-100 (PBT). For enzymatic antigen retrieval, slides were incubated in 147 u/mL hyaluronidase (Sigma) in PBS pH 6. 7 for 1 hr at 37°C followed by 0. 1% (w/v) pepsin (Sigma) in 0. 01N HCl for 30 min at 37°C for anti-Col2a1 or 0. 1% (w/v) pepsin in 0. 5M acetic acid for 2 hr at 37°C for anti-Col1a1, according to the protocol of Egerbacher et al. (2006). Slides were then rinsed 3 × 10 min in PBT, blocked for 30 min in 10% sheep serum and incubated in primary antibody (under a parafilm coverslip) in a humidified chamber overnight at 4°C. The following day, slides were rinsed 3 × 5 min in 1X PBT, and then incubated in secondary antibody (under a parafilm coverslip) in a humidified chamber overnight at 4°C. Slides were then rinsed 3 × 10 min in PBT and coverslipped with Fluoromount G containing DAPI (Southern Biotech). Primary and secondary antibodies were diluted in 10% sheep serum in PBT to the following concentrations: anti-COL2A1 (II. II6B3, Developmental Studies Hybridoma Bank, University of Iowa; 1: 20), anti-COL1A1 (LF-68, Kerafast; 1: 100), AF568-conjugated goat-anti-rabbit IgG (A11011, Invitrogen; 1: 500) and AF488-conjugated goat-anti-mouse IgG (A11001, Invitrogen; 1: 500). All immunostaining was carried out in triplicate (on three separate stage-matched individuals), and negative controls were conducted by following the same staining protocol but in the absence of primary antibody. 5-ethynyl-2' -deoxyuridine (EdU – ThermoFisher Scientific) retention experiments were conducted to label the nuclei of cell that have undergone S-phase DNA synthesis and their progeny (Salic and Mitchison, 2008). For skate hatchling EdU retention experiments, hatchlings were anaesthetized in MS-222 in seawater (150 mg/L MS-222 buffered with 300 mg/L sodium bicarbonate) and given a single intraperitoneal (IP) microinjection of 2 μL of a 5 mM EdU solution in 1X PBS with a Picospritzer pressure injector. Animals were then recovered in seawater and reared in a flow-through seatable for 1,5, 10 or 40 days, at which point animals were euthanized and fixed as described above. For adult skate EdU pulse-chase experiments, eight adults weighing between 750–850 g were given three 5 mL IP injections of 22 mM EdU in 1X PBS (a dose of 27. 7 mg EdU/injection), with 48 hr between each injection. Animals were anaesthetized in MS-222 in aerated seawater (150 mg/L MS-222 buffered with 300 mg/L sodium bicarbonate) prior to injection, and after injection, animals were recovered in aerated seawater before being returned to their tank. Two animals were euthanized, dissected and fixed as described above 3 days, 1 month, 2 months and 5. 5 months after the final EdU injection. EdU detection was carried out on 8 μm paraffin sections using the Click-iT EdU Cell Proliferation Kit (ThermoFisher Scientific) according to the manufacturer’s instructions. After detection, slides were coverslipped with Fluoromount G containing DAPI, imaged, then de-coverslipped in water and stained with modified Masson’s trichrome. Given the large size of the metapterygium and the relative sparsity of certain label-retaining cell types (e. g. chondrocytes in adult sections), cell counts were performed across five successive sections through the region of the metapterygium indicated in Figure 1 for adult skates, and three successive sections through the equivalent region of the metapterygium in hatchlings. For counts of EdU+ perichondral cells in skate hatchlings, the sum total of EdU+ cells for each individual were plotted against chace time in MS Excel, and a polynomial trend line was added. For paraffin embedding and sectioning of adult tissues for mRNA in situ hybridization, tissues were rehydrated into diethyl pyrocarbonate (DEPC) -treated water and demineralised for 24 hr in Morse Solution (5 g sodium citrate dihydrate, 12. 5 mL formic acid and 37. 5 mL DEPC water). Demineralised tissues were then dehydrated stepwise into 100% ethanol before infiltration, paraffin embedding and sectioning as described above. Chromogenic mRNA in situ hybridization on paraffin sections for Leucoraja erinacea Col2a1 (GenBank MT254563) and Agc (GenBank MT254564) was carried out according to the protocol of O' Neill et al. (2007), with modifications according to Gillis et al. (2012). mRNA in situ hybridization by chain reaction (HCR) was carried out according to the protocol of Choi et al. (2018) with the following modifications: slides were pre-hybridized for 30 min at 37°C; for the hybridization step, 0. 8 μL of each 1 μM probe stock was used per 100 μL of hybridization buffer; and hairpins were used at 4 μL of hairpin stock per 100 μL of amplification buffer. Probe sets for Leucoraja erinacea Col2a1 (GenBank MT254563), Sox9 (GenBank MT254560), Sox5 (GenBank MT254561) and Sox6 (GenBank MT254562) and hairpins were purchased from Molecular Instruments. Molecular Instrument probe lot numbers are as follows: Col2a1 (PRB574), Sox9 (PRB571), Sox5 (PRB572) and Sox6 (PRB573). All mRNA in situ hybridization slides were coverslipped with Fluoromount G containing DAPI. Orthology of the Leucoraja erinacea Col2a1, Agc, Sox9 and Sox6 sequences used for probe design was confirmed by phylogenetic analysis. Full or partial coding sequences were translated using ORFfinder (NCBI), and multiple sequence alignments with aggrecan, clade A collagen, SoxD and SoxE protein family members were constructed using Clustal Omega (Sievers and Higgins, 2018). The alignments were trimmed with TrimAl (Capella-Gutiérrez et al., 2009) and subsequently used to infer evolutionary relationships with maximum likelihood method in IQ-TREE v1. 6. 12 (Nguyen et al., 2015). ModelFinder (Kalyaanamoorthy et al., 2017) implemented in IQ-TREE was used to find the best-fit substitution model based on the Bayesian information criterion (BIC) for each protein alignment. The branch supports for ML analyses were obtained using the ultrafast bootstrap (UBS) (Minh et al., 2013) with 1000 replicates. Phylogenetic trees (Figure 3—figure supplement 1–4) were prepared using iTOL v5 (Letunic and Bork, 2019) and bootstrap values below or equal to 75% are shown. The following amino acid sequences were retrieved from GenBank for inclusion in our phylogenetic analyses: Mouse (Mus musculus) Sox5, XP_006506994; human (Homo sapiens) Sox5, NP_008871; chick (Gallus gallus) Sox5, XP_015145677; rat (Rattus norvegicus) Sox5, XP_006237666; zebrafish (Danio rerio) Sox5, XP_021330769; cow (Bos taurus) Sox5, XP_005207008; frog (Xenopus tropicalis) Sox5, XP_031753364; dog (Canis lupus familiaris) Sox5, XP_022267096; elephant fish (Callorhinchus milii) Sox5, XP_007895487; mouse (Mus musculus) Sox6, XP_006507558; human (Homo sapiens) Sox6, NP_001354802; rat (Rattus norvegicus) Sox6, XP_006230149; zebrafish (Danio rerio) Sox6, NP_001116481; chick (Gallus gallus) Sox6, XP_025006442; frog (Xenopus tropicalis) Sox6, XP_031755685; cow (Bos taurus) Sox6, XP_024831142; dog (Canis lupus familiaris) Sox6, XP_022263574; elephant fish (Callorhinchus milii) Sox6, XP_007885710; mouse (Mus musculus) Sox8, NP_035577; human (Homo sapiens) Sox8, NP_055402; rat (Rattus norvegicus) Sox8, NP_001100459; chick (Gallus gallus) Sox8, NP_990062; cow (Bos taurus) Sox8, XP_002698019; frog (Xenopus tropicalis) Sox8, XP_002932315; dog (Canis lupus familiaris) Sox8, XP_022275986; horse (Equus caballus) Sox8, XP_005599176; elephant fish (Callorhinchus milii) Sox8, XP_007901694; Mouse (Mus musculus) Sox9, NP_035578; human (Homo sapiens) Sox9, NP_000337; chick (Gallus gallus) Sox9, NP_989612; rat (Rattus norvegicus) Sox9, NP_536328; zebrafish (Danio rerio) Sox9, NP_571718; cow (Bos taurus) Sox9, XP_024836864; frog (Xenopus tropicalis) Sox9, NP_001016853; dog (Canis lupus familiaris) Sox9, NP_001002978; horse (Equus caballus) Sox9, XP_023507898; mouse (Mus musculus) Sox10, NP_035567; human (Homo sapiens) Sox10, NP_008872; chick (Gallus gallus) Sox9, XP_015139949; rat (Rattus norvegicus) Sox10, NP_062066; zebrafish (Danio rerio) Sox10, NP_571950; cow (Bos taurus) Sox10, NP_001180176; frog (Xenopus tropicalis) Sox10, NP_001093691; dog (Canis lupus familiaris) Sox10, XP_538379; horse (Equus caballus) Sox10, XP_023487097; fruit fly (Drosophila melanogaster) Sox100B, NP_651839; fruit fly (Drosophila melanogaster) Sox102F, NP_001014695; mouse (Mus musculus domesticus) aggrecan, AAC37670; human (Homo sapiens) aggrecan, AAH36445; cow (Bos taurus) aggrecan, AAP44494; rat (Rattus norvegicus) aggrecan, AAA21000; zebrafish (Danio rerio) aggrecan, XP_021326217; elephant fish (Callorhinchus milii) aggrecan, XP_007906559; frog (Xenopus tropicalis) aggrecan, XP_017948155; horse (Equus caballus) aggrecan, XP_005602856; mouse (Mus musculus domesticus) Col2α1, NP_112440; rat (Rattus norvegicus) Col2α1, XP_006242370; human (Homo sapiens) Col2α1, NP_001835; cow (Bos taurus) Col2α1, NP_001001135; chick (Gallus gallus) Col2α1, XP_025001042; zebrafish (Danio rerio) Col2α1a, NM_131292; dog (Canis lupus familiaris) Col2α1, NP_001006952; horse Col2α1 (Equus caballus), XP_005611139; frog (Xenopus tropicalis) Col2α1, NP_989220; elephant fish (Callorhinchus milii) Col2α1, XP_007908719; sea lamprey (Petromyzon marinus) Col2α1a, ABB53637; sea lamprey (Petromyzon marinus) Col2α1b, ABB53638; mouse (Mus musculus domesticus) Col1α1, CAI25880; human (Homo sapiens) Col1α1, BAD92834; zebrafish (Danio rerio) Col1α1, AAH63249; dog (Canis lupus familiaris) Col1α1, NP_001003090; cow (Bos taurus) Col1α1, AAI05185; frog (Lithobates catesbeianus) Col1α1, BAA29028; mouse (Mus musculus domesticus) Col1α2, NP_031769; human (Homo sapiens) Col1α2, AAH42586; chick (Gallus gallus) Col1α2, XP_418665; dog (Canis lupus familiaris) Col1α2, NP_001003187; frog (Xenopus laevis) Col1α2, AAH49287; zebrafish (Danio rerio) Col1α1, NP_892013; human (Homo sapiens) Col3α1, AAL13167; dog (Canis lupus familiaris) Col3α1, XP_851009; frog (Xenopus laevis) Col3α1, AAH60753; cow (Bos taurus) Col3α1, NP_001070299; mouse (Mus musculus domesticus) Col5α2, NP_031763; human (Homo sapiens) Col5α2, NP_000384; chick (Gallus gallus) Col5α2, XP_015144688; dog (Canis lupus familiaris) Col5α2, XP_535998; cow (Bos taurus) Col5α2, XP_581318; rat (Rattus norvegicus) Col5α2, NP_445940; sea urchin (Strongylocentrotus purpuratus) ColP2α, NP_999675; tunicate (Ciona intestinalis) fCol1, XP_026690723; acorn worm (Saccoglossus kowalevskii) fibrillar collagen, ABB83364. The sea lamprey aggrecan-like protein sequence (ENSPMAP00000001826) was retrieved from P. marinus Ensembl genome assembly (Pmarinus_7. 0) based on BLAST searches for sequence conservation. Similarly, tunicate clade A fibrillar collagen gene (ci0100150759) was retrieved from JGI C. intestinalis genome assembly (C. intestinalis V2. 0). The Leucoraja erinacea Sox5 sequence corresponds with the 3’ untranslated region (UTR) of the Sox5 transcript, and so orthology could not be confirmed as described above. However, this sequence showed significant homology with the 3’ UTR of predicted Sox5 transcripts from other chondrichthyan species (Rhincodon typus, Amblyraja radiata and Callorhinchus milii) by BLAST. For cartilage injury experiments, adult skates ranging in weight from 500 to 750 g were anaesthetized in MS-222 in aerated seawater (150 mg/L MS-222 buffered with 300 mg/L sodium bicarbonate) until the animals failed to respond to noxious stimulus (e. g. a pinch with forceps). Anaesthetized animals were given a pre-operative analgesic (0. 25 mg butorphanol by intramuscular injection) and then moved from the anesthesia bath to an operating table, where their gills were perfused with anesthetic seawater for the duration of the procedure (~5 min). A small (~2 cm) surgical incision was made through the dorsal surface of the fin, ~3 cm from the base of the metapterygium, and a wedge of cartilage was removed from the metapterygium using a 4 mm biopsy punch. Following biopsy, the incision was sutured, animals were given a postoperative dose of antibiotic (15 mg ceftazidime by intramuscular injection) and then recovered in aerated seawater until fully awake before returning to their holding tank. No animals died as a result of the procedure. Two animals were collected one-week post-operation, and then at monthly intervals for the following year. All animals were euthanized, dissected, fixed and processed for histological analysis as described above. Tissue samples from adult skates collected one-week post-biopsy were imaged by X-ray microtomography (microCT) at the Cambridge Biotomography Centre (Department of Zoology, University of Cambridge). Samples were scanned using a Nikon XTH225 ST scanner, at 100kV and a 120microamps beam current. All microscopy was performed with a Zeiss Axioscope A1 and Zen software. All images were processed and plates prepared using Adobe Photoshop CC and Adobe Illustrator CC.

For our joints to move around freely, they are lubricated with cartilage. In growing mammals, this tissue is continuously made by the body. But, by adulthood, this cartilage will have been almost entirely replaced by bone. It is also difficult for adult bodies to replenish what cartilage does remain - such as that in the joints. When growing new cartilage, the body uses so-called progenitor cells, which have the ability to turn into different cell types. Progenitor cells are recruited to the joints, where they transform into cartilage cells called chondrocytes, which generate new cartilage. But adults lack these progenitor cells, leaving them unfit to heal damaged cartilage after injury or diseases like osteoarthritis. In contrast, certain groups of fishes, such as skates, sharks and rays, produce cartilage throughout their life - indeed their whole skeleton is made of cartilage. So, what is the difference between these cartilaginous fishes and mammals? Why can they generate cartilage throughout their lives, while humans are unable to? And does this mean that these adult fish are better at healing injured cartilage? Marconi et al. used skates (Leucoraja erinacea) to study how cartilage develops, grows and heals in a cartilaginous fish. Progenitor cells were found in a layer that wraps around the cartilage skeleton (called the perichondrium). These cells were also shown to activate genes that control cartilage development. By labelling these progenitor cells, their presence and movements could be tracked around the fish. Marconi et al. found progenitor cells in adult skates that were able to generate chondrocytes. Skates were also shown to spontaneously repair damaged cartilage in experiments where cartilage was injured. Marconi et al. have identified the skate as a new animal model for studying cartilage growth and repair. Studying the mechanisms that skate progenitor cells use for generating cartilage could lead to improvements in current therapies used for repairing cartilage in the joints.

lay_elife

Opening credits and series recap. JACK : (VO) Torchwood : Outside the government, beyond the police. Tracking down alien life on earth and arming the human race against the future. The 21st century is when everthing changes, and you gotta be ready. EXT. WOODS - NIGHT -1812. The woods are dark and foggy. A pretty blonde woman enters shot and looks behind her towards a young soldier. MARY : Nearly there, we've been right busy since you lot were billeted here. This your first time ? The others been teasing you, that it ? My name's Mary. Mary starts to undo the man's jacket button. Mary : Mary, like the virgin. The soldier slaps her hard across the face. MARY : Religious man are ya ? The soldier slaps her again and she smiles at him incredulously and wipes her mouth. Moving closer she puts her hand over his cheek. MARY : I'm not your bloody hound ! She scratches his face and runs into the trees. The soldier chases her. SOLDIER : Whore ! Mary carries on running deeper into the trees. A shrill sound makes her cover her ears. She sees a light among the trees. She pauses, looks behind her then runs towards the light. The soldier is still running after her and he sees the light and stops to stare for a second. The soldier slowly draws his pistol and hears the same shrill noise - like a steam horn. The lights go out with a rush of wind. The soldier grits his teeth and walks quickly in the direction of Mary, pistol held out in front of him. He sees Mary who stands a short distance away, she turns as he lowers the pistol and walks towards her. SOLDIER : Do whores have prayers ? He stops, raises the gun and shoots her calmly. Mary smiles. EXT. A BUILDING SITE - DAY - PRESENT The Torchwood SUV travels down a dusty track then stops between police cars. Owen exits the passenger door then goes to the boot to collect equipment. Gwen exits the rear of the car and walks to the front of the SUV to wait. Jack is using his wrist cuff to scan then walks away, followed by Gwen and Toshiko. They turn off the path and walk into a red tent. Shot continues tracking past them along the path. Mary stands on the edge of the police cordon. She wears modern clothes and has a modern haircut but is otherwise unchanged. She watches the proceedings with interest. OPENING CREDITS EXT. A BUILDING SITE - DAY - CONTINUOUS An aged skeleton lies in a shallow hole hidden by the tent. Jack crouches near while Owen is in the hole examining the skeleton. JACK : Once, just once I'd like to walk into one of these tents and find it's a party, you know with food and drink, people dancing, a girl crying in the corner. Gwen stands near to Jack who continues scanning an alien device which sits near the body. It is a metal object, like a large pair of pincers. GWEN : Is it alien ? JACK : And how ! I'm picking up traces of illmanite peroxine, even dark matter. Toshiko crouches to take a look. GWEN : Any idea what it is ? JACK : Not a clue, could be a weapon or a really big stapler. How's our friend doing there ? Jack turns his attention to Owen who is analysing the skeleton. OWEN : She's dead ! JACK : Yeah, thanks Quincy. She ? OWEN : Judging by the size of her skull. JACK : How long have they been here Tosh ? TOSHIKO : From the depth they found them, a 196 years, eleven to eleven and half months. The earth's been disturbed so I'm afraid I can't be more accurate. GWEN : What killed her, the stapler ? OWEN : Nah, see those shattered ribs ? I reckon she was shot. The skeleton has a large hole in the centre of the rib cage. JACK : Well let's get her back to the hub and find out. He gets up and leaves the tent. Owen starts to climb the short ladder out of the hole and Gwen helps him up. GWEN : Need a hand ? Ooo you so ligh t! You're like a girl. OWEN : I'm not light. I'm wiry, fat girls go mad for it. But I guess I don't need to tell you that. Owen smiles at Gwen and leaves. Gwen turns smiling to Toshiko. Her smile fades as she looks at Toshiko who looks unhappy but smiles tightly then continues scanning with a tool which looks like a sonic screwdriver. INT. THE HUB - TOSHISKO'S WORK STATION - DAY Gwen and Owen are under the desk, a football lies on Toshiko's keyboard. As Toshiko enters they stand up and move away, Owen holding the football. OWEN : Err... I'm really sorry but I think your computer might be dead. TOSHIKO : You're kidding ! What happened ? OWEN : Ok, so she said I was no good at sport. Owen points at Gwen, speaking obliviously to Toshiko's panic. OWEN : Hello ! So I said, well throw something to me and I'll... Toshiko cuts him off sharply. TOSHIKO : What happened to the computer ! OWEN : Oh I, kicked out the plug. TOSHIKO : What ! It was running a translation program I'd written. Translated every scrap of alien language we've got and broken it down into binary threads to see if there was common derivation. Toshiko is upset, trying to explain her concerns to people who couldn't careless. Owen looks at Gwen. OWEN : That's a bit of a mouthful. Gwen laughs, but her smile quickly fades when she sees Toshiko's expression. GWEN : Sorry private joke. Stupid joke ! TOSHIKO : We're supposed to be professionals ! We've got a job to do. GWEN : She's right... You're right Tosh, I'm sorry. OWEN : Do you know what, Tosh ? Sometimes I think that even that stick up your arse has a stick up its arse ! Owen walks away, bouncing the football. Gwen, bothered by Toshiko's lack of humour and being shouted at, begins working at her computer. INT. A BAR - EARLY EVENING Toshiko sits at the bar drinking, mulling over her bad day and wondering about the situation between Owen and Gwen. A little jealous. Mary walks over to her. MARY : So the guy over there has been staring at me all evening and I've told him he's wasting his time but he won't listen. So I come over to talk to you because I know how this ends. He gets a punch in the neck and I get barred, and I've already been barred from about 20 pubs and I don't want to get barred from this one because they do these nice olives on the tables. TOSHIKO : Oh... Right. Ok then. MARY : Cool, let me get you a drink. TOSHIKO : Really there is no need. MARY : (To bartender) JD 'n coke, and Toshiko what do you want ? TOSHIKO : I didn't tell you my name MARY : Oh yeah, that was the other thing. I kind of know who you are. TOSHIKO : I think you're mistaking... MARY : Toshiko Sato, born in London 1975, moved to Osaka when you were 2, back to the UK, 1986. Parents were in the RAF. Grandfather worked at Bletchley Park, very impressive ! University ... blah blah. Snapped up to government science think tank when you were 20, recruited to Torchwood 3 years ago. I saw you at the building site this morning. What was that you had in the case ? TOSHIKO : How do you know about Torchwood ? Mary lights a cigarette, comfortable and chatting happily. She knows Toshiko won't tell her to go away. MARY : Oh, there's stuff on the internet but you have to dig really deep, plus we pick up bits and pieces from police work, radio scanners... TOSHIKO : We ? MARY : Scavengers. Collectors. Just like you. Mary sits down with her drink. TOSHIKO : How many of them are you ? MARY : Mmm... Listen, don't think it's in any way organised. It's really just a disparate bunch of IT guys who live with their mothers. TOSHIKO : I shouldn't talk to you. MARY : So go... Toshiko makes no move to leave. A little later on, still in the pub with Mary. They now sit at a table in the centre of the room. Toshiko is tipsy, talking earnestly about Torwood to Mary. TOSHIKO : What's most amazing are the similarities with our own culture but that can be horrible because we find lots of weapons and it just makes you think, my god everything wages war. It's not just a trait of ours but a trait of existence. It makes you feel so hopeless, but then there are times... We found this thing, it was about A4 size and had all these symbols on it, and it took me about three months to translate. It was a letter someone had written to his family, to his children, to say how much he was missing them and it just made me cry because even across these unimaginable distances there are fundamentals that stay exactly the same and there's no one to talk to about this. I mean the guys at work they're great but they don't see it the way I do. Toshiko pauses to drink her wine. TOSHIKO : I could be fired just for telling you that. Mary takes a box from her bag and places it on the table. MARY : I want to show you something. She takes a pendant from the box. TOSHIKO : It's a pendant. MARY : Put it on. Toshiko does so and suddenly she can hear peoples thoughts, words jumble at first and she has to breathe shallowly for a second because of the shock. The following are thoughts : MAN 1 : I'll just have one more and drive slow... WOMAN 1 : Does coffee count as food if it's got sugar in it... WOMAN 2 : If he touches his groin one more time I'm gonna smack him in the face... MAN 2 : I should of said I was hammered when she asked me what the time was earlier, that would have made 'em all laugh. TOSHIKO : (To Mary) What are they ? I can hear voices. MAN 3 : Bloody sudoku, so bloody smart. MAN 4 : Did I send that email ? Did I click the reply to all ? MARY : They're people's thoughts... WOMAN 3 : He doesn't touch me anymore. TOSHIKO : What ? MARY : They're people's thoughts Toshiko... WOMAN 3 : I wish I'd shagged that single dad I met at the parents' evening. MAN 5 : What's that Asian girl doing ? Is she having a fit or something ? TOSHIKO : It's... They're so loud. MAN 5 : She's cute though. Is that her girlfriend, Marcus Rowe reckons he's done it with two lesbians, lucky sod. How'd it work ? TOSHIKO : That man over there, with... I can hear him... MAN 5 : I mean does one of them sit on the side or something ? MARY : Ok Toshiko I need you to focus. MAN 6 : No way, I ain't showin' that to me doctor. MARY : Home in on my voice, shut everything else out. Toshiko shuts her eyes in concentration. MARY : Just me, there is no one else but me. Can you hear me ? Toshiko nods, Mary stops talking aloud and uses her thoughts to talk to her. MARY : Can you hear me now ? Ok I want you to home in on just my thoughts. Ignore everything else. TOSHIKO : There's just so much... MARY : You can do it. It takes practise but this is what you have to learn, now what am I thinking ? TOSHIKO : This is so hard. MARY : Follow my voice. What am I thinking ? TOSHIKO : You're thinking that... MARY : That I want to kiss you. Toshiko opens her eyes, snatches the pendant from her neck and breathes deeply. MARY : I'm sorry, sometimes you can't control. TOSHIKO : I know. It's fine. Where did you get it ? MARY : It's, been in the family for a long time. TOSHIKO : I've never seen anything like it. It's incredible ! MARY : It's more than incredible. With this you can read people's minds, it levels the pitch between man and God. TOSHIKO : Is it alien ? MARY : I guess. Toshiko gives it back to her, but Mary stops her. MARY : No, I want you to keep it. TOSHIKO : I can't Mary. MARY : Please. I've kept it too long. After awhile it gets... You hear too much. It changes how you see people. TOSHIKO : I'll have to show it to the others. Mary laughs very lightly. TOSHIKO : What ? MARY : Nothing. MARY : Just I bet you won't. TOSHIKO : And you know this from finding my CV on the internet ? MARY : No. Because I know the pendant. TOSHIKO : Well you're wrong, because I will... MARY : Yeah. But you won't. INT. THE HUB - NEXT DAY Toshiko enters reception. She presses the button and while the door to the hub opens she takes the pendant from her bag. Ianto walks over and Toshiko hides the pendant behind her back. IANTO : Good morning. TOSHIKO : Yeah, hi Ianto. Ianto goes into his office behind the counter and Toshiko puts the pendant on. She enters the central hub, Gwen is at her desk, Owen walks from the autopsy room. OWEN : (Thoughts) What the hell would produce such a perfect circular puncture ? Maybe some sort of wooden stake... OWEN : Hi Tosh. OWEN : (Thoughts) She better not go into one about the computer again. GWEN : Morning. GWEN : (thoughts) Sergeant giving it all, ooh, Gweny's dined call her old mates, should put a weevil in his bathroom. TOSHIKO : I've got something to show you. OWEN : Sure. OWEN : (thoughts) please don't make us sit through another slideshow about the Incas or whatever it was. I want a biscuit. GWEN : Have I got time for a pee first ? GWEN : (thoughts) Oh sweetheart the jeans & boots thing has really kinda had its day. TOSHIKO : I err... I found this thing. OWEN : (thought) what's she talking about. She can be dead weird. I wonder what she'd be like in bed, Catholic, be grateful I bet. TOSHIKO : Ok I don't know whether this comes under actual technology... GWEN : (thought) That's Owen I can smell him on me after that shag in his car this morning. That's twice now, does that make this an arrangement or it has to be more than two times surely, as long as we keep it to just the two times we're fine. What's Tosh looking at ? GWEN : You okay Tosh ? OWEN : (thought) She did that thing where she ran her tongue across my teeth. I should've worn different trousers, I'm gonna have to sit down 'til this subsides a bit. TOSHIKO : Yeah, no fine. GWEN : So, what is it you want to show us ? GWEN : (thoughts) I wonder if I could get Owen to come down to the volt. No I couldn't have s*x in front of a weevil. Couldn't even do it in front of Trent Kendle's cat. TOSHIKO : Forget it... It... I found this article, I'll bring it in tomorrow. Toshiko cannot tell them about the pendant. She can no longer think straight having found out for certain about Gwen and Owen. OWEN : No worries. OWEN : (thought) Well thanks for that rambling trip to nowhere Tosh. Later Toshiko sits at her desk, thinking. Ianto enters, tidying cups. IANTO : (thoughts) Can't imagine the time when this isn't everything. Pain so constant but my stomach's full of rats. Feels like this is all I am now. There isn't an inch of me that doesn't hurt. IANTO : I'm about to brew some of Jack's industrial strength coffee, would you like a cup ? TOSHIKO : I'm, fine. Thanks Ianto... Toshiko realises how much pain Ianto is still in, how well he hides it, and she speaks quietly to him. She takes the pendant off. EXT. STREET OUTSIDE TOSHIKO'S HOUSE - EVENING Mary is sitting on the wall opposite her house, waiting for Toshiko to get home. TOSHIKO : Might've known you'd have my address as well. MARY : Did you tell them ? TOSHIKO : No. I didn't ! She walks across the road and enters her house, leaving the door open. Mary follows. INT. TOSHIKO'S KITCHEN Toshiko storms around angrily and give Mary a dirty look when she enters, shutting the door behind her. MARY : What made you change your mind ? You listened to them didn't you ? See I told you, isn't it incredible ? Some of the stuff you hear. Toshiko gets the pendant out of her bag. TOSHIKO : What is this thing ? She throws the pendant on the table. TOSHIKO : Why did you give it to me ? MARY : I told you... TOSHIKO : The things I heard. What they thought of me ! They really... Oh these are the people that are supposed to like me ! MARY : They do like you! People are complicated. They... Ok I should have warned you about this, it isn't like reading someone's diary. Toshiko goes to the fridge and as she shuts the door Mary sees photographs - Tosh and Owen, some friends. MARY : The stuff you've been hearing is so deep, so personal. Stuff they're not even aware they're thinking. TOSHIKO : You think you know someone and then suddenly you see them for real, and they're b*st*rd little kids ! Toshiko is mainly upset about Owen rather than anything else. MARY : Not everything, not everyone. Mary takes her jacket off and picks up the pendant. She puts the pendant back on Toshiko once on she holds Toshiko's face, standing close to her. TOSHIKO : I wouldn't say your thoughts were exactly pure. MARY : At least they're consistent. Mary takes a step back, Toshiko looks down, upset and confused. MARY : No agenda, no resentment. TOSHIKO : They pity me... You don't pity me. MARY : Why would I ? TOSHIKO : What you're thinking now... That's pretty graphic. MARY : That wasn't my thought. TOSHIKO : What ? MARY : I wasn't thinking anything. That wasn't my thought, must've been yours. TOSHIKO : That one, there, that's yours. MARY : Yeah... That was mine. TOSHIKO : I... Umm I certainly seem to be enjoying myself. MARY : You would. You will. Toshiko kisses her passionately. INT. TOSHIKO'S BEDROOM The room is modern, tidy and chic. Toshiko is in bed knees up to her chest, hugging the sheets to her, wondering why she just slept with Mary - out of character for her. Mary enters wearing a robe and leans on the doorway smoking and holding a glass and an eggcup. MARY : You have no ash trays, not in your whole house. Not one. TOSHIKO : What are you using ? MARY : I think it's an egg cup. Mary sits on the bed next to her, Toshiko sits up and turns her back to her. MARY : You ok ? Freaking out a little ? Mary, unconcerned, looks through the things on Toshiko's bedside cabinet. Picks up a card. MARY : Your birthday is July right ? TOSHIKO : You're the expert... MARY : Isn't it a little late to have your cards up... She reads from the card in her hand. MARY : Lots of love Owen. I'm guessing that's Owen from work. Owen from the building site yesterday morning. Owen from the photo on your fridge. TOSHIKO: Put that down ! MARY : All I'm saying is that I don't want to get in the way of anything. TOSHIKO : There's nothing to get in the way of. Gets up and storms into her walk-in wardrobe. MARY : I see. You wanna talk about it ? TOSHIKO : Not really. MARY : It's ok. Wouldn't be the first time I'd been a rebound shag. Toshiko comes back into the room, fastening a robe around her. TOSHIKO : You weren't. Ok. Nothings happened. Nothing will ever happen. One of the delightful things I've found out, thanks to this bloody pendant. She throws the pendant at Mary and it lands on the floor at the far side of the room. Mary goes to pick it up and she brings it back over to Toshiko who is now lying on the bed, foetal position facing away from Mary. Mary lies down behind her speaking into her ear and dangling the pendant in front of her. MARY : It's not all bad. Some of the things it can do are extraordinary. TOSHIKO : What good could ever come of that ? MARY : You need to work that out for yourself. INTERCUT WITH : EXT. A BUSY CARDIFF STREET Toshiko stands looking around uncertain. MARY : (VO) You need to go somewhere public, somewhere crowded. TOSHIKO : What am I looking for ? MARY : It will find you... Toshiko sits up and turns to face Mary. TOSHIKO : I'm sick of these riddles. What's going on. Where did you get this ? MARY : I told you. TOSHIKO : Who are you Mary ? Is that even your real name ? MARY : Ok, here's another name. Philoctetes. I'm Philoctetes. Toshiko standing in the Cardiff street puts on the pendant slowly. She is immediately assaulted by hundreds of thoughts which slowly start to separate. WOMAN 4 : ...Sasha wants a latte... WOMAN 5 : I could bandage my hand since I shut it in car door. That would explain why the signatures don't match. MAN 6 : That gives me an hour before Lisa gets back to dress up. Got to be careful she's starting to notice her tights are getting baggy round the crotch. WOMAN 7 : All big eyed and giggly and I'm sat there with my boobs like something out of the national geographic. WOMAN 8 : 6 cigarettes today and all them post coital, glorious. MAN 7 : Ahh, Mr. Bond I've been expecting you. Toshiko laughs at the man's thought then sees another person - a woman who notices her looking. WOMAN 9 : Some people should actually be prohibited from wearing a thong. What's that girl looking at ? A thought attracts her attention from the others and she sees a man carying a long bag. NEIL : I'm gonna kill them. I'm gonna kill them. I'm gonna kill them. I'm gonna kill them. Lay their bodies out afterwards and I'll lie next to them. So I'll have to do myself lying down. Should've practised that. A woman walks into Tosh. TOSHIKO : (thought) Sorry. Toshiko loses the man for a minute then finds him again and begins to follow. NEIL : Lawrence comes in and finds this he'll know that what I've been doing is right and what he's been doing is trespassing. I won't miss anything, I won't miss this city, I won't miss this body. I won't miss anything. INT. A HOUSE - CONSECUTIVE Neil arrives at the door. A boy on his game boy opens the door and wanders off again. NEIL : Alright Danny ? CAROL : I want him back at 6 and I mean 6 this time. Kelly's given me 90 minutes after hours at the tanning salon as a wedding present. Besides, you're breaking the law bringing him home late. My dad reckons I could have you arrested, so think on. Danny enters. CAROL : Hey put your shoes on and go to the toilet. What's that face ? DANNY : I don't wanna go. CAROL : Well you have to. Lawrence'll be here in a minute and we'll be looking at napkins. DANNY : It's borin'. CAROL: Take your space invaders. Lawrence bought him that, haven't heard a peep out of him now. Mind that's Lawrence all over, dead thoughtful. Oh and no Cola, don't want him coming home all excited. Neil puts the bag down on the hallway floor. CAROL : I thought you'd stop going fishing ? Neil pulls out a shotgun. CAROL : Oh my God, what do you think you're doing ? Grabs her son and hugs him to her. NEIL : I was thinking of the Isle of Wight. You remember we had that chalet around when Danny was walking and the chalet was full of spiders and you called me your hero because I wasn't scared. I'd just pick 'em up and throw them out. CAROL : Oh my god Neil. You're scaring me ! NEIL : It was this perfect little memory, we were happy together, because we were together and all this nonsense with Lawrence it's fine. I forgive you because I'm looking at the bigger picture now. Neil loads the shotgun and starts to pull it up to aim. CAROL : Oh my god, oh my god. No no please don't ! NEIL : It's ok, it's just like falling asleep, really. CAROL : No don't, don't do this to Danny please ! NEIL : Then we will be together forever. Neil collapses on the floor. As he falls Toshiko stands behind him brandishing a golf club. TOSHIKO : It's ok. You're ok now. INT. THE HUB - AUTOPSY ROOM - DAY Owen is by the bed with the skeleton on it. Gwen and Jack are on the balcony. Owen is throwing things at Gwen because she is singing. OWEN : Stop singing ! GWEN : The leg bone's connected to the hip bone... OWEN : Please stop singing. Anything to stop you singing. Jack laughs. OWEN : I dunno what you're laughing at ! Owen throws things at him. OWEN : Stop singing. Please don't sing, please don't sing. Not listening, lalala. Right, okay. Owen throws things at Toshiko as she enters the room. TOSHIKO : What's going on ? GWEN : You know the skeleton we found at the building site. Well, Amanda Burton here has just completed the post mortem. OWEN : Ok I can explain... GWEN : As you may remember at the building site, Owen said this was a woman killed by a single gunshot. OWEN : Yeah ok I'd been there like a minute. GWEN : Since then he has had to tweak some of his initial conclusions. The first being that this isn't in fact a woman but a man. OWEN : A young man. A very girly man. GWEN : But still, ultimately, a man. Then there was the cause of death, Owen said GSW, uh uh, the correct answer was... OWEN : Unidentified trauma but... TOSHIKO : Unidentified trauma ? GWEN : Umm. You see it in RTA's when something like a steering column or a post goes into a body at great velocity. But the one thing that could be ruled out was... OWEN : Gunshot wound... Taps the balcony bar. GWEN : Gunshot wound ! Was there in fact any part of your prognosis that was right ? OWEN : I got that it was a... skeleton. GWEN : Yes you did ! Puts on finger on her nose and points the other at him Jack laughs and walks away with his mobile phone to make a call. GWEN : Yes you did ! Where did you train ? Where did you train ? Did you train... OWEN : Look... [SCENE_BREAK] INT. JACK'S OFFICE Jack he wanders into his office Followed by Tosh. He dials a number on his mobile and wanders round while it rings. TOSHIKO : Jack... umm... So you know anything about Greek mythology ? JACK : (Into phone) Security Visa, 45895 Harkness. Jack sits behind his desk then speaks to Toshiko. JACK : A little, why ? TOSHIKO : You ever heard of Philoctetes ? It came up in a pub quiz. JACK : You went to a pub quiz ? TOSHIKO : Yeah. No I love pub quizzes, down at the Prince of ... Tights. Jack looks at her, not believing her but not questioning her reasons. JACK : Philoctetes was an archer recruited to fight in the Trojan War, he got in an argument and was marooned on the island of Limnos for about ten years. TOSHIKO : Just left there ? Jack doesn't answer and Toshiko begins to wander out. Jack calls after her before she reaches the door. JACK : Hey what's happening with that list for UNIT ? TOSHIKO : Hmm...Oh yeah I'm still working on it. JACK : Right well you know... when you're ready. Toshiko pauses looking at Jack, wanting to talk but then walks away. JACK : (on phone) Prime minister, is this a secure line ? Can you tell me why Torchwood operations have become part of your security briefings to the leader of the opposition ? The deal is... INT. A CAFE IN CARDIFF MARY : This is incredible This is the most incredible thing I ever heard! They should make an action figure of you ! TOSHIKO : You were right about the pendant. I see it now. It can be used for good. MARY : What did they say at work ? How did you explain it. TOSHIKO : I didn't tell them. MARY : I think that's wise. I'm sorry but I'm going to have to kiss you now. TOSHIKO : Mary, no... MARY : Listen. You do something unbelievably brave and sexy, I have to kiss you. I don't make the rules. Mary leans over the table and they kiss. Toshiko pulls away after a short time, self consciously. MARY : So what's happening with the thing you found on the building site ? TOSHIKO : Dunno my boss is dealing with that. MARY : I thought you did all the technological stuff. TOSHIKO : I do. But sometimes our jobs overlap a bit. I'm doing... there's an admin thing he has asked me to do. MARY : Don't you have a secretary for that ? TOSHIKO : It's actually quite complicated. MARY : So what's he found out ? TOSHIKO : I dunno. He's not said anything. MARY : Hmm... That's kind of strange. TOSHIKO : No, it isn't, it's fine... MARY : No, sure. I mean if he's keeping stuff from you there is bound to be a reason. Toshiko drinks her coffee thoughtfully, Mary's seeds of doubt taking hold. INT. THE HUB - AUTOPSY ROOM Owen is analysing the skeleton. Toshiko walks in carrying some coffee. TOSHIKO : Not still worrying about that are you ? OWEN : Ok so I'm thinking if it isn't a gun shot or a musket shot or whatever they had then, maybe it was some kind of ritual. Toshiko hands him a coffee OWEN : You are gorgeous ! Toshiko smiles clinging to the comment. Owen continues talking regardless. OWEN : So I started looking into devil worship and stuff from that era to see if there is anything about plucking out hearts, and would you believe it, there's nothing. They ate eyeballs, they drank blood, they had s*x with animals but they did not pluck out each others hearts 'cause obviously that would have been a bit weird. Toshiko laughs a little at his joke. TOSHIKO : Why are you so bothered ? Whoever did this is hardly a threat to society anymore. OWEN : Yeah I know. It's just that there's something... Does that remind you of anything ? Owen indicates the hole in the skeleton's chest. TOSHIKO : Erm, that bit in Alien when that thing bursts out of John Hurt. OWEN : I'm sorry I should've been more specific. Does that remind you of anything helpful ? TOSHIKO : No, sorry. OWEN : Right. Erm just go over there, do your computer stuff and think about shoes aye ? Thank you. TOSHIKO : (halfway up the stairs) Has Jack said anything to you about the hardware we found with the skeleton ? OWEN : No, why ? Toshiko puts the pendant on. OWEN : (Thought) Check any hospitals nearby, could've been someone dying in an operation. Owen drinks the coffee and pulls a face at the taste. OWEN : (thoughts) This has got sugar in it. TOSHIKO : No worries, just asking. Gwen enters and sits at the top of the stairs. OWEN : Hello. OWEN : (thoughts) Keep looking at the skeleton, don't look at her, no grinning. GWEN : Hey Tosh, that coffee going begging ? GWEN : (thoughts) What's the matter with him ? Why isn't he looking at me ? OWEN : There are copies of that Michael Hamilton statement on yer desk. He's still seeing cyberman outside his mother's house. OWEN : (thought) Don't think her palm on the bottom of my spine, her hand in my hair. TOSHIKO : I think I'll just go over there. Owen and Gwen ignore her. GWEN : Ok. I'll phone social services, see if there's a history of mental illnesses. GWEN : (thoughts) No, Gwen, this good. Can't go on, this is a good thing. Why the hell isn't he looking at me ? TOSHIKO : I think my desk is on fire. Toshiko leaves angrily, Gwen continues looking at Owen. Toshiko sees alien device found with the skeleton and starts to look at it, Jack comes down the stairs, he sees Toshiko and hangs around the door frame. JACK : So I've just come from a really interesting conversation with a detective inspector Henderson. TOSHIKO : Right. Jack enters the room and stands next to the alien device. JACK : Interesting because, firstly, the man had the biggest hands I'd ever seen and secondly because of the story he told me about you saving a woman and her kid from being murdered by her ex-husband ? TOSHIKO: Yeah. No I was going to tell you about that. JACK : So why didn't you ? TOSHIKO : I dunno. It wasn't a work thing. Just a thing. Stuff happens all the time that's not pertinent to here. JACK : You do this all the time ? So you secretly fight crime, is that it Tosh ? TOSHIKO : I didn't want it to look like I was showing off. JACK : The guy they arrested, Henderson said you heard him muttering to himself as he was walking along and that's what tipped you off. Jack starts to examine the alien device. TOSHIKO : Humm. Couldn't really work out what he was saying at first and then I, it was like, Jesus ! Toshiko tries to laugh off the story and Jack smiles, then starts talking seriously. JACK : That's weird, cos when I'm about to murder someone I'm really careful not to talk to myself about it while I'm on the street. TOSHIKO : No, sure. I mean that's lesson one. JACK : Humm. TOSHIKO : I was wondering how you were getting on with this ? She nods indicating the alien device. JACK : It's ongoing. TOSHIKO : Can you dismantle it ? JACK : Like I said, it's ongoing. Toshiko turns away and touches the pendant, as she does so Jack looks up but doesn't see her touch the pendant. Toshiko tries to read his mind but she can't. Toshiko turns to look at him, wondering why she can't read Jack's thoughts, unnerved by the silence. JACK : What ? Have I got something on my face ? Is it food ? TOSHIKO : No sorry, I zoned out. JACK : Well listen that was a good save Tosh. Well done. INT. TOSHIKO'S KITCHEN Mary unpacks a plastic shopping bag onto Toshiko's kitchen table. Toshiko stands with her back to Mary holding the pendant. MARY : Ok so I've got crisps, I've got coffee, real coffee. Wine... TOSHIKO : I'm giving them the pendant. MARY : Right lets... TOSHIKO : Your right it's not like reading someone's diary, it's so much worse. Toshiko turns to face Mary. TOSHIKO : And it makes me feel dirty, and ashamed and now I'm spying on my friends. MARY : Some friends. TOSHIKO : What's that supposed to mean ? MARY : They pity you. They exclude you; they've got you doing bloody admin. TOSHIKO : So ? No I've made up my mind. MARY : Toshiko, don't do this. TOSHIKO : So they'll probably want to talk to you. Why do you care ? It's the pendant they're interested in, they don't care about you ! MARY : If I go in that place I won't come out again. TOSHIKO : What are you talking about ? They're not Nastarsi. Look I'll get my boss to come... Toshiko goes to get her mobile from her bag. MARY : Put the phone down ! Mary speaks in a voice that is not her own - a low echoing roar. Toshiko moves away from her bag nervously. Mary's voice returns to normal when Toshiko moves. MARY : Ok, I'll show you. Mary closes her eyes and blue tinged light streams from her face and her body disappears in cracks to reveal a tall, elegant, semi-transparent creature with streaks of light floating from her back. She has long fingers and spiky tentacles sprout from her face like hair. MARY : (Thought) This is why you can't tell them ! Toshiko moves towards Mary and reaches out to touch her hand. TOSHIKO : You're cold. Who are you ? MARY : Still the person you kissed, the person you caressed. Mary changes back and Toshiko stares at her in wonder. MARY : Say something. TOSHIKO : So I'm shagging a woman in an alien. MARY : Which is worse ? TOSHIKO : Well I know which one my parents would say. I read your thoughts. I didn't see this. What else are you keeping from me ? MARY : You think there could be anything bigger than this ? The freedom that you have. When I first got here I found it almost obscene. My world was savage, enforced worship in temples the size of cities, execution squads roaming the streets. Descent of any kind meant death or transportation to what they called a feral outpost. TOSHIKO : And the pendant ? MARY : It's how my people communicate. It's how we've communicated for centuries. Speaking orally, using a prearranged and finite number of words. It's so archaic ! And kind of gross to look at ! The machine you found is a transporter. It brought me here, it can get me home again, I need it back before you dismantle it. TOSHIKO : Won't you be in danger ? MARY : 200 years have passed. There'll be a new government, there'll have been 20 new governments by now. TOSHIKO : Then why hasn't someone come back for you ? MARY : I've been forgotten. Like Philoctetes on Limnos. TOSHIKO : Let me take you to Torchwood. Maybe we can help you. Fix the transporter, get you back home. MARY : You won't ! You'll examine me. Assess whether or not I'm useful, whether I'm a danger, then lock me in a cell. They're not interested in understanding alien cultures. It's just as well you haven't got the technology to reach other planets yet ! Yours is a culture of invasion. Do you really I'm going to walk, hands raised in surrender into that ? Toshiko doesn't answer, knowing the truth of Mary's words. EXT. A STREET Toshiko stands alone wondering what to do, listening to people's thoughts as she walks. MAN 8 : Silence when the door opens, can't do another night with the silence. WOMAN 10 : His hands were cold. MAN 9 : Giving it all we got, calling social services just because of a couple of bruises. MAN 10 : Worst case scenario, lives 'til she's really old, and the whole inheritance goes on sheltered accommodation. Toshiko rubs her head, the thoughts crowding in until one thought comes through clearly and she stops. WOMAN 11 : Calling it granddads little secret. Toshiko pulls the pendant off. INT. THE HUB - AUTOPSY ROOM Owen is in the autopsy room with the skeleton on the table. He leaves the room and goes to his station and takes off his white coat. He starts putting on his coat to leave then notices something on the screen and sits down. He starts to search the Cardiff General Hospital records, a list of names appears. INTERCUT WITH : INT. TOSHIKO'S BEDROOM Toshiko is lying on the bed. Mary is on the armchair in the corner. TOSHIKO : I can't stand it anymore. The weight of it. The Depravity. The fear. It fills me up. INTERCUT WITH : Jack standing by a clock tower in the night watching over the city. TOSHIKO : It's in my mouth, my hair, my eyes. Owen sees something useful and highlights a name on the search. OWEN : Marna ? He pulls up the autopsy report. TOSHIKO : Like I'm drowning in ink ! OWEN : What am I doing, what I am doing ? Heart removed. Owen looks at a post mortem picture of a girl. OWEN : In the post mortem, past operation. Owen suddenly connects his memory. OWEN : Lowry. Owen enters a Torchwood password '45895' and types Lowry into the search. TOSHIKO : And even when I don't have the pendant on, even when there is nothing... Jack stands by the tower, overlooking the road, still watching. TOSHIKO : I can't forget the things I've seen. Things I've heard. Owen continues searching. OWEN : Heart removed... He types another name. OWEN : Heart removed... He types another name, sees the pictures all with their heart removed. He keeps typing in names, the cases moving back through time. OWEN : Removed... How far back does it go ? TOSHIKO : It's like a curse. Something the Gods sent to drive someone mad. I had hope, I'd see something. Jack watches the city. TOSHIKO : A little random act of kindness, make me think we were safe. Owen at his computer. TOSHIKO : Some essential good in us. Owen's computer screen reads: Cause of death - a hole punctured through the chest. The heart gone. Unsolved. OWEN : This is impossible. TOSHIKO : There isn't, it's like one of the weevils. Track through the hub cellblock, Janet is in one of the cells, reacting to the telepathy. TOSHIKO : (VO) Gets inside you, it's just like this great drawling scream. Janet jumps up at her cell wall, growling. OWEN : This is completely impossible ! Owen dials a number on the phone. TOSHIKO: You were right, everything you said about us. Jack's phone rings and he answers. TOSHIKO : How we are frightened. Callus... JACK : Owen... OWEN : You need to see this. He hangs up the phone. Toshiko is increasingly upset, crying now. TOSHIKO : And I can't be a part of it any longer. I don't know what to do, tell me what to do... MARY : Get me into Torchwood ! INT. THE HUB - NIGHT Toshiko enters the hub with Mary behind her. MARY : In Xanadu did Kubla Khan / A stately pleasure-dome decree / Where Alph, the sacred river, ran Through caverns measureless to man / Down to a sunless sea. Toshiko finds the table where Jack had kept the transporter on, only to find it is missing. MARY : So where is it, lover ? TOSHIKO : Stay here. Jack, my boss, has got it. Toshiko goes to the stairs. As she passes Mary grabs her arm, pulling her to face her. MARY : Be quick. I've got a long journey ahead of me. Strokes Toshiko's face. MARY : I might need to something to eat before I go. JACK : (From the balcony.) This what you're looking for ? Holds up the transporter. TOSHIKO : Jack ! Jack starts walking around the balcony and down the stairs to them, telling a story as he moves. JACK : Friend of mine, lets call him Vincent. That was his name after all. Regular guy, girlfriend. Likes his sport, likes a beer. Starts acting a little strange, a little distracted. Suddenly he disappears for a couple of months. He comes back and we've got to start calling him Vanessa. Since then I've always been a little nervous when a friend behaves out of character. He looks at Mary who stares back impassively. JACK : I'm sorry we haven't been introduced. He leans over the stair rail. JACK : Jack Harkness ! My guess is your not from around these parts ! Now this Jack holds up the transporter standing before them, a little way up the stairs. JACK : This, huh. Is incredible. Walks around the back of the station so he comes out behind Toshiko and Mary. JACK : You know what it is ? TOSHIKO : It's a transporter. Mary was a political prisoner, she was exiled here. Look, Jack... JACK : You got half of that right. Mary, it is Mary isn't it ? You wanna tell her the really interesting bit ? Mary doesn't answer. JACK : No ? Ooh chatty isn't she ? I don't know how you got a word in edgeways, Tosh. It's a two man transporter. But whatever you people may be, you may be squids for all I know. A two squid transporter, room for one prisoner and one guard! You wanna tell us what happened to the guard Mary ? MARY : I killed him ! But I was disturbed. INTERCUT WITH : Flashback to the woods in 1812. Mary approaches the alien and it takes over her body. The cog door in the hub closes and the other members of Torchwood enter slowly. Mary looks at them then dismisses them, retuning her attention to Jack. MARY : Then another came, a soldier. Gwen enters the room behind Jack, Owen behind Mary. MARY : He tried to shoot me. Flashback to Mary being shot by the soldier. Mary walks towards him and punches him in the chest, withdrawing her hand holding his heart. MARY : (VO) So I plunged my new human hand into his chest and plucked out his heart ! Ianto is standing by Owen now. OWEN : And that's what you've been doing ever since. MARY : This form needs to fed. Mary walks between Jack and Owen, wandering among her audience as she talks. OWEN : All the punctures, all about the size of a fist. All those people, you killed all those people. MARY : I fled before any more soldiers came, I had so much to explore! And how I loved this body, so soft so wicked. Mary touches herself suggestively. MARY : Such powers body has in this world ! Within a few years the forest had gone. Transporter was safely buried under the spread of the city, I didn't care. I wasn't exactly in a hurry to get home. JACK : And you've been killing ever since ! MARY : I knew there might come a time when my situation here became complicated. Toshiko puts on the pendant. MARY : But I was safe as long as I knew where the transporter was. GWEN : (thought) The way she looks at you with those eyes. She's like an animal. OWEN : (thoughts) When they brought that girl into the hospital. I'd only been qualified 6 months I wanted to throw up. JACK : And then the machine was uncovered. MARY : As soon as the air touched its surface I could feel it. OWEN : (thought) She's just talking. She's not even frightened of us, she is just talking. Mary continues talking, unheard under the thought. MARY : ...My beautiful Toshiko... OWEN : (thought) I'm gonna go for it... TOSHIKO : Owen, no ! Mary suddenly moves quickly, beyond human speed. She grabs Toshiko from behind and puts a knife to her throat. The others stand, arms outstretched ready for any opportunity. JACK : Let her go ! Mary ! Let her go ! MARY : Toshiko tell them to give me the transporter. TOSHIKO : I can't Mary ! Toshiko struggles against the knife. OWEN : (thought) Ridiculous, we're unarmed. We're just shouting at her ! IANTO : (Thought) Not again, please not again ! GWEN : (Thought) Knife has incisors on the blade it'll tear Tosh's throat out. Mary looks over at Gwen. MARY : How's this ? I'll exchange Toshiko for that one. Mary looks at Gwen, so does Jack. MARY : Your choice. OWEN : (Thought) No, no, not Gwen. GWEN : Just put the knife down... MARY : Did you hear him ? He didn't want to did he ? OWEN : (thought) She read my thoughts. She actually read my thoughts. GWEN : (Thought) What happened did he not what to, what did he say ? TOSHIKO : Please don't... MARY : That's what they think of you. That's who you've been working for for all these years. OWEN : That's not true Tosh, don't listen. MARY : But not me. Whatever I've done, it doesn't change the way I feel about you ! We have a connection Toshiko, something real... JACK : (telepathy) Toshiko, don't move. Don't do anything until I say. JACK : Ok you want the transporter. We want Toshiko ! I think that's a fair swap. GWEN : (thought) I've been trained for this hostage situation. For gods sake Gwen think. JACK : Give me the knife and I'll give you the transporter myself. OWEN : (thoughts) He's just going to let her go. Jack offers the transporter to Mary who looks at Jack then pushes Toshiko away. IANTO : (Thought) It's a bluff that thing can't stillwork. Mary walks towards Jack and takes the transporter from him. Jack still stands on the other side of it, holding it close to Mary. MARY : You smell different to them. Jack smiles at her, flirting. JACK : That's nothing, it's when you compare teeth with a British guy. That's when it's really scary. MARY : What are you ? JACK : I don't know. MARY : And you would have put me in a cage ! Mary notices that the transporter has clicked into life. MARY : What's happening ? JACK : Oh that ? Jack releases the transporter which is now locked onto Mary. He steps back out of the way. JACK : I reprogrammed it for you. It's set to enable. Mary vanishes with the transporter in a long flare of disappearing light, flying through the roof of the hub. They all look up following her trajectory. JACK : Sort of now... TOSHIKO : Where did she, has she gone home ? JACK : I reset the coordinates. TOSHIKO : Where to ? JACK : To the centre of the sun. It shouldn't be hot. I mean we sent her there at night and everything. TOSHIKO : You killed her ! JACK : Yes ! Jack walks away unconcerned while Toshiko cries while trying to be strong. Understanding. She looks up at the roof. INT. THE HUB - LATER Owen and Gwen are talking on the balcony. OWEN : Just ask her... GWEN : I will Owen, just give 'er a bit of time. She looks over at Toshiko in the office speaking to Ianto. OWEN : Just say something to her. GWEN : Owen I don't know what you want me to d, she's been through enough. OWEN : She is really creeping me out... GWEN : Owen, don't be selfish about this alright. Toshiko leaves the office and walks up to Gwen. GWEN : When did you have this, I dunno, ability ? TOSHIKO : Just a couple of days. OWEN : What did you hear ? TOSHIKO : A lot of it was noise... emotions, references I wouldn't understand. OWEN : Yeah... and the rest ? TOSHIKO: The rest was none of my business. OWEN : No it wasn't. Owen walks away angrily. TOSHIKO : I don't know where this leaves us... GWEN : Me neither... We can't really take the moral high ground with this. This thing between me and Owen it... TOSHIKO : No Gwen, what I did was an invasion. I wasn't in control. I realise that now. Even so I, I can't... I have to live with this... not what I heard, what I did to you. GWEN : And my betrayal ? TOSHIKO : What do you mean ? Gwen looks down at Owen working on his computer. GWEN : I'm living with mine. This should be my wake up call. I should stop. But I won't! what does that say about me ? TOSHIKO : I'm not really in a position to make judgements... GWEN : This is what I'm saying Tosh... neither am I. Toshiko starts to walk away. GWEN : Don't let this, it doesn't matter... sorry, forget it. TOSHIKO : What ? GWEN : Don't let this put you off. The last couple days you've had a look about you. Love suited you ! EXT. NEAR MILLENNIUM CENTRE - NIGHT Jack and Toshiko sit on a bench, talking. Toshiko looks at the pendant in her hands. TOSHIKO : It's funny such a small thing could be the most powerful piece of technology we've ever found. It could tear down governments, wipe out armies. What do we do with it ? JACK : Your call. TOSHIKO : It's a curse. Drops the pendant and crushes it with her boot. TOSHIKO : Why couldn't I read your mind ? JACK : I dunno, 'though I could feel you scrabbling around in there. He laughs, looking away at the passers-by. TOSHIKO: I got nothing. It's like you were... I dunno, dead. Jack's smile fades. JACK : I want that list for UNIT on my desk tomorrow. Or I'll... What do bosses do in situations like these ? You know, regular bosses. Do I get to beat people ? Laughs, Toshiko laughs too. TOSHIKO : We've got rules for that. JACK : Red tape ! TOSHIKO : Jack, something Mary said... probably the only honest thing she ever did say, I asked her why she gave it to me, and she said after a while it gets to you. Changes how you see people. Jack puts his hand on Toshiko's on her knee concerned for her. TOSHIKO : ...how can I live with it ? JACK : There are some things we're not supposed to know. You got a snapshot, nothing more. TOSHIKO : I don't mean about Gwen and Ianto and Owen. I mean, the whole world. Toshiko is crying slightly, Jack stands up and puts his hand on her hair. TOSHIKO : Doesn't matter. He puts his finger under her chin and lifts her head gently, then he wipes a tear from her eye with his finger. He walks away leaving Toshiko to sit and think. He is unable to say anything to comfort her so he doesn't bother. Credits : Daniela Denby-Ashe - Mary / Tom Robertson - Soldat / Ravin J Ganatra - Neil / Eiry Thomas - Carol / Shaheen Jafargholi - Danny / Paul Kasey - Weevil.

After meeting an usual woman named Mary at a bar, Toshiko is given a strange alien pendant. She is upset, yet amazed by what she now has - the ability to read people's thoughts. As the rest of the team puzzle over a centuries-old skeleton, Tosh discovers that her newfound ability is as much of a curse as it is it a blessing, and begins to question her commitment to Torchwood.

summ_screen_fd

Nitric oxide reductases (NORs) are membrane proteins that catalyze the reduction of nitric oxide (NO) to nitrous oxide (N2O), which is a critical step of the nitrate respiration process in denitrifying bacteria. Using the recently determined first crystal structure of the cytochrome c-dependent NOR (cNOR) [Hino T, Matsumoto Y, Nagano S, Sugimoto H, Fukumori Y, et al. (2010) Structural basis of biological N2O generation by bacterial nitric oxide reductase. Science 330: 1666–70. ], we performed extensive all-atom molecular dynamics (MD) simulations of cNOR within an explicit membrane/solvent environment to fully characterize water distribution and dynamics as well as hydrogen-bonded networks inside the protein, yielding the atomic details of functionally important proton channels. Simulations reveal two possible proton transfer pathways leading from the periplasm to the active site, while no pathways from the cytoplasmic side were found, consistently with the experimental observations that cNOR is not a proton pump. One of the pathways, which was newly identified in the MD simulation, is blocked in the crystal structure and requires small structural rearrangements to allow for water channel formation. That pathway is equivalent to the functional periplasmic cavity postulated in cbb3 oxidase, which illustrates that the two enzymes share some elements of the proton transfer mechanisms and confirms a close evolutionary relation between NORs and C-type oxidases. Several mechanisms of the critical proton transfer steps near the catalytic center are proposed. Bacterial denitrification is one of the examples of anaerobic respiration in which nitrate (NO3−) is stepwisely reduced to dinitrogen (N2) [1]–[3]. During denitrification, the key intermediate step of the reduction of nitric oxide (NO) to nitrous oxide (N2O) is catalyzed by a membrane-bound enzyme nitric oxide reductase (NOR) according to the following scheme: 2NO+2e−+2H+→N2O+H2O. Bacterial NORs perform fundamental chemistry and are the largest source of N2O, a greenhouse gas and an ozone-depleting substance, released into the atmosphere [1]. This enzyme also has an important role in the evolution of the respiratory system. NOR belongs to the superfamily of O2-reducing heme-copper oxidases (HCOs) and is believed to be evolutionary linked to a proton pump cytochrome c oxidase (CcO). Both enzymes may have evolved from a common ancestor [2]. The ancestral oxidase was probably involved in NO reduction, but later switched to oxygen reduction and additionally acquired the ability of proton pumping, although this issue is still open to debate [3]–[9]. After the structure of CcO was solved more than a decade ago [10], [11], that system became the focus of numerous experimental studies, which produced a number of X-ray structures from different organisms and a wealth of mutational, biochemical and spectroscopic data, as well as theoretical and simulation ones (for recent reviews, see refs. [12]–[14]). In contrast, the information about NORs was limited, but the first structure of cytochrome c-dependent NOR (cNOR) from Ps. aeruginosa has been recently determined by Shiro and co-workers [15], and that provides a basis for studies aimed at describing the mechanism of NO reduction at the atomic level. cNOR consists of two subunits, NorB and NorC, and contains four redox active metal centers, namely hemes b, b3 and c and a non-heme iron (FeB). The latter and the iron of heme b3 form the binuclear (BN) center, a site of the NO reduction. The crystal structure revealed that FeB has three His and one Glu ligands and that a tightly bound Ca2+ ion is bridging hemes b and b3. Although the function of Ca2+ is not yet fully clear, it is interesting to note that it has the same binding position as in a recently determined structure of the microaerobic respiratory enzyme cbb3 oxidase [16], which is a C-type HCO able to reduce NO to N2O in low-oxygen conditions [17], [18]. For the NO reduction reaction protons have to be delivered to the BN center, which is buried inside membrane. Previous experiments with the whole-cell [19] and liposome-reconstituted [20], [21] cNORs demonstrated that protons utilized in the catalytic reaction are taken up (on a ms timescale) from the periplasmic side (i. e. the same side as electrons), which suggests that the NO reduction reaction is non-electrogenic and therefore cNOR is not a proton pump. In order to explain the functioning of cNOR, it is necessary to understand the detailed mechanism of the proton delivery to the BN center. Since proton transfer (PT) can occur efficiently only when the donor and acceptor groups are immediately close to each other, the long-distance proton translocations in proteins (e. g. proton pumping across the membrane or proton delivery from the bulk to the buried active site) require specialized proton-conducting pathways, which involve protein ionizable groups and intermediate water molecules as proton-binding sites (see e. g. refs. [22]–[28]). Analysis of the cNOR crystal structure yielded two independent H-bonded networks, designated as Channels 1 and 2, which are formed by the resolved water molecules and the charged/polar residues [15]. These channels were proposed as potential PT pathways. However, since X-ray crystallography provides only static snapshots of the protein structure, which are averaged over many unit cells, in general such structures even at high resolution show only a few water molecules (at the most stable positions) inside the protein but miss many dynamic ones. The proposed proton pathways did not provide a continuous connection from the surface to the active site (i. e. protonic “gaps” were present), and in particular the pathways near the catalytic center, where no water molecules were resolved, remained elusive. As we mentioned, in such situations the connectivity is expected to come from the intervening water molecules. Thus, water in cNOR could play a very important role in the enzyme function and has to be fully characterized. Molecular dynamics (MD) simulations of membrane proteins within an explicit membrane/solvent environment (for some recent works and reviews see refs. [28]–[35]) can provide important information about the water dynamics, such as a “real” level of hydration and specific water positions inside the protein, and are most valuable in the cases when the water/PT channels are not yet described at the atomic level. For example, MD simulations have been recently used to explore the water dynamics in different regions in CcO and greatly contributed to the understanding of the details of PT channels in that enzyme [36]–[42]. We note that a study with an explicit membrane/solvent by Olkhova et al. [39] suggested a large number of water molecules within the PT channels in CcO, in contrast to simulations which utilized different kinds of reduced models or truncated systems. In this work we performed MD simulations of cNOR. We focused on the water dynamics, with the aim to identify the water channels and H-bonded networks that could serve as pathways for the proton delivery to the active site. The obtained information will be important for further elucidation of the mechanisms of the proton translocation and NO reduction in cNOR. We performed an all-atom MD simulation of cNOR in the explicit lipid/water environment (Figure 1a). The details of the system setup and simulation and analysis protocols are provided in the Text S1. Briefly, the initial system was prepared from a 2. 7 Å resolution crystal structure of the cNOR from Ps. aeruginosa (PDB ID 3O0R) [15]. A simulation system is shown on Figure 1a: cNOR was embedded into the pre-equilibrated POPE (palmitoyl-oleoyl-phosphatidylethanolamine) lipid bilayer membrane and a solvent box of water molecules. The total size of the simulation system was ∼110,000 atoms. The main purpose of introducing the lipid bilayer in MD simulation is to model cNOR in situ, i. e. in the environment as close to its natural as possible. POPE is the major lipid component of bacterial membranes [43]. Explicit membrane provides additional stability to the protein in MD simulations and allows a correct description of the protein-solvent and protein-lipids interactions. MD simulations were carried out in NAMD [44] with the CHARMM force field [45], [46]. After minimization and equilibration parts, production runs were performed at a constant temperature, pressure, and surface area (NPAT ensemble) for 300 ns, providing reasonable conformational sampling of the protein. Stability of the simulated protein-membrane complex was assessed from the analysis of several parameters along the MD trajectory (Figure S1). The root-mean-square deviation (RMSD) of the helical Cα atoms is below 2 Å while RMSD of Cα atoms in a transmembrane (TM) region is ∼1. 2 Å. The RMS fluctuations (RMSFs) calculated for each residue also illustrate that the TM region is very stable while the outer and inner domains exhibit, as expected, larger motions. Finally, the area/lipid, which was calculated using the Voronoi analysis tool [47], remains close to the experimental value for the POPE lipids [48], also indicating a stable simulation of the protein-membrane complex. One of the proposed PT pathways (Channel 1 in Figure 3 in ref. [15]) goes through a large hydrophilic region, which is located on the periplasmic side of the enzyme at the interface of a TM region (NorB subunit) and an outer soluble domain (NorC subunit) (Figure 1). Four residues, namely Glu135, Asp198, Lys53c, and Glu57c, were designated as Channel 1 [15]. [Subscript “c” indicates residues of the NorC subunit, while residues of the NorB subunit are numbered without additional subscripts. ] In MD simulations we observed that Channel 1 indeed connects the protein surface to propionates of heme b3 (the distance ∼16 Å) via a number of ionizable residues and water molecules and supports formation of the H-bonded networks (see below). Our analysis provides important additional details about Channel 1 (Figure 2a). MD results indicate that the following three residues participate in the HB networks in that region: Arg134, Lys199, and Glu70c, and therefore they have to be included in Channel 1. All seven ionizable residues are highly conserved in cNORs. Together, they line up a large hydrophilic channel, and their sidechains assist in the formation of the H-bonded water chains. Channel 1 has a connection to the bulk solvent between two helices, TM VI of NorB (with Asp198 and Lys199) and α2 of NorC (with Lys53c and Glu57c). The entrance site formed by the amino acids Glu57c, Lys53c, and Arg134 (Figures 2a and S2) remains rigid due to three stable salt bridges: Glu57c-Lys53c, Arg134-Asp198, and Lys53c-Asp198 (Figure S3). These residues partially block water influx. However, water molecules still occasionally cross through that site, and thus can serve as intermediate proton sites (Figure S4). Also, the dynamic HB networks involving sidechains of Glu57c and Asp198 and waters at both sides of the entrance, i. e. in the bulk and inside the Channel 1 cavity, are observed at any time of MD trajectory. Therefore it is possible that one of these residues could be directly involved in PT by picking up protons from the bulk and releasing them to the water chain inside Channel 1. The mutagenesis experiments with P. denitrificans cNOR [Pia Ädelroth, unpublished data] showed the importance of Asp185 (equivalent to Asp198 in Ps. aeuginosa cNOR) for the enzymatic activity and proton uptake, and provide partial support to this proposal. From the entrance region the proton pathway proceeds further through the dynamic water chains. Water channels in cNOR have “irregular” shape and lack simple symmetry (like, e. g. straight TM channels in aquaporins or ion channels). Therefore to perform meaningful statistical analysis in each region we selected water molecules within a reasonable distance cutoff (typically 4. 5 Å) near the sidechains of the pathway' s residues. We verified that with such definition water molecules “inside the pathway” were not skipped. Our calculations show that Channel 1 is very well hydrated: in MD simulation ∼20 water molecules are observed in this hydrophilic region (Figure 2c), which is higher than ∼12 molecules resolved in the X-ray structure. This result can be explained by the presence of mobile water molecules, which were not resolved in X-ray crystallography. To provide some quantitative representation, we have calculated volume occupied by water molecules during simulation (“water density”, see e. g. refs. [49], [50]) in different regions of cNOR. Figure 2 illustrates water spatial distribution in Channel 1 as obtained from MD simulation, showing both a 3D water volume map (Figure 2a, isosurface at 25% occupancy) and a 2D contour plot (Figure 2b, an XY-plane projection of the water density; see figure caption for details). A few observations can be made from these figures. (i) Water density representation shows the extent of the hydrophilic regions and confirms a stable connection from the bulk to the active site heme. (ii) Water molecules form an extensive water cluster between propionates of heme b3 (PropA and PropD). [Please note that compared to the previously reported cytochrome c oxidase structures the active site heme in cNOR, i. e. heme b3, is flipped and the order of the propionate groups A and D is different. ] (iii) After the entrance region, the channel goes into a water-filled cavity. An important finding is that further the pathway splits into two branches: one path leads via a water chain (5–6 water molecules) directly to PropA, while another – via Glu70c and a short chain (2–3 water molecules) at the other side of that residue – to PropD. The terminal region of both paths is the water cluster near heme b3 propionates. The existence of two branches in Channel 1 could be observed in the MD simulation, but was not evident from the static X-ray structure. This feature provides a possibility of PT over different pathways and probably adds to the robustness of the proton uptake via Channel 1. (iv) When water density is plotted at a higher occupancy level, one obtains positions of the water sites that are occupied almost permanently during the simulation. One example is a crystallographic water molecule, Wat65, which remains bound near Ca2+ for the entire MD trajectory. Such “permanent” water sites in general superimpose well with the positions of waters resolved in the X-ray structure (indicated by purple spheres on Figure 2b). Figure 2a also presents a typical configuration of the H-bonded networks forming in Channel 1, while Video S1 and multiple MD snapshots on Figure S5 illustrate their time-dependent dynamics. The average lifetime of a hydrogen bond (HB) in the water chains is in the ps range due to rotating and/or moving water molecules. It can be seen that water molecules in Channel 1 have high mobility and exchange rates and, as a result, the forming H-bonded networks are constantly “fluctuating” (similar to the H-bonded networks in CcO [39], [40]). Continuous HB paths between the bulk and heme b3 propionates do form, and their consistency is limited by the intervening water chains, namely a chain from Asp198 to Glu135 (probability ∼25–35%) and a chain from Asp198 to Glu70c (probability >60%). But it is important that such connections are forming at all times, and thus can assist efficient proton translocation [51]. Participation of the Channel 1 residues in the H-bonded networks can be assessed quantitatively by calculating the number of surrounding water molecules and formed HBs (Table S1). In particular, these results, in addition to visual analysis, suggest that Glu70c could play an important role in the proton uptake process. It is desirable to verify its involvement in the PT pathway by site-directed mutagenesis experiments. From the inspection of the X-ray structure, Hino et al. identified another cavity, which contains many crystallographic waters, and proposed it as a second possible proton-conducting pathway (Channel 2 in Figure 3 in ref. [15]). In MD simulation we observed a large hydrophilic region formed by the residues Arg416, Thr66c, Glu77c, Gln411, and Gln415, with an exit to the bulk beyond the latter (Figure 3a). On average, there are 10 to 12 water molecules in the cavity. However, we found that water molecules from this cavity cannot pass to the water cluster near heme b3, and further to the active site. Two loops, and more specifically two glycine residues Gly340 and Gly69c, are in close contact en route to heme b3 and, together with a ring of Tyr73c, disrupt a possible water chain. A close steric contact between two loops remains for the entire length of the MD trajectory, as evidenced by the Gly-Gly distance (Figure 3c), which stays around 3. 5–4 Å (i. e. similar to the distance in the crystal structure). Water densities (Figures 3a and b) clearly show a wide gap with no substantial density between the upper hydrophilic cavity and the water cluster. We do not completely rule out a possibility that mobile water molecules can occasionally cross the gap region; however, no such crossings or continuous HB networks were observed in 300 ns. Moreover, the proton translocation through the region with no polar/charged residues or water molecules would encounter high activation barriers. Thus, our results do not support the previously proposed Channel 2 as an alternative pathway for proton delivery to the active site. The exact functional role of this hydrophilic cavity in cNOR is not clear. A careful analysis of MD trajectories revealed another plausible proton pathway, which we designated as the (periplasmic) Channel 3 (see Figures 1 and 4). This pathway involves the residues Glu135, Glu138, Arg57, Asn54, and Asn60c. The first three are highly conserved in all NORs and oxidases, while Asn54 is conserved in cNORs. In the X-ray structure, three water molecules are resolved in a cavity formed by these residues. During the initial part of the MD simulation this region has no connection to the periplasmic surface (Figures 4a, b). The calculated water density clearly shows that the cavity is completely separated from the bulk solvent and that two asparagine residues, Asn54 and Asn60c, effectively work as a gate, blocking water access from the outside. However, after ∼165 ns in the MD simulation the Asn54-Asn60c gate opens and a new water channel is formed (Figures 4c, d). A continuous water density then extends up to two important residues, Glu135 and Glu138, and the H-bonded networks involving mobile water molecules and amino acid sidechains readily form. The number of waters in the hydrophilic region, and in particular around the sidechain of Glu138, significantly increases with the gate opening and remains high even after the gate closes back (Figure 5c). Figure 5 also shows minimal distances between the Asn54-Asn60c and Glu138-Asn60c pairs in the MD simulation, along with the representative snapshots of the gate region. Clearly, the gate is closed when two Asn are H-bonded. Sidechains of Glu138 and Asn60c exhibit large-amplitude rotations (Figure S6), in particular Glu138 can take several conformations, and the initial event leading to the gate opening seems to be a rotation of Glu138 to the “up” position after ∼135 ns and the formation of a HB to Asn60c. Soon after that a strong HB between two Asn is broken. As a result, a helix TM II (with Asn54 on a top) slightly tilts away, and that opens water access to the internal cavity. An overlay of the open and closed configurations (Figure 5f) shows that the required structural changes are rather small: two Asn move away only by a few Å, but that is enough to break a HB between them and to open access to the internal cavity for water molecules from the outside. The gate is open for ∼60 ns, after which the HB between Asn60c and Asn54 is re-formed; the HB between Glu138 and Asn60c breaks prior to that. The explicit gate opening/closing process and formation of the dynamic water chains in Channel 3 are illustrated by Video S2. We would like to emphasize that similar events were also observed in the extended simulation as well as in independent runs (Figure S7), indicating that such events can occur in cNOR on a 100-ns timescale, which is much shorter than the experimentally measured rate of the proton uptake (∼25 ms) [20], [21]. This suggests that such structural reorganizations due to protein fluctuations are feasible during catalysis in cNOR and that Channel 3, in principle, can provide a pathway for a water-mediated proton uptake. We propose to examine the role of Asn54 and Asn60c in the Channel 3 gating by the mutagenesis experiments. A newly found channel is consistent with previous experimental data. Two key residues, Glu135 and Glu138 (Glu122 and Glu125 in P. denitrificans cNOR), were shown by site-directed mutagenesis to be essential for the enzymatic activity and were proposed to be a part of the proton input pathway [52]–[54], though their exact positions predicted with the homology-based model (namely, on a protein outer surface) [21] turned out to be incorrect. With the cNOR structure available now, it is known that Glu135 is a ligand to Ca2+. That explains why its substitution with Asp still showed a level of activity close to the wild type (i. e. Ca2+ coordination was kept) while a substitution with Ala or Gln resulted in a loss of activity (most likely caused by a Ca2+ dissociation). The structural function of Glu135 also makes its direct participation in PT problematic: it is unlikely that Glu135 can get protonated or that the protons coming from the periplasm can be transferred through a densely packed region occupied by the Ca2+ ion and its ligands. The substitution of Glu138 with Ala and Asp resulted in a loss of activity, while a mutation to Gln showed some, though significantly reduced activity [53], [54]. These results could indicate that the length of the sidechain is more important than retaining a negative carboxylic group at that position. The observation fits into the above suggested mechanism of the Channel 3 opening and “activation” of the proton pathway, which includes a Glu138 sidechain rotation to the “up” position to form a HB to Asn60c, thus helping to break a HB between two Asn. In contrast to Glu135, Glu138 can actively participate in the PT process. A Glu122Asp mutation in P. denitrificans caused a significant pKa shift of a presumed nearby proton donor group [54], and Glu138 seems to be the best candidate for that role. The proton pathway beyond Glu138 is also offered by our MD results. After the gate opening, Glu138 is well hydrated, with typically 5 to 8 water molecules near its sidechain (Figures 4c, d and 5c). We observed the H-bonded water chains leading from this site toward the water molecules bound near BN, thus avoiding the Ca2+ site (see the corresponding discussion below). A key finding is that the suggested novel channel in cNOR is equivalent to the putative PT pathway (the “periplasmic cavity”) in a recently determined structure of cbb3 oxidase [16]: a comparison of two regions shows that their positions are identical (Figure 6). Moreover, the important residues which form this hydrophilic cavity, namely Glu135, Glu138 and Arg57 in cNOR and Glu122, Glu125, Arg57 in cbb3, are conserved. The periplasmic cavity in cbb3 oxidase was suggested to be an exit pathway of the pumped protons or a pathway for proton uptake from periplasm when the enzyme is involved in NO reduction [16]. The fact that for NO reduction cbb3 uses protons from the periplasmic side of the membrane has been recently confirmed by the experimental work of Lee et al. [18]. We note that such cavity is not found in other structurally known HCOs and that aa3 oxidases (A-type HCOs) are incapable of NO reduction, while ba3 oxidases (B-type HCOs) can reduce NO but much slower than cbb3 [3], [5], [9]. The presence of a plausible PT pathway in the equivalent region in cbb3 oxidase is an additional argument for the functional importance of Channel 3 in cNOR. The finding that two enzymes likely have common elements of the PT mechanism, along with other common structural factors, such as the identical position of Ca2+, fits nicely into the phylogenetic pictures that draw C-type HCOs as the closest evolutionary relatives of NORs. We have also analyzed the region equivalent to Channel 1 in the cbb3 structure [16]. It seems that the corresponding region cannot provide a pathway for proton translocation in cbb3 because: (i) some of the charged residues present in Channel 1 in cNOR, namely Lys199, Lys53c, Glu57c, and Glu70c, are either missing or located far away in cbb3, (ii) a coil with several hydrophobic residues is located in the central part of that region and splits it into two parts; the water distribution is disconnected too [to be published], (iii) a second Ca2+ site is located at the position equivalent to the entrance to Channel 1 in cNOR and most likely blocks proton transfer. We have shown that Channel 1 and Channel 3 can connect the periplasmic surface to the region near heme b3. Its propionates together with a nearby water cluster and Glu138 are the likely intermediate proton acceptor groups. (It is less likely that PropA can get protonated since it serves as a ligand to Ca2+.) It is worth mentioning that in CcO one of the active site heme propionates is thought to be the likely proton loading site for the pumped protons [55]–[57]. The idea about the functional importance of protonated water clusters inside proteins is also not new. For example, in CcO a protonated water cluster was suggested as a proton storage site in the D-channel [58], while in bacteriorhodopsin a protonated water cluster is a presumed proton release group [59], [60]. An important question is how protons are delivered to the catalytic center when they are needed for the NO reduction, i. e. what are the structural elements critical for the final PT steps? The distance (>8 Å) is still long for direct PT, but no water molecules were resolved in the vicinity of the BN center. So the further proton path was not clear from the X-ray structure, and intermediate water molecules are expected to play important role. In a working enzyme, water will be produced at the active site as a byproduct of the catalytic NO reduction. In contrast to the crystal structure, the MD simulation reveals the presence of water molecules near the BN center (Figure S8) and describes their distribution (Figure 7). The exchange rate of waters is much lower compared to the channels discussed above. Water molecules are found persistently at several positions and keep these positions for 20–50 ns or longer (Figure S9); such water molecules might serve as intermediate proton sites. Figure 7 depicts a representative configuration of water molecules in that region, along with the calculated water density (see also Figure S10). It can be seen that one permanent water site is located between two irons of the BN center (i. e. where NO ligands will bind during the enzymatic cycle), another corresponds to the water molecule bound between FeB and Glu280, and two more water sites are located between FeB and PropA. It is interesting that in a recent high-resolution structure of Th. thermophilus ba3 oxidase [61] two water molecules were resolved at the identical positions. Analysis of the water dynamics and distribution offers several possible paths for the final PT steps to the BN center (Figure 7): High-resolution crystal structures of CcO and subsequent mutational studies identified a number of critical residues in the proton pathways from the cytoplasm to the active site (K and D channels). However, in cNOR most of these residues are replaced by hydrophobic residues. The crystal structure of cNOR neither provides an obvious water channel from the cytoplasmic side of the membrane nor a H-bonded network in the regions that correspond to the K and D proton channels in CcO (see Figure 4 in ref. [15]). Similarly, our MD simulation shows no water in those regions (Figure 8), with the exception of a hydrophilic cavity below the active site with three glutamates, Glu211, Glu280, and Glu215. Thus, in cNOR there is no proton pathway from the cytoplasmic side. This is consistent with the experimental observations that cNOR is not electrogenic and has no proton-pumping activity, and that the electrons and protons for the catalytic reaction are supplied from the periplasmic side. The position of the above-mentioned small hydrophilic region overlaps with the terminal part of the K-pathway in cytochrome oxidases. That could indicate a beginning of the K-channel formation in the evolutionary steps leading to the appearance of proton pathways from the cytoplasm and eventually to the proton pumping in other HCOs. A very recent structural characterization of a single-subunit quinol-dependent NOR (qNOR) from G. stearothermophilus [64] surprisingly revealed the existence of the water channel from the cytoplasmic side at the position equivalent to the canonical K-pathway and absence of the periplasmic pathways found in cNOR. It will be interesting to test by calculations if a similar cytoplasmic channel can be formed in cNOR as the result of selective mutations. We have performed a 300 ns MD simulation of cNOR, based on its first crystal structure, and fully characterized water inside the protein. Our simulations have revealed two potential PT pathways from the periplasmic side, Channels 1 and 3. Both pathways are supported by the continuous distribution of water molecules and formation of the dynamic H-bonded networks within the channels, as well as by the highly conserved nature of the participating residues and previous experiments, which had shown functional importance of some of these residues. Since cNOR is not involved in a vectorial proton translocation (pumping against the gradient), a robust gating mechanism, as those suggested in CcO [56], [57], [62], [65], [66], is not required, and chemical protons have to arrive at the active site in one way or another. So, in principle, both pathways may be used. From our MD results we cannot unambiguously establish what the exact role of each channel is or how they are synchronized. In our opinion, Channel 1 is probably the main pathway for the proton uptake since both static and dynamic structures clearly show extensive H-bonded networks and water chains, and the path toward the catalytic site seems to be more straightforward. Meanwhile, Channel 3 is revealed only by the dynamic simulations (and the water channel is formed only for a part of the simulation), some protein structural rearrangements are required there to allow for channel formation, and the path from Glu138 to the active site goes through an intermediate hydrophobic region. A further discussion about the details of the proton uptake mechanism in cNOR should be based on additional experimental evidences and explicit PT calculations. We would like to emphasize that MD simulations provide important information about the dynamics of water molecules and H-bonded networks and, as a result, about locations of potential proton pathways. However, classical MD simulations alone cannot describe explicit proton translocation, which is an intrinsically quantum mechanical process. The energetics of PT along different pathways has to be addressed by mixed QM/MM methods [25], [56], [67]–[69], and this will tell whether each pathway is feasible. The key issues in such calculations are the energies of charge formation at different sites along the translocation path and activation barriers of individual PT steps. In our calculations we observed a fairly high number of mobile water molecules (which could not be resolved in the X-ray structure) in the cNOR hydrophilic cavities. Similar results were previously reported in analogous MD studies (with explicit membrane/solvent, at ambient temperatures) of systems like proton pumps cytochrome c oxidase [39], bacteriorhodopsin [28], [70], bc1 [71], voltage-gated proton channel Hv1 [27], [72] and calcium pump [73], [74], whose function relies on the water-assisted proton translocation. Therefore such simulations, although they are computationally expensive, can be used for the detailed characterization of water inside membrane proteins and for the identification of potential proton pathways, which in many cases are critical for protein function. Finally, several common structural features, namely the position of the Ca2+ binding site and similarity of Channel 3 in cNOR and the periplasmic cavity in cbb3 oxidase, indicate the evolutionary relationship between the two enzymes. The likely loss of Channel 1 in cbb3 oxidase might be the key step during the molecular evolution leading to the establishment of the PT pathway from the cytoplasm, while a less effective Channel 3 was probably kept as a proton exit pathway for proton pumping. Our results have implications on the development of PT pathways in HCOs and the evolution of respiratory enzymes in general – a topic which remains a subject of intense debate.

Denitrification is an anaerobic process performed by several bacteria as an alternative to aerobic respiration. A key intermediate step is catalyzed by the nitric oxide reductase (NOR) enzyme, which is situated in the cytoplasmic membrane. Proton delivery to the catalytic site inside NOR is an important part of its functioning. In this work we use molecular dynamics simulations to describe water distribution and to identify proton transfer pathways in cNOR. Our results reveal two channels from the periplasmic side of the membrane and none from the cytoplasmic side, indicating that cNOR is not a proton pump. It is our hope that these results will provide a basis for further experimental and computational studies aimed to understand details of the NOR mechanism. Furthermore, this work sheds light on the molecular evolution of respiratory enzymes.

lay_plos

"I lay on my straw, but I could not sleep. I thought of the occurrences of the day. What chiefly struck me was the gentle manners of these people; and I longed to join them, but dared not. I remembered too well the treatment I had suffered the night before from the barbarous villagers, and resolved, whatever course of conduct I might hereafter think it right to pursue, that for the present I would remain quietly in my hovel, watching, and endeavouring to discover the motives which influenced their actions. "The cottagers arose the next morning before the sun. The young woman arranged the cottage, and prepared the food; and the youth departed after the first meal. "This day was passed in the same routine as that which preceded it. The young man was constantly employed out of doors, and the girl in various laborious occupations within. The old man, whom I soon perceived to be blind, employed his leisure hours on his instrument, or in contemplation. Nothing could exceed the love and respect which the younger cottagers exhibited towards their venerable companion. They performed towards him every little office of affection and duty with gentleness; and he rewarded them by his benevolent smiles. "They were not entirely happy. The young man and his companion often went apart, and appeared to weep. I saw no cause for their unhappiness; but I was deeply affected by it. If such lovely creatures were miserable, it was less strange that I, an imperfect and solitary being, should be wretched. Yet why were these gentle beings unhappy? They possessed a delightful house (for such it was in my eyes), and every luxury; they had a fire to warm them when chill, and delicious viands when hungry; they were dressed in excellent clothes; and, still more, they enjoyed one another's company and speech, interchanging each day looks of affection and kindness. What did their tears imply? Did they really express pain? I was at first unable to solve these questions; but perpetual attention, and time, explained to me many appearances which were at first enigmatic. "A considerable period elapsed before I discovered one of the causes of the uneasiness of this amiable family; it was poverty: and they suffered that evil in a very distressing degree. Their nourishment consisted entirely of the vegetables of their garden, and the milk of one cow, who gave very little during the winter, when its masters could scarcely procure food to support it. They often, I believe, suffered the pangs of hunger very poignantly, especially the two younger cottagers; for several times they placed food before the old man, when they reserved none for themselves. "This trait of kindness moved me sensibly. I had been accustomed, during the night, to steal a part of their store for my own consumption; but when I found that in doing this I inflicted pain on the cottagers, I abstained, and satisfied myself with berries, nuts, and roots, which I gathered from a neighbouring wood. "I discovered also another means through which I was enabled to assist their labours. I found that the youth spent a great part of each day in collecting wood for the family fire; and, during the night, I often took his tools, the use of which I quickly discovered, and brought home firing sufficient for the consumption of several days. "I remember, the first time that I did this, the young woman, when she opened the door in the morning, appeared greatly astonished on seeing a great pile of wood on the outside. She uttered some words in a loud voice, and the youth joined her, who also expressed surprise. I observed, with pleasure, that he did not go to the forest that day, but spent it in repairing the cottage, and cultivating the garden. "By degrees I made a discovery of still greater moment. I found that these people possessed a method of communicating their experience and feelings to one another by articulate sounds. I perceived that the words they spoke sometimes produced pleasure or pain, smiles or sadness, in the minds and countenances of the hearers. This was indeed a godlike science, and I ardently desired to become acquainted with it. But I was baffled in every attempt I made for this purpose. Their pronunciation was quick; and the words they uttered, not having any apparent connexion with visible objects, I was unable to discover any clue by which I could unravel the mystery of their reference. By great application, however, and after having remained during the space of several revolutions of the moon in my hovel, I discovered the names that were given to some of the most familiar objects of discourse: I learned and applied the words _fire_, _milk_, _bread_, and _wood_. I learned also the names of the cottagers themselves. The youth and his companion had each of them several names, but the old man had only one, which was _father_. The girl was called _sister_, or _Agatha_; and the youth _Felix_, _brother_, or _son_. I cannot describe the delight I felt when I learned the ideas appropriated to each of these sounds, and was able to pronounce them. I distinguished several other words, without being able as yet to understand or apply them; such as _good_, _dearest_, _unhappy_. "I spent the winter in this manner. The gentle manners and beauty of the cottagers greatly endeared them to me: when they were unhappy, I felt depressed; when they rejoiced, I sympathized in their joys. I saw few human beings beside them; and if any other happened to enter the cottage, their harsh manners and rude gait only enhanced to me the superior accomplishments of my friends. The old man, I could perceive, often endeavoured to encourage his children, as sometimes I found that he called them, to cast off their melancholy. He would talk in a cheerful accent, with an expression of goodness that bestowed pleasure even upon me. Agatha listened with respect, her eyes sometimes filled with tears, which she endeavoured to wipe away unperceived; but I generally found that her countenance and tone were more cheerful after having listened to the exhortations of her father. It was not thus with Felix. He was always the saddest of the groupe; and, even to my unpractised senses, he appeared to have suffered more deeply than his friends. But if his countenance was more sorrowful, his voice was more cheerful than that of his sister, especially when he addressed the old man. "I could mention innumerable instances, which, although slight, marked the dispositions of these amiable cottagers. In the midst of poverty and want, Felix carried with pleasure to his sister the first little white flower that peeped out from beneath the snowy ground. Early in the morning before she had risen, he cleared away the snow that obstructed her path to the milk-house, drew water from the well, and brought the wood from the out-house, where, to his perpetual astonishment, he found his store always replenished by an invisible hand. In the day, I believe, he worked sometimes for a neighbouring farmer, because he often went forth, and did not return until dinner, yet brought no wood with him. At other times he worked in the garden; but, as there was little to do in the frosty season, he read to the old man and Agatha. "This reading had puzzled me extremely at first; but, by degrees, I discovered that he uttered many of the same sounds when he read as when he talked. I conjectured, therefore, that he found on the paper signs for speech which he understood, and I ardently longed to comprehend these also; but how was that possible, when I did not even understand the sounds for which they stood as signs? I improved, however, sensibly in this science, but not sufficiently to follow up any kind of conversation, although I applied my whole mind to the endeavour: for I easily perceived that, although I eagerly longed to discover myself to the cottagers, I ought not to make the attempt until I had first become master of their language; which knowledge might enable me to make them overlook the deformity of my figure; for with this also the contrast perpetually presented to my eyes had made me acquainted. "I had admired the perfect forms of my cottagers--their grace, beauty, and delicate complexions: but how was I terrified, when I viewed myself in a transparent pool! At first I started back, unable to believe that it was indeed I who was reflected in the mirror; and when I became fully convinced that I was in reality the monster that I am, I was filled with the bitterest sensations of despondence and mortification. Alas! I did not yet entirely know the fatal effects of this miserable deformity. "As the sun became warmer, and the light of day longer, the snow vanished, and I beheld the bare trees and the black earth. From this time Felix was more employed; and the heart-moving indications of impending famine disappeared. Their food, as I afterwards found, was coarse, but it was wholesome; and they procured a sufficiency of it. Several new kinds of plants sprung up in the garden, which they dressed; and these signs of comfort increased daily as the season advanced. "The old man, leaning on his son, walked each day at noon, when it did not rain, as I found it was called when the heavens poured forth its waters. This frequently took place; but a high wind quickly dried the earth, and the season became far more pleasant than it had been. "My mode of life in my hovel was uniform. During the morning I attended the motions of the cottagers; and when they were dispersed in various occupations, I slept: the remainder of the day was spent in observing my friends. When they had retired to rest, if there was any moon, or the night was star-light, I went into the woods, and collected my own food and fuel for the cottage. When I returned, as often as it was necessary, I cleared their path from the snow, and performed those offices that I had seen done by Felix. I afterwards found that these labours, performed by an invisible hand, greatly astonished them; and once or twice I heard them, on these occasions, utter the words _good spirit_, _wonderful_; but I did not then understand the signification of these terms. "My thoughts now became more active, and I longed to discover the motives and feelings of these lovely creatures; I was inquisitive to know why Felix appeared so miserable, and Agatha so sad. I thought (foolish wretch!) that it might be in my power to restore happiness to these deserving people. When I slept, or was absent, the forms of the venerable blind father, the gentle Agatha, and the excellent Felix, flitted before me. I looked upon them as superior beings, who would be the arbiters of my future destiny. I formed in my imagination a thousand pictures of presenting myself to them, and their reception of me. I imagined that they would be disgusted, until, by my gentle demeanour and conciliating words, I should first win their favour, and afterwards their love. "These thoughts exhilarated me, and led me to apply with fresh ardour to the acquiring the art of language. My organs were indeed harsh, but supple; and although my voice was very unlike the soft music of their tones, yet I pronounced such words as I understood with tolerable ease. It was as the ass and the lap-dog; yet surely the gentle ass, whose intentions were affectionate, although his manners were rude, deserved better treatment than blows and execration. "The pleasant showers and genial warmth of spring greatly altered the aspect of the earth. Men, who before this change seemed to have been hid in caves, dispersed themselves, and were employed in various arts of cultivation. The birds sang in more cheerful notes, and the leaves began to bud forth on the trees. Happy, happy earth! fit habitation for gods, which, so short a time before, was bleak, damp, and unwholesome. My spirits were elevated by the enchanting appearance of nature; the past was blotted from my memory, the present was tranquil, and the future gilded by bright rays of hope, and anticipations of joy."

The monster notices the care and concern the family has for each other, and he senses that there is a mood of despair among the younger family members. The family suffers from poverty and a lack of food. Originally a well-to-do family from France, the De Lacey's have been exiled from France to Germany. The monster learns the French language from the family and practices those words by himself. Desiring to keep his cottagers happy, the monster becomes an aid to the family by secretly hauling wood to the cottage and performing repairs, all under the cover of darkness. He begins to follow a routine of daily activity and time passes from winter to spring.

booksum

View rankings table here and interactive graphics here. Drinking and driving make for an incredibly deadly combo. The risk of death by car accident is over eleven times greater for drivers impaired by alcohol. And, if you don’t value your own life enough to swap for a soda or catch a ride, you have to at least consider all the innocent people you are putting in danger on the roads around you. According to MADD, “In 2016, 10,497 people died in drunk driving crashes – one every 50 minutes – and 290,000 were injured in drunk driving crashes” Or maybe money motivates you. Drunk driving accidents, injuries, and deaths on average are costing every American adult over $500 and the U.S. as a whole $132 billion each and every year! This is quite possibly the costliest, deadliest problem our country has ever faced. Read on to see which ten states are in the most trouble. Click here for our detailed methodology. The 10 Deadliest States for Drunk Driving #10 – Texas Best Ranking Factor: DUI Arrests – 35th Worst Ranking Factor: DUI Fatalities – 9th Of the total car crash deaths in Texas in 2016, 1,670 – 44 percent – of them involved an alcohol-impaired driver. Thankfully, the Lonestar State is trying to corral its drunk drivers. In 2016, there was an impressive total of 68,371 DUI arrests made in Texas. As you can see in the below news report, even Texas judges who beg for leniency are getting arrested for drinking and driving. Hopefully the extra efforts from law enforcement make a difference, because according to our studies, the public roadways in Texas are some of the deadliest in America. Texas has ranked in the top five states every year since 2011 for having the worst drivers in the country. #9 – New Mexico Best Ranking Factor: DUI Arrests – 29th Worst Ranking Factor: DUI Death Cost – 6th Like its neighbor, Texas, New Mexico is working to stop drunk drivers, ranking in the better half of the U.S. for its number of DUI arrests. However, the families of the 148 people killed on New Mexico roads in 2016 by drivers impaired by alcohol must feel this is far from enough. Residents of this “Land of Enchantment” aren’t just paying in deaths and injuries for this selfish behavior either. Each New Mexico state resident is paying an average of $223.45 every year in taxes to help cover the cost of drunk driving fatalities. This must be extremely disenchanting for the over two million people living in this state. #7 (Tie) – Nebraska Best Ranking Factor: DUI Death Cost – 34th Worst Ranking Factor: DUI Arrests – 5th You can’t live “the good life” if you’re killed by a drunk driver or put in prison for being a drunk-driving murderer. Nebraska’s strengths and weaknesses are the exact opposite of New Mexico’s. The tax cost might be on the lower end with each resident paying $154.16 to cover drunk driving deaths, but only 7,057 DUI arrests were made in 2016. Far too many Nebraska drivers are getting away with being drunk behind the wheel considering 38 percent of all the 2016 traffic deaths in this state involved a driver impaired by booze. The scariest part? Nebraska was ranked 26th on this countdown last year... talk about a huge jump in the wrong direction! #7 (Tie) – Idaho Best Ranking Factor: DUI Death Cost – 25th Worst Ranking Factor: DUI Laws – 3rd Unfortunately, Idaho’s best ranking factor comes from the fact that state residents are paying $165.17 a year in taxes due to their drunk drivers. The Gem State isn’t putting a high enough value to all its treasures considering its lax drunk driving laws. Sobriety checkpoints are illegal by Idaho state law, and it wasn’t until March 26, 2018 that this lenient state started to require ignition interlock devices for all DUI offenders. Thanks to insufficient laws, Idaho has 4th DUI offenders caught driving, blowing three times the legal limit. It’s a good thing these teen “heroes” where there to help: Thankfully this story has a good ending, but it isn’t safe for teens to be following and filming a drunk driver around as she takes out mailboxes and nearly causes car crashes. Idaho drunk drivers need to be stopped long before it gets to this treacherous point. #6 – Rhode Island Best Ranking Factor: DUI Arrests – 33rd Worst Ranking Factor: DUI Laws – 3rd Although it’s the smallest state in America, Rhode Island is still making a big impact when it comes to deadly drunk driving in our country. Little Rhody might be making a good amount of DUI arrests, which equaled over 10 percent of the total arrests in 2016, but the arrests themselves aren’t going to do any good without laws to back them up. The State Supreme Court has made sobriety checkpoints illegal in Rhode Island, there aren’t license revocation laws, child endangerment isn’t a felony, and the lookback period is only five years. Hopefully, Rhode Island officials realize the dangers of drunk driving and take action to strengthen their state laws soon. #5 – Maine Best Ranking Factor: DUI Penalties – 38th Worst Ranking Factor: DUI Fatalities – 4th With 42 percent of all its traffic deaths involving a driver impaired by alcohol, Maine is illustrating the way life shouldn’t be. Thankfully, Maine takes drunk driving seriously with a Zero Tolerance law and penalties that come from both the state courts and the Bureau of Motor Vehicles including suspended licence, mandatory ignition interlock device, driver’s education courses, fines, and possible jail time. Regrettably, these drunk driving penalties weren’t enough to keep this 23-year-old from being struck and killed by a repeat drunk driving offender: Jumping from 10th place to 5th in under a year, Maine is quickly headed in the wrong direction in regards to drunk driving. #4 – Montana Best Ranking Factor: DUI Penalties – 40th Worst Ranking Factor (Tie): DUI Death Cost & DUI Laws – 1st It’s pretty sad that the state landing in 5th last year and one spot higher this year, wins 1st place for the worst in the U.S. for not just one, but TWO drunk driving categories. Montana residents pay the highest amount in the entire country – a whopping $431.65 each year in taxes – to help cover the cost of all the drunk driving deaths in their state. Plus, “The Treasure State” has the most lenient drunk driving laws and statutes in the entire country with no sobriety checkpoints and lax or non-existent laws regarding ignition interlock devices, license revocation, and refusing a breathalyzer. Not only has Montana landed in the top five on this countdown, but it got 6th place on our Most Dangerous Highways study and was awarded 1st for being home to the Worst Drivers in the nation. #3 – South Dakota Best Ranking Factor: DUI Penalties – 48th Worst Ranking Factor: DUI Arrests – 1st With 46.55 percent of the total traffic deaths being caused by a driver impaired by alcohol, there should be far more than just 15.14 percent of all the arrests being for a DUI. South Dakotans must feel it’s so unlikely to get arrested for drunk driving, that it’s worth the risk. The DUI penalties in South Dakota might be on the strict side, ranking in the top five for that category, but these penalties aren’t always enforced. For example, when Scott Larson got his 9th DUI, instead of 10 years in prison he got 18-24 months in a court program thanks to some good ole South Dakota mercy: It’s even legal in “The Mount Rushmore State” to sell alcohol to someone who is only 18 years old as long as they are with a parent, guardian, or spouse over the age of 21. #2 – North Dakota Best Ranking Factor: DUI Penalties – 18th Worst Ranking Factor (Tie): DUI Death Cost & DUI Arrests – 2nd Sadly, North Dakota not only ranks in the worst half of the U.S. for all five of the categories in this drunk driving study, but it also ranks a top ten worst state in three of the five categories. North Dakota state residents pay $399.76 annually to cover the cost of drunk driving deaths in their state. It’s good that the North Dakota police force is catching drunk drivers, but it’s clearly a problem when 17.66 percent of all the arrests taking place in the state are for drunk driving. Considering it has ranked in the top five for being one of deadliest states for drunk driving four years in a row now, we really hope that “The Peace Garden State” starts waging war against its drunk drivers. #1 – Wisconsin Best Ranking Factor: DUI Death Cost – 31st Worst Ranking Factor: DUI Penalties – 3rd Thanks to its excessively high number of drunk driving deaths and DUI arrests and due to having some of the most lenient drunk driving laws and penalties, Wisconsin has earned the title: Deadliest State for Drunk Driving. As you can see in the below CBS report, Wisconsin is the only state in America to not criminalize 1st time drinking and driving offenders. “People know the first offense is a slap on the wrist,” addressing the issue Wisconsin Senator, Chris Larson says, “it’s a moving violation.” With some of the shortest license suspensions for drunk driving, cheapest DUI fines in the country, and sobriety checkpoints being prohibited by the state, Wisconsin is clearly not doing enough to stop drunk drivers. LifeSafer says, “In every class, there are star students and laggards, and so is it with anti-DUI efforts. Some states do everything they can to address the issue of drinking and driving. And some states are, well, Wisconsin.” Ranking 4th, 3rd, 2nd, and now 1st for having the most dangerous roads in the U.S. due to drunk drivers, The Badger State needs to make some big changes (and fast) to make roads safer for all those who live there. Trends and Highlights View the interactive graphics here. Seven of the 10 states from our countdown landed on this deadliest list last year: As you can see, Rhode Island was the only state to make an improvement from last year, and Maine made the greatest jump in the wrong direction. Six of the 10 states on this deadliest list had their worst ranking factors land in the top three for the entire country: Idaho: DUI Laws – 3rd DUI Laws – 3rd Rhode Island: DUI Laws – 3rd DUI Laws – 3rd Montana: DUI Death Cost & DUI Laws – 1st DUI Death Cost & DUI Laws – 1st South Dakota: DUI Arrests – 1st DUI Arrests – 1st North Dakota: DUI Death Cost & DUI Arrests – 2nd DUI Death Cost & DUI Arrests – 2nd Wisconsin: DUI Penalties – 3rd Lenient drunk driving laws and far too many traffic deaths caused by drunk drivers are two of the biggest reasons the above ten states landed on this list. Not once did DUI Laws or DUI Death Rank come up as best ranking factors. The most common worst ranking factors were DUI Death Cost, DUI Arrests, and DUI Laws, each showing up three times on our deadliest list this year. Drive Safe and SOBER! NEVER driving while impaired by alcohol is the key to end drunk driving, but you can take it a step further and learn ways to spot drunk drivers and alert the cops. Here are the top ten things drunk drivers do behind the wheel: When your plans involve alcohol, here are some tips to keep yourself and all those around you safe: Drinkinganddriving.org says, “Since the September 11 terror attacks, over 200,000 men women and children have died in America as a result of drunk driving... It is time to stand up and admit that as a culture, we drink together and LET each other drive away.” Methodology The rankings for this comprehensive drunk driving study came from the most up-to-date reports from the following leading resources: All 50 states plus D.C. received a final score based on five main categories that each had its own intricate calculation: For each category, the highest rankings were awarded to the worst states in the country. The higher a state ranked in each category, the lower final score it received. Lowest Scores = Deadliest States for Drunk Driving Our study implemented weighted measures to demonstrate the impact each manifestation of a category can have on a person, community, and state. Each weight is representative of the severity of the category. Category Weight Fatality Rank 0.35 Cost per Fatality by State 0.10 DUI Arrests 0.25 DUI Penalties 0.20 Types of Laws and Statutes 0.10 These weighted scores are necessary to deduce the most dangerous state. The final rankings, which determines the worst state, are not perfectly correlated with the final scores of the non-weighted rankings, which determines the worst state per category. Complete Rankings: Most Dangerous States for Drunk Driving (Weighted + Unweighted Tables) – To view and sort the table by category, click on header columns. – Click the box to expand the table for weighted and unweighted rankings – Click here for the full stats and sources for each category. For all media inquiries, please email: Joshua Barnes 2018 Most Dangerous States for Drunk Driving Unweighted State Fatality Rank Cost Per Fatality Rank DUI Arrests DUI Penalties Types of Laws/Statutes Total Score Rank Wisconsin 8 31 8 3 8 58 2 North Dakota 16 2 2 18 8 46 1 South Dakota 3 9 1 48 3 64 4 Montana 12 1 4 40 1 58 2 Maine 4 21 6 38 27 96 12 Rhode Island 11 28 33 5 3 80 6 Idaho 23 25 9 22 3 82 7 Nebraska 13 34 5 28 27 107 16 New Mexico 13 6 29 23 16 87 8 Texas 9 10 35 24 16 94 11 California 22 32 22 7 16 99 13 Hawaii 31 22 11 10 16 90 9 South Carolina 10 3 24 40 16 93 10 Wyoming 42 19 3 9 3 76 5 Alaska 2 39 10 50 27 128 25 New Hampshire 15 15 13 36 27 106 15 Massachusetts 7 34 44 12 8 105 14 Vermont 4 7 31 39 27 108 18 Connecticut 6 14 36 21 37 114 22 District of Columbia 1 15 51 18 27 112 20 North Carolina 18 26 26 16 27 113 21 Colorado 27 37 7 17 47 135 32 Pennsylvania 43 33 15 2 16 109 19 Nevada 29 36 18 18 27 128 25 Kentucky 46 27 17 1 16 107 16 Ohio 18 49 48 3 16 134 31 Arizona 31 46 16 6 51 150 39 Missouri 24 11 21 33 47 136 33 New Jersey 26 39 34 24 8 131 27 Washington 24 43 19 43 16 145 36 Michigan 39 44 27 14 2 126 24 Louisiana 16 12 44 43 16 131 27 Oregon 29 41 12 50 8 140 34 Minnesota 28 50 14 45 8 145 36 Iowa 36 17 22 47 3 125 23 Arkansas 33 30 40 8 37 148 38 Mississippi 45 4 32 13 37 131 27 Oklahoma 46 13 19 26 37 141 35 Indiana 46 28 40 11 8 133 30 Tennessee 40 19 25 36 37 157 42 West Virginia 43 7 36 27 37 150 39 Maryland 35 47 30 30 37 179 48 Florida 37 23 47 30 16 153 41 Delaware 18 48 49 35 47 197 49 Kansas 50 18 38 15 37 158 43 Virginia 34 24 39 42 37 176 46 New York 37 38 43 32 27 177 47 Alabama 41 5 46 34 37 163 44 Georgia 49 44 42 28 8 171 45 Illinois 21 42 50 49 47 209 51 Utah 51 51 27 45 27 201 50 State Fatality Rank Cost Per Fatality Rank DUI Arrests DUI Penalties Types of Laws/Statutes Total Score Rank 2018 Most Dangerous States for Drunk Driving Weighted State Fatality (Weighted Rank) Cost Per Fatality (Weighted Rank) DUI Arrests (Weighted Rank) DUI Penalties (Weighted Rank) Types of Laws/Statutes (Weighted Rank) Total Score Rank Wisconsin 2.80 3.10 2.00 0.60 0.80 9.30 1 North Dakota 5.60 0.20 0.50 3.60 0.80 10.70 2 South Dakota 1.05 0.90 0.25 9.60 0.30 12.10 3 Montana 4.20 0.10 1.00 8.00 0.10 13.40 4 Maine 1.40 2.10 1.50 7.60 2.70 15.30 5 Rhode Island 3.85 2.80 8.25 1.00 0.30 16.20 6 Idaho 8.05 2.50 2.25 4.40 0.30 17.50 7 Nebraska 4.55 3.40 1.25 5.60 2.70 17.50 7 New Mexico 4.55 0.60 7.25 4.60 1.60 18.60 9 Texas 3.15 1.00 8.75 4.80 1.60 19.30 10 California 7.70 3.20 5.50 1.40 1.60 19.40 11 Hawaii 10.85 2.20 2.75 2.00 1.60 19.40 11 South Carolina 3.50 0.30 6.00 8.00 1.60 19.40 11 Wyoming 14.70 1.90 0.75 1.80 0.30 19.45 14 Alaska 0.70 3.90 2.50 10.00 2.70 19.80 15 New Hampshire 5.25 1.50 3.25 7.20 2.70 19.90 16 Massachusetts 2.45 3.40 11.00 2.40 0.80 20.05 17 Vermont 1.40 0.70 7.75 7.80 2.70 20.35 18 Connecticut 2.10 1.40 9.00 4.20 3.70 20.40 19 District of Columbia 0.35 1.50 12.75 3.60 2.70 20.90 20 North Carolina 6.30 2.60 6.50 3.20 2.70 21.30 21 Colorado 9.45 3.70 1.75 3.40 4.70 23.00 22 Pennsylvania 15.05 3.30 3.75 0.40 1.60 24.10 23 Nevada 10.15 3.60 4.50 3.60 2.70 24.55 24 Kentucky 16.10 2.70 4.25 0.20 1.60 24.85 25 Ohio 6.30 4.90 12.00 0.60 1.60 25.40 26 Arizona 10.85 4.60 4.00 1.20 5.10 25.75 27 Missouri 8.40 1.10 5.25 6.60 4.70 26.05 28 New Jersey 9.10 3.90 8.50 4.80 0.80 27.10 29 Washington 8.40 4.30 4.75 8.60 1.60 27.65 30 Michigan 13.65 4.40 6.75 2.80 0.20 27.80 31 Louisiana 5.60 1.20 11.00 8.60 1.60 28.00 32 Oregon 10.15 4.10 3.00 10.00 0.80 28.05 33 Minnesota 9.80 5.00 3.50 9.00 0.80 28.10 34 Iowa 12.60 1.70 5.50 9.40 0.30 29.50 35 Arkansas 11.55 3.00 10.00 1.60 3.70 29.85 36 Mississippi 15.75 0.40 8.00 2.60 3.70 30.45 37 Oklahoma 16.10 1.30 4.75 5.20 3.70 31.05 38 Indiana 16.10 2.80 10.00 2.20 0.80 31.90 39 Tennessee 14.00 1.90 6.25 7.20 3.70 33.05 40 West Virginia 15.05 0.70 9.00 5.40 3.70 33.85 41 Maryland 12.25 4.70 7.50 6.00 3.70 34.15 42 Florida 12.95 2.30 11.75 6.00 1.60 34.60 43 Delaware 6.30 4.80 12.25 7.00 4.70 35.05 44 Kansas 17.50 1.80 9.50 3.00 3.70 35.50 45 Virginia 11.90 2.40 9.75 8.40 3.70 36.15 46 New York 12.95 3.80 10.75 6.40 2.70 36.60 47 Alabama 14.35 0.50 11.50 6.80 3.70 36.85 48 Georgia 17.15 4.40 10.50 5.60 0.80 38.45 49 Illinois 7.35 4.20 12.50 9.80 4.70 38.55 50 Utah 17.85 5.10 6.75 9.00 2.70 41.40 51 State Fatality (Weighted Rank) Cost Per Fatality (Weighted Rank) DUI Arrests (Weighted Rank) DUI Penalties (Weighted Rank) Types of Laws/Statutes (Weighted Rank) Total Score Rank Previous Rankings: View rankings table here and interactive map here and more interactive charts here. Downing a few drinks may sound like an appealing way to kick back and relax, but drunk driving can have fatal repercussions. In 2013, 28.7 million people admitted to driving under the influence of alcohol; that’s more than the population of Texas! Every two minutes, a person is injured in a drunk driving crash. Every day in the United States, another 27 people die as a result of drunk driving crashes. Not only can drunk driving injure you and others, but it can also hurt your bank account and those of your fellow hard-working Americans. Drunk driving costs each American adult over $500 a year and the entire U.S. $132 billion a year. If you’ve ever wondered about the chances of encountering drunk drivers in your state and how much they cost you as a taxpayer, keep reading to learn about the most dangerous states for drunk driving. Review your auto policy every six months and compare three to four policies. Don’t allow the price to be the only factor. Use our FREE comparison tool to get started today! Methodology We based our drunk driving rankings on the following categories: Cost per Fatality by State – Drunk Driving Fatalities per 1,000,000 Population and Taxpayer Subsidy per Drunk Driving Fatality by State – Drunk Driving Fatalities per 1,000,000 Population and Taxpayer Subsidy per Drunk Driving Fatality by State Drunk Driving – Percentage of Fatal Crashes that Involved Alcohol – Percentage of Fatal Crashes that Involved Alcohol DUI Arrests – Arrests of Minors and Adults that were Driving Under the Influence (“DUI”) related per 1,000,000 Population – Arrests of Minors and Adults that were Driving Under the Influence (“DUI”) related per 1,000,000 Population DUI Penalties – State Penalties for 1st-time DUI Offenders and Repeat DUI Offenders ranked against the Nation’s Average Penalties – State Penalties for 1st-time DUI Offenders and Repeat DUI Offenders ranked against the Nation’s Average Penalties Laws/Statutes – Percentage of Drunk Driving Fatalities by State per State’s amount of Drunk Driving Laws and Statutes We weighted the categories below and tallied for a total score. Find out more details about our categories here. The worst states received the lowest score. Category Weight Fatality Rank 0.35 Cost per Fatality by State 0.10 DUI Arrests 0.25 DUI Penalties 0.20 Types of Laws and Statutes 0.10 For example, South Dakota was the state with the worst rating for DUI arrests (total DUI arrests per population) in 2015 and received only seven points for this category. The District of Columbia, however, had the lowest amount of DUI arrests and earned a score of 255. We compiled all of our information using the following sources: Top 10 Most Dangerous States for Drunk Driving Our article will focus on the top 10 most dangerous states for drunk driving. However, you can access the rankings for all 50 states, plus the District of Columbia. Simply click here to view the rest of the data. Although Utah didn’t make this year’s top ten list, it’s worthwhile to note that they are in the process of trying to lower their blood alcohol limit from.08 to.05. That would be the lowest limit in America. That change will be worth watching for next year’s study. #10 - Maine Best Ranking Factor: DUI Penalties: 35th Worst Ranking Factor: DUI Arrests: 5th DUI Penalties: 35thDUI Arrests: 5th Coming in at #10, Maine has the fifth highest amount of DUI arrests. Perhaps that’s why it also ranks higher for DUI Penalties. A penalty for the first offense did not have a mandatory minimum, which contributed to Maine ranking better for DUI Penalties. This state — known for lighthouses, lobsters, L.L. Bean, and famous writers — may not boast much good news when it comes to DUIs, but hopefully next year, we’ll get to kick Maine off the list. #8 - Wyoming (tie) Best Ranking Factor: Fatality Rank: 30th Worst Ranking Factor: DUI Arrests: 3rd Fatality Rank: 30thDUI Arrests: 3rd Wyoming sits right behind North Dakota (2nd place) and South Dakota (1st place) as the worst for DUI arrests. Fifty-four percent of Wyoming’s population (586,107 total population) was arrested for a DUI in 2015. You read that right! Wyoming is also in 4th place for Laws/Statutes. Yikes. Thankfully, their fatalities aren’t as numerous as some other states, but “The Equality State” still has much to improve on when it comes to DUIs. #8 - Idaho (tie) Best Ranking Factor: Cost per Fatality: 28th Worst Ranking Factor: DUI Penalties: 3rd Cost per Fatality: 28thDUI Penalties: 3rd Sliding in at the 24th spot, Idaho also did decently well for Fatality Rank. It could have been much worse! Other than DUI Penalties, Laws/Statutes needs some work, as Idaho placed 7th in this category. Their motto may be, “It is forever,” but hopefully that won’t be the case for DUIs! We would love to see a positive shift in their scores going forward. #7 - Missouri Best Ranking Factor: Laws/Statutes: 49th Worst Ranking Factor: Fatality Rank: 5th Laws/Statutes: 49thFatality Rank: 5th Missouri was one of three states — the other two being South Dakota and Vermont — that all shared one thing in common: no mandatory minimum sentencing for a DUI’s second and third offense. However, the laws against drunk driving don’t seem to be saving lives, which is surprising, as there are only two DUI laws that Missouri doesn’t enforce. Or maybe what Missouri ranks in fatalities is why they have so many laws against drunk driving. “The Show-Me State” shows some promise of inhibiting DUIs, but the people of Missouri need to carefully follow the laws to decrease fatalities. #5 - South Dakota (tie) Best Ranking Factor: DUI Penalties: 47th Worst Ranking Factor: Laws/Statutes, DUI Arrests: 1st In South Dakota, it looks like the laws enforced to fight drunk driving are not making an impact, as SD also has the worst score for DUI arrests. But heavy penalties may make people think twice before driving drunk. After all, this state shares the 47th spot for DUI Penalties with Alaska. Both South Dakota and Alaska have a $4,000 minimum fine for a DUI’s third offense, which is the second highest minimum fine in the country. Indiana and Texas hold the number one spot for the most expensive minimum fine for a third offense at $10,000. #5 - Montata (tie) Best Ranking Factor: Fatality Rank: 27th Worst Ranking Factor: Cost per Fatality: 1st Fatality Rank: 27thCost per Fatality: 1st Montana’s total score of 15 points tied with South Dakota’s. But since this state is the only one that tops North Dakota with a $435.65 taxpayer subsidy, Montana ranks the worst in Cost per Fatality. There may be more daredevils in Montana costing their fellow taxpayers the highest price per fatality in the U.S., which is not surprising considering this state is home to famed daredevil Evel Knievel. #4 - Rhode Island Best Ranking Factor: DUI Arrests: 37th Worst Ranking Factor: Fatality Rank: 1st DUI Arrests: 37thFatality Rank: 1st Rhode Island may only be 1,214 square miles total, but it still packs a punch. People flock to major metro areas like Providence. More people and congested streets could equal trouble. On the downside, Rhode Island tied with Hawaii for first place in fatalities. Unfortunately, Rhode Island also got second place for Laws/Statutes. Although this state is best at keeping down DUI arrests, its people need to brush up on the laws before hitting the road. #3 - North Dakota Best Ranking Factor: DUI Penalties: 22nd Worst Ranking Factor: Cost per Fatality, DUI Arrests: 2nd DUI Penalties: 22ndCost per Fatality, DUI Arrests: 2nd North Dakota might be massive as far as land mass is concerned (the 19th largest in the U.S.), but it only has a total population of 756,927. At a close 21st place, Fatality Rank was North Dakota’s second best ranking factor. But both the Cost Per Fatality and DUI Arrest numbers are too high. As a taxpayer, each North Dakota state resident has to pay an average subsidy estimate of $400.30 because of drunk driving fatalities. North Dakota, please don’t drink and drive! #2 - Wisconsin Best Ranking Factor: Cost per Fatality: 31st Worst Ranking Factor: Fatality Rank: 3rd Cost per Fatality: 31stFatality Rank: 3rd It’s not surprising that Wisconsin holds the second spot in our top ten, considering the role alcohol plays in this state: Copious amounts of alcohol are expected at tailgating parties for the Green Bay Packers. Wisconsinites love brandy. If they plan on sobering up, they switch to beer. Cheeseheads are all about the beer and cheese tastings; there’s no need for the fancy wine and cheese parties. Along with Wyoming and Idaho, “The Badger State” also holds third place in a Worst Ranking Factor. Overall, Wisconsin didn’t do well in the worst ranking categories: It’s also seventh in DUI Penalties and ninth in DUI Arrests. Step it up, guys! #1 - Hawaii Best Ranking Factor: Laws/Statutes: 35th Worst Ranking Factor: Fatality Rank: 1st Now we’ve made it to number one on our list. Unfortunately for Hawaii, this list isn’t for top vacation spots. If you’ve dreamed of saying “aloha” to this vacation destination, keep in mind that you may be in jeopardy while on the road, as Hawaii ranks number one for drunk driving fatalities. Consequently, Hawaii’s best ranking factor is their laws and statutes against drunk driving. The only law they don’t have on the books is an increased penalty for refusing to take a breathalyzer test. This law could help a little, but there’s still much to do to prevent people from driving drunk. Beautiful Hawaii, let’s see some major improvements by next year! Highlights and Trends North Dakota, South Dakota, and Wyoming are neighbors and share the top three of the top four spots for DUI Arrests. It’s worth noting here that these three neighboring states all ranked on last year’s top 10 list. They also shared three out of the top six for DUI Arrests. What’s more, this study from 2016 (see also, this study from 2015 and this study from 2014) have these three states ranked high for alcohol consumption. The most recent study points out cultural factors may be in play as well geography — rural western states tend to drink more beer. We can conclude that the higher rate of consumption is leading to more arrests based on this year-to-year trend. Next, small states with a large population generally have a higher fatality rank. Rhode Island and Hawaii both share the number one spot for Fatality Rank. Rhode Island is the smallest U.S. state in land area, and Hawaii is the eighth smallest state. With a population of 1,431,603 as of 2015, Hawaii is the 13th most populated state, and Rhode Island has a population of 1,056,298. There are 26 states — over half of the U.S. — with no mandatory minimum sentencing for a DUI’s first offense. No mandatory minimum sentencing means that a judge could sentence a defendant to “0 days” or a fine of “$0.” Although this might be rare, the possibility of lower penalties might not discourage drunk driving like we would like. Eight out of the top 10 most dangerous states for drunk driving held first through third places in their worst factors, except for Maine and Missouri, which both got fifth place in their category. Don’t Drink and Drive You can make an impact by choosing not to drink and drive. And if you live in one of these most dangerous states, be vigilant and encourage your friends and family to practice safe driving and — better yet — self-control with alcohol consumption. Here are some tips if you’re drinking with friends or family: Plan ahead by collecting car keys when guest arrive Drink responsibly / pace yourself when drinking Cut off drinks an hour before your gathering ends Always offer non-alcoholic drinks Always have food along with drinks Arrange for alternate transportation (i.e., taxi, Uber, Lyft, designated driver, etc) See the NHTSA’s SafeRide app Offer to let people stay with you if necessary State Farm gives these tips if you’re on the road. Watch for drivers who are: “Making wide turns Weaving, swerving, drifting, or straddling the center line Almost striking an object or vehicle Driving on the wrong side of the road Driving at a very slow speed Stopping without cause Braking erratically Responding slowly to traffic signals Turning abruptly or illegally Driving after dark with headlights off” Call 9-1-1 if you see these signs while driving or if you have a friend who you know is driving under the influence and cannot be immediately stopped. Complete Rankings: Worst on Combating Drunk Driving – To view and sort the table by category, click on header columns. – Click here for the full stats and sources for each category. For all media inquiries, please email: Joshua Barnes Our study implemented weighted measures to demonstrate the impact each manifestation of a category can have on a person, community, and state. Each weight represents the severity of the category. Definitions for each category can be found here. State Fatality (Weighted Rank) Cost Per Fatality (Weighted Rank) DUI Arrests (Weighted Rank) DUI Penalties (Weighted Rank) Types of Laws/Statutes (Weighted Rank) Total Score Rank Hawaii 0.35 2.20 2.50 1.00 3.50 9.55 1 Wisconsin 1.05 3.10 2.25 1.40 2.40 10.20 2 North Dakota 7.35 0.20 0.50 4.40 0.90 13.35 3 Rhode Island 0.35 3.00 9.25 1.80 0.20 14.60 4 Montana 9.45 0.10 1.75 3.40 0.30 15.00 5 South Dakota 4.55 0.70 0.25 9.40 0.10 15.00 5 Missouri 1.75 1.40 5.25 2.40 4.90 15.70 7 Idaho 8.40 2.80 3.25 0.60 0.70 15.75 8 Wyoming 10.50 1.50 0.75 2.60 0.40 15.75 8 Maine 4.55 2.20 1.25 7.00 2.40 17.40 10 South Carolina 1.75 0.40 5.75 7.60 2.20 17.70 11 Arizona 3.85 3.90 5.00 2.20 3.80 18.75 12 Pennsylvania 11.20 2.80 4.00 1.20 0.60 19.80 13 Colorado 7.70 3.60 1.00 4.80 4.00 21.10 14 Ohio 5.95 4.90 6.75 1.40 3.10 22.10 15 Texas 2.80 1.10 9.50 8.20 0.50 22.10 15 North Carolina 16.10 1.90 3.00 0.40 1.10 22.50 17 Nevada 4.20 3.70 8.75 5.00 1.00 22.65 18 Michigan 9.45 4.00 7.25 0.20 2.60 23.50 19 Arkansas 5.25 2.40 10.00 3.00 3.30 23.95 20 Washington 2.80 4.40 4.25 8.00 4.50 23.95 20 Maryland 6.65 4.60 7.75 4.00 1.40 24.40 22 Louisiana 2.45 0.80 12.00 7.20 2.10 24.55 23 New Mexico 16.10 1.00 2.75 3.60 1.20 24.65 24 Tennessee 8.40 1.50 6.25 5.60 3.00 24.75 25 Nebraska 12.60 3.30 5.50 3.00 1.70 26.10 26 Massachusetts 3.50 3.80 11.50 7.40 1.30 27.50 27 Oklahoma 15.40 1.20 7.00 1.80 2.90 28.30 28 Mississippi 10.15 0.30 9.00 5.20 3.70 28.35 29 New Jersey 5.95 4.00 8.25 5.40 5.00 28.60 30 Minnesota 10.85 5.00 1.50 9.20 2.20 28.75 31 California 11.90 3.40 3.75 6.20 3.60 28.85 32 Kentucky 17.50 2.40 4.75 0.80 3.40 28.85 32 Connecticut 6.65 0.80 10.25 8.80 2.60 29.10 34 New Hampshire 11.55 1.50 3.50 8.40 4.20 29.15 35 Vermont 17.15 2.10 4.50 3.80 1.80 29.35 36 New York 8.40 3.40 11.00 2.80 3.90 29.50 37 West Virginia 12.60 0.60 8.00 4.20 4.30 29.70 38 Oregon 5.25 4.20 8.50 10.20 2.00 30.15 39 Alabama 7.70 0.50 11.25 6.60 4.10 30.15 39 Alaska 14.35 4.60 1.75 10.00 0.80 31.50 41 District of Columbia 11.90 1.30 12.75 4.40 1.90 32.25 42 Illinois 1.40 4.30 12.50 9.80 4.40 32.40 43 Kansas 13.65 1.80 7.50 6.20 4.70 33.85 44 Virginia 13.30 2.40 9.75 7.80 1.50 34.75 45 Iowa 16.10 1.90 6.25 9.00 1.60 34.85 46 Georgia 14.35 4.40 10.75 5.80 3.10 38.40 47 Utah 17.85 5.10 6.00 5.80 5.10 39.85 48 Florida 14.00 2.70 11.75 8.60 4.80 41.85 49 Indiana 14.35 3.20 10.50 9.40 4.50 41.95 50 Delaware 15.40 4.80 12.25 6.80 2.80 42.05 51 This photo released by the Iron County Sheriff's Department in Michigan shows a car driven by a 19-year-old man that crashed into a tree on April 21, 2013. Investigators said the driver was drunk and speeding. (Photo: AP) Fueled by higher-than-average alcohol consumption and plenty of bad weather, Northern states in the Midwest and West are the most dangerous for drunken driving, according to a survey obtained by USA TODAY. North Dakota ranked first in fatalities and in driving-under-the-influence arrests in 2015, according to the study by CarInsuranceComparison.com, a site that allows people to compare insurance companies. Montana was second, with the highest cost per fatality and types of laws, according to the report. Idaho, Wisconsin, South Carolina and South Dakota rounded out the riskiest states for impaired driving, according to the study. “I think that the combination of higher than average alcohol consumption and a higher chance of running into dangerous driving conditions with sleet, snow, and ice during the winter months could be the reason that we're seeing so many of those northern states rank poorly,” Tyler Spraul, who directed the study, told USA TODAY. The Dakotas, Idaho and Wisconsin each ranked among the highest consumption of alcohol in 2009, according to a study by the National Institute on Alcohol Abuse and Alcoholism. In North Dakota, state police and sheriffs are cracking down on underage drinking during the season for prom and graduation this year.” “If we find them consuming or in possession of alcohol, they could end up in court and ordered to pay fines,” Bismarck Police Lt. Jason Stugelmeyer said this month. Montana is focused on discouraging impaired driving, after having 33 people die in traffic accidents during January, February and March, which double the number during the same period in 2015. Nearly three-quarters of the state's fatalities during the last decade were because of impaired driving or failing to use seatbelts, according to the state Transportation Department. The percentage of adults who reported driving after drinking too much during the previous month was 3.4% in Montana, compared to 1.9% nationwide, the Centers for Disease Control and Prevention said from a 2012 survey. “We are seeing an early surge in fatalities with every indication that things could get worse,” Mike Tooley, state transportation director, said last month. Meanwhile, Utah, where more than two-thirds of the residents are Mormons who eschew alcohol, ranked lowest for drunken-driving incidents, according to the study. That state was followed closely by Indiana and Florida, which tied, Georgia and Minnesota. The rankings were based on statistics from the FBI, the National Highway Traffic Safety Administration, the advocacy group Mothers Against Drunk Driving, the Kids Count Data Center and DrivingLaws.org. For example, Mothers Against Drunk Driving reported a $132 billion cost in 2009 from drunken-driving incidents, with about half based on monetary costs and the rest on quality-of-life losses, based on research from the Pacific Institute for Research and Evaluation. The categories that contributed to CarInsuranceComparison.com's rankings included: --Driving fatalities, which counted for 35% of a state’s score. The category counted motorists with a blood-alcohol content of 0.08% or higher, which violates all state laws, and a lesser amount of alcohol. --Arrests for driving-under-the-influence, which counted for 25% of a state’s score. The category included minors and adults at least 18 years old, divided by the population. --Penalties for driving-under-the-influence, which counted for 20%v of a state’s score. The category was based on jail time, license suspensions, and maximum fines for first, second and third offenses. --The cost per fatality, which counted for 10% of a state’s score. --Types of laws to discourage drunken driving, which counted for 10% of a state’s score. Read or Share this story: http://usat.ly/26w26Kh

Be careful on the roads in North Dakota. The state is the most dangerous in the country when it comes to drunk driving, according to an analysis by CarInsuranceComparison.com. It considered five factors: a state's drunk-driving-related deaths and arrests in 2015, its penalties and laws, and its cost per fatality; each measure was then weighted, with fatality rank being the most prominent factor (see full methodology here). North Dakota was followed by Montana, Idaho, Wisconsin, South Carolina, and South Dakota. Why? Tyler Spraul, the study's director, cites "higher than average alcohol consumption," for one. Indeed, USA Today flags a study by the National Institute on Alcohol Abuse and Alcoholism that found North and South Dakota, Idaho, and Wisconsin were among 2009's biggest alcohol consumers. Spraul further tells USA Today "a higher chance of running into dangerous driving conditions with sleet, snow, and ice during the winter months could [also] be the reason that we're seeing so many of those northern states rank poorly." Potentially undermining that argument: The wintry states of Alaska and Minnesota ranked 46th and 47th, respectively. They were followed by Georgia, Indiana, and Florida, with Utah 51st (the District of Columbia was included). See where your state ranks here.

multi_news

"THE INVASION" BY DERRICK SHERWIN FROM A STORY BY KIT PEDLER First broadcast - 16th November 1968 Running time - 23 minutes 44 seconds [SCENE_BREAK] 1. WAREHOUSE (PACKER watches the guards load the unconscious forms of ZOE and ISOBEL into two of the crates.) PACKER: Load it onto the return van. (JAMIE sees the guards pick up the crates.) JAMIE: Zoe's there! (JAMIE starts to charge out of his hiding place.) DOCTOR: (Dismayed.) No, Jamie, wait. (JAMIE rounds the corner and tackles PACKER to the floor.) PACKER: (Shouting.) GUARDS! GUARDS! (JAMIE lets go of PACKER and he and the DOCTOR start to run away. Meanwhile, the other guards and some friends of theirs hear PACKER's cries and turn towards them. The DOCTOR tries to lead JAMIE away but their way is blocked by more guards. PACKER draws his revolver as he pulls up off the floor and gloats at the DOCTOR and JAMIE, and their raised hands of surrender.) PACKER: Like rats in a trap... You really don't learn, do you? This is private property. A restricted area. JAMIE: Where's Zoe? Where have you taken her?! PACKER: Be quiet! JAMIE: Look, we heard them scream. If you hurt her... PACKER: BE QUIET! (JAMIE lunges for Packer's throat, but PACKER catches JAMIE and easily hurls him back across the wall. PACKER intends further damage to JAMIE's well-being until VAUGHN steps into the room.) VAUGHN: Packer! You really must try and control this violent streak in your nature, Packer. Although I must admit, the situation is provoking. (To the DOCTOR and JAMIE.) So here you are again. You really are beginning to try our patience, you know. JAMIE: (Nodding at PACKER.) And he's beginning to try mine. DOCTOR: Jamie, Jamie. (To VAUGHN.) We came here looking for two friends of ours. VAUGHN: Two young ladies? Yes? JAMIE: So he admits they are here. VAUGHN: (Laughs.) Ha ha. Correction - they were. It would seem that you have been chasing each other's tails. They came here - looking for you. DOCTOR: Where are they now? VAUGHN: Why, they left of course. JAMIE: Aye, carted off by two of your thugs in a box. (VAUGHN smiles.) VAUGHN: Oh really... JAMIE: Look it's true. We heard them scream and I saw some of Zoe's clothes trapped in the lid. VAUGHN: You really do have a very fertile imagination, young man. JAMIE: Now listen you, I'm telling... I definitely saw those... DOCTOR: Jamie, Jamie. (To VAUGHN.) It would set our minds at rest if we could take a look inside those boxes. VAUGHN: Then you shall. (To PACKER.) Packer, I assume the only crates going out of here are the empty ones being returned to the factory. PACKER: Yes sir, they're loading now, sir. VAUGHN: Then you shall inspect them at your leisure. After you. DOCTOR: How kind. (VAUGHN gestures for them to lead the way. On the way out, he turns and signals to PACKER. PACKER turns on a small wristwatch radio he is wearing.) PACKER: This is Packer. Get the return van moving - immediately. Do you understand? Immediately! [SCENE_BREAK] 2. OUTSIDE (The DOCTOR, JAMIE, and VAUGHN emerge into open air to find they've just missed the train carrying the boxes.) VAUGHN: Ah, just too late, I'm afraid. Such a pity. Still, all is not lost - I am myself going down to the factory today. Care to join me? We can meet the train on its arrival. DOCTOR: That's very civil of you. VAUGHN: My pleasure. (The three turn back into the building.) [SCENE_BREAK] 3. UNIT AEROPLANE (The BRIGADIER is on the radio to his surveillance crews.) BRIGADIER: How long ago did they go in? BENTON: (OOV.) Some time ago, sir. Tracy followed them as far as the official boundary of the railway sidings. They went in about an hour ago. BRIGADIER: Is Tracy still round there? BENTON: (OOV.) Yes sir. BRIGADIER: Right, stick around for a while... BENTON: (OOV.) Just a minute, sir. BRIGADIER: What is it, Benton? BENTON: (OOV.) The Doctor and the boy. They're coming out sir... with Vaughn. [SCENE_BREAK] 4. CAR (The DOCTOR and VAUGHN climb into VAUGHN's very large private car. JAMIE darts round the car and takes the front seat, to the annoyance of PACKER, who wanted it.) [SCENE_BREAK] 5. UNIT AEROPLANE BRIGADIER: Benton, what's happening? WHAT'S HAPPENING? BENTON: (OOV.) Oh, sorry sir. Nothing really, they've all got into Vaughn's private car... oh, they're driving off, sir. BRIGADIER: Is any force being used? BENTON: (OOV.) No, not that I can see. Packer was there but it looked very friendly to me. Shall we follow? BRIGADIER: No, I have them tracked more discretely. Over and out. (He signs off, and then turns to Sergeant WALTERS.) BRIGADIER: Sergeant Walters. WALTERS: Yes sir? BRIGADIER: Alert all area patrols in section three. (He turns to TURNER.) BRIGADIER: Did you hear all that, Jimmy? WALTERS: Yes sir. BRIGADIER: Look, we'll get you aboard a chopper and you can rendezvous with the tracking agent. WALTERS: All right, sir. Are there any specific instructions? BRIGADIER: No, we're playing a bit by ear for the moment, so try to keep out of trouble. Should the Doctor contract us for help, I have his signal relayed straight to you. WALTERS: Right, sir. (He walks off towards the exit.) [SCENE_BREAK] 6. IE'S COUNTRY HQ (VAUGHN's car drives to the main gate of the countryside IE compound the DOCTOR, ZOE and JAMIE left earlier. The guards open the gates and allow the car to drive through. As they close the gates, they see a helicopter hovering nearby, watching them.) [SCENE_BREAK] 7. HELICOPTER (Inside the chopper, the Captain is on the radio.) TURNER: They've just gone into the IE area, sir. Should we follow? [SCENE_BREAK] 8. UNIT AEROPLANE BRIGADIER: No, circle the area. We can't really do anything until we get a request from the Doctor. TURNER: (OOV.) Standing by, sir. BRIGADIER: Oh, and Jimmy, keep out of sight. If Vaughn's private army see you, they might have the wind, and they might make things rather unhealthy for the Doctor and the boy. Over and out. [SCENE_BREAK] 9. IE'S COUNTRY HQ (VAUGHN's car parks next to the main building and PACKER, VAUGHN, JAMIE, and the DOCTOR climb out. The security guards in front of the building all give a loud militaristic salute as VAUGHN passes them.) [SCENE_BREAK] 10. LIFT (Inside the building, VAUGHN leads the party to the end of a corridor and a lift.) JAMIE: Hey, what's happening? I thought we were looking for Zoe and Isobel. VAUGHN: All in good time, young man. The train on the indicator will take some time to get here. In the meantime, I'd rather like to talk to you. DOCTOR: Oh really? VAUGHN: Yes... Those circuits you gave me, they're rather fascinating. I'd like to know more about them. (The door opens.) VAUGHN: This way gentleman. DOCTOR: Thank you. VAUGHN: Packer. (When the lift reaches its destination, the four people file out, but VAUGHN and PACKER lag behind - PACKER in the doorway, VAUGHN in the corridor.) VAUGHN: Be a good fellow and see if Professor Watkins is finished, will you? PACKER: Very good, Mr. Vaughn. (The lift door starts to close but PACKER stops it.) VAUGHN: You might offer him a little encouragement. (To the DOCTOR and JAMIE, walking off.) Coming gentlemen. (PACKER climbs back into the lift and smiles.) [SCENE_BREAK] 11. VAUGHN'S OFFICE (JAMIE, the DOCTOR, and VAUGHN enter VAUGHN's office. JAMIE gapes in astonishment because this office is exactly the same as the one in London - the video screens, the swirly patterned carpet, the desk, the window (with a different view, of course), everything.) JAMIE: Hey!? Doctor, it's... VAUGHN: Confusing, isn't it? JAMIE: Exactly the same as your office in London. VAUGHN: In all basic assertions - yes it is. That's the secret of my success, you see: uniformity, duplication. My whole empire is based on that principle. The very essence of business efficiency. Do sit down. DOCTOR: Oh, how kind. (The DOCTOR sits down - JAMIE turns and stares out of the window at the different view.) VAUGHN: I must say I'm rather glad that we have this opportunity to talk. DOCTOR: Really? VAUGHN: I should of course be angry with you. You've thwarted my elaborate security precautions twice. I'd like to know why. DOCTOR: Oh, that's quite simple. I hate computers, and refuse to be bullied by them. VAUGHN: Your young friend Zoe... JAMIE: Well, what about her? VAUGHN: She appears to have the same instincts. She so confused one of my computers, she ruined its memory store. JAMIE: (Turning from the window.) So that's why your thugs dragged her and Isobel away. VAUGHN: My dear young man, I wasn't angry with her. On the contrary, I found the incident quite amusing. She's a remarkable girl. And you, Doctor, are obviously a man of no mean scientific ability. DOCTOR: Why do you say that? VAUGHN: Oh, my research department found these... (He pulls the TARDIS circuits from a breast pocket.) ...quite fascinating, particularly the illogical factor in their construction. Are they your own invention? (The DOCTOR gives a noncommittal answer, with his eyes fixing themselves on something interesting on the ceiling.) VAUGHN: Ah, you're determined to guard your secret, I see. (He puts the circuits down.) VAUGHN: I don't blame you. I promise not to pry any more. In fact, I'll do all I can to help you. DOCTOR: Oh, how kind. VAUGHN: Now you say you came here looking for Professor Watkins because you thought that he might be able to help you. DOCTOR: Yes. VAUGHN: Then I'll "try" to persuade him to put aside his work and um... concentrate his efforts on your behalf. Make yourself at home. (VAUGHN stands and walks out of the room.) JAMIE: Look, you're not going to trust him, are you? What about Zoe and Isobel? DOCTOR: I haven't forgotten them, Jamie. JAMIE: They're in that box, I'm sure of it. DOCTOR: Jamie, we're not going to help Zoe by annoying Mr. Vaughn, are we. JAMIE: I don't think you can annoy that man even if you want to. He's being as nice as pie. DOCTOR: He's too nice. JAMIE: Why should he be? DOCTOR: I don't know. He was a little too interested in these circuits for my liking. JAMIE: Hey, you think he knows about the TARDIS then? DOCTOR: (Surprised.) Well, I don't see how he could do. JAMIE: Perhaps this Professor Watkins will be able to tell us what's going on around here. DOCTOR: Yes, that's what's worrying me. JAMIE: What? DOCTOR: If... if Vaughn has anything to hide, why is he letting us talk to Watkins. [SCENE_BREAK] 12. LABORATORY (PACKER is talking with a bearded man (WATKINS).) PACKER: I'd think about it if I was you, Professor. After all, she is a pretty girl. It'll be a shame to spoil all that. WATKINS: What a vicious sadist you are, Packer. I don't believe you anyway. PACKER: I don't make idle threats, Professor, as you well know. If you want to see that girl again in one piece, I suggest you do as Mr. Vaughn asks. WATKINS: How do I know you haven't already harmed her. If you got her... (VAUGHN enters the room.) VAUGHN: You can take my word for that, Professor. WATKINS: Your word. (Laughing.) Oh, I know how much value I can put on that. VAUGHN: Harsh words, Professor, harsh words. (To PACKER.) Any progress? (VAUGHN crosses the laboratory to examine a tangle of technology on the table.) VAUGHN: So you haven't yet completed the machine. WATKINS: No, and I don't intend to. VAUGHN: Oh I think you will, Professor. Much as I detest violence... I find it difficult to restrain Packer's... er... indisputable talent for persuasion. Your niece is in our custody, and unless you do as I ask, I shall hand her over to Packer. The choice is yours... WATKINS: (Thinks.) If I do co-operate, will you let her go? VAUGHN: (Laughs.) Oh no, she's our guarantee, but she'll come to no harm. WATKINS: All right. But I must see Isobel first. VAUGHN: Of course. Oh, there is just one other thing. (WATKINS looks hard at him.) VAUGHN: Don't look at me so suspiciously, my dear Professor. It's merely that there are some friends of yours that wish to see you. WATKINS: Friends? VAUGHN: (Purrs.) Yes, they're going to great pains to seek you out. WATKINS: But I haven't been allowed visitors since I came here. Why are you suddenly considerate? I might tell them everything. VAUGHN: Everything? My dear Professor, you know nothing that can harm me. And besides, there's always Isobel to consider, isn't there. PACKER: Or would you rather leave her to me. VAUGHN: Now, now, Packer. The Professor's going to do exactly as we ask, aren't you. (WATKINS looks totally defeated.) [SCENE_BREAK] 13. VAUGHN'S OFFICE (The DOCTOR and JAMIE are looking out the window in VAUGHN's office at something interesting in the compound.) JAMIE: Can you see? DOCTOR: Yes, Jamie, I can see. Let's have a closer look at that, shall we? (The DOCTOR takes an extending telescope from a pocket and rests it on JAMIE's shoulder to get a better look.) DOCTOR: Now just hold still a minute. Keep your shoulder still. (Through the lens he sees three large white spheres resting on buildings.) DOCTOR: That's odd. That's very odd. JAMIE: What is it? DOCTOR: It looks like a deep-space radio communication system. What's it doing here? JAMIE: Don't ask me. You're the brains. (JAMIE then sees something else.) JAMIE: Hey, a helicopter. Could it be the Brigadier's lot? (He hears something.) JAMIE: Shh. (Understanding the meaning, the DOCTOR and JAMIE turn round just in time to see PACKER enter.) PACKER: Come on. (The DOCTOR and JAMIE pass him to leave the room.) [SCENE_BREAK] 14. UNIT AEROPLANE (Sergeant WALTERS is manning the radio.) WALTERS: Hold on, sir, he's just here. (He turns as the BRIGADIER approaches.) WALTERS: Sir, it's Captain Turner. BRIGADIER: (Into the microphone.) Yes, Jimmy. Any news? TURNER: (OOV.) No, sir. Not a sign of them. BRIGADIER: Right, stay in the area. We daren't make a move until we hear from the Doctor. Over and out. [SCENE_BREAK] 15. LABORATORY (WATKINS, the DOCTOR, and JAMIE are alone in WATKINS' lab.) WATKINS: Yes, of course, I remember Anne Travers telling me about you. She was a student of mine, you know, some years ago - brilliant girl, brilliant. DOCTOR: Yes, yes, they... they went to America, didn't they? WATKINS: Oh, Travers was getting past it, a little you know, and Anne had persuaded him to go with her. DOCTOR: Oh yes. (The DOCTOR looks around the room, still nervously and suspiciously.) WATKINS: What are you doing here? DOCTOR: Well, it's a long story... [SCENE_BREAK] 16. VAUGHN'S OFFICE (VAUGHN stands in his office, confiding events to PACKER.) VAUGHN: This strange Doctor has been on another planet, Packer. PACKER: That's not possible! VAUGHN: It is a fact, Packer. He has some sort of machine. Now let's see if we can learn more about it shall we? Switch on, Packer... with sound. (PACKER throws a switch, and a view of the lab and the DOCTOR's voice appear on the monitors in the room, and Packer smiles as he realises this is the reason VAUGHN wanted them to talk alone.) DOCTOR: (OOV.) ...so we went to see Mr. Vaughn. He said you were working on something important. PACKER: I see... That's why you want to leave them alone together. VAUGHN: Exactly, Packer. [SCENE_BREAK] 17. LABORATORY (The DOCTOR has spotted something on a wall.) JAMIE: But the Doctor needs some help with some electronic circuits that the TARDIS has. WATKINS: Really? JAMIE: Aye, aye... DOCTOR: Jamie, Jamie... I don't think the Professor wants to hear about that. WATKINS: On the contrary, from what Anne told me of the TARDIS, it's most intriguing... I'd like to hear a lot more about... DOCTOR: Professor, I... think that I should warn you that Anne Travers allowed her imagination to run a little wild. WATKINS: (Turning to JAMIE.) You mean to say that there is no such machine. JAMIE: Of course there is... DOCTOR: Jamie, I think that there are other more important things to talk about at the moment. (While saying this, the DOCTOR points to the camera, from out if its shot. JAMIE and WATKINS catch on.) JAMIE: Oh, I see, yes. DOCTOR: Professor, tell us about what you're doing. (The DOCTOR fumbles in his pockets for something.) WATKINS: Oh, it's very simple, really. I've been developing some new kind of teaching Machine. DOCTOR: Oh yes? A teaching machine? JAMIE: What's it called? WATKINS: I call it the Cerebraton Mentor. The main difference from the other teaching machines is that it is able to induce emotional changes in the subject. JAMIE: Oh. (The DOCTOR suddenly places a small piece of metal up against the camera's box.) [SCENE_BREAK] 18. VAUGHN'S OFFICE (The picture and sound from the monitor disappear, to be replaced by static.) PACKER: Could it have broken down? VAUGHN: Let's check. (VAUGHN, via the controls on his desk, checks other cameras to see if it's just the screen that's broken down. All the others are working perfectly.) VAUGHN: All the other monitors are functioning. PACKER: Yeah? VAUGHN: Our friend, the Doctor, is a resourceful man. No wonder our allies fear him. PACKER: (Surprised.) Our allies know him?! VAUGHN: Oh yes, I was ordered to destroy him. First I must know the secrets of this extraordinary machine of his. PACKER: If you've been ordered to destroy him... (VAUGHN rounds on PACKER and shouts loudly:) VAUGHN: I DON'T TAKE ORDERS. I GIVE THEM! Now, I think the time has come to stop playing games with this Doctor! (He turns to walk out of the office.) [SCENE_BREAK] 19. LABORATORY (The DOCTOR is looking over the machine.) WATKINS: But what can we do? If, as you say, they have Isobel and your young friend, then we are entirely at their mercy. DOCTOR: Oh no, not entirely. There's my friend the Brigadier, remember. WATKINS: Do you think he can help us? DOCTOR: Possibly. But quickly, what is Vaughn up to? What's he doing here? WATKINS: I don't know more than you do. He's a ruthless man, without morals or principles. His object, I'm sure, is to get complete control of the electronics industry of the world! DOCTOR: (Faintly.) I wonder... I've got a nasty feeling that he's aiming somewhat higher than that... (JAMIE, listening at the door, turns rapidly about.) JAMIE: Doctor, quick, somebody's coming. (Before the DOCTOR can undo his work to the camera, VAUGHN enters.) VAUGHN: Don't bother, Doctor. Allow me. (VAUGHN looks at the piece of metal on the camera.) VAUGHN: Very ingenious. A simple magnet. I congratulate you. (He hands the magnet back to the DOCTOR.) DOCTOR: (Staring straight ahead.) Oh how kind. VAUGHN: However, you must surely realise that it has forced me to consider other methods to make you talk. JAMIE: What are you going to do? VAUGHN: Your friend, Zoe, is due to arrive here shortly. JAMIE: So you have got her! Listen, if you have harmed her in any way... (He moves forward to grab VAUGHN but PACKER and a guard grab him.) VAUGHN: You may still be adolescent enough to make idle threats, young man, but I can assure you, I am not. (To the DOCTOR.) I want your travel machine, Doctor. Otherwise, I shall hand your friend Zoe over to Packer. It's a simple choice, and it shouldn't take you longer than an hour to consider. Take them away, Packer. (PACKER escorts the DOCTOR and JAMIE, giving them a little push as they pass him.) DOCTOR & JAMIE: All right, all right. PACKER: (To the guard.) Leave them to me, I'll take them. VAUGHN: Now, Professor, I suggest you continue with your work. [SCENE_BREAK] 20. CORRIDOR (The DOCTOR, JAMIE and PACKER are walking down the corridor.) DOCTOR: Oh dear. You know Jamie, I've always been rather scared of lifts. JAMIE: Why? DOCTOR: I don't even like to start them. You'll have to push the button for me. PACKER: Keep your mouth shut and do as you're told. DOCTOR: (Significantly.) Yes, Jamie. Do as you're told. (JAMIE nods recognition and steps into the lift.) JAMIE: Oh, I see... PACKER: Be quiet! (The DOCTOR suddenly turns round.) DOCTOR: Packer! Mr. Packer, I obviously can't let you harm Zoe, so I'll have to tell you all you want to know - now. PACKER: You mean, you're willing to talk? DOCTOR: Yes, I'm sure Mister Vaughn... (Looking over PACKER's shoulder.) Oh, there he is now... (PACKER turns round to look at VAUGHN, but VAUGHN isn't coming. The DOCTOR instantly pushes Packer to the floor...) JAMIE: Quick, Doctor. (...and leaps into the lift with JAMIE, who instantly closes the door and starts the lift.) [SCENE_BREAK] 21. LIFT DOCTOR: Your knife... Give me your knife. (JAMIE gives him his knife. He uses it to prise the cover off the lift's control panel.) JAMIE: What are you going to do? DOCTOR: Try to break the circuit. (The DOCTOR looks set to yank the wires.) JAMIE: What would that do? DOCTOR: It will either stop the lift, or send us out of control. JAMIE: But we're six floors up. DOCTOR: Well, we've got to take that risk. Hold tight, here we go. (He pulls the wires and the lift shudders to a sudden stop.) [SCENE_BREAK] 22. VAUGHN'S OFFICE (PACKER is reporting the escape to VAUGHN in the office, and VAUGHN is not at all pleased.) VAUGHN: Why are you so gullible, Packer? PACKER: Just wait until I get a hold of them... I do... VAUGHN: You'll do nothing! I want them in one piece. (A call comes over PACKER's wrist radio.) PACKER: Packer here. Have you got 'em? (He listens.) PACKER: Well, what happened!? (Listens again.) PACKER: STUCK! HOW!? (Listens.) PACKER: WELL, GET ON WITH IT MAN! (To VAUGHN, hardly able to believe what he is saying.) The lift is struck between the fourth and fifth floors. VAUGHN: So I gathered, Packer. PACKER: Well, it's obviously a mechanical fault... (He breaks off, while VAUGHN looks on.) PACKER: Wait a minute... HE did it! (Referring to the DOCTOR.) VAUGHN: Yes, Packer. Our clever Doctor has outwitted you. Oh, then that wouldn't be too difficult, would it? PACKER: Well, it won't do him much good, will it? After all, he can't get anywhere, can he? VAUGHN: No, but then why do it? (He thinks to himself.) VAUGHN: He might be playing for time. But I doubt it. [SCENE_BREAK] 23. LIFT DOCTOR: There was a fifty-fifty chance, Jamie. JAMIE: Yes, but what good has it done us. I mean, we're stuck here in this lift now. DOCTOR: No, no it's the lift that's stuck. Not us. Look! (He draws JAMIE's attention to the emergency door at the top of the lift.) JAMIE: Hey, where does that lead to? DOCTOR: Out into the lift shaft, I imagine. Quickly, on your back. (He bends down to allow JAMIE to climb up to it.) JAMIE: You know something? You're a clever wee chappie! [SCENE_BREAK] 24. CORRIDOR (PACKER is talking into the wrist radio whilst surrounded by guards.) PACKER: Switch over to the emergency circuits. I want that lift operational in three minutes! (He turns to speak to the guards assembled in front of him.) PACKER: I want all lift exits covered. I want a man on each floor. MOVE! (The guards do so - rather fast. When they clear, we can see VAUGHN approaching PACKER.) VAUGHN: Don't panic, Packer. Our birds can't fly away. [SCENE_BREAK] 25. LIFT SHAFT (JAMIE emerges onto the top of the lift and sees that they have a long climb ahead of them. He then helps the DOCTOR to scramble out of the lift.) DOCTOR: Thank you, Jamie. (He looks up at the huge shaft.) DOCTOR: Oh my word! Well, that's a long way up, isn't it? Oh well, we'd better get cracking before they realise what's happened. First I'd better shut this. (He then closes the emergency door, and then leads the way up the ladder on the side of the lift shaft.) JAMIE: Hey, Doctor? I've just been thinking. What happens if they get the lift going before we reach the top? DOCTOR: Oh, that's simple. JAMIE: What? DOCTOR: We get squashed. JAMIE: What! Well, come on! (They start to climb - the DOCTOR first, and then JAMIE.) [SCENE_BREAK] 26. CORRIDOR (PACKER gets a report on his wrist radio.) PACKER: (Into radio.) Right! (To VAUGHN.) They've switched over the circuits. The lift's working! VAUGHN: (Bored.) How splendid! Bring it up here. PACKER: (Into the wrist radio.) Bring it up to the sixth floor. [SCENE_BREAK] 27. LIFT SHAFT (As they climb, JAMIE hears a clanging noise.) JAMIE: Hey, that's not what I think it is, is it? DOCTOR: I'm very much afraid it is. Come on! (They start climbing faster, but JAMIE looks down and sees that the lift is rising quickly.) JAMIE: We'll never make it! DOCTOR: Yes, we will! Come on! (The lift suddenly stops.) JAMIE: It stopped! DOCTOR: They must be checking it. Come on, they may guess where we are and come after us. [SCENE_BREAK] 28. LIFT (The lift doors open and PACKER enters, gun ready. He is followed by VAUGHN.) PACKER: They vanished! They just vanished! VAUGHN: (Roaring.) CHECK THE OTHER FLOORS! (PACKER steps outside the lift and speaks into his radio.) PACKER: This is Packer. Check all the lift exits and get me the engineer. (He listens to the reply.) PACKER: Hello? Yes? (He listens.) PACKER: Did the lift stop at any other floors on the way up? (He listens again.) PACKER: You're certain. (And again, and then turns to VAUGHN.) PACKER: Right, the engineer's certain... VAUGHN: (Cutting him off.) The shaft terminates in the roof, doesn't it, Packer? PACKER: Yes, sir. VAUGHN: Why didn't you think of that? PACKER: (A little fearfully.) I'll get them! VAUGHN: (Angrily.) Call me when you do, Packer. I'll be in my office, and PLEASE DON'T FAIL THIS TIME, THERE'S A GOOD FELLOW. (VAUGHN walks out. PACKER talks into the radio again.) PACKER: This is Packer. Get some men on the roof. They're still in the lift shaft. (He listens.) PACKER: NO! Don't take the lift. Those two might still be... (He stops and thinks.) PACKER: Hey, wait a minute! (Into the radio.) I'll take the lift. Tell the engineer to take it right to the top. (The lift doors close, and PACKER stands in the centre of the lift with his arms folded, with a grim/furious look on his face.) [SCENE_BREAK] 29. LIFT SHAFT (The DOCTOR and JAMIE are nearing the top of the shaft. On the wall is chalked "Kilroy Was Here!".) JAMIE: Are we nearly there, Doctor? (The lift starts moving again.) JAMIE: It's started again! DOCTOR: Hurry, Jamie! Hurry! (They continue to climb faster and faster.) [SCENE_BREAK] 30. ROOF (The DOCTOR and JAMIE climb out through a door onto the open rooftop of the IE building. They crawl on the ground for a little bit before they straighten up on their knees. Both are exhausted from the climb.) DOCTOR: Come on, Jamie. JAMIE: Wait a minute, Doctor. DOCTOR: No, come on. Let's see where we are. (They stand up and walk over to the edge. The DOCTOR looks down.) JAMIE: Now where? DOCTOR: Down there. (JAMIE looks six floors down.) JAMIE: Oh no! DOCTOR: We can't go down the lift again, can we? Hello. (He spots something that we can't see.) DOCTOR: There's a fire escape. Come on. (They run over to it and the DOCTOR begins climbing down. JAMIE swallows and follows.) [SCENE_BREAK] 31. VAUGHN'S OFFICE (PACKER is reporting the DOCTOR's escape.) VAUGHN: Oh, Packer, you do disappoint me. PACKER: They must have gone down the fire escape. VAUGHN: And, of course, you didn't think to have a guard on that! PACKER: Well, normally... (VAUGHN shouts him down.) VAUGHN: YOU'RE A STUPID INCOMPETENT! I WANT THAT DOCTOR! PUT THE WHOLE COMPOUND ON ALERT! HAVE EVERY AVAILABLE GUARD ON THE JOB! FIND HIM PACKER! FIND HIM! [SCENE_BREAK] 32. TRAIN STATION (The DOCTOR and JAMIE hide in an alleyway formed by a wall and a railcar. They crouch down and crawl under the train, hiding from guards passing by. They come up from the other side and then climb into the door of the car, closing it behind them, while another alarm starts.) [SCENE_BREAK] 33. RAILCAR (The railcar is filled with crates like the ones which ZOE and ISOBEL were in.) JAMIE: Hey, Doctor. Do you think that this is the train that Zoe and Isobel came on? Vaughn said that one had empty crates on it, didn't he? DOCTOR: Yes. JAMIE: Well, we'll soon see. Let's peak inside. (They push one of the crates open, and to JAMIE's disappointment, it's full of a cobwebby substance.) JAMIE: Oh no, these are full. (PACKER's voice comes from outside.) PACKER: (OOV.) I want all these trains searched from top to bottom! MOVE! DOCTOR: Jamie! Hide! Quickly, Hide! (The DOCTOR dashes off to the left, off-screen, and JAMIE opens up the box to climb inside and hide beneath the lid - just in time, for as JAMIE's lid closes, the door is opened and light is thrown into the railcar.) [SCENE_BREAK] 34. CRATE (JAMIE lies next to something covered by a sheet and he hears PACKER's voice.) PACKER: (OOV.) You two, search this one. (JAMIE lies still nervously, and then even more nervously as he feels something shift beneath him. He turns and looks and sees that the cobwebby substance he's on top of is stirring silently...)

The Doctor and Jamie return to the IE factory in search of Zoe and Isobel, where they meet with Professor Watkins and fall foul of Vaughn.

summ_screen_fd

Egypt's embattled revolution faces one of its sternest tests on Saturday, as the country goes to the polls to elect its first post-Mubarak president amid a whirlwind of popular disillusionment and an unprecedented constitutional crisis. As heavyweights from across the political spectrum queued up to condemn what they described as a "blatant coup" by the old regime – a reference to three critical legal moves over the past week that appear designed to entrench military rule – and protesters returned once again to Tahrir Square, Nobel peace laureate Mohamed ElBaradei told the Guardian that his nation was on the verge of seeing a "new emperor" ascend to the throne. "We are in a total mess, a confused process that – assuming good intentions – has led us nowhere except the place we were at 18 months ago [when Mubarak fell], but under even more adverse conditions," said the opposition figurehead who withdrew from the presidential race earlier this year, arguing that a fair vote could not be held while the country remained in the grip of a violent junta. "We are going to elect a president in the next couple of days without a constitution and without a parliament. He will be a new emperor, holding both legislative and executive authority and with the right to enact laws and even amend the constitution as he sees fit." Following an emergency late-night meeting, the Muslim Brotherhood – whose candidate Mohamed Morsi is pitted against Ahmed Shafiq, a former air force commander and Mubarak's final prime minister, in the two-day presidential run-off vote – announced it was ignoring widespread calls to quit the electoral game in light of the constitutional uncertainty and vowed instead to fight on at the ballot box. "We are now facing a heinous coup, shamelessly aborting the revolution," claimed the political Islamists in a statement, as confirmation came through that the Brotherhood-dominated parliament had been dissolved by Mubarak-appointed judges. "Everyone knows we are facing a defining moment and a critical turning point," the statement continued, before going on to call for a "million-man march" to polling stations in an effort to hand Morsi victory and revitalise the struggle for change. "We will continue," Essam al-Erian, a senior Brotherhood official, told the Guardian. "This is our battle, the battle of the people, and voting for Mohamed Morsi means victory for the revolution." He went on to argue that a Morsi withdrawal would hand legitimacy to Shafiq and surrender the opportunity to expose electoral fraud, adding that "now that we have democracy, we must guard it". But many would question what sort of democracy Egypt is really enjoying, ahead of elections that are being conducted in a carefully manipulated climate of fear and division. Some high-profile commentators, including ElBaradei and author Alaa al-Aswany, have announced they will not be casting a ballot, echoing a grassroots boycott movement that has been steadily building up steam in recent days. ElBaradei, often viewed as a unifying figure by both secular and Islamist forces, issued one of his harshest condemnations yet of the Brotherhood and demanded to know why they were taking part in the poll at all. "The Brotherhood have not served themselves well – they have scared people right, left and centre with some of the extremist views put forward from them and other Islamist groups," he argued. "The Brotherhood should have realised that the vote they got at the parliamentary elections was not a true reflection of their support in the street – it was the product of a specific set of political conditions at the time. They should have reached out to other segments of society and built a broad coalition but they haven't done that – they started by saying we want to be part of big cake but they ended up wanting to have the whole cake for themselves." ElBaradei also spoke out against the "leaderless" vision of Egypt's revolutionary youth, claiming that the failure to delegate authority and produce a unifying set of demands had proved a "mortal mistake". On Wednesday Egypt's judiciary issued a decree extending the military's power to arbitrarily investigate and detain civilians, a move which Amnesty International labelled as "legally sanctioned abuse". With no constitution or parliament in place to check presidential power, critics fear that a Shafiq victory will deliver Egypt an army-backed autocrat with even stronger powers than Mubarak. But some analysts believe the Supreme Council of Armed Forces (Scaf), which assumed control of the country when Mubarak was overthrown in February of last year, has overplayed its hand. "In many ways the military have got more actual hard power right now in Egypt than they've had at any time since 1954," said Michael Hanna, an Egypt expert at the Century Foundation. "The day after Mubarak was toppled, the military enjoyed honorary sainthood and nobody wanted to question them. With that kind of soft power they could have found a way to protect their authority and privileges, but instead they went down a route which has ensured the role of the armed forces is now a live issue in the body politic in a way that it never was before." As darkness fell on Friday night, defeated pro-revolutionary election candidate Abdel Munim Aboul Fotouh declared that "the regime has not gone" and led a protest march across the capital which culminated in Tahrir Square. But with only hours to go before the polling stations opened their doors, demonstrator numbers were relatively thin compared to previous revolutionary rallies. "The revolutionaries are stuck because the Brotherhood are loath to join any protests – doing so would be a boon for Shafiq – and street mobilisations just help reinforce the counter-revolutionary narrative," said Hanna. "Ultimately protests in Tahrir have been normalised to the extent that they're no longer a menace to the status quo." CAIRO — Voters cast ballots on Saturday for the first competitively elected leader in Egypt ’s history, even as a last-minute grab for power by its ruling generals raised questions about whether the election would be a milestone in the transition to democracy or a facade obscuring the re-emergence of the old order. Voters faced a stark choice between two faces of the past: Ahmed Shafik, a former air force general and stalwart of former President Hosni Mubarak who promised to restore order and thwart the rise of an Islamist theocracy, or Mohamed Morsi, a veteran of the once-outlawed Muslim Brotherhood campaigning as a defender of the revolution against a return of the Mubarak-era autocracy. The ruling military council that took power after Mr. Mubarak’s ouster 16 months ago had pledged that this weekend’s two-day presidential runoff would be the final step in the transition to civilian government before the generals were to cede power. The day before the vote, however, they dissolved the democratically elected, Islamist-led Parliament that had been the chief accomplishment of the revolt so far. Acting on a rushed ruling by a court of Mubarak-appointed judges, they declared they would be the sole lawmakers, even after a new president is elected. And they began drawing up a new interim constitution that would define the power of the president whom voters were choosing on Saturday. “This is the end stage of the whole transition,” said Mahmoud Ismail, 27, a political activist in the Nile Delta who said he would vote for Mr. Morsi. “To be or not to be.” The military’s seizure of Parliament precluded the possibility of the Islamists of the Muslim Brotherhood taking control of both the legislature and the presidency. Now, if Mr. Morsi wins, he will face a prolonged struggle for power against the generals, while Mr. Shafik — who had long been considered one of Mr. Mubarak’s likely successors — could emerge as a new military-backed strongman unrestrained by either a constitution or Parliament. Mr. Shafik, Mr. Mubarak’s last prime minister, has made no public comment on the dissolution of Parliament. He cast his ballot on Saturday in the style of his former boss, arriving at a polling place in an upscale suburb surrounded by a heavy guard of military and police officers. The lines were pushed aside and guards immediately closed the facility for his private use. Crowds of his supporters were waiting both inside and outside the polling place. “The Brotherhood is dissolved,” they chanted, cheering at the dissolution of the Brotherhood-led Parliament. State media reported that a cameraman in a military vehicle filmed Mr. Shafik’s trip to the ballot box, apparently to preserve it for posterity. Mr. Morsi of the Muslim Brotherhood sought Saturday to cast the election as the last chance to beat back the full revival of the Mubarak government. He waited in line for more than two hours in the nearly 100-degree heat to cast his vote in the Nile Delta town of Zagazig, where he used to teach engineering. “God is great,” a throng of supporters cried as he emerged, and he shouted over them to salute those killed while demonstrating against Mr. Mubarak. “Today is the day of the martyrs,” he declared. “There is no place at all for Mubarak’s helpers.” Across Egypt, the streets were calm, but enthusiasm was low and turnout had fallen sharply from the first round. The two candidates, however, revved up the battle-tested political machines that helped them advance past last month’s first round of voting. Mr. Morsi turned to the Brotherhood’s system of local cells and charities built over 84 years of preaching and politics. Mr. Shafik, who surged to roughly tie Mr. Morsi in the first round of voting, scarcely a month after he announced his campaign, relied instead on the network of local power brokers, including retired army officers and former leaders in Mr. Mubarak’s defunct ruling party. David D. Kirkpatrick reported from Cairo, and Kareem Fahim from Menoufia, Egypt. Mayy El Sheikh contributed reporting from Cairo, and Dina Salah Amer and Liam Stack from Menoufia. Egyptians expressed wariness Saturday as they lined up in sweltering heat to vote in the runoff election for a replacement for ousted autocrat Hosni Mubarak. In sharp contrast to recent elections, the mood in Cairo and across the country was largely tense as Egyptians contemplated the polarizing choice between Mohamed Morsi, a conservative Islamist, and Ahmed Shafiq, who was Mubarak’s last prime minister. With the country’s lower house of parliament dissolved, the constitution suspended and the revolution pronounced all but dead, the outcome of the presidential vote that continues Sunday could not be more consequential. “This is a decisive moment, but nobody feels confident about anything,” said Samia, 45, a Shafiq supporter who asked to be identified by her first name because of her government job. “It’s like rolling the dice and hoping for the best.” The runoff began two days after a court ruling led to the dissolution of the lower house of Egypt’s Islamist-dominated parliament, a move that activists and some leading political figures described as a soft military coup. A small movement of boycotters urged voters to spoil their ballots in what they saw as an illegitimate election under military rule. The once-repressed Muslim Brotherhood has thrived since the revolt that overthrew Mubarak. The group dominated the parliament and took a sizable share of the seats in a body tasked with writing a new constitution. After the dissolution of the lower house of parliament this week, though, the military junta assumed all legislative powers. Military chiefs are soon expected to appoint a new constitutional assembly and issue a decree outlining the powers of the presidency. That means the Brotherhood’s sole hope of remaining politically powerful in the short run lies with Morsi. On Saturday, the Brotherhood appeared to be laying the groundwork to cry fraud if Shafiq is pronounced the winner. The group released a statement listing alleged violations, including military conscripts voting illegally and the arrest of revolutionaries holding pictures of slain protesters outside polling stations. However, the Brotherhood did not claim widespread fraud and expressed confidence that Morsi would win. The group’s political wing had harsh words for Egypt’s military. Dissolving parliament “confirms the desire of the military to take all authority against popular will,” a statement from the Brotherhood’s Freedom and Justice Party said. It said only a popular referendum could disband parliament. The statement called the move “an attack on the great Egyptian Revolution” and warned that the party would not honor the military’s decree about presidential powers. “Egyptians, defend your will, continue to vote heavily,” the statement said. “Remember the blood of the martyrs and the pain of the injured and do not allow in any way the return of the tyrannical and despotic regime that has always despised the Egyptian people.” Egypt’s transition has been marred by division among liberals, leftists and Islamists as well as what many see as the military rulers’ intention to protect their economic and political interests at the cost of democracy. This week, the Justice Ministry effectively put the nation under martial law, authorizing the military police to arrest any citizen suspected of a wide range of crimes. After voting in his home province of Sharqiya, Morsi promised a crowd of supporters, “I will lead you to the new Egypt and stability.” “With our soul, with our blood, we’re with you, Morsi,” men chanted, thronging around the black sport-utility vehicle that carried the candidate. In contrast, Shafiq slipped in through a side door of his polling station in suburban Cairo. The school was sealed off while he voted, and the former prime minister left with little fanfare. Turnout appeared high in some parts of the country but relatively low in the capital. Polling stations opened at 8 a.m., and some Egyptians waited more than an hour to slip their ballots into plastic bins. Final results are expected Thursday. About 150,000 security officers guarded polling stations across the country. The balloting comes about 16 months after millions of Egyptians revolted against Mubarak’s repressive rule. But since his ouster, many here have grown weary of unending protests, flashes of violence and a faltering economy. In Cairo’s working-class neighborhood of Imbaba, veiled women lined up outside a schoolhouse. “Before, everyone was happy,” said Fatima Soliman, 50. “Now nobody is happy.” When asked who she would vote for, she scanned the crowd warily and whispered, “Shafiq.” To the north, “No to Shafiq” was scrawled in black paint at a school being used as a polling station in the suburban Cairo neighborhood of Shubra el-Kheima. Inside, voters trickled in under the watchful eye of military officers. Morsi Ismail, 70, said he was thrilled that parliament had been disbanded. The Brotherhood, he said, “was trying to control all our institutions.” Rudji Mohamed, a 39-year-old upholstery repairman, was unwilling to say who he voted for but made it clear he was disheartened by the dissolution of parliament. “We lived under repression for 30 years,” he said. Special correspondents Hassan el Naggar and Ingy Hassieb contributed to this report.

Egyptians began voting today to determine who will be the first president in the post-Mubarak era, but the mood is anything but celebratory. This week's stunning move by military leaders to dissolve parliament and essentially consolidate power has overshadowed the race between the two candidates, which is in itself what the Washington Post calls a "polarizing choice." Voters will pick either former Mubarak Prime Minister Ahmed Shafiq or Muslim Brotherhood candidate Mohamed Morsi. "We are in a total mess," Nobel laureate Mohamed ElBardei tells the Guardian. "We are going to elect a president in the next couple of days without a constitution and without a parliament. He will be a new emperor, holding both legislative and executive authority and with the right to enact laws and even amend the constitution as he sees fit." The New York Times' David Kirkpatrick sees it a little differently, writing that the winner will initially "compete with the generals for power and influence." Voting ends tomorrow.

multi_news

The model and actor was found dead at his home in Montreal from an apparent suicide Rick Genest, the model known as Zombie Boy, whose heavily tattooed body made him a muse for Lady Gaga, Marc Quinn and numerous fashion designers, has died aged 32. Dulcedo Management, the Montreal talent and model agency that represented Genest, confirmed his death on Facebook. “The whole Dulcedo family is shocked and saddened by this tragedy,” they wrote, describing Genest as someone who “charmed all hearts” and who was loved by all who met him. “Thank you ZB for these beautiful moments in your company and for your radiant smile,” they added. He was found dead at his home in Montreal on Thursday. Police sources told Radio-Canada that his death is being classified as a suicide. Facebook Twitter Pinterest Zombie Boy in London in 2016. Photograph: CPG Photography Ltd/Rex/Shutterstock As news of his death broke, tributes poured in to the artist – who went from living on the streets of Montreal as a squeegee kid to high-end modelling shows in Paris and Berlin. Lady Gaga, who had cast Genest in her 2011 Born This Way video, wrote on Twitter: “The suicide of friend Rick Genest, Zombie Boy is beyond devastating. We have to work harder to change the culture, bring mental health to the forefront and erase the stigma that we can’t talk about it. If you are suffering, call a friend or family today. We must save each other.” Gaga’s stylist, Nicola Formichetti, who used Genest as a model when he was creative director of Thierry Mugler, said he was “absolutely heartbroken”. A Facebook account appearing to belong to Genest hinted at depression in a recent post. The May post shows a photo of Genest sitting in a hospital bed, wearing the Kill Me shirt that he often wore for interviews and photos with a tongue depressor hanging from his mouth. The post echoed a 2012 interview with the UK’s Evening Standard, in which they noted that Genest was reticent to delve into details of his private life. After Genest mentioned – seemingly apropos of nothing – “Depression is a strong thing…” he refused to elaborate when pressed by the reporter. In a TedX talk last year, titled Normal is an Illusion, Genest described growing up in a small town in Quebec that was riddled with political and racial issues. “French versus English, Mohawks versus the White, very few Blacks and even less other nationalities in between,” he told an audience in Austria. His parents’ strict religious beliefs kept him sidelined from most school celebrations, forbidden from taking part in what they saw as pagan rituals; carving pumpkins at Halloween, crafting Christmas tree decorations and painting Easter Eggs. Amid the jocks and preps of high school, he found his place among what he described as the “least favourite of all subcultures”: the goths. “Every day was a battle of final demise, suffering in agony in a world of the ignorant and chipper, bullied by most and befriended by few,” he said. As a teenager, he was diagnosed with a brain tumour. Doctors warned him that the surgery to remove the tumour would leave his face disfigured for life. After a six-month wait for the surgery, doctors presented him with another option; a new laser technology that would leave his face intact but which was a gamble, as it had only been carried out successfully on one other person in North America. Shortly after his 15th birthday, he told the audience in Austria, “I stepped out of the hospital as the second person in North America to survive this new state-of-the-art procedure.” Years later, he had a falling out with his parents and left home for Montreal, spending the next five or six years sleeping rough, earning money by washing windshields and dodging cops while soaking up the city’s punk scene. It was around that time that his tattoos began to multiply. “I gradually started resembling a human doodle board, riddled with tattoos,” he said during his TedX talk. Eventually he met tattoo artist Frank Lewis. The pair devised a theme of a rotting corpse to blend his many tattoos into a tattoo that would cover his whole body, with blackened eye sockets, flesh withering off bones and cockroaches crawling up his neck. As he turned 21, he got his most eye-catching tattoo; a skull tattooed on his face. At 22, he shaved off his two-foot high mohawk to tattoo an image of his brains on his scalp. His foray into international fame came soon after, when the UK’s Bizarre Magazine saw photos of him on MySpace and travelled to Montreal to meet him. From there, photos of him began to spread and his career snowballed. As well as his work with Gaga and Formichetti, he also modelled for Jay-Z’s fashion label Rocawear and appeared in the Keanu Reeves samurai film 47 Ronin. Acclaimed British artist Marc Quinn created a sculpture of Genest in 2011. Genest explained his phantasmagoric appearance to Wonderland magazine in 2012: “The zombie concept is also often used as a metaphor for runaway consumerism. Rebelling from this notion is the very meaning of punk. The origins of the zombie creature came about from stories of people being buried alive in times of plagues and such crises; that would come out the other side transformed.” He added: “Zombies, to many, represent a pervasive xenophobia. As in my life, I was often out-casted, hated or misunderstood.” • In the UK, Samaritans can be contacted on 116 123 or email jo@samaritans.org. In the US, the National Suicide Prevention Lifeline is 1-800-273-8255. In Australia, the crisis support service Lifeline is 13 11 14. Other international suicide helplines can be found at www.befrienders.org. Rick “Zombie Boy” Genest has led an unconventional life, to say the least. We caught up with Lady Gaga’s comrade-in-arms to talk about being the new face of Rocawear and his philosophy on life. Since you have become famous, have people’s reactions to you on the street changed? Well, first some things stayed the same. My life style was already nomadic, living out of my backpack; working odd jobs, meeting cool people, as well as often being stopped and photographed. This hasn’t changed. What changed was the masses’ reaction to me. Prior, I had my place amongst those who understood me and had the luxury of privacy. Now I often feel that every walk of life either has a question or an opinion about the way I breathe air – although I do seize this opportunity to raise awareness for tolerance, acceptance and embracing our differences. What’s the philosophy behind your tattoos? The zombie concept is also often used as a metaphor for runaway consumerism. Rebelling from this notion is the very meaning of punk. The origins of the zombie creature came about from stories of people being buried alive in times of plagues and such crises; that would come out the other side ‘transformed’. Zombies, to many, represent a pervasive xenophobia. As in my life, I was often out-casted, hated or misunderstood. You’ve just become the face of Rocawear. Can you tell us more about that? Growing up in the city as a teenager, I have always embraced urban culture and style. It is a great honor to represent what I live, breathe, and bleed for as long as I have. I’m excited to be involved with Rocawear’s re-launch across Europe for Spring Summer ’13. You and your art have become synonymous with Lady Gaga. How do you feel about that? I would defiantly have to say that we both share love for art, the love for revolution, and fearlessness of self-expression. You’ve recently filmed a part in 47 Ronin with Keanu Reeves, is acting something you have always wanted to do? Since my childhood, I had a profound interest in pirates. I recall in particular the movie Hook, starring Robin Williams, as well as pirate LEGO. As life went on, I had spent the greater part of my time living in likeness to the lost boys and pirates. What would be your ideal film role? I’ve had a chance to play as a punk rocker, a carnie and a pirate. My ideal film role yet would be as a deranged psycho in a horror picture. Are you planning on any more tattoos? I’m still working on my tattoo project. As well as finishing my bodysuit, I soon wish to sharpen my teeth, with likeness to those of a reptile. What do you have planned for 2013? In 2013, I will be starting to work on a music project, but more details would ruin the surprise! Words: Siobhan Frew www.rocawearbrand.co.uk Image copyright Getty Images Lady Gaga has paid tribute to model Rick Genest, who has died at the age of 32. Rick was known as Zombie Boy, due to bone and organ tattoos across his body, including skull features on his face. He was found dead at his home in Montreal, Canada and it was widely reported that he had taken his own life. Rick appeared in Gaga's Born This Way video in 2011 and the singer says the loss is "beyond devastating". Image copyright Getty Images Image caption Lady Gaga painted her face like Rick's in her Born This Way video "We have to work harder to change the culture, bring mental health to the forefront and erase the stigma that we can't talk about it," she wrote on Twitter. "If you are suffering, call a friend or family today. We must save each other." She followed this by sharing a photo of herself and Rick - and urging fans who might be struggling with their mental health to "reach out". Rick modelled for French fashion designer Thierry Mugler, appeared in Vogue Japan and was once the face of Jay-Z's Rocawear fashion label. He was 32 at the time of his death. His body was found six days before his 33rd birthday. Rick's management posted a tribute on Facebook, saying they were "shocked and pained" by his death. "Rico was loved by all those who had the chance to meet him and know him," said the post. "We received the unfortunate news at the beginning of the afternoon and members of the team have come to support his family and relatives in this dark and difficult moment. "We want to present our deepest condolences... Thank you zb for these beautiful moments in your company and for your radiant smile." If you are in the UK, you can call the Samaritans on 116 123 if you are struggling with mental health issues. You can also get help at the BBC Advice pages. Follow Newsbeat on Instagram, Facebook and Twitter. Listen to Newsbeat live at 12:45 and 17:45 every weekday on BBC Radio 1 and 1Xtra - if you miss us you can listen back here. Rating is available when the video has been rented. This feature is not available right now. Please try again later. World-renowned Montreal model, Rick Genest, better known as Zombie Boy, died Wednesday at the age of 32 in Plateau Mont-Royal, His agency, Dulcado Management, reported on Facebook Thursday. In the post, the agency said it was “shocked and saddened by this tragedy.” The post paid tribute to him by writing and called him an “icon of the artistic scene” who “charmed everybody’s hearts.” Genest was struggling with mental health problems and took his own life on Wednesday, Radio-Canada reported. Montreal police refused to confirm the information. Genest, who had formerly been a Montreal squeegee kid, catapulted to fame in the fashion industry by virtue of his totally tattooed body, including a rendering of brains on his skull. He also appeared in Lady Gaga’s Born this Way music video. The superstar tweeted after the reports of his death surfaced, imploring her followers to bring the issue of mental health to the forefront. The suicide of friend Rick Genest, Zombie Boy is beyond devastating. We have to work harder to change the culture, bring Mental Health to the forefront and erase the stigma that we can’t talk about it. If you are suffering, call a friend or family today. We must save each other. pic.twitter.com/THz6x5JlpB — Lady Gaga (@ladygaga) August 3, 2018 The suicide of friend Rick Genest, Zombie Boy is beyond devastating. We have to work harder to change the culture, bring Mental Health to the forefront and erase the stigma that we can’t talk about it. If you are suffering, call a friend or family today. We must save each other.pic.twitter.com/THz6x5JlpB Montreal model and artist Rick Genest, also known as Zombie Boy, has died, police sources have told Radio-Canada. Genest was 32. Genest came to the public's attention after tattooing his body from head to toe, including images of a skull and brain on his head. He holds a Guinness World Record for most insect tattoos (176) and another for most human bone tattoos (139). His body-art attracted fashion designers and he modelled at high-end shows in Paris and Berlin. In 2011, he appeared in Lady Gaga's music video for the song Born This Way. Police sources told Radio-Canada that ​Genest died in Montreal's Plateau–Mont Royal borough around 5 p.m. ET on Wednesday. The sources said police are classifying his death as a suicide. The Quebec coroner's office says it is still investigating the cause of death. The Montreal paramedics organization, Urgences-santé, said it could not comment on the circumstances surrounding his death, but said it had been related to a fall. Genest's manager, Karim Leduc, said in an interview with CBC Montreal's Homerun Friday that he believed the police had been too quick in their classification of the death. "The circumstances are quite mysterious and the pieces of the puzzle just don't fit together perfectly," Leduc said. He believes Genest's death was an accident. Lady Gaga tweeted Thursday night that the news is "beyond devastating." The suicide of friend Rick Genest, Zombie Boy is beyond devastating. We have to work harder to change the culture, bring Mental Health to the forefront and erase the stigma that we can’t talk about it. If you are suffering, call a friend or family today. We must save each other. <a href="https://t.co/THz6x5JlpB">pic.twitter.com/THz6x5JlpB</a> —@ladygaga From the streets, to worldwide fame Genest was born in the Montreal borough of LaSalle, grew up in the off-island suburb of Châteauguay and spent part of his life living on the streets. He got his first tattoo at 16 and left home the following year. About 90 per cent of his body was eventually tattooed, and most of it is by Montreal tattoo artist Frank Lewis, according to the Guinness Book of World Records. The extent of Genest's tattoos led him to work in sideshows and then garner an online following. "He was someone who was a prisoner of that image," said Pénélope McQuade, host of Radio-Canada's Les échangistes, who interviewed Genest in 2016. The British artist Marc Quinn is slated to create a 3.5-metre bronze sculpture of Genest for London's Science Museum next year. "I find him rather amazing. He is an artist. His artwork is himself," Quinn told the Guardian newspaper in March. The last post on Genest's official Facebook page, which has almost one million followers, is a poem called The Well. Fans have posted an outpouring of grief in the comments. Where to get help: Canada Suicide Prevention Service: 1-833-456-4566 In Quebec (French): Association québécoise de prévention du suicide: 1-866-APPELLE (1-866-277-3553) If you're worried someone you know may be at risk of suicide, you should talk to them, says the Canadian Association for Suicide Prevention.

Lady Gaga is mourning her friend and muse Rick Genest-better known as "Zombie Boy." The 32-year-old Canadian model, who went from the Quebec punk scene to catwalks around the world after his head-to-toe tattoos of bones, insects, and exposed brains made him famous, was found dead at his Montreal home Wednesday and police sources tell the CBC he died by suicide. Genest, who got his first tattoo at 16, died six days before his 33rd birthday. He appeared in Gaga's 2011 "Born This Way" video, was once the face of Jay-Z's fashion label, and modeled for French designer Thierry Mugler, among others, the BBC reports. Genest's agency, Dulcedo Management, said in a Facebook post that it was "shocked and saddened by this tragedy," the Montreal Gazette reports. Gaga tweeted that the suicide was "beyond devastating." "We have to work harder to change the culture, bring Mental Health to the forefront and erase the stigma that we can't talk about it," she said, posting a photo of herself with Genest. "If you are suffering, call a friend or family today. We must save each other." The Guardian notes that Genest, whose body was 90% covered in tattoos, likened the zombie theme to rebelling against consumerism in a 2012 interview. "Zombies, to many, represent a pervasive xenophobia," he said. "As in my life, I was often out-casted, hated, or misunderstood."

multi_news

Protein promiscuity is of considerable interest due its role in adaptive metabolic plasticity, its fundamental connection with molecular evolution and also because of its biotechnological applications. Current views on the relation between primary and promiscuous protein activities stem largely from laboratory evolution experiments aimed at increasing promiscuous activity levels. Here, on the other hand, we attempt to assess the main features of the simultaneous modulation of the primary and promiscuous functions during the course of natural evolution. The computational/experimental approach we propose for this task involves the following steps: a function-targeted, statistical coupling analysis of evolutionary data is used to determine a set of positions likely linked to the recruitment of a promiscuous activity for a new function; a combinatorial library of mutations on this set of positions is prepared and screened for both, the primary and the promiscuous activities; a partial-least-squares reconstruction of the full combinatorial space is carried out; finally, an approximation to the Pareto set of variants with optimal primary/promiscuous activities is derived. Application of the approach to the emergence of folding catalysis in thioredoxin scaffolds reveals an unanticipated scenario: diverse patterns of primary/promiscuous activity modulation are possible, including a moderate (but likely significant in a biological context) simultaneous enhancement of both activities. We show that this scenario can be most simply explained on the basis of the conformational diversity hypothesis, although alternative interpretations cannot be ruled out. Overall, the results reported may help clarify the mechanisms of the evolution of new functions. From a different viewpoint, the partial-least-squares-reconstruction/Pareto-set-prediction approach we have introduced provides the computational basis for an efficient directed-evolution protocol aimed at the simultaneous enhancement of several protein features and should therefore open new possibilities in the engineering of multi-functional enzymes. Proteins are capable to perform molecular tasks with impressive efficiency and, often, with exquisite specificity. Nevertheless, many proteins possess weak promiscuous functions, which are more or less related to the primary activity, but involve different substrates or different chemical alterations [1]–[5]. Protein promiscuity has been extensively studied in recent years due to its important biotechnological applications [6]–[12], to its role in adaptive metabolic plasticity [13]–[15] and also because of its fundamental connection with molecular evolution. Indeed, promiscuity in modern proteins is plausibly a vestige of the broad specificity of primordial proteins [1]. Furthermore, as briefly elaborated below, promiscuity likely plays an essential role in the development of new functions through divergent evolution [3], [5], [8], [16]–[20]. Development of new functions does occur during evolution, sometimes with impressive speed. In most cases, the process involves gene duplication as a necessary step. It has been repeatedly noted, however, that random accumulation of mutations in a gene is unlikely to create a new function. It is generally assumed, therefore, that a sufficient level of the new (initially promiscuous) activity must be present before the duplication event. In this way, natural selection can act on one of the gene copies to enhance the new function, while the original function is retained by the other copy. However, optimization of a functional site for a given molecular task likely interferes with the efficient performance of the protein for a different task based on the same site. Consequently, enhancement of the promiscuous activity prior to gene duplication may be expected to cause a decrease in primary activity that could conceivably compromise organism survival. As a solution to this conundrum, a “weak trade-off” scenario has been proposed [5]: enhancement of the promiscuous activity is assumed to be accompanied with only a moderate decrease in primary function and, therefore, a generalist protein (significant levels of both activities) can be formed prior to gene duplication without seriously impairing organism fitness. This weak trade-off explanation is certainly supported by a number of laboratory evolution experiments [5]. However, the possibility that natural evolution may actually avoid or bypass primary/promiscuous activity trade-offs (i. e., a “no trade-off” scenario as opposed to a “weak trade-off” scenario) should be seriously taken into account, since bifunctional enzymes with the capability to catalyze efficiently two different biochemical reactions based on the same active site are known and have been recently characterized in detail [21], [22] and experimental studies have supported that the trade-off between high activity and tight specificity can be greatly relaxed [23]. Here, we aim at assessing the patterns of primary/promiscuous activity modulation in the mutational space actually explored by natural evolution when recruiting a promiscuous activity for a new function. The approach we propose involves essentially three steps: A unique global optimum cannot be defined when dealing with a multi-objective optimization problem, such as, for instance, enhancing a promiscuous activity while keeping the level of the primary activity as high as possible. However, a set of several optimal solutions can be defined using the Pareto criterion: a solution (protein variant in this case) belongs to the set of optimal solutions (the so-called Pareto set) if it is not dominated by any other solution. The dominance relationship is defined as follows: a solution a dominates a solution b if it shows enhanced performance for all optimization objectives. In the specific case of interest here, variant a dominates variant b if primary-activity (a) >primary-activity (b) and simultaneously promiscuous-activity (a) >promiscuous-activity (b). The construction of the Pareto set of non-dominated solutions is illustrated with a simple example in Figure 1A. The Pareto set includes the solutions with optimal trade-offs between the different objectives and has been used extensively in economics, while its application to protein design has only been explored in recent years [29]–[31]. In the specific case of interest here, determination of the Pareto set should immediately clarify the main features of the modulation of the primary and promiscuous activities within a given mutational space. For instance, if the starting variant (the “wild-type” protein, for instance) already belongs to the Pareto set, enhancement of the promiscuous activity necessarily implies a decrease in primary function and the trade-off will be weak (Figure 1B) or strong (Figure 1C) depending of the general slope of the Pareto set in the plot of promiscuous activity versus primary activity. On the other hand, if the starting variant does not belong to the Pareto set, simultaneous optimization of both activities is in principle feasible (Figure 1D) and primary/promiscuous trade-offs can be avoided to some extent. To test the approach proposed, we have chosen the three basic activities associated with the thioredoxin fold: reduction of disulfide bridges, formation of disulfide bridges and isomerization (reshuffling) of disulfide bridges. The two latter activities are linked in vivo to protein folding processes [32], [33] (oxidative folding and rescuing of proteins with incorrect disulfide bridges) and, in the periplasm of bacteria, are performed by different proteins: DsbA and DsbC, respectively. By contrast, in the endoplasmic reticulum of eukaryotic cells, both disulfide-linked folding processes are catalyzed by the same protein: protein disulfide isomerase (PDI). PDIs are multidomain proteins that contain thioredoxin-fold domains [32]. Processes of disulfide reduction in vivo (obviously unrelated with folding) are typically catalyzed by single-domain thioredoxins, which may also show low in vitro levels of the protein folding activities associated to disulfide bridge formation and reshuffling. Indeed, it is tempting to speculate that low levels of these activities were already present in primordial thioredoxins and that, at some evolutionary point, were recruited for new-function development leading to the proteins involved in disulfide-bridge-linked protein folding [32]. The processes of thioredoxin-domain catalyzed reduction, formation (oxidation) and reshuffling of disulfide bonds are all dependent on the active-site CXXC motif. Reduction (see Figure 2) starts with the reduced enzyme and involves the nucleophilic attack of the thiolate form of the amino-terminal cysteine on the disulfide bridge of the substrate [34], [35]. The mixed-disulfide thus formed is resolved by the nucleophilic attack of the carbonyl-terminal cysteine. Oxidation, on the other hand (see Figure 3), involves a nucleophilic attack of the substrate on the disulfide bridge of the oxidized enzyme and the mixed-disulfide intermediate is resolved by attack from the free cysteine (in the thiolate form) of the substrate [36]. It is relevant that the oxidation and reduction processes involve opposite chemical changes in the substrate (break-up and formation of disulfide bridges) as well as different mechanisms for the resolution of the mixed-disulfide intermediate. Furthermore, the two processes may be expected to be linked to different values of the redox potential (as suggested by the redox potentials of thioredoxin and PDI: see Figure 6 in Hatahet & Ruddock [36]) and, as it has been extensively discussed in the literature, they likely have different molecular requirements in terms of the conformational changes during catalysis, the stability of cysteine thiolates and the modulation of the pK values of the catalytic groups [33], [36]–[39]. Clearly, disulfide reduction and catalysis of oxidative folding (involving formation of disulfide bridges) may be expected to strongly trade-off. Contrary to disulfide reduction and formation (Figures 2 and 3), disulfide-bridge reshuffling in misfolded proteins to yield the correctly folded state does not involve a net change in the oxidation state of the substrate and could in principle occur through cycles of catalyzed reduction/oxidation [36], [40]. Alternatively, the initial attack of the enzyme on a substrate disulfide bridge may yield a free cysteine that could attack another disulfide bridge thus starting a cascade of disulfide-bond rearrangments leading to the most stable configuration [36], [40]. The specific protein system we use in this work is E. coli thioredoxin, an enzyme involved in multiple reduction processes in vivo [35], [41] which, besides this primary (i. e. reductase) activity is able to catalyze, albeit with very low efficiency, disulfide-bridge-linked protein folding processes [42], [43] (promiscuous activities of E. coli thioredoxin). We apply the approach proposed (steps 1–3 above) to E. coli thioredoxin with the catalysis of oxidative folding as the promiscuous activity. Nevertheless, the variants thus obtained are also tested for the disulfide reshuffling activity. To find a set of positions likely linked to the emergence of disulfide-bridge-linked folding functions in the thioredoxin fold, we have used statistical coupling analysis (SCA) which works by comparing the amino acid distributions at different positions in a multiple sequence alignment (MSA) with the corresponding ones in a given sub-alignment [24]. To apply SCA to the study of primary/promiscuous activity modulation we propose selecting the sub-alignment on the basis of a function-related criterion related with the promiscuous activity. We thus start with a MSA derived from a sequence-database search using the E. coli thioredoxin sequence as query and select as sub-aligment those sequences belonging to thioredoxin-fold domains in proteins involved in protein folding in vivo. Actually, this selection step is made straightforward by the fact that these domains contain the active-site CGHC sequence, while thioredoxin reductases contain the CGPC sequence. In fact, the P34H mutation on the E. coli thioredoxin background has been shown to enhance significantly its “PDI-like” promiscuous activities [43]. The MSA we have used contains indeed a significant number of sequences with a histidine at position 34 (E. coli thioredoxin numbering) that, in most cases, belong to thioredoxin-fold domains of eukaryotic PDI' s. We thus obtained the statistical free-energies using the P→H substitution as the perturbation at position 34 (see Methods for details) and we expect these values to reveal networks of residues related with the emergence of the protein folding activities in the thioredoxin fold. However, SCA is based upon the perturbation of the 20 amino acid distribution at each position and actually provides a list of coevolving positions (see Figure 4A), while we are interested in specific mutations at these positions. Therefore, we included an additional layer of statistical analysis. For each given position, we considered the mutation from the amino acid present in E. coli thioredoxin (the “Ec” aminoacid) to the amino acid “X” defined as the amino acid different from “Ec” that has the highest frequency (largest number of occurrences in the sequence alignment) when there is a histidine at position 34. Then, for each of the 13 positions with the highest coupling free energies from the SCA analysis (Figure 4A), we calculated the following score: (1) where f is the frequency of occurrence of the amino acids in the sequence alignments and subscripts “H” and “P” refer to the condition for the calculation of the frequencies (histidine or proline at position 34, respectively). Large positive values for the score indicate that the P34H substitution shifts the statistics strongly towards amino acid X. We retained for experimental analysis the 10 positions (and the corresponding Ec→X mutations) for which the score was positive (see Figure 4B): I4V, D26E, W28Y, E30P, I38L, K57A, N59D, D61T, I75Y, L94R. It is important to note that the 10 positions selected form a well-defined, connected network surrounding the active site (see Figure 4C), a fact fully consistent with their likely role in the development of the new functions related with protein folding catalysis. We also wish to emphasize at this point that the immediate purpose of this work (see section below) is to probe the interplay between primary and promiscuous activities in the mutational space defined by the 10 mutations selected. Specifically, the derivation of a molecular-level picture of what each of these mutations is doing (a task that would require extensive structural work due to the potential non-additivity of the mutation effects), is beyond the scope of this work. A simple visual examination (see Figure 4D) of the sequences of the MSA used that include histidine at position 34 (most of them belonging to eukaryotic PDI' s) shows that different combinations of the 10 mutations selected occur in extant PDI' s. We conclude that natural selection does efficiently explore the mutational space defined by combinations of the 10 mutations. To assess how the interplay between the primary and promiscuous activities is modulated in this mutational space, we prepared a combinatorial library spanning the 10 mutations (i. e., 210 = 1024 variants) on the P34H background and determined the reductase and the catalysis of oxidative folding activities for 29 randomly selected variants. The primary activity has been probed by following the standard reduction assay for DTT-reduced thioredoxin which uses insulin as a model substrate (see Methods for details). Clearly, this assay reflects the reduction process of Figure 2. The catalysis of oxidative folding (promiscuous activity in E. coli thioredoxin) has been assessed using fully reduced ribonuclease A as substrate (see Methods for details). This assay might include some contribution from the reshuffling process since the first disulfide bridges formed need not be the correct ones. Nevertheless, it is expected to probe mainly the oxidation pathway (Figure 3), an expectation that will be supported by the results reported here. Most of the 29 variants screened show increased levels of the promiscuous activity with respect to both the background P34H variant and wt E. coli thioredoxin (Figure 5A). This result is consistent with the proposed role of the selected mutations in the emergence of the protein folding activities of the thioredoxin fold. What may perhaps be surprising, however, is that some of the variants also show an increased level of the primary activity, indicating the possibility of the simultaneous enhancement of the primary and promiscuous functions. In order to assess the full range of function modulation achieved by the combinatorial library we have carried out a fit of the experimental data based on the equation: (2) followed by a reconstruction of the entire library using the values of the fitted parameters. The meaning of the symbols in equation 2 is as follows: Ak is the dependent variable (activity) with k being a label that identifies the type of activity (i. e., k = “primary” or k = “promiscuous”) = 1; δi is an independent variable that may take the values 0 or 1, corresponding to the absence o presence of the mutations at position i; pik is a measure of the effect of the mutation at position on the activity Ak; δij = δi·δj is an independent variable that takes a value of 1 when mutations at positions i and j occur simultaneously (and takes a value of zero otherwise); pijk is a measure of the effect of the coupling between mutations at positions i and j on the activity k. Equation 2 embodies a comprehensive model that includes the effects of individual mutations (pik values) as well as the possibility that mutation effects are non-additive (pijk≠0). It involves, however, 110 fitting parameters (10 pik parameters and 45 pijk parameters for each of the two activities), while the number of experimental values to be fitted is only 58 (i. e., the values of the two activities –primary and promiscuous- for the 29 library variants studied). Having more fitting parameters than dependent variable values is a common occurrence in chemometrics, often addressed using partial least-squares [27], [28] (PLS), a dimensionality reduction approach akin to principal component analysis. Indeed, the widespread usefulness of the PLS approach is often credited to its ability to handle a large number of independent variables (i. e., fitting parameters) (see chapter 7 in Livingstone [44]. PLS thus uses latent variables (latent vectors): orthogonal combinations of the original variables that explain most of the variance in the original independent variable set and are also constructed to maximize their covariance with the dependent variables. The original variables may then survive the PLS dimensionality reduction, but they are combined in a few relevant latent vectors. In the case of interest here, once a PLS fit of equation 2 to the experimental data for the 29 variants studied (Figure 5A) has been performed (see Methods for details), it is straightforward to calculate the expected primary and promiscuous activity data for the whole library of 1024 variants. Of course, there remain two important issues related to the assessment of the uncertainty associated to such full-library reconstruction and to its experimental validation. To assess reconstruction uncertainty, we have used a bootstrapping approach involving PLS fits to replica sets obtained by randomly re-sampling from the original experimental set (see Methods for details). Full-library reconstructions resulting from the PLS analyses of 20 such replicas are given in Figure 5B. They clearly suggest that the mutation set derived from the statistical coupling analysis potential has a huge potential for modulating both, the primary and promiscuous activities. Experimental validation of the reconstruction (based on experimental measurements on the predicted Pareto set of optimal variants) is described in the following section. We derived an “optimistic” prediction of the Pareto set of optimal primary/promiscuous activities (Figure 5C) as the set of non-dominated solutions in the ensemble of reconstructions shown in Figure 5B. The 11 variants in this predicted set were prepared and their activities determined experimentally. There is an excellent qualitative agreement between prediction an experiment, in the sense that, for all the 11 variants, increased levels of both activities were found (Figure 5D). This agreement validates the reconstruction carried out on the basis of the PLS analysis of the 29-variants set. It is relevant to note at this point that the PLS-reconstruction/Pareto-prediction analysis leads to an expansion of our experimental variant set (from 29 variants to 40 variants) but in a manner that is not random. Actually, the 11 variants added to the experimental set allow us to move in the space of primary/promiscuous activities in the general direction of the simultaneous enhancement of both activities, as is visually apparent in Figures 5E and 5F. The Pareto set from the experimental data for the 29+11 = 40 variant set (Figures 5E and 5F) is still only an approximation to the Pareto set for the whole library, since additional cycles of PLS-reconstruction/Pareto-prediction could in principle lead to further enhancements in both activities. However, PLS-reconstruction starting with the 40-variant experimental data set suggests that additional improvements are expected to be small (see Figure 6), supporting that 40-variant Pareto set is likely close to the Pareto set of the full library. Furthermore, the main result of the analysis is already apparent with the 40-variant set: E. coli thioredoxin, as well as the background P34H variant for library construction, does not belong to the Pareto set and, therefore, simultaneous enhancement of the primary and promiscuous activities is feasible (and has been experimentally achieved: Figures 5E and 5F). Note that, in addition to the targeted simultaneous enhancement (implying enhanced levels for both activities), the experimental data set (as well as the PLS reconstructions of the full combinatorial library) indicates that the two activities can be modulated in an independent-like manner and includes “specialist” variants with a high level for one activity and low value for the other. Figure 7A highlights the modulation ranges experimentally achieved for the reductase and catalysis of oxidative folding activities (about 33-fold and 7-fold, respectively). One important issue is whether these ranges (in particular that of the promiscuous activity) are to be considered large or small. The answer to this question depends largely on the relevant context. Certainly, the modulation ranges we have found are much smaller than those reported in some protein design studies (consider, for instance, the 200-fold enhancement in engineered Kemp eliminase activity reported by Baker, Tawfik and coworkers [45]) and the ranges typically considered relevant in a biotechnological application context. It is important to note, however, that the approach we have used is aimed at assessing the patterns of primary/promiscuous activity modulation in the mutational space actually explored by natural evolution when recruiting the promiscuous activity for a new function. That is, the mutations included in our combinatorial library are those expected to be associated to the emergence of folding catalysis in the thioredoxin scaffold during the course of natural evolution. If the approach is successful, promiscuous activities approaching the levels of natural thioredoxin-scaffold folding catalysts should be reached. In an evolutionary/biological context, therefore, the promiscuous activity modulation achieved should be compared with the evolutionary significant modulation range estimated on the basis of activity data for a protein disulfide isomerase. Experimental data for bovine PDI are included in Figure 7 and indeed show an acceptable level of congruence with the Pareto set for the 40-variant variant set (Figure 7A) as well as with the corresponding PLS reconstructions (Figure 7B). Clearly, the modulation range achieved for the catalysis of oxidative folding is significant from a biological/evolutionary point of view. Interestingly, this does not appear to hold for the other folding-related activity of thioredoxin domains: the disulfide-reshuffling activity responsible for the rescue of misfolded proteins with incorrect disulfide bridges. Figure 7C is a plot of reshuffling activity (measured using disulfide-scrambled ribonuclease A as substrate: see Methods for details) versus catalysis of oxidative folding, including the 40-variant set, wild-type thioredoxin from E. coli, the P34H background variant and bovine PDI. Figure 7D is a similar plot including the PLS reconstructions based on the experimental data of Figure 7C. These two plots suggest that the combinatorial library used (based on the mutation set derived from SCA analysis: Figure 4) spans the evolutionary relevant range for the catalysis of oxidative folding, but not the corresponding range for the reshuffling activity. This result is actually consistent with some known features of the structure-function relationship in protein disulfide isomerases. PDIs have a multidomain structure usually described [46] in terms of four distinct domains (a b b′ a′), two of which (the a and a′ domains) display the thioredoxin-fold structure and the CXXC active site motif responsible for the catalysis of disulfide-linked process. The isolated a and a′ domains have been shown to introduce efficiently disulfide bridges into proteins [47], while additional domains are required for efficient catalysis of disulfide bond reshuffling in folded proteins [48]–[50], perhaps because the “inactive” b and b′ domains play a role in facilitating steps that involve difficult conformational changes [48]. Obviously, this “multidomain” effect cannot be reproduced by engineering based on a single-domain thrioredoxin scaffold. The primary/promiscuous plots presented so far (Figures 5,6 and 7), employ logarithmic activity scales in order to emphasize the order of magnitude of the modulations achieved. However, using linear scales in these plots (Figures 8A and 8B) reveals a surprisingly simple pattern: a linear-like Pareto set and a roughly triangular shape for the “cloud” of experimental data points below the Pareto set. This pattern is robust, being observed in the 40-variants data set and in the PLS reconstructions of the full combinatorial library. Note also that the observed pattern implies that essentially all the experimental data points are at or below a line connecting the expected maximum values for the primary and promiscuous activities and, therefore, that the experimental data points populate an area in the primary/promiscuous activity plot which is about half the maximum area accessible. The probability of this happening by chance if there is no correlation between the primary and promiscuous activities is on the order of (1/2) ∧NDP being NDP the number of experimental data points. This gives a negligible probability for NDP = 1024 even for NDP = 40. Finally, as we have already pointed out, the second-round of the library screening process was sharply focused to the Pareto set and that, as a result, the Pareto set of the 40-varaints experimental set is likely to be close to the Pareto set of the full library. We conclude from all this reasoning that the simple experimental pattern in Figures 8A and 8B is robust and is unlikely to have arisen by chance. It is natural then to seek a simple interpretation for such a simple, but intriguing pattern. As we elaborate below one simple explanation is provided by the so-called conformational diversity hypothesis. The conformational diversity hypothesis posits native proteins may exist in solution as different conformations in equilibrium and provides a plausible structural rationale for the existence of protein promiscuity [5], [51], [52]. In very simple terms, the most populated (i. e., dominant) conformation is responsible for the primary activity while alternative, low-population conformations perform the promiscuous activities. Mutations can shift the equilibria between the different conformations and thus modulate the balance between the primary and promiscuous activities. A linear-like Pareto set could thus be explained in terms of two optimal conformations, each being responsible for catalyzing efficiently only one of the activities. For instance, one conformation would achieve molecular optimization for the substrate reduction process (Figure 2) when the active-site disulfide is reduced, while an alternative conformation would achieve optimization for substrate oxidation (Figure 3) when the active-site disulfide is oxidized. Obviously, data points below the Pareto set would correspond to significant population of other conformations that are suboptimal in terms of activity. This interpretation is clarified below with a simple illustrative example. Consider three protein conformations: a0: a conformation with no activity; a1: the conformation responsible for the primary activity; a2: the conformation responsible for the promiscuous activity. The mol fractions of the three conformations must add up to unity: (3) Mutations may change these mol fractions and, obviously, the optimal primary/promiscuous activity situations will be achieved when X (a0) = 0 and, (4) Since activities should be proportional to the corresponding mol fractions, equation 4 defines a straight line in a plot of promiscuous versus primary activity. We refer to this line as the “trade-off line”. In the same plot, suboptimal situations in which X (a0) ≠0 will necessarily be represented by points in a triangular area defined by the trade-off line and the plot axes (see Figure 8D). Certainly, the plot in Figure 8D (showing the optimal trade-off line and a shaded triangular region corresponding to suboptimal situations) is an idealized representation. In practice, we must consider the possibility that the mutations used are unable to completely shift the equilibria towards the active conformations (i. e., they might be unable make the mole fraction of the inactive conformation strictly equal to zero). To provide an illustration of this situation, we have carried out a stochastic simulation of a 40-variant data set, assuming that conformation populations are proportional to statistical weights derived from flat distributions. That is, the mol fraction of a given conformation ai is given by wi/Σwi where wi is its statistical weight (derived from a random number generator in the [0,1] interval) and Σwi is the sum of the statistical weights for all the conformations. The result of this simulation (Figures 8E) is a roughly triangular-shaped cloud of data points with a linear-like Pareto set that approaches the trade-off line. For simplicity and illustration, the simulations included in Figure 8 assume that there is only one sub-optimal conformation and that it has zero primary and promiscuous activity levels. It is important to note, however, that the general result of the simulations is robust and it is obtained with different conformation models (see Figures 9A–C) including several sub-optimal conformations with non-zero activity levels. Apparently, all that is required for a linear-like Pareto set to be obtained in these simulations is a model with two optimal conformations with quite different capabilities to catalyze the primary and promiscuous processes. Actually, if the two optimal conformations are efficient at catalyzing both activities (and, therefore, there are no trade-offs!), the pattern of a linear-like Pareto set with a triangular-like data points cloud is not obtained (see Figure 9D for a representative simulation). Certainly, the simulations discussed above (Figures 8 and 9) are not meant to be taken as direct evidence in support of the conformational diversity. In fact, obtaining such direct evidence would require extensive structural and dynamic characterization (see, for instance [53] and [54]) which is beyond the scope of this work. Because of this we cannot rule out that other kinds of models (based, for instance, on modeling the mutation effects on the activities) could also explain the experimental results founds. Nevertheless, it is clear that the conformational diversity hypothesis provides a simple, Occam-razor explanation (since modeling of specific mutation effects is not involved) for an equally simple, but otherwise intriguing, experimental modulation pattern. The simultaneous enhancement of the primary and promiscuous activities we have discussed in the preceding sections is only one aspect (albeit a prominent one) of a general property of the set of mutations derived from the function-based statistical coupling analysis: the potential for originating a multiplicity of mutational paths leading to different types of function modulation patterns. To illustrate the idea (Figure 10) we use one of the full-library reconstructions derived from the PLS analysis of the 40-variants experimental set. Each of the mutational paths shown in Figure 10 has been constructed using the following procedure: a) An initial variant is chosen; b) the variants connected to the chosen one by single mutations are tested for a given activity-related condition; c) one variant among those that pass the test is randomly selected; d) the cycle a-c is repeated until no mutational steps are available. The paths shown in Figure 10A start with the background variant (i. e., the variant with no mutations) and mutational steps are allowed if promiscuous activity (catalysis of oxidative folding) is increased while the primary activity (reductase) is maintained above a certain threshold. These simulations illustrate the case in which there is selection for enhanced promiscuous activity while maintaining a level of the primary activity that does not compromise fitness. Several paths lead to a variant with increased promiscuous activity and still a significant level of primary activity. Interestingly, PDIs show a significant level of reductase activity (see published work [47], [48], [50] and Figure 7), perhaps because the catalysis of disulfide-linked folding likely involves steps in which incorrect disulfide bridges must be broken up. It is thus tempting to speculate that the no-trade-off paths in Figure 10A illustrate some of the actual function changes taking place in the evolution of these disulfide-linked folding catalysts. It is also interesting that some of the intermediate variants in the paths of Figure 10A have significantly increased levels of both activities, again emphasizing the possibility of the simultaneous enhancement of the primary and promiscuous functions. The Paths shown in Figure 10B use as starting point a variant with comparatively high values of both, the primary activity and the promiscuous activity (actually, a member of the Pareto set of optimal solutions) and mutational steps are allowed if the primary activity is decreased while the promiscuous activity remains above a given threshold. The paths in Figure 10B lead to a specialist protein with high promiscuous activity and low primary activity. These paths could be viewed as illustrating the molecular changes that could in some cases occur in one of the gene copies arising from the gene duplication event involved in the emergence of a new function. According the so-called balance hypothesis [55], single-gene duplication may actually be harmful because it immediately leads to a very large excess of a given protein, which may be deleterious. If imbalance is associated to an excessive level of the primary function, then the mutational paths in Figure 10B illustrate a potential of the mutational space explored by natural selection to efficiently restore balance. A final clarification should be made. The mutational paths in the illustrative simulations summarized in Figure 10 have been obtained assuming that all the single-mutation steps can be readily achieved, although some of them cannot be realized with a single-base substitution. However, these amino acid substitutions do occur during natural evolution (involving an intermediate amino acid) as clearly shown by the sequences in Figure 4D. In connection with this, it is important to note that the mutational space we have characterized is very likely a subspace of the full mutational space explored by natural selection in the evolution of disulfide-linked folding catalysts (the latter involving additional positions and several mutations at each position). Obviously, this fact only reinforces our conclusions. Current views on the relation between primary and promiscuous protein activities are derived to a significant extent from laboratory evolution experiments aimed at enhancing promiscuous functions. Many of these studies have found a decrease (often moderate) in primary activity concomitantly with the increase in promiscuous function, suggesting that the two activities trade-off. In this work, we have introduced an approach to determine how the interplay between the primary and promiscuous activities of a protein is modulated in the mutational space evolutionary linked to the emergence of a new function. Application of this new approach to the emergence of folding catalysts reveals a hitherto unexplored scenario: diverse patterns of primary/promiscuous activity modulation may occur as response to different types of evolutionary pressure, including no-trade-off paths involving the simultaneous enhancement of both activities. Some general remarks related with this result are appropriate: BLAST2 (1996–2003, W. Gish http: //blast. wustl. edu) was used to search the TrEMBL sequence database of October-2007 (http: //www. ebi. ac. uk/trembl) using the sequence of E. coli thioredoxin as query. The resulting sequences were aligned with the query sequence using the Smith-Waterman algorithm and only those with sequence identity with the query of 0. 3 or higher were retained for further analysis. We made no further attempt to correct or filter the alignment, since the results obtained from its analysis made clear sense from both, the structural and functional viewpoints (see Figures 4 and 5). Of the1440 sequences in the alignment used, 1264 had a proline at position 34 and 132 had a histidine at that position. Essentially all the sequences with histidine at position 34 belonged to eukaryotes and most of them were actually annotated as protein disulfide isomerases. Statistical coupling analysis of the sequence alignments based on the P34→H perturbation were performed using homemade programs, but in a manner identical to that described by Lockless and Ranganathan [24]. The robustness of this analysis is supported by the fact that the positions with high values for the statistical coupling energy also rank high in a simple covariance analysis [58] of the sequence alignments (see Figure S1 in Supporting Information). The combinatorial library of thioredoxin variant sequences on the P34H background was prepared by using gene assembly mutagenesis as we have previously described [59]. For ease of protein purification, the genes encoded a His6 tag at the N-terminal end (i. e., at a position roughly opposite to the active-site region). Purification of the thiredoxin variants, assessment of their purity and concentration measurements, were performed as previously described [59]. Bovine PDI was purchased from Sigma and used without further purification. Reductase activity of the thioredoxin variants was determined at 37°C by a turbidimetric assay of the thioredoxin catalyzed reduction of insulin [60]. Briefly, thioredoxin-variant (or PDI) solutions at pH 6. 5 (phosphate buffer 0. 1 M) in the presence of 2 mM EDTA and 0. 5 mg/mL insulin were prepared. The reactions were initiated by addition of DTT to a 1 mM final concentration and monitored by measuring the absorbance at 650 nm (A650) as function of time. Activity is calculated as the maximum value of the change of A650 with time, i. e., the maximum value for the derivative dA650/dt (see Figures S2 and S3 in Supporting Information for representative examples). Typically, for each variant, 3–4 experiments at different thioredoxin-variant concentrations (within the 0–5 µM range) were carried out and the specific activity values, together with its associated standard errors, were determined from linear fits to the activity versus concentration profiles: see Figure S4 in Supporting Information for representative examples and for further details. The catalysis of oxidative folding activity was determined by following the recovery of ribonuclease A (RNase A) activity from completely reduced RNase A following the procedure described by Lundström et al. [43]. Briefly, nitrogen-saturated solutions of thioredoxin variants (or PDI) in 0. 1 M phosphate buffer pH 7 in the presence of 1 mM EDTA and 100 µM oxidized glutathione were prepared. The reaction was initiated by addition of RNase A from a stock solution to a final concentration of 0. 4 mg/mL. After 1 hour incubation at 37°C, the RNase A activity was determined using the standard assay based on the hydrolysis of 2′-3′-cCMP. Typically, for each variant, 4 experiments at different thioredoxin-variant concentrations (within the 0–15 µM range) were performed and the specific activity values, together with its associated standard errors, were determined from linear fits to the recovered RNase A activity versus concentration profiles. Assays for the disulfide reshuffling activity were carried out in the same way, except that disulfide-scrambled RNase A was used and 100 µM reduced glutathione was included in the reaction solution. Fully-reduced and scrambled RNase A were prepared as described by Lündstrom et al. [43] See Figures S5 and S6 in Supporting Information for representative examples of the disulfide-linked folding assays and for further details. PLS analyses were carried out with the program Unscrambler X from CAMO software using the NIPALS algorithm. In all cases, the dependent variables were the logarithms of the values for the primary and promiscuous activities and were auto-scaled (i. e., they were subjected to mean subtraction followed by division by the standard deviation) prior to the analysis. Leave-one-out cross-validation was used and the number of latent variables retained was the optimum value suggested by the Unscrambler program on the basis of the mean square error of cross-validation. Actually, the PLS analyses were carried with 20 replica sets constructed from the original set through random re-sampling (bootstrapping) and the number of latent variables retained did depend on the replica set used; typical values, however, were on the order of 3–11 (i. e., much smaller than the numbers of dependent and independent variables involved). Illustrative plots experimental versus predicted activities are given in Figure S7 in Supporting Information.

Interpretations of evolutionary processes at the molecular level have been determined to a significant extent by the concept of "trade-off", the idea that improving a given feature of a protein molecule by mutation will likely bring about deterioration in other features. For instance, if a protein is able to carry out two different molecular tasks based on the same functional site (competing tasks), optimization for one task could be naively expected to impair its performance for the other task. In this work, we report a computational/experimental approach to assess the potential patterns of modulation of two competing molecular tasks in the course of natural evolution. Contrary to the naïve expectation, we find that diverse modulation patterns are possible, including the simultaneous optimization of the two tasks. We show, however, that this simultaneous optimization is not in conflict with the trade-offs expected for two competing tasks: using the language of the theory of economic efficiency, trade-offs are realized in the Pareto set of optimal variants for the two tasks, while most protein variants do not belong to such Pareto set. That is, most protein variants are not Pareto-efficient and can potentially be improved in terms of several features.

lay_plos

CROSS REFERENCE TO RELATED APPLICATION This application is related to application Ser. No. 806,394, filed Dec. 9, 1985, which is commonly assigned. BACKGROUND OF THE INVENTION This invention relates to the art of measurement for detecting the state of motion of an internal moving medium of a living body, and more particularly to a novel technique which is effectively, applicable to an ultrasonic diagnosis apparatus used for measurement of the moving speed (referred to hereinafter simply as speed) of an internal moving medium of a living body or measurement of the moving speed and moving speed dispersion (referred to hereinafter simply as speed dispersion) of such a moving medium. An ultrasonic pulse-Doppler method has been put into practical use for the measurement of the speed of an internal moving medium of a living body, for example, a visceral organ such as the heart or a fluid such as blood or humor in a circulatory organ. A prior art, ultrasonic diagnosis apparatus utilizing the ultrasonic pulse-Doppler method described above is disclosed in, for example, Jpaanese Unexamined Patent Publication No. 58-188433 (JP-A-58-188433) laid open on Nov. 2, 1983. In the cited patent publication, an autocorrelator is employed for converting a high-frequency signal reflected from an internal moving medium of a living body into complex signals and then computing autocorrelation between the complex signals, and a speed operator is employed to compute the speed of the moving member on the basis of the computed autocorrelation. However, the necessity for incorporation of the autocorrelator has required a complicated circuit structure resulting in a bulky overall size of the ultrasonic diagnosis apparatus. In the prior art, ultrasonic diagnosis apparatus disclosed in the cited publication, delay line cancellers are employed together with the autocorrelator for detecting the speed of the internal moving medium of the living body on the basis of the high-frequency signal received by an ultrasonic probe and including a Doppler shift frequency. Numerical formulas used for carrying out a series of signal processing on the received high-frequency signal by the delay line cancellers and autocorrelator do not include amplitude terms, and the speed of the moving medium is computed under the assumption that the amplitude of the received high-frequency signal is constant. Consider now the case of detection of the speed of blood flow, as an example. In this case, it is limitatively assumed that the second power of the amplitude (energy) of the Doppler shift signal is proportional to the number of blood cells scanned by the ultrasonic beam, and the number of blood cells scanned by the ultrasonic beam is constant regardless of the rate of blood flow. However, the number of blood cells scanned by the ultrasonic beam is not always constant regardless of the rate of blood flow. This is because turbulence may occur in the blood flow, and there is a concentration difference between red blood cells and white blood cells. Thus, the amplitude of the Doppler shift signal is not always constant. Further, although the power of the ultrasonic signal attenuates while the ultrasonic signal is received after it is transmitted into the living body, the amount of beam attenuation varies depending on the tissue structure of the scanned portion of the living body. Therefore, in the case of a moving medium such as the heart where the momentum is large, the state of the tissue structure, through which the ultrasonic beam passes, varies continuously, and it is unable to always receive a high-frequency signal including a Doppler shift signal having a constant amplitude. Thus, the prior art manner of signal processing, according to which the average speed is computed under the assumption the amplitude of the received signal is always constant, has been defective in that an error is inevitably included in the result of computation. SUMMARY OF THE INVENTION It is an object of the present invention to provide an ultrasonic diagnosis apparatus which can measure the speed of an internal moving medium of a living body using a simplified circuit structure not employing the autocorrelator incorporated in the prior art apparatus. Another object of the present invention is to provide an ultrasonic diagnosis apparatus which can measure the speed dispersion is addition to the speed of an internal moving medium of a living body using a simplified circuit structure not requiring the autocorrelator. The measurement of the speed dispersion is made in the case where the moving member is a fluid in a living body such as blood or humor. According to the present invention, an ultrasonic beam transmitted toward and reflected from an internal moving part of a living body is received and amplified, and, after conversion of the reflected wave into complex signals, the speed of the moving part is immediately computed by a speed operating circuit on the basis of the complex signals. In addition to the measurement of the speed of the moving member, the speed dispersion can also be computed by a speed dispersion operating circuit. Further, the speed operating circuit and the speed dispersion operating circuit are preferably those operable at a high speed so that individual points lying in the depthwise direction of the directed ultrasonic beam can be measured in real time. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a block diagram showing the general structure of a preferred embodiment of the ultrasonic diagnosis apparatus according to the present invention. FIGS. 2 to 5 illustrate how the speed and speed dispersion are computed in the embodiment of the present invention. FIG. 6 is a block diagram showing the detailed structure of one form of the speed operating circuit shown in FIG. 1. FIG. 7 is a block diagram showing the detailed structure of another form of the speed operating circuit shown in FIG. 1. FIG. 8 is a block diagram showing the detailed structure of one form of the speed dispersion operating circuit shown in FIG. 1. DESCRIPTION OF THE PREFERRED EMBODIMENTS A preferred embodiment of the present invention showing an application of the present invention to an ultrasonic diagnosis apparatus adapted to acquire information of an internal moving medium of a living body according to an ultrasonic pulse-Doppler method, will now be described in detail with reference to the accompanying drawings. In the drawings, the same reference numerals are used to designate the same functional parts. Referring to FIG. 1, an ultrasonic beam transmitted toward an internal moving medium of a living body from an ultrasonic probe 1 is produced by a transmitter circuit 2. Pulses of the transmitted ultrasonic beam are reflected from the internal moving medium of the living body to be received by the ultrasonic probe 1. The received high-frequency signal including internal information of the living body is amplified by a high-frequency amplifier circuit 3. A crystal oscillator 4 generates a high-frequency synchronizing signal. This high-frequency synchronizing signal is converted by a synchronizing circuit 5 into a reference signal having a frequency corresponding to the recurrence frequency of the ultrasonic pulses transmitted from the ultrasonic probe 1. The phase of the reference signal generated from the synchronizing circuit 5 is shifted by 90° by a phase shifter 6. The 90°--phase shifted reference signal from the phase shifter 6 is mixed with the amplified received signal in a first mixer 7 which acts to provide information of the moving direction of the moving medium which is, for example, blood flow. On the other hand, the reference signal from the synchronizing circuit 5 is directly mixed with the amplified received signal in a second mixer 8. A first canceller 14 and a second canceller 15 extract only Doppler components having information of the internal moving medium of the living body from the output signals of the first and second mixers 7 and 8 respectively. A speed operating circuit 16 computes the speed of the internal moving medium of the living body on the basis of the canceller output signals representing the extracted Doppler components. A speed dispersion operating circuit 17 computes the value of speed on the basis of the value of the speed computed by the speed operating circuit 16. The received signal is thus converted by the speed operating circuit 16 and speed dispersion operating circuit 17 into a signal indicative of the speed and a signal indicative of the speed deviation. An operation selector switch 18 is provided to determine whether the speed only is to be computed or whether both the speed and the speed dispersion are to be computed. This operation selector switch 18 is changed over under control of an operation selection setting circuit 19. The operation selector switch 18 may be disposed between the speed operating circuit 16 and the speed dispersion opreating circuit 17. A detector 20 detects the received signal including the internal information of the living body and amplified by the high-frequency amplifier circuit 3 so as to extract a sectional image signal of the internal moving medium of the living body. This extracted sectional image signal is written in a digital scan converter (abbreviated hereinafter as a DSC) 21. The high-frequency component of the output signal of the first mixer 7 is removed by a low-pass filter 10, and the output signal of the low-pass filter 10 is applied to a sample and hold circuit 11. In order to extract a one-dimensional Doppler shift component from the output signal of the low-pass filter 10 in a usual manner, a gate pulse signal produced by a sampling pulse generator 9 is applied to the sample and hold circuit 11 thereby extracting the signal representing the Doppler shift of the internal moving medium of the living body. After being smoothed by a band-pass filter 12, the Doppler shift signal is applied to a frequency analyzing circuit 13 there the one-dimensional Doppler shift signal is extracted as by high-speed Fourier transformation. This one-dimensional Doppler shift signal is also written in the DSC 21. The signals indicative of the internal information of the living body, written in the DSC 21, are converted into a video signal in the DSC 21 and read out as a television signal to be displayed on a display unit 22 such as a CRT monitor or a television monitor. Among the internal information of the living body, the speed and speed dispersion are computed according to arithmetic formulas or expressions described later. In order to compute the speed and speed dispersion of an internal member at a certain predetermined depth, it is necessary to detect amounts of Doppler shift occurring in a plurality of ultrasonic waves reflected from the predetermined depth. For conveniences of description, it is supposed herein that the number of received signals is two. The ultrasonic beam is transmitted from the ultrasonic probe 1 toward an internal moving medium of a living body at a time interval of Δt, and ultrasonic waves reflected from the internal moving 66 medium of the living body are received by the ultrasonic probe 1. When a time-dependent change of each reflection point is taken into consideration, the intensity at the reflection point of the Doppler signal extracted from each of the received signals is represented by the value, at a certain time, of a periodic function representing a certain intensity and having a period of Doppler shift. In order to detect the intensity represented by the periodic function and resulting from the Doppler shift (that is, the kinetic momentum of the internal moving medium of the living body), the combination of the phase shifter 6 and the mixers 7, 8 produces signals having a phase difference of 90° on the basis of the received signals. The intensities a, b, c and d of Doppler shift, at a certain depth, of the four signals in total are given by phases at time t and (t+Δt) as shown in FIG. 2. More preciselly, the intensities a and c, at time t, of the functions having the period (frequency) f d of Doppler shift and having the phase difference of 90° therebetween are expressed respectively as follows: a=l sin 2πf.sub.d t (1) c=l cos 2πf.sub.d t (2) where l is the absolute value of the intensity of the signal received at time t. Similarly, the intensities b and d at time (t+Δt) are expressed respectively as follows: b=l sin 2πf.sub.d (t-Δt) (3) d=l cos 2πf.sub.d (t-Δt) (4) On the basis of these expressions (1) to (4), the absolute intensities l t and l.sub.(t+Δt) of the functions at times t and (t+Δt) are expressed respectively as follows: l.sub.t =(a.sup.2 +c.sup.2).sup.1/2 (5) l.sub.(t+Δt) =(b.sup.2 +d.sup.2).sup.1/2 (6) These absolute intensities l t and l.sub.(t+Δt) are proportional to the flow rate of a fluid, for example, blood in the living body. How to compute the moving speed of the fluid in the living body will next be described. FIG. 3 illustrates that, at times t and (t +Δt), the Doppler waves have respective angular displacements θ and δ which are expressed as follows: ##EQU1## It is to be noted that the values of θ and δ determined by the expressions (7) and (8) lie within the ranges of -90°&lt;θ&lt;90° and -90°&lt;δ&lt;90° respectively. Therefore, in order to determine the values of θ and δ so that they lie within the ranges of 0°&lt;θ&lt;360°, 0°&lt;δ&lt;360°(9), the value of θ is computed on the basis of a (=l sin 2πf d t), c (=l cos 2πf d t) and c&#39; [=l cos (π-2πf d t)], as shown in FIGS. 4 and 5. Similarly, the value of δ is computed on the basis of b [=l sin 2πf d (t-Δt)] and d [=l cos 2πf d (t-Δt)]. That is, depending on whether the sine and cosine components of the signals having the phase difference of 90° are positive or negative, the value of x is sin -1 x lies is various ranges as shown in the following Table I: TABLE 1______________________________________sin coscomponent component Range of x______________________________________Positive Positive 0° &lt; × &lt; 90°Positive Negative 90° &lt; × &lt; 180°Negative Negative 180° &lt; × &lt; 270°Negative Positive 270° &lt; × &lt; 360°______________________________________ On the basis of Table I, the values of θ and δ given by the expressions (7) and (8) respectively are computed. The displacement angle Δθ of θ in the minute length of time of change Δt is computed according to the following expression (10) on the basis of the expressions (7) and (8): Δθ=θ-δ (10) The angular velocity ω is expressed as follows: ω=Δθ/Δt =(δ-θ)/Δt (11) From the above expression (11), the frequency f d of the Doppler shift is expressed as follows: f.sub.d =2π·ω=2π·(δ-θ)/Δt Since this Doppler shift frequency f d is proportional to the speed of the fluid flow in the living body, the moving direction of the fluid and the speed of the fluid flow in the living body can be computed on the basis of the Doppler shift frequency f d. The speed 16 shown in FIG. 1 computes the speed of the fluid flow in the living body according to the speed-computing expressions described above. The detailed structure of one form of the speed operating circuit 16 is shown in FIG. 6. Referring to FIG. 6, the values of the sine component, [a given by the expression (1) and b given by the expression (3)], of the signals received at times t and (t+Δt) are sequentially supplied to and temporarily latched in that order in a first latch circuit 101. Similarly, the values of the cosine component, [c given by the expression (2) and d given by the expression (4)], of the signals received at the times t and (t+Δt) are sequentially supplied to and temporarily latched in that order in a second latch circuit 102. A first delay circuit 103A is connected to the output of the first latch circuit 101 to provide the value of the sine component of the signal extracted from the preceding ultrasonic beam received from the same depth, this is, the value of a given by the expression (1). A second delay circuit 103B is connected to the output of the second latch circuit 102 to provide the value of the cosine component of the signal extracted from the preceding ultrasonic beam received from the same depth, that is, the value used for computation of sin -1 shown in Table I. An operator 104, which is in the form of a ROM (a read-only memory), has a table for computing the absolute value, [the value of l t given by the expression (5)], of the intensity of the received signal on the basis of the value of the sine component [a given by the expression (1)] and the value of the cosine component [c given by the expression (2)]. A third delay circuit 105 is connected to the output of the ROM 104 to provide the absolute value, [the value of l.sub.(t+Δt) given by the expression (6)], of the intensity of the signal extracted from the preceding ultrasonic beam received from the same depth. A second ROM 106 has a table for computing the value of the angle δ according to the expression (8) and Table I on the basis of the data outputs of the latch circuits 101, 102 and ROM 104. A third ROM 107 has a table for computing the value of the angle θ according to the expression (7) and Table I on the basis of the data outputs of the delay circuits 103A, 103B and 105. A fourth ROM 108 has a table for computing the value of the displacement angle Δθ according to the expression (10) on the basis of the data outputs of the ROM&#39;s 106 and 107. A Δt setting circuit 109 sets the time internal Δt of the transmission of the ultrasonic beam from the ultrasonic probe 1. A fifth ROM 110 has a table for computing the value of the angular velocity ω given by the expression (11) on the basis of the data output of the ROM 108 and the value of the setting of the time interval Δt of ultrasonic beam transmission. The operation of the speed operating circuit 16 having such a construction will be described with reference to FIG. 6. The data b of the sine component of the signal extracted from the reflected wave received at time (t+Δt) is supplied from the canceller 14 shown in FIG. 1 to the first latch circuit 101, and the data d of the cosine component of the signal extracted from the received wave is supplied from the canceller 15 to the second latch circuit 102. From the latch circuits 101 and 102, the data b and d of the sine and cosine components of the signal are supplied to the first ROM 104, and, from this ROM 104, the absolute value l(t+Δt) of the intensity of the received ultrasonic beam is supplied to the second ROM 106. Since the data b and d of the sine and cosine components of the received signal have been supplied from the first and second latch circuits 101 and 102 respectively to the second ROM 106 to be used for the computation of the value of the angle δ according to the expression (8), the computed value of the angle δ at time (t+Δt) is supplied from the second ROM 106 to the fourth ROM 108. The data output of the first ROM 104 or the absolute value l t of the intensity of the signal contracted from the preceding ultrasonic beam received at time t has been supplied to the third delay circuit 105 to be delayed by the setting of the time interval Δt of ultrasonic beam transmission. absolute value l t of the intensity of the signal extracted from the preceding ultrasonic beam received at time t is supplied from the third delay circuit 105 to the third ROM 107. Since the data a and c of the sine and cosine components of the signal extracted from the preceding ultrasonic beam received have been supplied to this ROM 107 from the first and second delay circuits 103A and 103B respectively to be used for the computation of the value of the angle θ according to the expression (7), the computed value of the angle θ at time t is supplied from the third ROM 107 to the fourth ROM 108 to be used for the computation of the displacement angle Δθ according to the expression (10). This computed value of Δθ is supplied to the fifth ROM 110 together with the value of Δt supplied from the Δt setting circuit 109 to be used for the computation of the angular velocity ω according to the expression (11), and the computed value of ω is supplied to the speed dispersion operating circuit 17 and DSC 21. Another form of the speed operating circuit 16 is shown in FIG. 7. Referring to FIG. 7, the speed operator 16A includes a microprocessor 201, a ROM 202, a RAM (a random access memory) 203 and an interface 204 connected together by a bus line. In this speed operating circuit 16A, the expressions (1) to (11) are computed according to a programmed software. The detailed structure of one form of the speed dispersion operating circuit 17 shown in FIG. 1 will be described with reference to FIG. 8. The computation of the speed dispersion σ carried out in this speed dispersion operating circuit 94 is expressed as follows: ##EQU2## where v i [i : 1 to n (an integer)] is the moving speed computed by the speed operating circuit 16, and N is the average speed. Referring to FIG. 8, buffers 300A to 300H store data of the moving speed v i. These buffers 300A to 300H are, for example, such that each of them has a capacity enough to store speed data extracted from the reflected wave of one ultrasonic beam. An address generator 301 generates addresses of the buffers 300A to 300H. These buffers 300A to 300H are selected under control of a controller 302 which is included in a control apparatus controlling the entire system. An ordinary operator 303 executes the computation of the speed dispersion σ according to the expression (12). Another ordinary operator 304 computes the average speed N according to the expression (13). The operation of the entire ultrasonic diagnosis apparatus embodying the present invention will now be described with reference to FIG. 1. The ultrasonic pulse beam transmitted from the ultrasonic probe 1 is generated by and applied from the transmitter circuit 2. The transmitted ultrasonic pulse beam is reflected from an internal moving medium of a living body, and the reflected ultrasonic beam is received by the ultrasonic probe 1. The received signal including the internal information of the living body is amplified by the high-frequency amplifier circuit 3. The stable high-frequency signal generated from the crystal oscillator 4 is converted by the synchronizing circuit 5 into the reference signal having the frequency corresponding to the recurrence frequency of the ultrasonic pulse beam, and the reference signal is applied to the mixer 8. The received signal amplified by the high-frequency amplifier circuit 3 is applied to the mixer 8 to be mixed with the reference signal. Also, the reference signal is applied to the phase shifter 6 to be shifted by 90° in its phase, and the output signal of the phase shifter 6 is mixed with the signal received by the ultrasonic probe 1 and amplified by the high-frequency amplifier circuit 3, so as to provide information of the moving direction of the internal moving medium of the living body. In order to extract the Doppler components bearing the kinetic information of the internal moving medium of the living body, the output signals of the mixers 7 and 8 are applied to the cancellers 14 and 15 respectively. The individual signals including only the Doppler components extracted by the cancellers 14 and 15 are used for the computation according to the aforementioned procedure in the speed operating circuit 16 to find the value of the speed v i. The speed deviation operating circuit 17 makes necessary computations according to the aforementioned procedure to find the value of the speed dispersion σ. The computed values of the speed v i and speed dispersion σ are stored in the DSC 21. Whether the computation to find the speed v i only is to be executed or the computation to find both the speed v i and the speed dispersion σ is to be executed is determined by the operation selection setting circuit 19, and the operation selector switch 18 is opened or closed. Further, the detector 20 detects the signal indicative of the sectional image of the internal moving medium of the living body from the living-body signal received by the ultrasonic probe 1 and amplified by the high-frequency amplifier circuit 3, and the output signal of the detector 20 is also stored in the DSC 21. The low-pass filter 10 removes the high-frequency component of the output signal of the mixer 7, and the output signal of the low-pass filter 10 is applied to the sample and hold circuit 11. In order to extract the Doppler shift component from the output signal of the low-pass filter 10, the gate pulse signal generated from the sampling pulse generator 9 is applied to the sample and hold circuit 11. As a result, the signal indicative of the Doppler shift of the internal moving medium of the living body is extracted in the sample and hold circuit 11. The output signal of the sample and hold circuit 11 is smoothed by the band-pass filter 12 and is then applied to the frequency analyzing circuit 13 which extracts the one-dimensional Doppler shift signal. This extracted Doppler shift signal is also stored in the DSC 21. The data stored in the DSC 21 are supplied to the display unit 22 to be displayed thereon. The pattern of the picture information displayed on the display unit 22 is selected under control of the system control apparatus (not shown). All the ROM&#39;s of the speed operating circuit 16 may have tables of the results of individual computations written therein so that the speed v i can be computed at a higher speed. Also, the speed dispersion operating circuit 17 may have a table of the results of computation of the average speed N so that the average speed used for the computation of the speed dispersion of the moving medium can be computed at a higher speed on the basis of the speed information stored in the buffers. It can be seen from the foregoing description that the embodiment of the present invention illustrated in FIGS. 1 to 8 provides the following advantages: (1) The speed of an internal moving medium of a living body can be computed in such a manner as to also follow up any change in the intensity of the signal reflected from the moving part, as shown by the expressions (1) to (11). Therefore, the result of computation of the speed according to the present invention is more accurate than that computed according to the prior art method. Thus, more accurate data of the speed of the internal moving part of the living body can be obtained. (2) The operating circuits 16 and 17 including ROM&#39;s are only required for computing the speed and speed dispersion respectively, thereby dispensing with the use of the autocorrelator essentially required in the prior art diagnosis apparatus. Therefore, the size of the diagnosis apparatus of the present invention can be reduced, and the cost can also be reduced. (3) Since the operating circuits 16 and 17 including ROM&#39;s as described above are used for computing the speed and speed dispersion respectively, the speed and speed dispersion an be computed faster than prior art devices. (4) By virtue of the advantages (1) to (3), the speed or both the speed and the speed dispersion of an internal moving medium of a living body scanned by an ultrasonic beam can be measured and computed for each depth at a high speed by the diagnosis apparatus of simple construction. Therefore, a greater amount of accurate information can be obtained for the diagnosis of an internal organ of a living body, so that the accuracy of diagnosis can be greatly improved. While a preferred embodiment of the present invention has been described in detail by way of example, it is apparent that the present invention is in no way limited to such a specific embodiment, and many changes and modifications may be made therein without departing from the subject matter thereof. It will be understood from the foregoing detailed description that, according to the present invention, the speed or both the speed and the speed dispersion of an internal moving medium of a living body scanned by an ultrasonic beam can be measured and computed for each depth at a high speed by a diagnosis apparatus of simple construction employing a speed operating circuit or the combination of a speed operating circuit and a speed dispersion operating circuit only. Therefore, a greater amount of accurate information can be obtained for the diagnosis of an internal organ of a living body, so that the accuracy of diagnosis can be greatly improved.

An ultrasonic diagnosis apparatus comprises an ultrasonic probe for transmitting an ultrasonic pulse beam toward an internal moving medium of a living body at a constant recurrence frequency, a converter for mixing the received high-frequency signal with a set of complex reference signals having a frequency n times as high as the recurrence frequency of the transmitted ultrasonic pulse beam and having a complex relation therebetween, thereby converting the high-frequency signal into complex signals, a speed operating circuit for computing the moving speed of the internal moving medium on the basis of the complex signals, and a display unit for displaying the moving speed in real time. The apparatus may include a speed dispersion operating circuit for computing the dispersion of the moving speed computed by the speed operating circuit in order to display both the moving speed and the dispersion thereof on the display unit.

big_patent

In budding yeast, the Pif1 DNA helicase is involved in the maintenance of both nuclear and mitochondrial genomes, but its role in these processes is still poorly understood. Here, we provide evidence for a new Pif1 function by demonstrating that its absence promotes genetic instability of alleles of the G-rich human minisatellite CEB1 inserted in the Saccharomyces cerevisiae genome, but not of other tandem repeats. Inactivation of other DNA helicases, including Sgs1, had no effect on CEB1 stability. In vitro, we show that CEB1 repeats formed stable G-quadruplex (G4) secondary structures and the Pif1 protein unwinds these structures more efficiently than regular B-DNA. Finally, synthetic CEB1 arrays in which we mutated the potential G4-forming sequences were no longer destabilized in pif1Δ cells. Hence, we conclude that CEB1 instability in pif1Δ cells depends on the potential to form G-quadruplex structures, suggesting that Pif1 could play a role in the metabolism of G4-forming sequences. At the chromosomal level, in addition to coding regions and epigenetic modifications, the biological information also resides in DNA secondary structures, but this layer remains to be further deciphered. Biophysical and structural studies have long established that in vitro DNA can adopt diverse structures different from the canonical Watson-Crick conformations [1]. However, for a long time, the hypothesis that these structures occur in the native chromosomal context, as an integral part of the functional architecture of a chromosome, has been regarded with a certain skepticism. One example of such a non canonical DNA structure is the G-quadruplex, also named G-tetraplex or G4 DNA. These structures form in vitro in guanine-rich sequences that contain four tracts of at least three guanines separated by other bases, and are stabilized by G-quartets that form between four DNA strands [2]. Under physiological conditions, long runs of G4-forming sequences promote the formation of highly stable structures that can form spontaneously in vitro and, once formed, are very resistant to thermal denaturation. It is also important to consider that sequences that form G4-DNA slowly in vitro may be more prone to fold in vivo owing to the action of proteins that promote and/or stabilize their formation, such as the beta subunit of the ciliate Oxytricha telomere binding protein complex [3], [4]. Evidence for in vivo formation of G4 DNA has emerged in recent years. Notably, G4 DNA has been observed by electron microscopy from transcribed human G-rich DNA arrays in bacteria [5] and has been detected at the end of the ciliate Oxytricha telomeres by immunochemistry [6], [7]. As a complementary approach, genome-wide bioinformatic analyses have identified regions that have the potential to form G4 DNA within evolutionary diverse model systems, from bacteria to human. For example, in the human genome, more than 300,000 distinct sites have the potential to form G4 DNA [8], [9]. These sequences are highly over-represented in the promoter regions of diverse organisms, including human [10], yeast [11] and bacteria [12]. In addition, potential G4-forming sequences are found in G-rich arrays such as telomeres, rDNA or G-rich micro- and minisatellites. Hence, it has been suggested that their presence might affect transcriptional or post-transcriptional events when the G4 forming sequence is within the transcribed region [11], [13]. G4 DNA has also been proposed to participate in telomere capping, DNA replication and recombination [14]. However, it remains to be determined how and to what extent these secondary structures affect these processes and how they are maintained through DNA replication despite causing a structural impairment to the various nucleic acid processing enzymes. It is clear that DNA goes through a single strand configuration locally during processes like DNA replication, transcription or repair, and many models argue that this single stranded stage favors G4 DNA formation [14]. In vitro, several DNA helicases, such as the human BLM, WRN, FANCJ and the S. cerevisiae Sgs1, can unwind G4 structures. They preferentially unwind G4 DNA over partially duplex DNA, forked DNA or Holliday junction substrates, and their helicase activity is inhibited in presence of G4 DNA ligands [15]–[18]. In Caenorhabditis elegans the FANCJ homolog dog-1 is involved in the maintenance of G-rich regions by preventing intrinsic instability and loss of these regions [19], [20]. However, considering that different G-rich sequences can adopt very diverse secondary structures, and that in numerous instances genes encoding helicases are not essential, the questions of how many and which class of helicases are indeed able to process efficiently these secondary structures formed in guanine-rich regions in a given organism remains to be addressed. Also, until now, very few in vivo systems exist to study the involvement of helicases in processing these structures and assay artificially designed variant substrates. In the present study, which was aimed at characterizing the mechanism (s) of rearrangement of tandem DNA repeats, we uncover an unexpected function of the Pif1 helicase with regards to processing G4 structures. Pif1 is a member of a conserved family of 5′-3′ DNA helicases, with distant homology to the RecD bacterial helicase. The S. cerevisiae Pif1 protein is important both for maintenance of mitochondrial DNA [21], [22] and as a negative regulator of telomerase-mediated telomere lengthening [23], [24]. Here we report that Pif1 also affects stability of the G-rich CEB1 minisatellite when it is inserted into a yeast chromosome. In contrast, mutations in other helicases, including the S. cerevisiae RecQ homologue Sgs1, had no effect on CEB1 stability. In vitro, CEB1 formed G4 structures that were efficiently unwound by Pif1. Finally, mutation of the CEB1 repeats such that they were no longer able to form G4 structures made them insensitive to Pif1. Thus we demonstrated that one of the functions of the Pif1 helicase is to process G4 structures. As sequences with the ability to form G4 DNA are found throughout the yeast genome, beyond acting on intrinsically instable repeats, we propose that the processing of G4 structures by Pif1 may facilitate DNA replication, transcription and/or repair. We previously developed yeast strains to study the genetic instability of a natural 1. 8 kb allele of the human minisatellite CEB1 inserted in the S. cerevisiae genome (Figure 1A). This allele (called CEB1-1. 8) is composed of a tandem array of 42 polymorphic repeats of sizes varying between 36 and 43 base pairs (bp) [25] (Figure S1). In our standard assay, which measures the frequency of allele size variation after growth for seven generations at 30°C, approximately 0. 3% of wild-type (WT) cells exhibit a change in CEB1 size (contractions and expansions). Using this system, we reported that CEB1-1. 8 was strongly destabilized in the absence of the Rad27/FEN1 endonuclease (42% instability) [26]. Recently, it was reported that the lethality caused by inactivation of the essential helicase/endonuclease Dna2, which participates with Rad27 in the maturation of Okazaki fragments, could be rescued by inactivation of the DNA helicase Pif1 [27]. These results prompted us to test if Pif1 also had an effect on the maintenance of CEB1 arrays in our system. Remarkably, in the absence of Pif1 (pif1Δ), the frequency of rearrangement by contractions or expansions of the parental allele increased 20-fold compared to WT cells (6% instability; Table 1, Figure 1B). As a control, a pif1Δ CEB1-1. 8 strain containing a multicopy plasmid that expressed the WT PIF1 gene under the control of the PIF1 promoter did not exhibit CEB1 instability. Together, these results demonstrate that the absence of Pif1 destabilizes the CEB1-1. 8 minisatellite at a rate of ∼1% per cell per generation. CEB1 instability was not specific to tracts inserted at the ARG4 locus as CEB1-1. 8 inserted at the ADP1 locus in chromosome III was stable in the presence of Pif1 but was rearranged in its absence (3. 6% instability; 7/192). The difference in stability between the two chromosomal locations is not statistically significant (Fisher' s Exact test, p = 0. 28). To determine if the helicase activity of Pif1 was required to stabilize the CEB1-1. 8 allele, we examined the stability of CEB1-1. 8 in strains carrying the pif1-K264A or pif1-K264R mutations, which inactivate Pif1 ATPase/helicase activity [28]. In both mutants, the frequency of CEB1 rearrangement was increased approximately 10-fold over the WT level (3. 2%; Table 1, Figure 1B). Thus, the helicase activity of Pif1 has a role in the stabilization of the CEB1 repeats during vegetative growth. Compared to the pif1Δ mutant, the frequency of size variants was approximately two-fold lower in both of the helicase-inactive mutants. This suggests that while ATPase/helicase activity is totally inactive in helicase-dead pif1-K264A mutant (see below, Figure 2F), the pif1-K264A polypeptide which retains wild type level of DNA binding [24], may act within a complex of proteins sufficient to partially protect CEB1 repeats from damage or recombinational repair. To characterize the internal structures of CEB1-1. 8 rearrangements obtained in the pif1Δ cells, we sequenced nine CEB1 contractions and compared them to the parental motif. As shown in Figure 1C and Figure S1, the sequenced contractions from pif1Δ cells were all different from each other. Three were simple deletions, one was a double deletion and five were complex events. To determine whether or not the destabilization of CEB1-1. 8 in pif1Δ cells was dependent on homologous recombination, we tested the stability of CEB1-1. 8 in pif1Δ rad52Δ and pif1Δ rad51Δ double-mutants. In both strains, rearrangement of CEB1-1. 8 occurred at close to WT levels, strongly reduced compared to pif1Δ cells (Figure 1D and Table 1). We conclude that the molecular events leading to CEB1 rearrangement are repaired by homologous recombination, similar to what is seen in the absence of Rad27 [25]. To determine if the effects of pif1Δ on CEB1 stability are a secondary consequence of the increased telomere length or mitochondrial DNA depletion that are characteristic of pif1Δ cells, we examined CEB1-1. 8 stability in mutants that affect either telomere length or maintenance of mitochondrial DNA. The deletion of the RIF1 gene results in telomere lengthening [29], a phenotype likely due to the enhanced access of telomerase to the telomere [30]. RIF1 inactivation did not destabilize CEB1-1. 8 (Table 1), indicating that long telomeres are not sufficient to destabilize CEB1-1. 8 repeats. Pif1 is present as two isoforms, one targeted to the nucleus and one to mitochondria. The pif1-m1 mutation prevents the synthesis of the mitochondrial isoform, resulting in mitochondrial deficiency but leaving nuclear Pif1 functions intact. In pif1-m2 cells, only the mitochondrial form is detected by western analysis [28], and this strain has normal mitochondrial function and long telomeres. However, telomere lengthening and de novo telomere addition are not as elevated in pif1-m2 cells as in a pif1Δ strain suggesting that some nuclear function is retained in the pif1-m2 allele [23]. As expected, CEB1-1. 8 was not destabilized (1/192) in pif1-m1 cells (Table 1). Surprisingly, CEB1 was also stable in pif1-m2 cells (1/384) (Table 1), a result that can be explained if pif1-m2 cells retain sufficient nuclear Pif1 to carry out its role in maintaining CEB1 stability. To test if a low level of the Pif1-m2 polypeptide could be active in the nucleus, we examined complementation of the pif1-m2 telomere phenotype by over expressing the pif1-m2 protein from its own promoter on a multi-copy 2 µ plasmid in pif1Δ cells. Telomeres were shorter in the strain over-expressing the pif1-m2 construct than in the control pif1Δ cells (data not shown). These results support our interpretation that in pif1-m2 cells, there is sufficient nuclear Pif1 protein to stabilize CEB1, although it is insufficient to sustain normal length telomeres. A similar observation was recently reported in the fission yeast S. pombe. As in budding yeast, the Pif1 homolog Pfh1p is present as a mitochondrial and a nuclear isoforms. However, expression of the mitochondrial-only isoform is able to complement pfh1p nuclear defects, even though the protein is not detectable in the nucleus at the protein level by western blot [31]. We investigated if the inactivation of other helicases would also affect CEB1-1. 8 stability. We previously showed that in a dna2-1 strain, CEB1-1. 8 was modestly destabilized (1. 8% instability) [26]. The viability of the DNA2 deletion in combination with the deletion of PIF1 [27] allowed us to examine the behavior of CEB1 in the complete absence of DNA2. As indicated in Table 1, in the pif1Δ dna2Δ CEB1-1. 8 strain, the frequency of CEB1 size variation was estimated at 4. 7%, a value significantly higher than in wild-type cells (p<0. 01, Fisher' s Exact Test), but not different than in the pif1Δ single mutant (p = 0. 48, Fisher' s Exact Test). This result indicates that the complete absence of Dna2 neither suppresses nor enhances the effects of Pif1 inactivation. Next, we examined the inactivation of Rrm3, a 5′-3′ DNA helicase that is closely related to Pif1 [32]. As shown in Table 1, deletion of the RRM3 gene did not destabilize CEB1-1. 8. Moreover, the frequency of rearrangement of CEB1 was not statistically different in the pif1Δ rrm3Δ (3. 9%) and pif1Δ (6. 0%) cells (p = 0. 2, Fisher' s Exact Test). We tested three additional helicases with well characterized roles in genome stability for the effects on CEB1-1. 8 stability. We examined the RecQ homolog Sgs1 helicase involved in multiple aspects of DNA recombination and repair [33]–[38], Srs2, a 3′ to 5′ helicase that disassembles abortive recombination intermediates [39], and the Mph1 helicase that plays a role in DNA repair [40]. Inactivation of these helicases did not destabilize the CEB1-1. 8 array, and the inactivation of both Pif1 and Sgs1 helicases (pif1Δ sgs1Δ strain), induced the same CEB1 instability as the pif1Δ strain (Table 1). We conclude that the role of Pif1 in stabilizing CEB1-1. 8 is specific for Pif1, rather than a general function of DNA helicases involved in DNA repair or recombination. We examined CEB1 alleles of various sizes, a shorter allele CEB1-0. 6 (14 repeats) and two longer alleles, CEB1-3. 0 (65 repeats) and CEB1-3. 5 (75 repeats). The two longer alleles were destabilized in pif1Δ cells, with instability increasing with the size of the array (Table 2, Figure 3C). For comparison, we performed similar studies in the rad27Δ cells. In all cases CEB1 rearrangements occurred at a lower frequency in the pif1Δ cells than in rad27Δ cells [25]. In the case of CEB1-1. 8, for which the largest sample of cells was examined, its instability was approximately 5-fold higher in rad27Δ than in pif1Δ cells. Next, we examined the instability of four natural yeast minisatellites that are normally found in the coding regions of the DAN4, FLO1, HKR1 and NUM1 genes [41]. This set represents a large variety of motifs in term of size (18 to 192 bp) and repeat units (10–30). All of these motifs were altered in rad27Δ but not in pif1Δ cells (Table 2). Likewise, the GC-rich hRAS1 human minisatellite [42] was not altered when propagated in pif1Δ cells (0/384 colonies). Finally, using the plasmid assay developed by Kokoska et al. (1998), we compared the behavior of four microsatellite sequences composed of 1,4, 5 and 8 nucleotide motifs and a triplication of a 20 nucleotides motif in wild-type, pif1Δ and rad27Δ haploid cells (see Table 2 for sequence of motifs). As previously reported [43], the rearrangement frequencies in the wild-type strain were on the order of 10−5–10−6 and were stimulated more than 10,000 fold in rad27Δ cells (Table 2). However, no significant increase in instability was detected in pif1Δ cells. Thus, in contrast to the strong and ubiquitous effects of Rad27 on minisatellite and microsatellite stability [41], [43], [44], the absence of Pif1 destabilized only the CEB1 arrays. DNA oligonucleotides containing at least four successive runs of three or more guanines have been shown to fold into intramolecular G4 DNA in presence of physiological concentrations of monovalent cations [45]. Examination of the CEB1 repeat sequence revealed the presence of 3 to 5 triplets of guanines localized on the same strand in each repeat of the CEB1-1. 8 allele (Figure 2A and Figure S1). It suggests that this minisatellite may form G4 structures, even if its primary sequence does not fit perfectly the d (G3+N1–7) 4 consensus used for most bioinformatic analyses. To test this hypothesis, we examined in vitro the formation of secondary structures using a single-stranded oligonucleotide that mimicked a complete CEB1 repeat (39Ceb) or a control sequence in which five of the guanines had been mutated (39Cebm) (Figure 2A). Four complementary assays were performed to detect the formation of G4 structures: First, 39Ceb and 39Cebm oligos were incubated in presence of 100 mM NaCl or KCl in conditions that favor G4 DNA formation. We measured the absorbance at 295 nm of 39Ceb and 39Cebm oligos at increasing temperatures. Indeed, an inverted transition corresponding to a conformational change associated with the temperature increase was observed with the 39Ceb oligo at a melting temperature (Tm) of ≈48°C in NaCl and 55°C in KCl, while no clear transition was seen with the 39Cebm sequence (Figure 2B and Table S2). Truncated versions of this motif were also analyzed (Table S2). Second, thermal differential spectra (TDS), which measure the difference between UV absorbance spectra of the oligonucleotide measured at a temperature above Tm (unfolded state) and below Tm (folded state), provides a clear signature for each type of nucleic acid structures including G4 DNA [46]. We measured the TDS in K+ buffer for 39Ceb and 39Cebm. As shown in Figure 2C, 39Ceb exhibits the typical pattern of a G4 structure with two positive maxima at 240 and 275 nm and a negative minimum around 295 nm [46]–[48] while 39Cebm exhibited a different signature, which does not correspond to quadruplexes. Third, we measured the circular dichroism (CD) spectra of the two oligonucleotides under experimental conditions that mimick the helicase assays (see below; briefly oligonucleotides were incubated at 140 µM strand concentration for 48 hours in 1 M NaCl). A positive maxima around 260 nm and a negative minimum around 240 nm was observed in the CD spectra of 39Ceb, an observation in agreement with the formation of parallel G4 structures (Figure 2D) [49], [50]. In contrast 39Cebm did not exhibit a CD spectra characteristic of any G4 structure found so far. Furthermore, when prepared under these conditions, the quadruplexes were extremely stable, as shown by temperature-independent CD profiles between 25°C and 90°C. This demonstrates that these structures are extremely heat resistant (no melting transition was observed by absorbance at 295 nm when the sample was prepared with this protocol; data not shown). Finally, 39Ceb and 39Cebm oligonucleotides were analyzed by polyacrylamide gel electrophoresis under native conditions where G4 structures are expected to show different mobility compared to unstructured oligonucleotides. No migration anomaly was found for 39Ceb when incubated in 100 mM LiCl, which does not stabilize G4 secondary structures [51] (data not shown). When 39Ceb is incubated in a sodium buffer at high strand concentration (Figure 2E; conditions identical as for helicase experiments, see below), bands of very low mobility were clearly visible. Intermolecular G4 structure formation was revealed by slow migrating bands as compared to the migration pattern of 39Cebm mutated control (Figure 2E). These higher order species likely correspond to bimolecular, tetramolecular (or higher) G4 structures. These experiments were repeated at lower strand concentration (50 nM or 4 µM), both in sodium and potassium. As expected for multimers (dimers, tetramers or species of even higher stoichiometry), concentration-dependent profiles were obtained (Figure S2). In conclusion, in all assays, the oligonucleotides containing the G-strand of the CEB1 motif exhibited the hallmarks of G4 structure formation in vitro whereas the 39Cebm control sequence did not. Depending on buffer conditions, strand concentration and incubation protocol, a variety of different quadruplex structures could be obtained with this sequence, arguing for the possible formation of multiple quadruplexes in vivo. If CEB1 also forms G4 DNA in vivo, Pif1 might inhibit CEB1 rearrangements by unwinding these structures. The prediction of this model is that Pif1 should be able to unwind these structures. To test this prediction oligonucleotides containing one CEB1 repeat were incubated in vitro using conditions that favor the formation of intermolecular G4 structures (see Materials and Methods). The G4-DNA substrate was first incubated in the presence of decreasing amount of purified recombinant Pif1. Upon 15 minutes incubation at 35°C, 5 nM Pif1 was enough to unwind 50% of the 20 fmol (2 nM) G4-DNA, while at least 20 times more Pif1 was necessary to unwind 20 fmol (2 nM) of a double-stranded oligonucleotide substrate (Figure 2F, G). The unwinding of both substrates required Pif1 helicase activity as no unwinding is observed in absence of ATP, or when the substrate is incubated in presence of saturating amount of the pif1-K264A helicase-dead mutant (Figure 2F). The rate of G4-DNA unwinding was also faster than unwinding of the double-stranded DNA substrate (Figure 2H, I). Indeed, 100 nM Pif1 was able to unwind 20 fmol (2 nM) of G4-DNA substrate in less than 5 minutes, while the enzyme was only able to unwind about 40% of the double-stranded substrate over the entire time course. These results demonstrate that Pif1 is more efficient at unwinding G4-DNA structures than regular double-stranded DNA. The in vitro experiments demonstrating the propensity of the CEB1 repeat to form G4 structures and the ability of Pif1 to unwind these structures led us to consider that Pif1 might unwind G4 structures in CEB1 in vivo. If this model is correct, mutations in CEB1 that eliminate its ability to form G4 structures might render it insensitive to Pif1. For these experiments, we developed a method combining both in vitro and in vivo steps to construct long (>1 kb) synthetic CEB1 alleles (see Text S1). We generated two categories of synthetic CEB1 arrays based on two different repeat units. The first category, named synthetic-CEB1-WT, was based on the repetition of the most common motif of the natural polymorphic CEB1-1. 8 allele (Figure S3, A, D). The second category, named CEB1-Gmut, was made from oligonucleotides in which 5 dispersed G bases were changed to either C, A or T in order to disrupt the original 5 G-triplets on the G-rich strand (Figure S3, A, E). In vitro analysis of the secondary structures of CEB1-Gmut oligonucleotides demonstrated that, as expected, they were unable to form G4 structures (39Cebm, Figure 2 and Table S2). The rearrangement frequency of the synthetic-CEB1-WT arrays (1. 0,1. 3,1. 7,1. 9 and 2. 3 kb long) and of the synthetic-CEB1-Gmut arrays (0. 7,1. 7,2. 5 and 3. 8 kb long) in WT, pif1Δ and rad27Δ cells is reported in Table 3 and summarized in Figure 3. As observed for the natural CEB1 alleles, the rearrangement frequency of the synthetic-CEB1-WT arrays was low in WT cells and increased in a size dependent manner in both pif1Δ and rad27Δ cells. In all cases, the frequency of instability for similarly sized alleles was higher in the synthetic-CEB1-WT arrays than in the natural CEB1 alleles. We attribute this difference to the greatly reduced polymorphism of the synthetic allele. However, the most striking result was that mutations in G4 prone motifs strongly decreased the frequency of their rearrangement in pif1Δ cells. We observed only one rearrangement of the CEB1-Gmut-1. 7 allele among the 383 colonies analyzed (0. 2%) while the synthetic-CEB1-WT-1. 7 allele was rearranged in 38/343 pif1Δ colonies (11%) (Figure 3 and Table 3). Similarly, the large synthetic-CEB1-Gmut-3. 8 array, which contains approximately 97 repeats, yielded only a few rearrangements in the pif1Δ and WT strains (4% and 2%, respectively; this difference was not statistically different, p = 0. 18, Fisher' s Exact Test). In contrast, CEB1-Gmut arrays rearranged in rad27Δ cells and the frequency of rearrangement increased in a size dependent-manner (Table 3). Thus, the synthetic and natural CEB1 alleles behaved similarly while the artificial CEB1 arrays containing mutation of G4-prone sequences were stabilized in pif1Δ but not in rad27Δ cells. These results strongly support our proposal that formation of G4 structures within the CEB1 array is responsible for their instability in vivo and that this secondary structure is processed by the Pif1 helicase. In previous studies, we reported that human CEB1 repeats inserted into the yeast genome are highly unstable in absence of the Rad27 endonuclease and slightly unstable in a dna2-1ts mutant [25], [26]. Since Rad27 and Dna2 are involved in the processing of flap structures during Okazaki fragment maturation [52], we concluded that CEB1 instability was likely due to the accumulation of unresolved flap structures during replication. We proposed that these intermediates would form recombinogenic structures that are repaired by homology-dependent strand displacement and annealing (SDSA) [53]. Here we show that inactivation of Pif1 also resulted in CEB1 instability. As in rad27Δ cells, the CEB1 rearrangements in pif1Δ cells had a high frequency of complex events (Figure 1C; [25]). In addition, in both mutants, CEB1 rearrangements depended on Rad52/Rad51-dependent homologous recombination (Table 1). These similarities suggest that the repair of the lesion leading to CEB1 rearrangement in the absence of either Pif1 or Rad27 occurs by SDSA, although the recombinogenic lesion may be different (for example a single-strand gap or a double-strand break). In pif1Δ and rad27Δ cells, the frequency of rearrangements increased with the size of the allele (Figure 3C; [25]). In rad27Δ cells, this increased instability may reflect the increased probability that longer arrays are more likely to contain more than one improperly processed flap. Similarly, in pif1Δ cells, long CEB1 minisatellites could form G4 structures with a higher probability, especially if quadruplexes involve G-tracts from adjacent repeats. Alternatively, lesions in small alleles could be rare or more often resected into the non-repeated flanking sequences, leading to the preferential restoration of the parental sequence by homologous recombination in G2 cells using the intact sister chromatid as a template [53]. Whereas all micro- and minisatellites sequences tested are unstable in rad27Δ cells ([43], [44] this study), only CEB1 was unstable in pif1Δ cells (Table 2). The CEB1 sequence is G/C rich (72%) with a high strand bias (23 G and 7 C per repeat of 39 bases). However, the instability of CEB1 in pif1Δ cells can not be attributed solely to its G/C rich sequence as the human hRAS1 minisatellite, which is also G rich (68%) with a strong bias (14 G and 5 C per repeat of 28 bases), was stable in the absence of Pif1. Each CEB1 repeat contains putative G4 signature motifs. Our biophysical analyses of CEB1 and hRAS oligonucleotides showed that the CEB1 motif readily formed G4 structures in vitro while hRAS1 did not (Figure 2 and Table S2). Moreover, synthetic CEB1 minisatellites in which the runs of guanine were mutated to disrupt their ability to form G4 structures were no longer unstable in pif1Δ cells. We propose that the recombinogenic lesions formed in the absence of Pif1 are unresolved intra- or inter-motifs G4 structures. Thus, while CEB1 alleles are unstable in both pif1Δ and rad27Δ cells, the events that initiate instability, unprocessed Okazaki fragments (in rad27Δ cells) or persistent G4 structures (in pif1Δ cells) are different (Figure 4). As a result, all tandem arrays are unstable in the absence of Rad27, including the synthetic G4-mutated CEB1 alleles, while only CEB1 was unstable in pif1Δ cells. What do our results suggest about the role (s) of Pif1 in the cell? Owing to the alternative use of a translation start site, PIF1 generates two isoforms, one with mitochondrial and one with nuclear functions. Several observations indicate that Pif1 is involved in the maintenance of mitochondrial DNA. Specifically, Pif1 increases the frequency of recombination between ρ+ and certain ρ− tandemly repeated mitochondrial genomes [21]. The loss of Pif1 is thought to trigger mtDNA breakage in specific regions, leading the authors to propose that Pif1 recognizes a specific but uncharacterized DNA topology [22], [54]. Although the ∼75 kb S. cerevisiae mitochondrial genome is AT-rich, it contains numerous G-rich stretches. We speculate that in the absence of mitochondrial Pif1, breaks occur due to defective processing of G4 structures and these breaks are repaired by recombination. Alternatively, G4 DNA can create a structural target for factors involved in DNA recombination. In the nucleus, Pif1 affects telomere length through direct inhibition of telomerase [23], [28] the specialized reverse transcriptase that lengthens telomeres in most eukaryotes. In vivo and in vitro data suggest that telomerase inhibition is achieved by direct displacement of telomerase from a DNA end [24]. Since Pif1 exhibits a marked preference for RNA-DNA hybrid unwinding in vitro [55], Pif1 is proposed to inhibit telomerase by unwinding the RNA-DNA hybrid formed between the telomerase RNA, TLC1, and the telomeric DNA end. Pif1-mediated removal of telomerase from DNA ends can explain the effects of pif1 mutations on both telomere length and de novo telomere addition [23], [56] as well as its inhibition of gross chromosomal rearrangements [57]. Human Pif1 (hPIF) may have similar functions as ectopic expression of hPIF causes telomere shortening and decreased telomerase processivity in vitro [58]. In addition, hPIF co-immunoprecipitates with telomerase subunits and telomerase activity [59]. Importantly for the present study, most telomeric DNA sequences, including yeast and human telomeric DNA, can form G4 structures in vitro. Moreover, G4 structures have been detected at ciliate telomeres in vivo [6]. In budding yeast, no evidence of the presence of G4 structures in the telomeric single stranded region has yet been reported, but proteins that bind or process G4 DNA in vitro are nevertheless present at yeast telomeres. In particular, in vitro studies have shown that the telomere binding protein Rap1 binds double-stranded telomeric DNA and promote the formation of G-quadruplex structures [60]. It is not known if this reaction occurs in vivo, but it is tempting to speculate that the formation of G4 DNA is necessary to promote the assembly of functional telomere. Alternatively or in addition to its ability to inhibit telomerase directly, Pif1 could counteract the formation of G4 structures in telomeric DNA, thus antagonizing the formation of proper telomere architecture. Consistent with this hypothesis, it has been shown that Pif1 overexpression compromises the viability of yeast strains with compromised telomere end protection [61]. Several studies suggest that Pif1 also has non-telomeric roles in replication and repair of nuclear DNA. First, in the rDNA, Pif1 helps maintain the replication fork barrier during replication [32]. Second, Pif1 is recruited to Rad52 DNA repair foci after gamma irradiation [62]. Third, lack of Pif1 suppresses the lethality of a Dna2 deletion, a helicase/endonuclease involved in the processing of Okazaki fragments by removing long 5′ flaps. Although the role of Pif1 in Okazaki fragment maturation is unclear, it is proposed to act by extending the flaps created by the lagging strand replicative polymerase at the junction of two consecutive Okazaki fragments [27]. Like Pif1, Dna2 is involved in telomere maintenance [63] and is able to process G4 DNA in vitro [64]. Thus, the two enzymes may act in concert to remove toxic intermediates, including G4-DNA, which could arise during lagging strand replication and, if not appropriately processed, promote formation of recombinogenic DNA lesions, such as double strand breaks. Finally, considering that in addition to G4-unwinding, Pif1 more efficiently unwinds RNA/DNA hybrids than DNA/DNA substrates [55], it is also to be envisaged that Pif1 plays a more general role in yeast cells when potential G4 structure can form, for example, during transcription. Budding yeast as well as all the other organisms encodes a large number of helicases. Current estimate in S. cerevisiae is approximately 120. This multiplicity raises the question of their specific substrate (s) and function (s), an issue which remains often unresolved and controversial. In S. cerevisiae, the RecQ homolog Sgs1 helicase was proposed to resolve G4 DNA, a conclusion primarily based on its ability, and more generally of members of the RecQ family, to resolve G4 DNA structures in vitro [16]. Compelling evidence for the involvement of Sgs1 in G4 DNA metabolism in vivo finally came from the survey of global gene expression analysis in absence of Sgs1 [11]. The authors found that the set of genes which expression level is affected in sgs1 mutant is biased towards genes that contain potential G4 forming sequences in their ORFs. To our surprise, the deletion of SGS1 had no effect on CEB1 stability (Table 1). The lack of in vivo redundancy between Sgs1 and Pif1 in this novel assay is interesting and allows several hypotheses. First, it is possible that Sgs1 and Pif1 do not recognize the same set of G4 structures. G4 forming sequences can give rise to secondary structures exhibiting very diverse sizes, topologies (parallel or anti-parallel) and arrangements (intra- or inter-molecular) [65], and these structures may be recognized or processed differently depending on helicase. Second, Sgs1 may not recognize the G4 substrates generated by CEB1 in vivo due to the polarity of the single strand region flanking the G4-DNA structure (Pif1 is a 5′-3′ helicase while Sgs1 has a 3′-5′ polarity). Third, it is likely that the numerous repeats in CEB1 that contain G4 forming sequences lead to the formation of highly stable structures in vivo that only some helicases are able to unwind. Finally, in the absence of more direct evidences for Sgs1 involvement in G4 DNA unwinding in vivo, there is also a possibility that Sgs1 plays a minor role in maintaining G4 DNA forming sequences. In multicellular organisms, the relationships between genomic instability, G-quadruplex structures and helicases functions have also been suspected. Studies in human cells deficient for the Werner, Bloom and RTEL helicases showed defects in telomere maintenance in vivo while G4 DNA is highly suspected to form at mammalian telomeres [66], [67] and a recent study reports the correlation between genomic stability and G4 DNA unwinding by the human FANCJ helicase [18]. Similarly, in Caenorhabditis elegans, the disruption of the RTEL homolog DOG-1 triggers deletions of polyguanine tracts matching the G4 DNA signature [20]. Finally, it should be mentioned that the inactivation of the potential Pif1 homolog in mice has no detectable phenotype, in particular regarding change in telomere length homeostasis [68]. In light of our present study, the stability of other repeated potentially G4 forming sequences in mice and mammalian cells should be examined. Also, taking advantage of the present yeast system allowing to test natural and synthetic substrates, we anticipate that further studies of pif1Δ cells will allow to uncover the multiple roles of this evolutionary conserved helicase, facilitate the characterization of G4 structures in vivo and finally enhance our understanding of the dynamics of G4 formation and function in vivo. The relevant genotypes and sources of haploid and diploid S. cerevisiae strains (S288C background) used in this study are indicated in Table S1. Examination of CEB1 instability during vegetative growth was done as previously described [25]. Individual colonies or colonies pools were analyzed by Southern blot depending on the rearrangement frequency (for rearrangement frequency >20%, individual colonies were privileged). Southern blots were performed using AluI digestion for natural CEB1 minisatellites and ApaI/SpeI for synthetic minisatellites and the corresponding membranes were hybridized with the radiolabeled CEB1-0. 6 and CEB1-synthetic probes, respectively. For the analysis of the yeast minisatellite instability (DAN4, FLO1, HKR1 and NUM1), Southern blots were performed using AluI digestion (which does not cut in these repeats). Membranes were hybridized with the radiolabeled purified PCR product of the corresponding minisatellite (primer sequences available under request). For the analysis of the human hRAS1 minisatellite instability, Southern blots were performed using ApaI/SpeI digestion and hRAS1 probe obtained from the p37Y8 plasmid (gift from D. Kirkpatrick). Detection of signals was done with a Storm PhosphorImager (Molecular Dynamics). For pools of genomic DNA from 12 or 16 colonies/wells, rearrangement is counted when the intensity of the rearranged minisatellite, quantified with ImageQuant software, corresponds to 1/12 or 1/16 of the total amount of signals measured in the lane. When several rearranged minisatellites migrate at the same size they are considered as clonal and are counted only once. The internal structure of rearranged alleles was determined by DNA sequencing as described previously [25]. Oligonucleotides were synthesized by Eurogentec (Belgium). Concentrations of all oligodeoxynucleotides were estimated using extinction coefficients provided by the manufacturer and calculated with a nearest neighbor model [69] under low salt conditions at 60°C in order to destabilize quadruplex formation. The sequences studied are shown in Table S2. Oligonucleotides chosen for non denaturing gel electrophoresis were first purified under denaturing conditions. Melting experiments were conducted as previously described [70]. Denaturation was followed by recording the absorbance at 240 or 295 nm [47], [71]. Melting experiments were typically performed at a concentration of 4 µM per strand. Thermal difference spectra (TDS) were obtained by difference between the absorbance spectra from unfolded and folded oligonucleotides that were respectively recorded much above and below its melting temperature (Tm). Circular dichroism (CD) spectra were recorded on a JASCO-810 spectropolarimeter using a 1 cm path length quartz cuvette in a reaction volume of 580 µl. Oligonucleotides were either i) prepared as a 4 µM solution in 10 mM lithium cacodylate pH 7. 2,100 mM NaCl or KCl buffer and annealed by heating to 90°C for 2 min, followed by cooling to 20°C or ii) preincubated for 48 hours at higher strand concentration (140 µM) in a 10 mM lithium cacodylate pH 7. 2,1 M NaCl buffer. Scans were performed at 25°C to 90°C over a wavelength range of 220–335 nm with a scanning speed of 500 nm/min, a response time of 1 s, 1 nm pitch and 1 nm bandwidth. Formation of G4-DNA was confirmed by non-denaturing PAGE. In this case, oligonucleotides were either directly observed by UV shadow (when incubated at high strand concentration) or 5′ labeled with T4 polynucleotide kinase. Prior to the incubation, the DNA samples were heated at 90°C for 10 min and slowly cooled (2 h) to room temperature (or 60°C for 48 hours). Oligonucleotides were first treated with 50 mM LiOH (to unfold quadruplexes) for 10 minutes followed by HCl neutralization. Samples were incubated at 10 nM or 4 µM strand concentration in Tris-HCl 10 mM pH 7. 5 buffer with 100–1000 mM Li+ or K+. 10% sucrose was added just before loading. Oligothymidylate markers (dT15, dT21, or dT30) or double-stranded markers (Dx9: 5′d-GCGATACGG+5′d-CCGATACGC Dx12: 5′d-GCGTGACTTCGG+5′d-CCGAAGTCACGC) were also loaded on the gel. Recombinant Pif1 was purified to homogeneity by affinity chromatography as described [55]. A Cy5-labeled oligonucleotide containing a 5′ poly (dA) tail followed by a CEB1 repeat (5′-Cy5-AAAAAAAAAAAGGGGGAGGGAGGGTGGCCTGCGGAGGTCCCTGGGCTG) was synthesized by Eurogentec (Belgium). For formation of the G-quadruplex, a solution of CEB1 oligo at 140 µM in 1 M NaCl was denatured 5 min at 100°C, then incubated at 65°C for 48 hours to promote formation of G4 intermolecular structures [72]. The double-stranded DNA control was made by annealing a 5′-Cy5-labeled 20 mer oligonucleotide to a 40 mer oligonucleotide, leaving a 20 nucleotide-long 5′ single-stranded DNA overhang. Briefly, 10 µM of each oligonucleotide were mixed in a buffer containing 10 mM Tris pH 8. 0 and 5 mM Mg2+. The mixture was denatured 5 minutes at 95°C and slowly let to cool to room temperature. The double-stranded DNA substrate was further purified from non annealed single-stranded DNA on a MiniQ anion exchange column. Helicase assays were carried out by incubating indicated amounts of Pif1 and 2 nM nucleic acid substrate at 35°C. Standard reaction buffer was 20 mM Tris pH 7. 5,50 mM NaCl, 100 µg/ml bovine serum albumin, 2 mM DTT, 5 mM Mg2+ and 4 mM ATP. For kinetic studies, reactions were started by addition of ATP in presence of 100 nM Pif1 and 2 nM substrate. 10 µl aliquots were withdrawn at indicated times and the reactions stopped by addition of 2 µl deproteinizing/loading buffer (6% Ficoll, 50 mM EDTA pH 8. 0,2. 5 µg/µl Proteinase K) and incubated further 15 minutes at 35°C. Reaction products were loaded on a 10% polyacrylamide non-denaturing gel and resolved by electrophoresis at 4°C and 10 V/cm in TBE 1× buffer. Gels were dried and scanned with a storm PhosphorImager (Molecular Dynamics) and quantified using ImageQuant software (GE Healthcare). Fisher exact test was performed using R software [73].

Changes in the primary DNA sequence are a major source of pathologies and cancers. The hereditary information also resides in secondary DNA structures, a layer of genetic information that remains poorly understood. Biophysical and structural studies have long established that, in vitro, the DNA molecule can adopt diverse structures different from the canonical Watson-Crick conformations. However, for a long time their existence in vivo has been regarded with a certain skepticism and their functional role elusive. One example is the G-quadruplex structure, which involves G-quartets that form between four DNA strands. Here, using in vitro and in vivo assays in the yeast S. cerevisiae, we reveal the unexpected role of the Pif1 helicase in maintaining the stability of the human CEB1 G-rich tandem repeat array. By site-directed mutagenesis, we show that the genomic instability of CEB1 repeats in absence of Pif1 and is directly dependent on the ability of CEB1 to form G-quadruplex structures. We show that Pif1 is very efficient in vitro in processing G-quadruplex structures formed by CEB1. We propose that Pif1 maintains CEB1 repeats by its ability to resolve G-quadruplex structures, thus providing circumstantial evidence of their formation in vivo.

lay_plos

Human monkeypox (MPX) occurs at appreciable rates in the Democratic Republic of Congo (DRC). Infection with varicella zoster virus (VZV) has a similar presentation to that of MPX, and in areas where MPX is endemic these two illnesses are commonly mistaken. This study evaluated the diagnostic utility of two surveillance case definitions for MPX and specific clinical characteristics associated with laboratory-confirmed MPX cases. Data from a cohort of suspect MPX cases (identified by surveillance over the course of a 42 month period during 2009–2014) from DRC were used; real-time PCR diagnostic test results were used to establish MPX and VZV diagnoses. A total of 333 laboratory-confirmed MPX cases, 383 laboratory-confirmed VZV cases, and 36 cases that were determined to not be either MPX or VZV were included in the analyses. Significant (p<0. 05) differences between laboratory-confirmed MPX and VZV cases were noted for several signs/symptoms including key rash characteristics. Both surveillance case definitions had high sensitivity and low specificities for individuals that had suspected MPX virus infections. Using 12 signs/symptoms with high sensitivity and/or specificity values, a receiver operator characteristic analysis showed that models for MPX cases that had the presence of ‘fever before rash’ plus at least 7 or 8 of the 12 signs/symptoms demonstrated a more balanced performance between sensitivity and specificity. Laboratory-confirmed MPX and VZV cases presented with many of the same signs and symptoms, and the analysis here emphasized the utility of including 12 specific signs/symptoms when investigating MPX cases. In order to document and detect endemic human MPX cases, a surveillance case definition with more specificity is needed for accurate case detection. In the absence of a more specific case definition, continued emphasis on confirmatory laboratory-based diagnostics is warranted. Since the global eradication of smallpox, the most important Orthopoxvirus infection in humans in terms of ongoing numbers of cases, morbidity, and mortality has been human monkeypox (MPX) [1,2]. Monkeypox virus (MPXV) is maintained by an enzootic cycle, with zoonotic introductions to humans often being followed by more limited human-to-human transmission [3,4]. The animal reservoir for MPXV remains unknown, but the virus has been isolated in the wild from a squirrel (Funisciurus anerythrus) and a sooty mangabey (Cercocebus atys) [5,6]. Infection with MPXV can lead to a smallpox-like illness characterized by a febrile prodrome, lasting 1–4 days, followed by a slowly progressing rash. The rash proceeds from macules to papules to vesicles to pustules to crusts and finally to desquamation. This occurs over a period of two to four weeks. At any given site on the body, the rash is generally in the same stage of development (e. g., all vesicles), and the lesions are typically circumscribed, umbilicated, deep-seated and firm. The rash has a centrifugal distribution, with a concentration of lesions on the extremities and face. As with smallpox, MPX lesions often appear on the palms of the hands and soles of the feet. Lymphadenopathy (inguinal, axillary, and/or cervical) is common in MPX patients and can occur prior to or at the onset of rash. Ocular infection with MPXV can lead to permanent corneal scarring and blindness [7–9]. Infection with varicella zoster virus (VZV) has a similar presentation to that of MPXV infection, and in areas where MPXV is endemic these two illnesses are commonly mistaken [10,11]. However, there are several features of illness that typically set one infection apart from the other. For example, VZV patients typically exhibit a short, mild period of febrile prodrome, or none at all, followed by a quickly evolving (1–2 day) pleomorphic rash (i. e., a rash for which neighboring lesions may be in different stages of development). VZV lesions also often have irregular borders, and are superficial on the surface of the skin (relative to those of MPX). In addition, varicella lesions often appear in a centripetal distribution on the body [9,12]. Although noted in rare occurrences, lesions on the palms of the hands and soles of the feet are not hallmarks of VZV infections [13]. VZV patients do not typically have pronounced lymphadenopathy, and, thus, the presence of lymphadenopathy is one distinguishing characteristic that can differentiate MPX from both smallpox and varicella. Additional illnesses that can be mistaken for MPX are other herpetic infections (in addition to VZV), drug eruptions, syphilis, yaws, scabies, and rickettsialpox [9]. Specimen collection followed by laboratory testing can be difficult to accomplish for all suspected cases in MPX endemic areas. A clinical case definition capable of enhancing the distinction between MPX and other illnesses would be useful to enable more accurate and expedient case detection, collection of higher-quality surveillance data, and improved patient management. MPXV is enzootic in western and central Africa, with the overwhelming majority of human infections reported each year from the forested areas of the Congo Basin of Democratic Republic of Congo (DRC) [2]. MPX is a nationally reportable disease in DRC and has been identified as one of the country’s priority diseases of epidemic potential. On a bi-weekly basis, notifications of suspected MPX cases from each of the country’s Health Zones are submitted to national public health authorities; few of the suspected cases are formally investigated (i. e., case investigation forms completed and diagnostic specimens collected). This study evaluates the diagnostic utility of two surveillance case definitions for MPX. Both definitions were herein applied to a cohort of suspected MPX cases that were identified over the course of a 42 month period via surveillance in one Province in DRC. This cohort is unique in that the dataset contained an in-depth list of signs/symptoms. The accuracy of case classification was determined using laboratory findings. We assessed clinical features of illness in patients with confirmed MPXV infection to identify characteristics distinctly associated with disease presentation and suggest modifications to the MPX surveillance case definition to improve specificity. Data from suspect MPX cases were obtained by investigation in accordance with national guidelines. Patients were identified as suspect MPX cases if they had a vesicular or pustular eruption with deep-seated, firm pustules and at least one of the following symptoms: fever preceding the eruption, lymphadenopathy (inguinal, axillary, or cervical), and/or pustules or crusts on the palms of the hands or soles of the feet. For suspect cases, a MPX-specific case investigation form was completed and, in most instances, two or more diagnostic specimens were collected from each suspected MPX case. The specimens were sent to the Institut National de Recherche Biomédicale (INRB) in Kinshasa for diagnostic testing. One specimen from each individual was tested at INRB for the presence of Orthopoxvirus DNA signatures using a real-time PCR assay [14]. If the initial PCR was negative for Orthopoxvirus, a second real-time PCR assay specific for VZV-specific DNA signatures was conducted (reagents provided by the United States Army Medical Research Institute of Infectious Diseases). DNA extracted at INRB and additional independent specimens, if available, were shipped to the Poxvirus Laboratory at the U. S. Centers for Disease Control and Prevention (CDC). At the CDC, DNA was extracted from original specimens and all specimens were tested with MPXV and VZV-specific real-time PCR assays [15,16]. An individual was classified as a laboratory-confirmed MPX case if at least one specimen was 1) positive with the Orthopoxvirus-specific assay, and/or 2) positive by MPX-specific real-time PCR. An individual was independently classified as a laboratory-confirmed VZV case if a crust specimen tested positive for VZV DNA signatures at INRB or if an original vesicular swab or crust specimen tested positive for VZV at CDC. These activities were determined to not be research by a CDC human subjects advisor. Suspect MPX cases in Tshuapa Province, DRC, with rash onset occurring between September 2009 and February 2014 were included in the analysis (N = 1025, Fig 1). These individuals were all assessed by a surveillance officer, who determined the individual met the surveillance case definition for a suspect MPX case. Individuals whose laboratory test results were suggestive of a coinfection with both MPXV and VZV, and those with incomplete or inconsistent laboratory results were excluded from analyses (n = 273). A total of 752 cases were included for further analyses; 333 laboratory-confirmed MPX cases, 383 laboratory-confirmed VZV cases, and 36 cases that were determined to not be either MPX or VZV (laboratory diagnosis undetermined). Clinical signs and symptoms were recorded as checkboxes—‘yes’, ‘no’, and ‘do not know’—on the case investigation form. If an individual case investigation form had a response (‘yes or ‘no’) for any signs/symptoms, but the absence of a response for another specific sign/symptom the variable was coded as “missing” for the specific sign/symptom without a response. Febrile prodrome status was determined by either a) selection of ‘yes/no’ for the febrile prodrome variable on the case investigation form, or, if that information was missing, by, b) presence of prodrome was ascertained by determining the time interval between onset of fever and that of rash, when the information was available. The presence of lymphadenopathy was determined using the individual lymphadenopathy-type fields (inguinal, axillary, cervical). If at least one category of lymphadenopathy was reported as present, then the individual was coded “yes” for lymphadenopathy; if all fields were “no”, the individual was coded “no” for lymphadenopathy. If one or more of the fields were missing and others were “no”, the individual was coded “missing” for lymphadenopathy. Two surveillance case definitions were evaluated in this study. According to Case Definition A, a suspect case is an individual with fever followed by a vesicular or pustular rash with the following symptoms: rash on palms, soles, and face; or the presence of 5 variola-like scars. Case Definition A has been recommended for use in endemic areas. Case Definition B was developed as a discriminatory case definition with the inclusion of several criteria that distinguish MPX from VZV. Case Definition B encompasses individuals who have a vesicular or pustular eruption with deep-seated, firm pustules and at least one of the following symptoms: fever preceding the eruption, lymphadenopathy (inguinal, axillary, or cervical), and/or pustules or crusts on the palms of the hands or soles of the feet. To evaluate the diagnostic accuracy of each case definition, the dataset was restricted to individuals who had all the information needed for classification using both case definitions (i. e., one dataset was used for the independent analyses of both definitions). A total of 645 of the 752 cases (85. 8%) had sufficient information to be included in the analyses of both case definitions; 314 laboratory-confirmed MPX cases, 305 laboratory-confirmed VZV cases, and 26 cases that were determined to be neither MPX nor VZV (Fig 1). Individuals met the criteria for Case Definition A if they had a febrile prodrome and either the presence of a) rash/scars on the face, palms, and soles, or b) more than 5 scars. Individuals met the criteria for Case Definition B if they had rash with deep-seated, firm lesions and either a) febrile prodrome, b) lymphadenopathy, or c) lesions on the palms of the hands or soles of the feet. This dataset was also used for the receiver operating characteristic analysis. The frequencies of each sign/symptom were calculated for all cases included in the dataset, and individually for laboratory-confirmed MPX and VZV cases. Associations between reported signs/symptoms and laboratory determined diagnoses were calculated using chi-squared and Fisher exact tests. Signs/symptoms that occurred with significantly different frequency (p<0. 05) between laboratory-confirmed MPX and VZV cases were further assessed for their individual sensitivity, specificity, PPV and NPV in relation to the confirmed diagnosis of MPX. For the analysis of the two case definitions, real-time PCR diagnostic test results (for MPX and VZV) were used as the ‘gold standard’ to establish MPX and VZV diagnoses. The sensitivity, specificity, positive predictive values (PPV), and negative predictive values (NPV) were computed for the two case definitions. A receiver operating characteristic (ROC) analysis was completed using ‘fever before rash’ and various summed frequencies of the 12 signs/symptoms that were identified as having high (>80%) sensitivities or specificities for laboratory-confirmed MPX cases. For example, individuals with ‘fever plus rash’ and one of these 12 signs/symptoms were categorized as having ‘1 criteria’; individuals with ‘fever plus rash’ and 12 signs/symptoms were ranked as ‘12 criteria’. Each of these 12 signs/symptoms counted equally to the sum. For example, if an individual reported two symptoms of ‘nausea’ and ‘cough’, they were categorized the same as an individual who reported two symptoms of ‘fatigue’ and ‘conjunctivitis’. ‘Fever before rash’ was identified as a mandatory sign/symptom because this has been consistently noted in the literature for MPX patients and also was observed at a frequency of 98. 1% for all suspect cases and 99. 1% for laboratory-confirmed MPX patients in the present analysis. The sensitivity, specificity, PPV and NPV for each model (with increasing number of signs and symptoms from 1 to 12) were assessed. All data analysis was performed using SAS version 9. 3. Characteristics of the Population: A total of 752 suspect cases were included in the analysis. Approximately 53% of suspect cases were male and 46% were female; this proportion remained consistent after laboratory case classification. The mean age of suspect cases was 17 years (median 13, range 0. 01–86). Of these suspect cases, 333 (44. 3%) individuals were classified as laboratory-confirmed MPX cases and 419 (55. 7%) were classified as laboratory-confirmed VZV (383) or undiagnosed (36) cases (Table 1). Performance of specific clinical characteristics: With the objective of improving the specificity and PPV of the case definitions under examination, associations between the reported clinical signs/symptoms and a laboratory-confirmed diagnosis of MPX (versus VZV) were investigated. Significant (p<0. 05) differences between laboratory-confirmed MPX and VZV cases were noted for the signs/symptoms of nausea, cough, lymphadenopathy (overall and each site), mouth ulcers, sore throat, malaise, fatigue, conjunctivitis, sensitivity to light, and bedridden (Table 2). Rash characteristics that were significantly different included same size, deep-seated, firm lesions, and the presence of lesions on the arms, legs, palms of the hands, soles of the feet, and genitals. Each of the significant signs/symptoms and rash characteristics occurred more frequently in laboratory-confirmed MPX cases than in laboratory-confirmed VZV cases. The majority of significant signs/symptoms (15/20) occurred in more than 50% in laboratory-confirmed MPX cases. Variables with high sensitivity for MPX (≥80%) were lymphadenopathy, fatigue, and the following rash characteristics: same size, deep-seated firm lesions, presence on the arms, legs, palms of the hands, and soles of the feet (Table 3). Nausea, conjunctivitis, bedridden, and lesions present on the genitals were signs/symptoms with a high specificity (≥80%), but none of these, individually, were found in laboratory-confirmed MPX cases at a frequency > 32%. Analysis of the case definitions: Two-by-two tables and diagnostic values for the two case definitions are presented in Tables 4 and 5. Two hundred ninety-one (92. 6%) laboratory-confirmed MPX cases satisfied Case Definition A; and 306 (97. 5%) laboratory-confirmed MPX cases satisfied Case Definition B; 245 (74%) and 303 (91. 5%) non-MPX cases [laboratory-confirmed VZV cases and undiagnosed (MPX and VZV negative) cases] satisfied Case Definitions A and B, respectively. The sensitivity of both case definitions was high, with the value for the Case Definition B a bit higher than that of Case Definition A (97. 45% vs. 92. 86%, respectively). Similarly, the specificity of both case definitions was low and the Case Definition A had a higher specificity (25. 98%) than Case Definition B (8. 46%). The PPVs were similar for both definitions (50. 25% for Case Definition B and 54. 92% for Case Definition A), as were the NPVs (77. 78% for Case Definition B and 78. 90% for Case Definition A). The NPV was higher than the PPV for both definitions. Receiver operating characteristic analysis: Using the 12 identified signs/symptoms with high sensitivity and/or specificity values, the ROC analysis tested the performance and accuracy of 12 models (with increasing numbers of signs and symptoms) (Table 6). In general, models with a greater number of signs/symptoms (>8 but <12) demonstrated excellent specificity (>90%) but low sensitivity (<40%). In contrast, models with a lower number of signs/symptoms (<7) had excellent sensitivity (>90%) but low specificity (<40%). The models for MPX cases that had the presence of ‘fever before rash’ plus at least 7 or 8 of the 12 signs/symptoms demonstrated a more balanced performance between sensitivity and specificity. There was greatly improved specificity for the models that included 7 (50. 76%) or 8 (70. 69%) signs/symptoms when compared to either Case Definition A (25. 98%) or B (8. 46%). The area under the curve for the model using these summed symptom counts was 0. 74 (Fig 2). The choice and utility of a case definition will be guided by its intended use. Case Definition A was designed to detect a single case of MPX illness, followed by an immediate outbreak response and control efforts. Case Definition B was designed to be a discriminatory definition used in the context of surveillance for disease, to better understand the extent and burden of disease in an endemic area. Both case definitions were characterized by high sensitivities but very low specificities. The high values for sensitivity were expected since the dataset represented patients who were diagnosed with suspected MPX virus infection (prior to laboratory confirmation). These characteristics indicate that both definitions are useful for screening purposes and are well-designed for outbreak detection. Given that MPX is an endemic, regularly occurring, reportable disease in Tshuapa Province, the case definitions should be sufficient to capture true MPX cases, such that local or national officials may want to launch an outbreak response if they observe an aberration or threshold in the number or rate of reported infections. For surveillance purposes, however, especially in resource-limited countries such as DRC, it is necessary for a definition to capture all true cases and at the same time minimize the number of false positives. Attributes of Case Definition B, including a low specificity and moderate PPV, are not optimal for the objectives of disease surveillance. Observations of a moderate PPV and high NPV for both case definitions is consistent with a disease with a low prevalence in the population. Although prominent and regularly occurring, MPX does have a relatively low incidence in Tshuapa Province with a cumulative incidence rate of 4. 8/10,000 over a four year period (data available upon request). Several signs/symptoms had a high sensitivity (lymphadenopathy, fatigue, and the following rash characteristics: same size, deep-seated firm lesions, presence on the arms, legs, palms of the hands, and soles of the feet), which indicates that these signs/symptoms may be useful in ruling out other febrile rash illnesses that may be circulating in an area of active MPXV transmission. Signs/symptoms with a high specificity (nausea, conjunctivitis, bedridden, lesions present on the genitals), on the other hand, may be useful in identifying true MPX cases. These four signs/symptoms and, also, sensitivity to light were characteristics that occurred more frequently in laboratory-confirmed MPX cases than VZV cases. However, none of these discriminatory signs/symptoms were found at a high frequency in MPX cases (<50%). Thus, these signs/symptoms may be eligible components of a case definition to identify MPX cases, however, they cannot be a mandatory component. Case definitions for MPX include characteristics of disease presentation specific to the rash itself. This analysis reinforced the inclusion of lesion size and surface presentation (deep-seated vs superficial). Notably, the “characteristic” lesion locations of palms of the hands and soles of the feet, although present in higher frequency in MPX cases than VZV cases, were prevalent in all suspect cases (91. 2% and 81. 3%, respectively) and were not helpful in increasing the specificity for MPX cases. Lesions on the palms and soles have been previously noted in VZV cases in central Africa [13], and the findings here indicate that this presentation may be more common than recognized before. The benefit of an ROC analysis is that one is able to evaluate the effect of increasing number of signs and symptoms on the sensitivity and specificity of case identification. Both laboratory-confirmed MPX and VZV cases presented with many of the same signs and symptoms. Instead of limiting the case definition to an all-or-nothing analysis, we chose to limit the ROC analysis to a subset of highly sensitive and specific signs/symptoms. As such, the analysis indicated that the combination of 7 or 8 signs/symptoms was the most optimal model to accurately predict laboratory-confirmed MPX cases. This analysis emphasized the utility of including the minimum 12 signs/symptoms (nausea, conjunctivitis, bedridden, lesions on genitals, lymphadenopathy, fatigue, lesions of the same size, deep-seated firm lesions, lesions present on the arms, legs, palms of the hands, or soles of the feet) on a MPX-specific case investigation, followed by the classification of a patient as a suspect MPX case if they possessed a combination of any 7 or 8 of these specific signs/symptoms plus ‘fever before rash’. Suspected MPX patients are rarely followed over the course of their infection. Patients are often only seen once, the data on clinical signs/symptoms is assessed and captured at that single time point, and no further follow-up is conducted. A definition such as the one suggested by the ROC analysis may allow for greater flexibility and utility in detecting true MPX cases at any given point during the course of the infection, since it allows for the presence of 7 or 8 signs/symptoms (versus 12). Additional evaluations to discern a specific suite of signs/symptoms that can be easily identified by healthcare personnel in endemic areas are warranted. This could be followed by modification of the surveillance investigation tool to incorporate the 12 signs/symptoms and evaluation of the utility of a new, modified case definition that accounts for the presence of 7 or 8 signs/symptoms when determining if a patient is a suspected MPX case. The population used in this analysis was a population of suspect MPX cases identified through surveillance for human MPX illness in DRC. A total of 137 cases were excluded from the final analysis due to incomplete data from case report forms (missing signs/symptoms). The majority of excluded cases were laboratory-confirmed VZV cases (78 or 72. 9% of those excluded), which may lead to a slight bias in the dataset that contains relatively more laboratory-confirmed MPX cases than were identified in the surveillance dataset. However, a similar proportion of laboratory-confirmed MPX and VZV cases were used for the analyses. Further, data from the suspect MPX cases was collected at one point in time during their illness. The dataset is unique in that it captured many signs/symptoms present for patient; similar datasets with a large number of patients/cases are not available for independent comparison. This data does not represent the spectrum of signs/symptoms that a patient may experience over the course of their illness, and, the result may be a limitation in the frequency of signs/symptoms for suspect cases. Nevertheless, this data reflected the range of presentations of signs/symptoms recognized in suspected MPX patients in an area with endemic disease. In order to document and detect endemic human MPX cases, a surveillance case definition with more specificity in accurate case detection is needed. In rural DRC there are increasingly limited resources, competing health priorities, and a lack of regional testing capacity, which emphasizes the need to easily and efficiently deploy a case definition to accurately identify true MPX patients and limit false positives. A single MPX case or the decision to launch an outbreak response requires considerable resources. According to national guidelines, once a MPX case is identified, the case should be isolated and contacts should be followed for 21 days. Strict recommendations regarding hygiene and infection control are instituted for the entire period of illness, which can last for four weeks. In the absence of a more specific case definition, continued emphasis on laboratory-based diagnostics is warranted. More rapid and efficient methods of diagnosing suspect MPX patients, via a regional surveillance laboratory or a clinical laboratory, are needed to better identify and care for patients followed by appropriate control measures. Disclaimer: The findings and conclusions in this report are those of the author (s) and do not necessarily represent the views of the Centers for Disease Control and Prevention.

Human monkeypox is the most significant Orthopoxvirus infection since the eradication of smallpox. The disease is endemic in Africa and the majority of cases occur in the Congo Basin. Correct identification of patients is critical to deployment of efficient control measures to prevent further transmission and appropriate care for the patient. An evaluation of two surveillance case definitions revealed that the definitions had high sensitivities but low specificities for correct case identification, which results in the identification of patients with other rash illnesses. Several signs and symptoms of laboratory-confirmed monkeypox cases were identified that could be used to aid in the development of surveillance case definitions to correctly identify cases. Laboratory diagnostics continue to be an important part of correct patient identification in order to control the disease and provide adequate care.

lay_plos

Background SSA administers the Old Age, Survivors, and Disability Insurance programs under Title II of the Social Security Act. About 96 percent of the nation’s work force is in social security-covered employment and pays tax on their annual earnings. When workers pay social security taxes, they earn coverage credits, and 40 credits—equal to at least 10 years of work— entitle them to social security benefits when they reach retirement age. In 1977, the Congress authorized the President to enter into totalization agreements with other countries. These bilateral agreements are intended to accomplish three purposes. First, they eliminate dual social security coverage and taxes that multinational employers and employees encounter when they operate and their workers temporarily reside and work for the corporation, usually no more than 5 years, in a foreign country with its own social security program. Under the agreements, U.S. employers and their workers sent temporarily abroad would benefit by paying only U.S. social security taxes, and foreign businesses and their workers would benefit by paying only social security taxes to their home country. Second, the agreements provide benefit protection to workers who have divided their careers between the United States and a foreign country, but lack enough coverage under either social security system to qualify for benefits, despite paying taxes into both systems. Totalization agreements allow such workers to combine (totalize) work credits earned in both countries to meet minimum benefit qualification requirements. Third, most totalization agreements improve the portability of social security benefits by removing rules that suspend benefits to noncitizens who live outside the benefit-paying country. By law, proposed agreements are sent to the Congress together with a report on the effects on the agreement. Under the statute, the agreement becomes effective on any date provided in the agreement after one House of the Congress has been in session 60 days, unless either House of the Congress adopts a resolution of disapproval. Table 1 shows agreements in effect and the years they became effective. To qualify for totalized U.S. social security benefits, a worker must have at least 6 but no more than 39 U.S. coverage credits. Benefit amounts are based on the portion of time a foreign citizen worked in the United States and, thus, are almost always lower than full social security benefits. The average monthly, totalized social security benefit at the end of 2001 was $162, compared with the average nontotalized monthly social security benefit of $825. In 2001, SSA paid about $173 million under totalization agreements to about 89,000 persons, including their dependents. Under U.S. law, immigrants may not work in the United States unless specifically authorized. Nevertheless, immigrants often do work without authorization and pay social security taxes. Under the Social Security Act, all earnings from covered employment in the United States count towards earning social security benefits, regardless of the lawful presence of the worker, his or her citizenship status, or country of residence. Immigrants become entitled to benefits from unauthorized work if they can prove that the earnings and related contributions belong to them. However, they cannot collect such benefits unless they are either legally present in the United States or living in a country where SSA is authorized to pay them their benefits. Mexico is such a country. SSA’s Process for Developing Agreements Is Not Thorough or Well- Documented A lack of transparency in SSA’s processes, and the limited nature of its review of Mexico’s program, cause us to question the extent to which SSA will be positioned to respond to potential program risks should a totalization agreement with Mexico take place. SSA officials told us that the process used to develop the proposed totalization agreement with Mexico was the same as for prior agreements with other countries. The process—which is not specified by law or outlined in written policies and procedures—is informal, and the steps SSA takes when entering into agreements are neither transparent nor well-documented. Current law does not prescribe how SSA should select potential agreement countries. According to SSA, interest in a Mexican agreement dates back more than 20 years. SSA officials noted that increased business interaction between the two countries due to the North American Free Trade Agreement (NAFTA) was a factor in the renewed negotiations. In addition, because there is a totalization agreement with Canada, our other NAFTA partner, SSA believed that equity concerns required consideration of an agreement with Mexico. In February 2002, SSA sought clearance from the Department of State to begin such negotiations. The law also does not specify which elements of other countries’ social security systems must be evaluated during totalization agreement negotiations. SSA officials met with Mexican officials to exchange narrative information on their respective programs. Senior SSA officials also visited Mexico for 2 days in August 2002. During their visit, these officials told us that they toured social security facilities, observed how Mexico’s automated social security systems functioned, and identified the type of data maintained on Mexican workers. SSA took no technical staff on this visit to assess system controls or data integrity processes. In effect, SSA only briefly observed the operations of the Mexican social security program. Moreover, SSA did not document its efforts or perform any additional analyses then, or at a later time, to assess the integrity of Mexico’s social security data and the controls over that data. In particular, SSA officials provided no evidence that they examined key elements of Mexico’ s program, such as its controls over the posting of earnings, and its processes for obtaining key birth and death information for Mexican citizens. Nor did SSA evaluate how access to Mexican data and records is controlled and monitored to prevent unauthorized use or whether internal and external audit functions exist to evaluate operations. Because all totalization agreements represent a financial commitment with implications for social security tax revenues and benefit outlays, a reasonable level of due diligence and analysis is necessary to help federal managers identify issues that could affect benefit payment accuracy or expose the nation’s system to undue risk. Our Internal Control Management and Evaluation Tool provides a risk assessment framework to help federal managers mitigate fraud, waste, abuse, and mismanagement in public programs, such as social security. A key component of this framework is the identification of internal and external risks that could impede the achievement of objectives at both the entity and program levels. Identified risks should then be analyzed for their potential effect and an approach devised to mitigate them. SSA did not conduct these types of analyses in previous agreements or in the case of the proposed Mexican agreement, despite documented concerns among Mexican government officials and others regarding the integrity of Mexico’s records, such as those for birth, death, and marriage, as well as its controls over assigning unique identification numbers to workers for benefit purposes. Such information will likely play a role in SSA’s ability to accurately determine Mexican workers’ initial and continuing eligibility for benefits under a totalization agreement. Totalization Agreements Will Increase Benefit Payments to Mexican Citizens A totalization agreement with Mexico will increase the number of Mexican citizens who will be paid U.S. social security benefits in two ways. First, the agreement will make it easier for Mexican workers to qualify for benefits. Second, it will remove some nonpayment restrictions that affect benefit payments to non-U.S. citizens’ family members residing in another country, thus providing U.S. social security benefits to more survivors and dependents of entitled Mexican workers. Under current law, a worker must earn sufficient coverage credits to qualify for benefits under the U.S. Social Security program. For example, a worker who was born in 1929 or later generally needs 40 coverage credits to be insured for retirement benefits. Credits are based on a worker’s annual earnings in social security-covered employment. At most, 4 credits can be earned per year so that it takes at least 10 years of covered earnings in the United States for a worker to accumulate the necessary 40 credits and become insured for retirement benefits. Currently, social security credits are earned by anyone who has worked in covered employment in the United States. This is true even if the person was unauthorized to work when he/she earned coverage credits. For example, noncitizens, including Mexicans, who are at least 62 years old and lawfully present in the United States, will receive retirement benefits today as long as they meet the coverage credit threshold. Even Mexican citizens who are not lawfully present in this country can receive social security benefits earned through unauthorized employment if they later return to live in Mexico. Similarly, under current law, noncitizen dependents and survivors can also receive social security benefits under some circumstances. Totalization agreements generally expand benefits to both authorized and unauthorized workers and create new groups of beneficiaries. This would be the case for a totalization agreement with Mexico if it follows the same pattern as all prior totalization agreements. Mexican citizens with fewer than 40 coverage credits will be permitted to combine their annual earnings under their home country’s social security program with their annual earnings under the U.S. Social Security program to meet the 40- credit requirement. In addition, more family members of covered workers will qualify for dependent and survivor benefits. Totalization agreements generally override Social Security Act provisions that prohibit benefit payments to noncitizens’ dependents and survivors who reside outside the United States for more than 6 months, unless they can prove that they lived in the United States for 5 years in a close family relationship with the covered worker. If a totalization agreement with Mexico is structured like others already in force, the 5-year rule for dependents and survivors will be waived. However, it is important to understand that not all unauthorized Mexican citizens who have worked in the United States will receive totalization benefits. Some will have earned at least 40 coverage credits and can receive social security benefits without a totalization agreement. Still others may have worked under false identities and may not be able to prove that they have the necessary coverage credits to be entitled to benefits. Others still may not accumulate sufficient credits under the Mexican social security system to totalize with their U.S. social security coverage. Poor Data Undermine the Reliability of SSA’s Cost Estimate The cost of a totalization agreement with Mexico is highly uncertain. In March 2003, the Office of the Chief Actuary (OCACT) estimated that the cost of the Mexican agreement would be $78 million in the first year and would grow $650 million (in constant 2002 dollars) in 2050. SSA’s actuarial cost estimate assumes the initial number of newly eligible Mexican beneficiaries was equivalent to the 50,000 beneficiaries living in Mexico today and would grow sixfold over time. However, this proxy figure is not directly related to the estimated millions of current and former unauthorized workers and their family members from Mexico and appears small in comparison to those estimates. Furthermore, even if the baseline estimate is used, a sensitivity analysis performed by OCACT shows that an increase of more than 25 percent—or 13,000 new beneficiaries—would produce a measurable impact on the long-range actuarial balance of the trust funds. Our review of cost estimates for prior totalization agreements shows that the actual number of beneficiaries has frequently been underestimated and far exceeded the original actuarial estimates. Actuarial Estimates Are Based on Varied Data Sources OCACT develops estimates of expected costs of totalization agreements by analyzing pertinent data from prior agreements, work visas issued, foreign corporations operating in the United States, and U.S. Census data. Because of extensive unauthorized immigration from Mexico, OCACT concluded that U.S. Census data, that would typically be used to estimate the number of new beneficiaries under an agreement, were not reliable. Instead, OCACT used the number of fully insured beneficiaries—U.S. citizens and others living in Mexico—currently receiving U.S. social security benefits as a proxy for the number of Mexican citizens who would initially receive totalized benefits. The principal basis for this assumption was a 1997 study of Mexican immigration patterns conducted by a private nonprofit organization. This study indicated that the percentage of Mexican immigrants who returned to Mexico after more than 10 years and, therefore, could qualify for benefits is roughly equal to the percentage that returned after staying 2 to 9 years and would not have the required credits. Thus, OCACT assumed that the potential totalized initial new beneficiaries would be equivalent to the 50,000 persons currently receiving benefits in Mexico. For the proposed Mexican agreement, both a short-term (covering the first 8 years of the agreement) and a long-term (covering 75 years) cost estimate were developed. The estimated cost to the Social Security Trust Funds would be about $78 million in the first year of the agreement. For the long-term cost estimate, OCACT projected that the number of beneficiaries would ultimately increase sixfold to 300,000 over a 45-year period after the agreement took effect and equal about $650 million (in constant 2002 dollars) in 2050. However, the actuarial analysis notes that the methodology was indirect and involved considerable uncertainty. As a rough check on the reasonableness of using current beneficiaries in Mexico for its cost estimate, OCACT analyzed totalized beneficiary data for Canadian citizens because Canada, like Mexico, is a NAFTA trading partner and shares a large contiguous border. After determining the ratio of Canadians receiving totalized versus fully insured benefits, OCACT applied this ratio to the number of Mexican-born U.S. social security beneficiaries and found that about 37,000 beneficiaries would be expected under the agreement initially, if the Canadian experience proves predictive of the Mexican outcome. According to OCACT, this comparison increased its confidence that the assumed 50,000 new beneficiaries under the agreement was within a reasonable range. Estimated Cost of Mexican Agreement Is Highly Uncertain Limited data about unauthorized workers make any estimate of the expected costs of a Mexican totalization agreement highly uncertain. A significant variable of any totalization agreement cost estimate is the identification of the number of potential beneficiaries. Estimates of the number of unauthorized Mexican immigrants living in the United States vary. The federal government’s estimate was published in January 2003 and comes from the former Immigration and Naturalization Service (INS). INS estimated that, as of January 2000, about 5 million, or 69 percent of all unauthorized immigrants in the United States, were from Mexico. INS’s estimate also indicated that this figure was expected to increase by about 240,000 persons annually. The INS estimate, however, does not include unauthorized Mexican workers and family members who no longer live in the United States and could also conceivably benefit from a totalization agreement. Economic disparity between the United States and Mexico has fostered longstanding immigration from Mexico to the United States dating back many decades. Various studies also show that fewer than a third of Mexican immigrants stay more than 10 years in the United States, the minimum amount of time needed to qualify for social security retirement benefits. For cost analysis purposes, little is known about the population of former immigrants who have returned to Mexico in terms of their age, work history, dependents, and social security coverage. These factors increase the inherent uncertainty of any long-range forecasts with regard to Mexico. It is under this backdrop that OCACT set about developing an estimate of the costs of the potential totalization agreement. We have several concerns about OCACT’s estimate of the number of expected beneficiaries and cost of an agreement with Mexico. First, the use of the 50,000 fully insured beneficiaries receiving benefits in Mexico as a proxy for individuals who might initially benefit from an agreement, does not directly consider the estimated millions of unauthorized Mexican immigrants in the United States and Mexico who are not fully insured and might receive totalized benefits. Furthermore, despite the availability of key data about earnings, work histories, years of employment, and dependents for the 50,000 fully insured beneficiaries, OCACT did not analyze this population to determine whether they represented a good proxy for individuals likely to qualify for totalized benefits. The cost estimate also inherently assumes that the behavior of Mexican citizens would not change after a totalization agreement goes into effect. Under totalization, unauthorized workers could have an additional incentive to enter the United States to work and to maintain the appropriate documentation necessary to claim their earnings under a false identity. Thus, a large number of Mexican citizens have likely earned some social security coverage credits through both authorized and unauthorized work to meet the 40-credit threshold requirement and are not directly accounted for in SSA’s estimate. Second, SSA’s reasonableness check using Canadian data faces similar questions. While Mexico and Canada are NAFTA partners and share a common border with the United States, there is a dramatic difference in the extent of unauthorized immigration from these two countries and, in our view, the Canadian experience is not a good predictor of experience under an agreement with Mexico. Recent INS data show that Mexican citizens account for about 69 percent of unauthorized U.S. immigrants, whereas Canadian citizens account for less than 1 percent, and all other totalization agreement countries combined account for less than 3 percent. It is this population of unauthorized immigrants that makes estimating the cost of a totalization agreement with Mexico particularly problematic. Finally, even though SSA’s actuarial analysis increases the number of beneficiaries sixfold over time, the expected 300,000 beneficiaries in 2050 represents only about 6 percent of the estimated number of unauthorized Mexicans in the United States today, and thus appears relatively low. Although it would be unreasonable to expect all unauthorized Mexicans in the United States to qualify for totalized benefits, the very large difference between estimated and potential beneficiaries underscores the uncertainty of the estimate and suggests that any difference between estimated and actual costs will be on the high side. Indeed, it would take only a relatively small increase in new beneficiaries from the original actuarial assumption of 50,000 initial new beneficiaries to have a measurable impact on the long-range actuarial balance of the trust funds. OCACT has estimated that the agreement would not generate a measurable impact on the long-range actuarial balance. However, a subsequent sensitivity analysis performed at our request shows that a measurable impact on the long-range actuarial balance of the trust funds will occur if the baseline figure is underestimated by more than 25 percent—just 13,000 additional beneficiaries above the estimated 50,000 new beneficiaries. Our analysis of past actuarial estimates of expected beneficiaries under totalization agreements shows that exceeding the 25 percent threshold has not been unusual, even in agreements where uncertainty about the number of unauthorized workers is substantially less. Our review of prior estimates shows that OCACT frequently either overestimated or underestimated the number of expected beneficiaries, usually by more than 25 percent (see table 2). In fact, where underestimates occurred, the differences were huge, involving several orders of magnitude. However, it is important to note that the number of estimated beneficiaries for prior agreements is substantially smaller than for the proposed Mexican agreement. Therefore, the differences in actual beneficiaries from estimated beneficiaries have a higher proportional impact. Furthermore, OCACT has not underestimated the number of expected beneficiaries for the agreements we analyzed since the 1991 agreement with Austria. Nevertheless, the numerous uncertainties and data gaps associated with the Mexican agreement elevate the risks associated with any cost estimate. Conclusions Totalization agreements between the United States and other countries often foster enhanced diplomatic relations and provide mutually beneficial business, tax, and other incentives to employers and employees affected by these agreements. At the same time, they impose a financial cost to both countries’ social security programs. SSA’s processes for entering into these agreements have been informal and have not included specific steps to assess and mitigate potential risks. Regardless of the country under consideration, sound management practices dictate that SSA managers have a risk management process in place to ensure that the interests of the United States and the Social Security Trust Funds are protected. Most totalization agreements have been with countries that are geographically distant to the United States, have developed economies, and represent only a fraction of the estimated unauthorized immigrants in the United States. Still, all agreements include some level of uncertainty, and require due diligence on SSA’s part to alleviate those uncertainties. An agreement with Mexico, however, presents unique and difficult challenges for SSA because so little is known about the size, work history, earnings, and dependents of the unauthorized Mexican population. Furthermore, a common border and economic disparity between the United States and Mexico have fostered significant and longstanding unauthorized immigration into the United States, making an agreement with Mexico potentially far more costly than any other. Thus, for the Mexican agreement, additional analyses to assess risks and costs may be called for. A revised approach for entering into totalization agreements with all countries would enhance the quality of information provided to the Congress, which is tasked with reviewing these vital long-term commitments. A more thorough prospective analysis will also provide a better basis for determining whether agreements under consideration meet the mutual economic and business needs of all parties. Finally, current solvency issues require the Congress to think carefully about future trust fund commitments resulting from totalization agreements. Having more timely and complete information on the benefits, costs, and risks associated with each agreement can only serve to better inform their decisions. Mr. Chairman, this concludes my statement. I would be happy to respond to any questions that you or other members of the Subcommittee may have. For information regarding this testimony, please contact Barbara D. Bovbjerg, Director, Education, Workforce, and Income Security Issues, on (202) 512-7215. Individuals who made key contributions to this testimony are Daniel Bertoni, Gerard Grant, William Staab, and Paul Wright. This is a work of the U.S. government and is not subject to copyright protection in the United States. It may be reproduced and distributed in its entirety without further permission from GAO. However, because this work may contain copyrighted images or other material, permission from the copyright holder may be necessary if you wish to reproduce this material separately.

Totalization agreements foster international commerce and protect benefits for persons who have worked in foreign countries. They eliminate dual social security taxes that multinational employers and their employees pay when they operate and reside in countries with parallel social security systems and fill gaps in benefit protection for persons who have worked in different countries. Because Mexicans are believed to represent a large share of the millions of unauthorized workers present in the United States, a totalization agreement with Mexico has raised concerns that they would become newly eligible for social security benefits. To shed light on the possible impacts, this testimony (1) describes the Social Security Administration's (SSA) processes for developing the agreement with Mexico, (2) explains how the agreement might affect the payment of benefits to Mexican citizens, and (3) assesses the cost estimate for such an agreement. SSA has no written policies or procedures it follows when entering into totalization agreements, and the actions it took to assess the integrity and compatibility of Mexico's social security system were limited and neither transparent nor well-documented. SSA followed the same procedures for the proposed Mexican agreement that it used in all prior agreements. SSA officials told GAO that they briefly toured Mexican facilities, observed how its automated systems functioned, and identified the type of data maintained on Mexican workers. However, SSA provided no information showing that it assessed the reliability of Mexican earnings data and the internal controls used to ensure the integrity of information that SSA will rely on to pay social security benefits. The proposed agreement will likely increase the number of unauthorized Mexican workers and family members eligible for social security benefits. Mexican workers who ordinarily could not receive social security retirement benefits because they lack the required 40 coverage credits for U.S. earnings could qualify for partial Social Security benefits with as few as 6 coverage credits. In addition, under the proposed agreement, more family members of covered Mexican workers would become newly entitled because the agreements usually waive rules that prevent payments to noncitizens' dependents and survivors living outside the United States. The cost of such an agreement is highly uncertain. In March 2003, the Office of the Chief Actuary estimated that the cost of the Mexican agreement would be $78 million in the first year and would grow to $650 million (in constant 2002 dollars) by 2050. The actuarial cost estimate assumes the initial number of newly eligible Mexican beneficiaries is equivalent to the 50,000 beneficiaries living in Mexico today and would grow sixfold over time. However, this proxy figure does not directly consider the estimated millions of current and former unauthorized workers and family members from Mexico and appears small in comparison with those estimates. The estimate also inherently assumes that the behavior of Mexican citizens would not change and does not recognize that an agreement could create an additional incentive for unauthorized workers to enter the United States to work and maintain documentation to claim their earnings under a false identity. Although the actuarial estimate indicates that the agreement would not generate a measurable long-term impact on the actuarial balance of the trust funds, a subsequent sensitivity analysis performed at GAO's request shows that a measurable impact would occur with an increase of more than 25 percent in the estimate of initial, new beneficiaries. For prior agreements, error rates associated with estimating the expected number of new beneficiaries have frequently exceeded 25 percent, even in cases where uncertainties about the number of unauthorized workers were less prevalent. Because of the significant number of unauthorized Mexican workers in the United States, the estimated cost of the proposed totalization agreement is even more uncertain than in prior agreements.

gov_report

It’s safe to say that Tomi Lahren of The Blaze is not a fan of Meryl Streep after her tweetstorm aimed at the actor’s Golden Globes acceptance speech regarding Donald Trump’s treatment of Hollywood, immigrants and the press. Meryl bout to get some "final thoughts" tomorrow. These entitled Hollywood crybabies still don't understand how out of touch they are! — Tomi Lahren (@TomiLahren) January 9, 2017 Lahren promises to cover Streep’s speech on the next installment of her popular segment, “Final Thoughts,” where she aims to dismantle her opposition’s views with a fast-paced monologue. Lahren couldn’t wait, though, and launched into a Twitter tirade that will likely serve as a preview for her show. It might be warm in LA, but make no mistake it's raining snowflakes. Out of touch, whiny, overpaid SNOWFLAKES! #GoldenGlobes — Tomi Lahren (@TomiLahren) January 9, 2017 These Hollywood elites wouldn't know average, every day hard-working Americans if we bit them in the ass. #GoldenGlobes — Tomi Lahren (@TomiLahren) January 9, 2017 Oh no!! What will @realDonaldTrump do without the support of the liberal Hollywood elite?! Oh I know, he will Make America Great Again. — Tomi Lahren (@TomiLahren) January 9, 2017 After the initial barrage, Tomi gathered herself and attempted a three part mic drop. Oh and Meryl, we are just fine with watching football. Thanks. #GoldenGlobes — Tomi Lahren (@TomiLahren) January 9, 2017 You're right about 1 thing Meryl, violence does incite violence kinda like the violence the DNC paid for at Trump rallies. Kinda like that.. — Tomi Lahren (@TomiLahren) January 9, 2017 Safeguard the truth Meryl? Like the truth in those 33k deleted emails? Or are we talking about Hillary's lies? Not sure, Meryl…. — Tomi Lahren (@TomiLahren) January 9, 2017 RELATED: There wasn’t a dry eye in the audience after the Golden Globes paid tribute to Carrie Fisher and Debbie Reynolds Lahren redirects Streep’s criticisms of Trump towards Hillary Clinton and the Democratic party after implying that America would be just fine without Hollywood. If these are just her initial reactions, after stewing for a day, her show is sure to be a can’t-miss. Not everyone liked Meryl Streep’s Golden Globes speech Beloved Meryl Streep somehow made Hollywood fall even more in love with her at the 2017 Golden Globes. Streep, who was honored with the Cecil B. DeMille award, accepted her trophy and gave a politically charged speech about President-elect Donald Trump and his tendency to target foreigners. Streep defended the press, Hollywood and immigrants, and called upon the late Carrie Fisher to help her close out her speech. "As my friend, the dear, departed Princess Leia, said to me, 'Take your broken heart, make it into art,'" she said. The Florence Foster Jenkins star never mentioned Trump by name. Golden Globes cameras panned across the audience to show various members tearing up and looking at Streep with awe. Meryl Streep gave a powerful, politically charged speech at the Golden Globes and, while it was obvious whom she was speaking about, never mentioned Donald Trump by name. In response, President-Elect Donald Trump made a series of tweets dismissing Meryl Streep as overrated. And thus the Hollywood vs. politics debate rages on, now perhaps in a discourse more churlish and on a platform more undignified than ever. Spurned by a petulant outburst by the incoming commander-in-chief, an obtuse tweet from John McCain’s daughter, teed-up yacking by Kellyanne Conway on Fox News, and a prevailing frustration over actors constantly espousing their politics in public arenas—a frustration that deserves to be validated—there is backlash over Meryl Streep’s acceptance speech at the Golden Globe Awards Sunday night. Enough. If we’re going to shine a spotlight on things that are tired, it is not the practice of celebrities talking about politics. It is the practice of complaining about that. Here’s a brief recap of the debate. Meryl Streep was given a lifetime achievement award Sunday night. Her speech commanded the room, with the Oscar winner eschewing the opportunity to express polite gratitude and toss off some witty quips to instead make a call to arms from her peers in Hollywood. She brought up the incident in which Trump appeared to mock a disabled reporter—something he denied doing again in an interview with The New York Times and in his tweet tirade, but which there is video evidence of—and used the anecdote as the catalyst for a plea for greater empathy, from the people in the highest position in the world to those sitting in the ballroom to us watching at home. Trump scoffed at the speech as pandering from a “Hillary flunky who lost big.” He called Meryl Streep, who was at the Globes to accept the Cecil B. DeMille Lifetime Achievement Award for a career that has included 3 Oscars, 9 Golden Globes, and the branding as the Greatest Actress of All-Time, “overrated.” (Perhaps, just perhaps, a person who purports to be in the business of uniting our nation could do better in that pursuit than disparaging Meryl Streep’s acting chops.) Trump’s message—that he was attacked unfairly—was amplified by his supporters, who continue to insist that politics should be kept out of the mouths of celebrities. Get The Beast In Your Inbox! Daily Digest Start and finish your day with the top stories from The Daily Beast. Cheat Sheet A speedy, smart summary of all the news you need to know (and nothing you don't). By clicking “Subscribe,” you agree to have read the Terms of Use and Privacy Policy Subscribe Thank You! You are now subscribed to the Daily Digest and Cheat Sheet. We will not share your email with anyone for any reason. Meghan McCain, daughter of Senator John McCain and frequent TV commentator, tweeted earlier in the night that, “This Meryl Streep speech is why Trump won. And if people in Hollywood don’t start recognizing why and how – you will help him get re-elected.” Presumably she was referencing a sense of exhaustion over people in privileged positions presuming that their opinions merit public forums, and that Hollywood’s vocal support of Hillary Clinton irritated voters enough to flee in the other direction—that the optics of Clinton’s high-powered, rich and famous friends meant she was out of touch with the needs of everyday people. It’s always been a confusing notion, particularly in comparison to mogul and reality star Donald Trump. There’s also the idea that they’re “just celebrities,” that they don’t have any expertise on these issues and have no right to speak authoritatively on them. To that, as journalist Glenn Greenwald aptly inquired in a tweet that’s being circulated Monday morning, “Why are actors less qualified to comment on political issues than, say, cable TV or talk radio hosts? Never understood this rationale.” Sure, like anyone who has passionate feelings on an issue and a microphone to share them from, people in Hollywood—liberals or otherwise—hope their message is heard by people with opposing views, that they digest it and consider it. But the idea that Meryl Streep was unaware that she was speaking to a ballroom of friendly ears misunderstands what I gathered to be her intention. She was speaking to a room full of celebrities whose politics she knows, about a frustration she knew they felt. Assumptions from tweets like McCain’s insinuating that Hollywood liberals don’t realize they’re in a bubble, or that Trump voters don’t like them, are ridiculous. Streep made a plea to stand up against bullying, for dignity in power, and advocated for the arts. The catalyst for her plea was the election of Donald Trump. Of course it was. Streep, in addition to much of the Globes attendees, made no secret of her support of Hillary Clinton and her lack of respect for Trump and what he stands for. Why would anyone be surprised that a Hollywood liberal made such a plea at an event celebrating Hollywood liberals? “She didn’t change anyone’s minds” is the critique most commonly charged against her speech Monday morning. She wasn’t trying to change minds, or campaign for anything. She was calling to action people whose politics she already knew aligned with her own, whom she hoped she could motivate to take a stand for something meaningful. Maybe that’s what made Sunday’s awards telecast seem so tonally off. These events are typically characterized by cheerleading: for Obama, for their shared politics, for Hillary or against Trump, and, most often, for themselves. That kind of positive, unanimous energy is what made room for silly distractions and goofy bits like the ones host Jimmy Fallon tried to stage. But when the room no longer wanted to cheer—politically or otherwise—and instead set a more somber tone, the producers didn’t know how to reconcile that. But to make the case that politics shouldn’t have been a part of the show entirely? C’mon. People who continually say Hollywood and awards shouldn’t be platforms for politics and social justice need a new script. Have you seen an award show in the last 40-some years? It’s nothing new, and it’s not going to change. Stretch back to Marlon Brandon inviting Sacheen Littlefeather on stage in 1973 all the way up to Boyhood’s Patricia Arquette urging for gender pay equality in 2014, with stops at every Jane Fonda, Vanessa Redgrave, Sally Field, Michael Moore, Leonardo DiCaprio, and George Clooney in between. If “know your audience” is a critique lobbied against Streep, “know your show” is applicable to those still upset about the confluence of Hollywood and politics. Hollywood and politics, by the way: a copulation that inseminated Donald Trump and birthed his public career, and a topic he seemingly can’t stop tweeting about. This is an industry that Trump has desperately wanted to be included in and welcomed by. There are the dozens and dozens of TV show and movie cameos he made, award shows he attended, and photos he took at parties with celebrities. He desperately wanted to win an Emmy and still complains that he didn't. All he has to show for his showbiz yearning is a Razzie award and an inauguration at which no one will perform. It’s no wonder he’s taken to calling Meryl Streep overrated. And it’s no wonder Meryl Streep feels compelled to speak out. Tweet with a location You can add location information to your Tweets, such as your city or precise location, from the web and via third-party applications. You always have the option to delete your Tweet location history. Learn more Add a location to your Tweets When you tweet with a location, Twitter stores that location. You can switch location on/off before each Tweet and always have the option to delete your location history. Learn more This Meryl Streep speech is why Trump won. And if people in Hollywood don't start recognizing why and how - you will help him get re-elected Conway accuses Meryl Streep of 'inciting people's worst instincts' Meryl Streep’s Golden Globes speech Sunday night was a disappointment, incoming counselor to the president Kellyanne Conway said Monday morning, stating that the actress offered a divisive message indicative of Hollywood’s “self-pity” instead of a unifying one. Streep, who was honored with a lifetime achievement award by the Hollywood Foreign Press Association at the Golden Globes awards show, devoted significant time in her acceptance remarks to criticizing Donald Trump. She accused the president-elect of bullying and said his speech last year in which he seemingly mocked a disabled person was the “one performance this year that stunned me.” Story Continued Below “She sounds like 2014. The election is over. She lost,” Conway said on Fox News’ “Fox & Friends.” “Everybody in that audience, with very few exceptions, was of a single, myopic mind as to how they wanted the election to go and how they expected the election to go. They lost, and I really wish she would have stood up last night and said ‘Look, I didn’t like the election results, but he’s our president and we’re going to support him.’” Conway accused Streep of “inciting people’s worst instincts” with her speech. Trump’s incoming counselor said the time would have been put to better usesearching for common ground with the incoming administration, something Conway suggested the president-elect “has actually done from moment one.” “But this is Hollywood. I think where there is self-pity, a lot more self-awareness would do them some charm,” she continued. “Talking about how vilified poor Hollywood is, in their gazillion-dollar gowns. Can I borrow a couple of those for the inaugural, please?” Trump shot back at Streep as well, taking to Twitter to label her “one of the most over-rated actresses in Hollywood” and a “Hillary flunky who lost big.” As he has in the past, Trump disputed the notion that he had mocked the reporter, The New York Times’ Serge Kovaleski, insisting that he was mimicking the nervousness of a reporter backing away from a story years after the fact. Trump had used a 2001 story by Kovaleski as the basis for his fictitious claim that thousands of Muslims celebrated the Sept. 11 terrorist attacks on rooftops in New Jersey. Trump insisted he had seen video of the incident, although none has ever been uncovered, and Kovaleski disputed Trump’s understanding of the event. On “Fox & Friends,” Conway wondered aloud why Streep had used her time on stage to attack Trump’s supposed mockery of a disabled individual and not to call attention to a recent case from Chicago in which four people were arrested last week for allegedly torturing a teenager with special needs and streaming the episode live on Facebook. Conway noted the races of those involved, four African-American suspects and a white victim, and said that if Streep is truly concerned about the disabled community, she should speak out about the incident in Chicago. “I’m glad that Meryl Streep has such a passion for the disabled, because I didn’t hear her weigh in and I didn’t hear her even use her platform last night, Ainsley, to give a shoutout to the mentally challenged boy who last week was tortured live on Facebook for half an hour by four young African-American adults who were screaming racial and anti-Trump expletives and forcing him to put his head in toilet water,” Conway told Fox News host Ainsley Earhardt. “So I’d like to hear from her today, if she wants to come and continue her platform on behalf of the disabled.”

Donald Trump was quick to jump on Meryl Streep after the actress spoke out against him during her Golden Globes acceptance speech, and now other reactions are trickling in. On Monday's Fox & Friends, Trump adviser Kellyanne Conway accused Streep of "inciting people's worst instincts" with her speech, Politico reports: "Everybody in that audience, with very few exceptions, was of a single, myopic mind as to how they wanted the election to go and how they expected the election to go. They lost, and I really wish she would have stood up last night and said 'Look, I didn't like the election results, but he's our president and we're going to support him.'" She also took Streep and others to task for acting like Hollywood is under attack from Republicans. "I think where there is self-pity, a lot more self-awareness would do them some charm," Conway said. "Talking about how vilified poor Hollywood is, in their gazillion-dollar gowns. Can I borrow a couple of those for the inaugural, please?" Meghan McCain echoed that thought process on Twitter: "This Meryl Streep speech is why Trump won. And if people in Hollywood don't start recognizing why and how-you will help him get re-elected." Not surprisingly, conservative pundits Ann Coulter and Michelle Malkin also weighed in on Twitter. Coulter noted, "Streep Lifetime Achievement Award decided by 93 people in Hollywood Foreign Press. Trump President of U.S, decided by 65 million Americans," while Malkin called Streep a "middle America-hater." E! notes that, of course, Streep's fellow celebrities are reacting quite differently, with many in the audience Sunday night moved to tears and others tweeting their adoration of Streep. On the Daily Beast, Kevin Fallon calls the backlash from conservatives "f---ing ridiculous." "People who continually say Hollywood and awards shouldn't be platforms for politics and social justice need a new script," he writes. "Have you seen an award show in the last 40-some years? It's nothing new, and it's not going to change."

multi_news

This paper shows that the various computations underlying spatial cognition can be implemented using statistical inference in a single probabilistic model. Inference is implemented using a common set of ‘lower-level’ computations involving forward and backward inference over time. For example, to estimate where you are in a known environment, forward inference is used to optimally combine location estimates from path integration with those from sensory input. To decide which way to turn to reach a goal, forward inference is used to compute the likelihood of reaching that goal under each option. To work out which environment you are in, forward inference is used to compute the likelihood of sensory observations under the different hypotheses. For reaching sensory goals that require a chaining together of decisions, forward inference can be used to compute a state trajectory that will lead to that goal, and backward inference to refine the route and estimate control signals that produce the required trajectory. We propose that these computations are reflected in recent findings of pattern replay in the mammalian brain. Specifically, that theta sequences reflect decision making, theta flickering reflects model selection, and remote replay reflects route and motor planning. We also propose a mapping of the above computational processes onto lateral and medial entorhinal cortex and hippocampus. This paper describes a dynamic Bayesian model of spatial cognition. Here we define spatial cognition as including the tasks of localisation (estimating where you are in a known environment), sensory imagery (constructing a virtual scene), decision making (deciding which way to turn to reach a goal), model selection (working out which environment you are in) and motor planning (computing a sequence of motor commands that will lead to a sensory goal). We show that all of these tasks can be implemented using statistical inference in a single probabilistic model. We note that the above formulation is slightly different to previous definitions by OKeefe and Nadel [1], Gallistel [2], and Redish [3] which stress the capacity of determining and performing a path from a current position towards a desired location. The model has hidden states comprising speed, direction and allocentric location, control variables comprising change in direction and speed, and sensory states representing olfactory, somatosensory and visual information. The model describes the dynamical evolution of hidden states, and provides a mapping from hidden to sensory states. Inference in the model is then implemented using a common set of ‘lower-level’ computations involving forward and backward inference over time. We propose that these computations are reflected in recent empirical findings of pattern replay in the mammalian brain [4], [5]. Specifically, we propose that theta sequences reflect decision making, theta flickering reflects model selection, and remote replay reflects route and motor planning. Our use of the terms ‘forward’ and ‘backward’ here relate to time and should not be confused with the direction of message passing in a cortical hierarchy [6]. Our approach falls into the general category of ‘map-based’ or ‘model-based’ planning [1], [7]–[10], or ‘model-based decision making’ [11]. The term ‘model-based’ refers to making and updating a representation of the world (such as a cognitive map). This is to be contrasted, for example, with ‘model-free’ approaches in which agents merely react to stimuli, after having previously learnt stimulus-response mappings through extensive exposure to an environment [12]. More generally, agents will use a variety of navigation strategies depending on their cognitive capabilities and familiarity with an environment. Spatial decisions can, for example, be classified [13] as being cue-guided (eg. move towards the red house), stimulus triggered (eg. turn left at the red house), route based (turn left at the red house then right at the blue house). There is a good deal of evidence showing that the brain has multiple decision making or control systems, each with its own strengths and weaknesses [14]–[16]. The usefulness of model-based planning is most apparent after an agent has sufficient experience to learn a model of an environment and when, subsequently, local changes to that environment are made which affect the optimal route to a goal [15]. In statistical terms, these would be referred to as nonstationarities. For spatial models this could be, for example, a hole appearing in a wall enabling an agent to take a shortcut, or a new object appearing preventing an agent taking a habitual route. Another strength of model-based control is that it can reduce learning time. Tse et al. [17], for example, studied decision making in rats and found that learning required fewer trials when it occurred against a background of prior knowledge. This allows new information to be assimilated into an existing schema or model. The model-based versus model-free distinction has become important for the study of decision making in general as the underlying neuroanatomical differences are being delineated [11], [15]. Khamassi and Humphries [18] argue that, due to the shared underlying neuroanatomy, spatial navigation strategies that were previously described as being either place-driven or cue-driven are better thought of as being model-based versus model-free. Daw et al. [15] propose that arbitration between model-based and model-free controllers is based on the relative uncertainty of the decisions and more recently, Pezzulo et al. [19] have embedded both types of decision making systems into a single ‘mixed instrumental controller’. This paper describes the computations underlying spatial cognition, initially, at a rather abstract level of manipulations of probability densities and then employs vector and matrix representations of variables and connectivities. Although we later on go on to describe how our model relates to underlying neuronal implementations, the model itself is not specified at a neuronal level. This style of modelling has many precedents in the literature. For example, Bousquet et al. [20] have conceived of the hippocampus as a Kalman filter. This requires that the hippocampus has an ‘observation model’ relating hidden states (places specified in allocentric coordinates) to sensory cues, and a dynamic model relating previous to current state via path integration. Kalman filtering then refers to the forward inference algorithm that combines path integral estimates of state with current sensory cues to provide optimal updates of the agent' s location. The main function of Kalman filtering in this context is therefore one of localisation. One of the key points of this paper is that if an agent has taken the trouble to construct a ‘dynamic model’ and an ‘observation model’ then they can be used for more than just localisation; the same models, when combined with additional inference steps, can also be used for model selection, decision making and motor planning and to construct sensory imagery. Other statistical treatments of hippocampal function address the issue of context learning [21]. Here, a context is defined in statistical terms as a stationary distribution of experiences. The problem of context learning is then reduced to one of clustering together an agent' s experiences into a finite number of contexts. This is addressed through the use of Hidden Markov Models (HMMs) and it is shown how this perspective explains experimental findings in rat navigation concerning sequence and reversal learning and place-cell remapping. Johnson et al. [22] provide a normative statistical model of exploratory behaviour called Information Foraging (IF). ‘Passive IF’ describes the temporal distribution of an agent' s sampling process (eg. spending longer investigating novel versus familiar objects) whereas ‘Directed IF’ describes its spatial distribution (eg. where it should move to next). Additionally, IF is conceived to apply both to the environment and the agent' s memory of the environment. Directed IF proposes a common hippocampal substrate for constructive memory (eg. scene construction), vicarious trial and error behaviour, model-based facilitation of memory performance, and memory consolidation. The IF framework samples spatial locations, or episodic memories using an information theoretic criterion. To compute this criterion it is necessary for the agent to possess an observation model of the sort described in our article below. A further statistical treatment of hippocampal function comprises a two-stage processing model of memory formation in the entorhinal-hippocampal loop [23]. The first stage, which is proposed to take place during theta activity, allows hippocampus to temporally decorrelate and sparsify its input, and develop representations based on an Independent Component Analysis. The second stage, which is proposed to take place during Sharp Wave Ripples [24], allows hippocampus to replay these new representations to neocortex where long term memories are held to be instantiated. This paper is concerned with computational processes underlying spatial cognition and we describe how the underlying computations may be instantiated in hippocampus and associated brain regions. The hippocampal formation is, however, implicated in a much broader array of functions [25], such as episodic memory, that our model does not address. Indeed one of the key differences between our approach and some other models of spatial cognition [10], [16] is that the approach we describe has no episodic component. Specifically, the sequences that are generated in our model are the result of online computation rather than memory recall. However, as we highlight in the discussion, the interactions between episodic memory and the computations we describe would be especially interesting to examine in future work. The paper is structured as follows. The computer simulations in this paper describe an agent acting in a simple two-dimensional environment. This environment produces visual, somatosensory and olfactory cues as described in the methods section on the ‘Environmental Model’. The agent then develops its own model of the environment as described in the ‘Probabilistic Model’ section. This describes the two elements of the model (i) a dynamical model describing the evolution of hidden states and (ii) a mapping from hidden states to sensory states. The section on ‘Spatial Cognition as Statistical Inference’ then describes how the various tasks of localisation, decision making (and sensory imagery), model selection and motor planning can be described in probabilistic terms. The section on ‘Forward and Backward Inference’ describes the common set of forward and backward recursions for estimating the required probability densities. The section on ‘Results’ describes an implementation of the above algorithms and provides some numerical results. The discussion section on ‘Neuronal Implementation’ then describes our proposal for how these algorithms are implemented in the brain and how functional connectivity among a candidate set of brain regions changes as a function of task. We conclude with a discussion of how the above computations might relate to pattern replay and what are the specific predictions of our model. Computer simulations are implemented in Matlab (R2012a, The MathWorks, Inc.) and are based on an agent navigating in a simple 2D environment depicted in Figure 1. The location of the agent is specified using orthogonal allocentric coordinates and its direction of heading (clockwise from positive) is. The environment contains two inner walls and four boundary walls. The agent is equipped with a touch sensor that detects the minimum Euclidian distance to a wall,. It is also equipped with a nose that detects olfactory input,. In this paper we consider a single olfactory source located at allocentric coordinates. We assume this source diffuses isotropically with scale parameter so that olfactory input at location is given by an exponential function (1) All of the simulations use a single olfactory source with, and. More realistic environments with multiple olfactory sources and turbulence [26] are beyond the scope of this paper. The agent is also equipped with a retina that is aligned with the direction of heading. The retina provides one-dimensional visual input,, from −45 to +45 degrees of visual angle around and comprises pixels. The retina provides information about the ‘colour’ of the walls within its field of view. In our simulations ‘colour’ is a scalar variable which we have displayed using colormaps for ease of visualisation. The scalar values corresponding to the various walls are 0. 14 (north border), 0. 29 (east border), 0. 43 (south border), 0. 57 (west border), 0. 71 (west wall), 0. 86 (east wall). These map onto the colours shown in Figure 1 using Matlab' s default colour map. Although classical laboratory navigation tasks do not involve walls with different colours, they employ extra-maze cues which enable experimental subjects to localize themselves. For the sake of simplicity, here we provide such visual information to the simulated agent by variation of wall colour. The environmental model of retinal input takes the values of and and produces using calculations based on the two-dimensional geometrical relation of the agent with the environment. This uses a simple ray-tracing algorithm. The agent then has its own predictive model of retinal input, described in the ‘vision’ section below, which predicts from and using a basis set expansion. The agent has similar models of olfactory and somatosensory input (see ‘Olfaction’ and ‘Touch’ below). Overall, the environmental model produces the signals, and which form the sensory inputs to the agent' s spatial cognition model (see next section). We write this as to denote sensory signals from the environment. For a sequence of signals we write. These sensory inputs are surrogates for the compact codes produced by predictive coding in sensory cortices [27]. We emphasise that the agent has its own model of sensory input (an ‘observation model’) which is distinct from the environmental input itself. The agent' s observation model is learnt from exposure to the environment. We investigate agents having a model comprising two parts (i) a dynamical model and (ii) an observation model. The dynamical model describes how the agent' s internal state, is updated from the previous time step and motor efference copy. The observation model is a mapping from hidden states to sensory states. Our probabilistic model falls into the general class of discrete-time nonlinear state-space models (2) where is a control input, is state noise and is sensory noise. The noise components are Gaussian distributed with and. This is a Nonlinear Dynamical System (NDS) with inputs and hidden variables. We consider a series of time points and denote sequences of sensory states, hidden states, and controls using,, and. These are also referred to as trajectories. The above equations implicitly specify the state transition probability density and the observation probability density. This latter probability depends on the agent' s model of its environment,. Together these densities comprise the agent' s generative model, as depicted in Figure 2 (top left). This section describes, initially at the level of manipulations of probability densities, how the various computations underlying spatial cognition can be implemented. It then describes a practical algorithm based on local linearisation. If an agent has a probabilistic model of its environment,, then the various tasks that together comprise spatial cognition are optimally implemented using statistical inference in that model. These inferences will be optimal in the sense of maximising likelihood. The various tasks - localisation, imagery, decision making, model selection and planning - all rely on the same statistical model. They are differentiated by what variables are known and what the agent wishes to compute. This is depicted in the panels in Figure 2 where shaded circles denote known quantities. Additionally, for each task, the information entering the system may be of a different nature. For example, for imagery, the inputs,, are virtual motor commands and for localisation they are motor efference copies. Similarly, during localisation and model selection the agent receives inputs from sensory cortices. For the simulations in this paper these come from the environmental model,. However, during decision making and motor planning these inputs do not derive from the agent' s environment but are generated internally and correspond to the agent' s goals. Text S2 describes how the required probability densities can be computed at the very general level of manipulations of probability densities. However, these operations cannot be implemented exactly. They can only be implemented approximately and there are basically two types of approximate inference methods. These are based either on sampling [39] or Local Linearization (LL) [40]. In this paper we adopt an LL approach although this is not without disadvantages. We return to this important issue in the discussion. The following subsections describe the forward and backward inference algorithms under LL assumptions. Readers unfamiliar with statistical inference for dynamical systems models may benefit from textbook material [38]. This section describes a preliminary learning phase in which an agent is exposed to an environment to learn the sensory mapping from states to observations. Here the agent is provided with the observations and also exact knowledge of the hidden states. More realistic simulations would also require the agent to infer the hidden states whilst learning. This is in principle straightforward but is beyond the scope of the current paper, as our focus is on temporal dynamics. We return to this point in the discussion. The olfactory and sensorimotor models use a 10-by-10 grid of basis cells giving 100 cells in all. We assume that the parameters governing the location and width of these cells have been set in a previous learning phase. The weight vectors and (see equations 11 and 12) were optimised using least squares regression and 225 training exemplars with uniform spatial sampling. The retinal model used the same number and location of basis cells. It additionally used 32 head direction cells each having a directional precision parameter. The conjunctive representation comprised 3200 basis cells. The weight vector (see equation 14) was optimised using least squares and a training set comprising 10,575 exemplars. These were generated from spatial positions taken uniformly throughout the maze. Visual input from the environmental model for multiple directions at each spatial location was used to create the training examples. At the end of this learning phase the agent is exquisitely familiar with the environment. A trained model can then be used to generate visual imagery. This is implemented by specifying a synthetic control sequence, running path integration and generating predictions from the model. For example, Figure 3A shows a control sequence that is used to generate the ‘north-east’ trajectory shown in Figure 3C. We also generated ‘north-west’, ‘south-west’ and ‘south-east’ trajectories by changing the sign of direction change,, and/or the initial direction,. To quantitatively assess the accuracy of these imagery sequences,, we compared them to the sequence of visual inputs that would have been received from the environmental model,. Figure 3D plots the proportion of variance explained by the agent' s model as a function of retinal angle. These plots were computed separately for each trajectory, and show that only activity in the central retina is accurately predicted. This is due to the increased optic flow in peripheral regions of the agent' s retina. The asymmetry in Figure 3D is due to the particular spatial arrangement and numerical values of the visual cues. These results suggest that it would be better to have a retina with lower spatial resolution in the periphery. This simulation shows how an agent can localise itself in an environment. The agent was located centrally and moved according to the south-east trajectory. Its exact path was computed using noiseless path integration and the appropriate environmental inputs were provided to the agent. In the discussion section below we propose a mapping of the forward and backward inference equations onto the hippocampal-entorhinal complex. We now report the results of two simulations. The first used the standard forward inference updates in equations 23 and 24. This corresponds to the algorithm that an agent with an intact hippocampus would use. The second, however, had a ‘lesioned hippocampus’ in that only the path integral updates in equation 23 were used (we set). This in effect removed the top down input from hippocampus to MEC (see ‘Localisation’ subsection in the discussion) so that path integral errors are not corrected by sensory input. In both cases the agent' s path updates,, were subject to a small amount of noise (with standard deviation 0. 01) at each time step. Figure 4 shows the results for single and multiple trials. Here, localisation with an intact hippocampus results in better tracking of the agent' s location. Localisation accuracy was assessed over multiple trials () and found to be significantly more accurate with, rather than without, a hippocampus (). The mean localisation error was 60 per cent smaller with a hippocampus. For the above simulations we disabled somatosensory input by setting. This was found to be necessary as this input is not a reliable predictor of location (the distance from a boundary is the same at very many locations in an environment). This simulation shows how an agent can make a decision about which direction to turn by computing likelihood ratios. To demonstrate this principle, we selected the ‘north-west’ and ‘north-east’ trajectories as two possible control sequences. The sensory goal was set equal to the sensory input that would be received at the end of the ‘north-east’ trajectory. This goal was set to be identical at all time points. The agent' s starting location was and with initial speed set to zero. The log of the likelihood ratio (see equation 28),, for model 1 versus model 2 was then computed at each time step. Figure 5 shows the accumulated as a function of the to time points along the trajectory. A of 3 corresponds to a probability of 95% [42]. This indicates that a confident decision can be made early on in the hypothesized trajectories. The degree to which each sensory modality is used in the above computations is determined by the relative values of observation noise covariances (see Text S4). These were initially fixed to the values described at the beginning of the simulations section. Whilst a confident decision could soon be reached using the above default values, decomposition of the LR into modality specific terms showed a strong contribution from both olfactory and visual modalities, but a somatosensory contribution that was initially rather noisy. This is due to small idiosyncrasies in the predictions of somatosensory values. We therefore experimented with the level of somatosensory noise covariance. Figure 5 was produced using a value of which means LR effectively ignores this contribution (although we also have, there are 20 visual inputs). This simulation shows how likelihood ratios can also be used to estimate what environment an agent is located in. We first trained an agent on the maze as described in the imagery section. We refer to this as environment one and the model, described by the set of estimated weights, as model one. We then trained the agent on a second environment and allowed it to develop a separate model. These are referred to as environment two and model two. The second environment was exactly the same as the first except that the east and west boundary walls had their colours swapped. We then placed the agent in the first maze and used the ‘north-east’ control trajectory,, and allowed the agent to compute the likelihood of observed data under its two models, and, as described earlier. The log of the likelihood ratio, for model 1 versus model 2 was then computed at each time step. Figure 6 shows the as a function of the number of time points along the trajectory. The degree to which each sensory modality is used in the above computations is determined by the relative values of observation noise covariances. These were fixed to the values described at the beginning of the simulations section. However, because the only difference between the two models is in their predictions of retinal input (due to the swapping of wall colours), the above computation is driven solely by vision. For the decision making example, described above, the likelihood of reaching the goal given the two trajectories is also differentiated by the olfactory inputs at the goal location (as the olfactory source is located in the south west corner and diffuses isotropically, there will be weaker input in the north east than north west corner). This explains the scaling differences in the likelihood ratios - decision making is easier, in this example, as it is guided by olfaction as well as vision. This is not generally the case, however, and only occurred here due to the specifics of the environments and goals (same olfactory sources at same locations in both mazes, different olfactory inputs at the two goals). This simulation gives an example of how route and motor planning can be implemented. The agent is placed in maze 1 at starting location, with initial speed and direction. This initial state,, is known with high precision (see equation 27). The initial distribution over motor controls has mean and precision (see equation 32). The covariance of the noise on the motor controls is set to (see equation 31). This specifies that the control signals for changes in acceleration (first element) are expected to be larger than those for direction (second element). For this simulation we augmented the sensory vector with observations of the agent' s speed. The sensory goal is multimodal with components for olfaction, touch, vision and speed. For olfaction, touch and speed we set, and. The goal is therefore to navigate to the point in space with olfactory code most similar to. The environmental location with this value is,. The observation noise covariance for speed was set to. A second aim is that the distance to the nearest boundary should be close to. A third aim is that the speed should be as near to as possible. That is, the agent should be stationary at the target. The visual component is set to correspond to an image of the left wall with all ‘yellow’ values. The desired goal trajectory,, is set to be equal to the goal at all time points. The degree to which each sensory modality is used in motor planning is determined by the relative values of observation noise covariance. We used the values described at the beginning of the simulations section. This means that motor planning is guided most by olfaction and touch, and least by vision. The estimated hidden states and inputs were then computed as shown in the earlier section on ‘Inference over Inputs’. Figure 7 shows the planned route traced out by forward and backward inference. For forward inference we are plotting the and elements of (see equation 24), and for backward inference the and elements of (see equation 30). The paths for backward inference are smoother and more direct. Figure 7 also shows the estimated motor control sequence. These sequences correspond to the mean from backward inference,, as described in the section on ‘Inference over Inputs’ (see equation 33). Simple decisions such as ‘turn left’ or ‘turn right’ can be implemented using the ‘decision making’ procedure described in the above section. This is a rudimentary form of planning. The route and motor planning described here is a more powerful approach that we envisage is engaged when the optimal route to a goal involves the chaining together of multiple decisions (eg. ‘turn left’, ‘straight on’, ‘turn right’). This section discusses how and where in the brain the above computational processes might be implemented. Our starting point here is Figure 8 which describes a candidate set of brain regions. Entorhinal cortex is partitioned into Lateral (LEC) and Medial (MEC) components, with the latter representing spatial and the former non-spatial information [43]. The LEC receives substantial input from perirhinal cortex which in turn receives major projections from temporal cortices, whereas the MEC receives substantial input from parahippocampal cortex which in turn receives projections from parietal cortices. The anatomical connectivity supporting this architecture is described in Figure 3 of [44]. We assume that temporal, parietal, parahippocampal and perirhinal cortices and the machinery that feeds into them, together produce a compact coding of spatial and non-spatial aspects of the agent' s environment. These processes are not explicitly modelled in this paper. Our simple and tentative mapping onto hippocampal neuroanatomy currently does not distinguish between CA3 and CA1, instead we consider a single hippocampal node encompassing the activity of CA3-CA1 place cells. Our model then comprises two hippocampal-entorhinal loops, one spatial and one non-spatial, as shown in Figure 8 (top left). The spatial loop proceeds from superficial MEC layers to CA3-CA1, and returns to deep layers of MEC. This partitioning into deep and superficial layers is consistent with known anatomy and previous functional models [45]. Anatomically, entorhinal-hippocampal connectivity is more complex with, for example, direct connections from EC layer three to CA1 [46], and return connections via proximal CA1 (CA1p) and distal Subiculum (SUBd) [47], but our model does not have this level of detail. The non-spatial loop proceeds from superficial LEC layers to CA3-CA1, and returns to deep layers of LEC. The sensory states of our spatial model,, are compact codes representing non-spatial information in the superficial layers of LEC. Predictions of these sensory states from the agent' s model,, are made via the CA3-CA1 to LEC pathway. In our model, the function of CA3-CA1 is to integrate spatial input from MEC with non-spatial input from LEC. This is consistent with a recent schematic model [48], where it is argued that this functionality is preserved across mammals. The mapping from CA3-CA1 to LEC generates the agent' s predictions of sensory states, whereas the mapping from LEC to CA3-CA1 implements the (approximate) inverse of this mapping. Together, these recurrent connections constitute the agent' s model of its environment,, and different models will be instantiated in different subsets of these connections. That populations of cells in LEC encode sensory prediction errors,, is supported by recent recordings in rats [49]. This study identified cells that fired at locations where objects had been located on previous trials (high prediction error), but did not respond when the object was actually present (no prediction error). As with other proposals that the brain may implement some form of approximate Bayesian inference [71], to formally test this idea it is necessary to have a proposal for how neural populations represent uncertainty. Ma et al. [72], for example, have shown how populations of cells can represent probability distributions using probabilistic population codes in which simple linear combinations of firing rates can implement Bayesian inference. Beck at al. [73] have shown how such a scheme can implement Kalman filtering. As we have locally linearised the dynamic and observation nonlinearities, the forward inference step in this paper closely corresponds to Kalman filtering. It therefore seems plausible that forward inference using EKF can be implemented using similar principles. Thus, although equations 23 to 26 perhaps seem rather removed from neurobiology there may well be a plausible neural implementation. It has yet to be demonstrated how the gamma recursions underlying backward inference could be implemented using probabilistic population codes. However, given that the gamma recursions comprise an implementation of Bayes rule followed by a marginalisation (see Text S2) whereas Kalman filtering is a marginalisation followed by Bayes rule (see Text S2) we imagine a similar instantiation is possible. The Beck at al. [73] approach assumes that trial-to-trial variability in population firing rates is in a class of distributions from the linear-exponential family. This includes distributions where cells have independent Poisson rates. There is good evidence to suggest that MTL cell firing is not independent and Poisson [74], but it is not known if their activity falls into the more general linear-exponential family. Other proposals as to how the brain might implement Bayesian inference are specific to the hippocampus. One proposal [75] suggests that higher certainty is encoded by spike patterns containing more spikes and where the spikes are closer together. If this is true then our perspective makes a number of simple predictions. For example, because backward inference produces higher certainty estimates than forward inference, backward replays should produce burstier spike trains. This should be simple to test using existing data [76]. An important part of our proposal is that the multiple tasks that together comprise spatial cognition can all be implemented using probabilistic inference in a single model. A caveat here is that our approach is restricted to goal-direction navigation. Whilst the forward inference in nonlinear dynamical systems that gives rise to the EKF algorithm, has a long history in estimates of localisation, there have been no proposals, to our knowledge, that also consider planning. However, in the machine learning literature, similar approaches for solving planning or control problems have been developed under the generic term ‘Planning as Inference’. For example, Attias [77] has proposed that planning problems can be solved using Bayesian inference. The central idea is to infer the control signals,, conditioned on known initial state, and desired goal states. Similarly, Toussaint [78] describes the estimation of control signals using a Bayesian message passing algorithm which defaults to the classic Linear Quadratic Regulator (LQR) for linear Gaussian dynamics. Proposals have been made regarding how this Planning as Inference framework maps onto neural architectures in the brain [79], [80]. A key difference to our proposal is that Toussaint solves a closed-loop (feedback) control problem. This finds a mapping from state-space to the optimal action, also known as the ‘policy’. In terms of the underlying generative model in Figure 2, this requires extra links from to. In this paper we solve an open-loop control problem. Our estimated control trajectory is a set of ballistic commands that cannot be updated in light of future information regarding the state of the system. Nevertheless, these commands can be rapidly computed at arbitrary time scales ‘on the fly’, and this type of control strategy may be sufficient for a compliant motor system. In our simulations the agent learnt to predict sensory input using a pre-developed set of place cells with fixed centres and widths. This allowed us to use a simple regression approach for learning the basis function weights, which is similar to the standard two-stage optimisation process in machine learning. In the first stage basis functions are estimated in an initial unsupervised learning phase (eg. based purely on MEC input), and basis function weights are learnt in a second, supervised learning phase [81]. Our simulations also assumed the agent had exact knowledge of its hidden state during learning, whereas more realistic simulations would also require the agent to infer these states. In principle this requires a straightforward implementation of the Expectation-Maximisation (EM) algorithm [38], [82] for learning in dynamical systems. A more powerful alternative which integrates out the dependence on model parameters in the forward and backward passes is Variational Bayes (VB) [83], [84]. Implementation of these VB schemes would mean that the maximum likelihood approach described in this paper would be replaced by a maximum evidence approach. Agents would implement decision making, model selection and motor planning by maximising the model evidence. Given that VB approximates the model evidence using free energy, the resulting scheme would then be broadly consistent with the Free Energy Principle [85]. A further detail here is that in previous applications of VB [83], [84], backward inference was implemented using the beta not the gamma recursions. In this paper we propose that it is the gamma recursions that are implemented in the brain, as they do not require storage of sensory observation sequences. The forward and backward algorithms are general purpose computations which may be implemented in a number of ways and this paper has focussed on an implementation based on local linearisation. The benefit of this is that the state probability distributions are Gaussian and so may be described with a small number of parameters; means and covariances. Additionally, there are analytic formulae for updating the parameters. A drawback of the LL approach is that the true probability distributions may be non-Gaussian. One possibility is that the distribution over the agent' s location may be multimodal. This will be the case when an agent is placed in a familiar environment at an unknown location where there are multiple locations consistent with sensory data. For this scenario inferential methods based on sampling, such as particle filtering, would be more appropriate [37]. A second concern is that a single iteration of forward and backward inference may not be sufficient to find the controls that maximise the planning likelihood. It may be possible to improve the estimated controls by running multiple forward and backward replays such that the linearisation takes place around a different and improved trajectory each time. This iterated local linearisation would be analogous to the iterative Local Quadratic Gaussian (iLQG) approach from control theory [86]. This second concern may also be addressed by treating space as discrete rather than continuous. In this perspective the agent is currently located in one of a finite number of ‘bins’ each of which may correspond to the support of a place cell. The optimal trajectory through these bins can then be computed by solving a discrete Bellman equation. Todorov has shown that this corresponds to backward inference in a hidden Markov model [87]. This computation relies on a recursive high-dimensional update that is perhaps readily suited to the massively recurrent nature of CA3. These computations would be consistent with earlier proposals that the hippocampus itself is suited for solving shortest path problems [88]. In regard to motor planning, this paper has described a forward and backward inference procedure which allows an agent to solve an open-loop control problem. This produces a control trajectory that is a set of ballistic commands that cannot be updated in light of future information regarding the state of the system. It is possible to augment the generative model to include extra links from states to actions, so that the agent instead learns a policy - a mapping from states to actions, as in [78]. This would then provide a solution to the closed-loop (feedback) control problem. However, it may be the case that the mammalian brain solves the closed-loop problem in two stages. First, the computational power of recurrent networks in CA3 could be used to implement forward and backward inference to solve the open-loop problem. Estimated trajectories would then be replayed to ventral striatum during quiet wakefulness or slow wave sleep. This is consistent with an earlier model [89] and the observation of ripple activity propagating to this region [90]. These replays would then be used to train up a habitual dorsal striatal decision making system (see [11] for a review of habitual versus flexible/deliberative systems and their anatomy). This is also consistent with proposals that for known environments, navigational control is gradually transferred from a flexible inferential system to a habitual system based on a hippocampo-striatal mapping [14]. Such a hippocampo-striatal model has previously been proposed by Foster et al. [29]. This paper has described how the various aspects of spatial cognition can be implemented using inference in a statistical model. It has not, however, addressed the broader cognitive control issues such as how internally generated goals are produced or when to switch between localisation versus model selection versus decision making modes. A recent computational framework [22], called Information Foraging (IF), however, does address some of these issues. This approach requires that agents compute the information that will be gained by making spatial decisions, which in turn requires the agent to have a probabilistic model of its environment. Thus, it would be possible for both IF and the Forward-Backward (FB) model to both use the same underlying probabilistic model, with perhaps IF deciding when to run an iteration of FB. This paper has proposed how model-based control may be implemented using spatial models implemented in hippocampal circuits. But it has not addressed how the control of decision making is arbitrated between, for example, model-based and model-free controllers. An influential proposal here [15] is that such arbitration is based on the confidence with which each system can make a decision. Thus, model-based and model-free systems can be combined by weighting each decision with their relative confidence. The ‘Mixed Instrumental Controller’ [19] also makes use of both types of decision making system. The model-based system incurs a fixed computational penalty reflecting the fact that model-based decisions require time to reach. If the estimated benefit of a model-based decision does not exceed this penalty then control is given to the model-free controller. The next and final section of this discussion summarises the specific predictions of the model proposed in this paper. To put these predictions in context we now briefly review two sets of empirical findings. These are, firstly, the observations of ‘theta sequences’ [91] which are sequential patterns of place cell firing occurring whilst rats move about in their environment and theta activity is recorded in hippocampus. The second set of observations are, again, sequential patterns of place cell firing but now occurring during sleep or quiet wakefulness and when Sharp Wave Ripples (SWRs) (henceforth ‘ripples’) [24] are recorded in hippocampus. The phenomenon of phase precession refers to the observation [92], [93] that place cells fire at gradually earlier phases of the hippocampal theta rhythm as rats move through their place fields. This is consistent with the notion of ‘theta sequences’ in which place cells fire in sequence within a theta cycle. Theta sequences have since been measured across cell-populations [91]. Additionally, theta sequences which sweep forward in advance of a rat' s current location have been observed and are especially noteworthy at decision points in maze navigation. For example Johnson and Redish [67] recorded the activity of neural ensembles in the dorsal hippocampal CA3 region of awake behaving rats running in a T-maze. They found that as rats reached a decision point, representations swept predominantly forward from the current location, first down the right path and then the left. This activity did not occur in both forward directions simultaneously: the representation first encoded one arm and then the other. Finally, Gupta et al. [4] have shown that theta sequences represent distances further ahead of a rat during acceleration and further behind during deceleration, and that these sequences represent the environment in ‘chunks’. A key feature of theta sequences is that they are time-compressed, occurring at about 5 to 10 times the speed of actual behaviour [91], [93], [94]. That is, were a rat to run through an environment at a typical speed, it could activate the same sequence of place cells, but would do so 5 to 10 times more slowly. We now turn to the discussion of ripple activity. In humans, episodic memories are thought to be encoded by the Medial Temporal Lobe (MTL) memory system. Information regarding these memories can then be transferred to neocortex [95]–[97] and a proposed mechanism of this transfer is the replay of episodes during later waking or sleep [27] so that neocortical synaptic plasticity can then act to strengthen cortico-cortical connections. This replay activity has been observed primarily in rodents using spatial navigation tasks [98] during ripples in Slow Wave Sleep (SWS) [99] and quiet wakefulness. There is evidence that this pattern replay is related to consolidation and transfer, as disrupting ripples impairs performance in a spatial memory task [100]. Place cell sequences observed during awake ripples have been observed to be played backwards. This is known as reverse replay. Foster and Wilson [76], for example, recorded from cell ensembles in dorsal CA1 hippocampus in awake behaving rats and detected reverse replays after a rat had run the length of a 1D track. Similar reverse replays that start immediately after navigation have been observed on other 1D tracks [101], a linear path through a 2D environment [102], a 2D open-field environment [103], and a two choice T-maze [104]. Place cell sequences observed during awake ripples have also been observed to be played forwards [101]. This is known as forward replay. Replay activity during ripples is also time-compressed, with sequences being replayed within the duration of a single ripple (50–250 ms). This corresponds to a compression factor of about 15 to 20 relative to the original behaviour [102]. The above forward and backward replays are also known as ‘local replays’ or ‘locally initiated replays’ so as to distinguish them from another phenomenon known as ‘remote replay’ or ‘remotely initiated replay’. This occurs when a rat replays an experience of one place whilst being physically located in another. In one experiment [105], rats were exposed to two different environments which had the same physical structure (allocentric layout) but differed in their set of visual cues. Replays of trajectories in one maze were observed whilst the rat was located in the other. Remote replay has also been observed [102], [104] where rats replayed activity corresponding to remote parts of the same environment. As is the case with local replays, remote replays can be forward or backward in time [104]. In general, replay activity during ripples can be forward or backward, whereas theta sequences are always forward. Jadhav et al. [106] have interrupted awake ripples during performance of a navigation task with alternating goals in a W-shaped maze. Ripple disruption was found to affect decision making on the outbound leg of the task, which required linking of past information with current location. However, it did not affect the inbound leg which required no such memory component therefore providing evidence that awake ripples support spatial working memory. Finally, Dragoi and Tonegawa [107] have observed ‘preplay’ activity. Here, the sequence of place-cell firing during a novel spatial experience occurred on a significant number of occasions during the resting or sleeping period prior to that experience. They propose that this activity organises hippocampal assemblies into dynamical structures ready for subsequent associations with sensory episodes. This section summarizes the predictions of our model (the ‘FB model’). We indicate where these predictions are unique to the proposed model and where they are shared by others. We have shown that the various computations underlying spatial cognition can be implemented using statistical inference in a single probabilistic model. Inference is implemented using a common set of ‘lower-level’ computations involving forward and backward inference over time. We have proposed a mapping of the above computational processes onto lateral and medial entorhinal cortex and hippocampal regions CA3-CA1. This proposed mapping is consistent with recent findings in rat electrophysiology, and other proposals that one function of the hippocampus that is preserved across mammalian species, is that it integrates spatial and non-spatial information. We have also proposed that these computations are reflected in recent findings of pattern replay in the mammalian brain. Specifically, that theta sequences reflect decision making, theta flickering reflects model selection, and remote replay reflects route and motor planning. Many of the underlying hypotheses can be tested using existing data.

The ability of mammals to navigate is well studied, both behaviourally and in terms on the underlying neurophysiology. Navigation is a well studied topic in computational fields such as machine learning and signal processing. However, studies in computational neuroscience, which draw together these findings, have mainly focused on specific navigation tasks such as spatial localisation. In this paper, we propose a single probabilistic model which can support multiple tasks, from working out which environment you are in, to computing a sequence of motor commands that will take you to a sensory goal, such as being warm or viewing a particular object. We describe how these tasks can be implemented using a common set of lower level algorithms that implement 'forward and backward inference over time'. We relate these algorithms to recent findings in animal electrophysiology, where sequences of hippocampal cell activations are observed before, during or after a navigation task, and these sequences are played either forwards or backwards. Additionally, one function of the hippocampus that is preserved across mammals is that it integrates spatial and non-spatial information, and we propose how the forward and backward inference algorithms naturally map onto this architecture.

lay_plos

Estimation of division and death rates of lymphocytes in different conditions is vital for quantitative understanding of the immune system. Deuterium, in the form of deuterated glucose or heavy water, can be used to measure rates of proliferation and death of lymphocytes in vivo. Inferring these rates from labeling and delabeling curves has been subject to considerable debate with different groups suggesting different mathematical models for that purpose. We show that the three most common models, which are based on quite different biological assumptions, actually predict mathematically identical labeling curves with one parameter for the exponential up and down slope, and one parameter defining the maximum labeling level. By extending these previous models, we here propose a novel approach for the analysis of data from deuterium labeling experiments. We construct a model of “kinetic heterogeneity” in which the total cell population consists of many sub-populations with different rates of cell turnover. In this model, for a given distribution of the rates of turnover, the predicted fraction of labeled DNA accumulated and lost can be calculated. Our model reproduces several previously made experimental observations, such as a negative correlation between the length of the labeling period and the rate at which labeled DNA is lost after label cessation. We demonstrate the reliability of the new explicit kinetic heterogeneity model by applying it to artificially generated datasets, and illustrate its usefulness by fitting experimental data. In contrast to previous models, the explicit kinetic heterogeneity model 1) provides a novel way of interpreting labeling data; 2) allows for a non-exponential loss of labeled cells during delabeling, and 3) can be used to describe data with variable labeling length. There is little consensus about the expected life spans of lymphocyte populations in health and disease. Labeling the DNA of dividing cells with deuterium has proved to be one of the most reliable and feasible ways to study the population dynamics of lymphocytes in healthy human volunteers and in patients [1], [2], [3]. Deuterium, in the form of deuterated glucose or heavy water, is used to measure the rate at which cells are dividing in vivo, without the need to interfere with these cellular kinetics. Deuterium is incorporated into newly synthesized DNA via the de novo pathway [4], and enrichment of deuterium (over hydrogen) in the DNA of cells is therefore related to cell division. During label administration, the fraction of deuterium-labeled nucleotides increases over time, and after label withdrawal, the fraction generally declines over time [2], [3]. Labeling DNA with deuterium in humans has a number of clear advantages over other labeling techniques such as with BrdU, including the absence of toxicity, the fact that the rate of incorporation of deuterium into the DNA is independent of the amount of nucleotides present, and a simpler mathematical interpretation of the data [5], [6], [4]. Several mathematical models have been proposed for estimation of cellular turnover rates from labeling data [1], [2], [7], [8], [9], [10]. In their study on deuterium labeling, Mohri et al. [2] found that the estimated rate of cell proliferation was typically smaller than the rate of cell death. Because the cell population under investigation was in steady state, the extra death must be compensated by a source of cells, for example from the thymus. This interpretation was challenged by the work of Asquith et al. [9], which pointed out that estimated proliferation and death rates do not have to be equal if the population is kinetically heterogeneous (i. e., different cells in the population divide and die at different rates). Because the labeled population preferentially contains cells that proliferate (and die) relatively rapidly, the estimated rate of cell death is in fact expected to be higher than the average proliferation rate [9]. Here we extend these studies and propose an alternative approach to estimate the rates of lymphocyte proliferation and death from deuterium labeling experiments. First, we show that the three most commonly used mathematical models lead to identical estimates of the average rate of cell turnover and only differ in their biological interpretation of the model parameters. Second, we formulate a novel mathematical model which explicitly takes into account kinetic heterogeneity of lymphocyte populations, and show how lymphocyte turnover rates can be calculated using this model. Several previously made experimental observations arise naturally from the new model. For example, we find that the rate of label loss during delabeling generally exceeds the rate of label accumulation during the labeling phase. Our model also explains the dependence of the rate at which labeled DNA is lost after label withdrawal on the duration of the labeling period [9]. As a proof of principle, we demonstrate that the newly developed model can fit artificially generated data, and correctly returns their underlying kinetic parameters. We also illustrate the usefulness of the new model by fitting it to several experimental datasets. The novel explicit kinetic heterogeneity model may offer alternative interpretations of how infections or treatments affect the turnover of human lymphocytes in vivo. Although different models have been proposed for interpretation of deuterium labeling data [2], [9] and are being debated in the literature, they are in fact mathematically identical, i. e., they predict mathematically identical labeling curves with one parameter for the exponential up and down slope, and one parameter defining the maximum labeling level. Following De Boer et al. [11], consider a cell population consisting of a fraction of cells with average turnover rate (i. e., an expected life span of days), and a fraction of cells that do not turnover at all on the time scale of the experiment. During the labeling phase, consider the fraction of unlabeled DNA in the sub-population with death rate. Because DNA is only lost by cell death, changes according to: During the delabeling phase the fraction of labeled DNA in that same population () is described by: because labeled DNA can only be lost by cell death. Since, the fraction of labeled DNA in the whole population is described by: (1) where is the duration of the labeling period. Given that only a fraction of all cells in the population are turning over (or dying) at rate, the average turnover rate of the whole population is [11]. Importantly, this approach does not require us to describe how new cells are formed, i. e., they could be generated by the thymus and/or by proliferation. As in our previous work [11], this model assumes that the source produces cells with labeled DNA during the labeling phase, and cells with unlabeled DNA during the delabeling phase. This is in contrast with the model by Mohri et al. [2] which allowed the source to produce both cells with labeled and unlabeled DNA during both the labeling and the delabeling period. The reason for this simplification is that the model by Mohri et al. [2] was over-parameterized, i. e., the different source constants cannot be reliably determined from most labeling data (see [2] and results not shown). Moreover, the simpler model with a source of cells with only labeled or unlabeled DNA, typically describes the data with similar quality as the more complicated models (e. g., [11]). Because the fraction of labeled nucleotides cannot exceed one, there is always a trivial asymptote at. The explicit asymptote defined in the above model (and those discussed later) implies that even after infinite labeling, the fraction of labeled nucleotides will be saturated at a level, which could be due to the presence of non-dividing cells. Extending the simple model given in Eqn. (1) by assuming sub-populations with different rates of cell proliferation and death, and possibly generation of new cells from a source (Figure 1), the fraction of labeled nucleotides in the whole population at time is given by: (2) where is the fraction of cells in population with death rate, and is the asymptote that would be approached if label would be administered indefinitely. The only requirement for the model defined by eqn. (2) is that cells within a given sub-population must have identical kinetic properties. For instance, in the absence of an acute infection, we expect that a clone of T cells with the same antigenic specificity may form a sub-population with identical kinetic properties (although there is no experimental evidence for that, see also Discussion section). In our model, new cells are produced by proliferation and from a source (Figure 1). For naive T cells, the source could represent production of cells by the thymus and for memory T cells the source could represent activation of resting cells [12], [11]. Even though the biological interpretation of the source may not always be clear, this forms no problem from a mathematical point of view, because the source term never enters the expression for the fraction of labeled DNA (see Eqn. (2) ). The “source” model that was previously proposed by Mohri et al. [2] considered one homogeneous cell population, but allowed for a source of unlabeled cells during the labeling phase, i. e.,, which also gives rise to an asymptote, defining the fraction of cells that can maximally become labeled (here where is the total number of cells in the population at equilibrium and is the number of cells with unlabeled DNA coming from the source per day during the labeling phase [2]). Mathematically, the source model is therefore identical to eqn. (1). Similarly, in the kinetic heterogeneity model devised by Asquith et al. [9], for the labeling phase and for the delabeling phase. Assuming one again obtains Eqn. (1) with. Therefore, all these models are mathematically identical and only differ in the biological interpretation of their model parameters (see also [13]). We propose to call all these models the “asymptote model”. Importantly, in all models the product can be interpreted as the average rate of cell turnover of the population as a whole [11], and therefore, all three models, when fitted to data, will deliver identical estimates of the average turnover rate, which is the parameter of key interest. There is an important drawback of this approach, however. By interpreting labeling data only in terms of the average turnover rate one may not be able to explain why the average turnover rate is different, for example, between healthy controls and infected patients, and what the consequences of such a difference may be. One would need a particular biological model to explain such a difference. However, our results show that multiple models could be consistent with the labeling data and therefore, model specific predictions arising from labeling data alone may not be robust to changes in the model assumptions. Because of its simplicity, the model given in eqn. (1) has two limitations. First, the asymptote level is a phenomenological parameter that depends on the length of the labeling period [9]. As a consequence, datasets with different labeling periods will likely give rise to different estimated asymptotes and different estimated average rates of cell turnover. Therefore strictly speaking, this model cannot be used to explain multiple datasets coming from the same experimental setup varying only in the length of the labeling period; the differences in the rate at which labeled DNA is lost would force either the asymptote or the estimated average turnover rate to be different for the different labeling periods (Den Braber et al. in preparation). Second, the model assumes that the increase in labeled DNA during the uplabeling phase, and the loss of labeled DNA during the delabeling phase can be described by single exponentials. This may be incorrect if cell populations with different turnover rates are labeled and subsequently lost. Under very general assumptions, we have formulated an alternative model that does not make these a priori assumptions. In our new model, a cell population consists of sub-populations each with different kinetic properties (see Figure 1 and eqn. (2) ). If the number of sub-populations is large (), the sum in Eqn. (2) can be replaced by an integral. The fraction of labeled nucleotides in the population then becomes (see Text S1 for derivation) (3) where is given by Eqn. (1) where is the frequency distribution of turnover rates, and is the probability that a randomly chosen cell in the population belongs to a sub-population with a turnover rate in the range. If the turnover rates in the population,, follow a gamma distribution, the change in the fraction of labeled DNA with time is given by: (4) where is the average rate of cell turnover in the population, is the shape parameter of the gamma distribution, and is the duration of the labeling period. For, the gamma distribution becomes an exponential distribution, and the rate at which the fraction of labeled DNA changes is simply: (5) This is an interesting model in which a single parameter predicts both the rate of uplabeling and downlabeling, and in which there is no asymptote below 100% for the level of labeled DNA, i. e., under continuous label administration all cells in the population will become labeled (Figure 2). Moreover, this model predicts that the initial rate at which labeled DNA is lost after label cessation depends on the duration of the labeling period, (see Text S1 for derivation). According to this model, short labeling experiments (,) will lead to 2-fold faster initial rates of decline in the fraction of labeled nucleotides than longer labeling experiments (,). Solutions (4) and (5) predict that the initial rate of increase in the fraction of labeled DNA is the average rate of cell turnover (see also Text S1). However, the increase in the fraction of labeled DNA does not appear to be exponential, as was implicitly assumed in the asymptote models discussed above. Similarly, during the delabeling period, the model predicts a non-exponential decline in the fraction of labeled DNA (Figures 2 and 3). In general, the initial rate of label loss during delabeling is given by: (6) where is the variance of the distribution of turnover rates in the population. In case when turnover rates follow a gamma distribution, the initial rate of loss of the label after short labeling periods depends on the shape parameter of the distribution,, while it does not after long labeling periods (). The rate of loss of labeled DNA slows down as less DNA remains labeled, which is most clearly seen when proliferation rates are distributed according to a very skewed gamma distribution (, Figure 3). This is a natural property of the explicit kinetic heterogeneity model as loss of labeled DNA is reflecting the distribution of the turnover rates of the different sub-populations, with labeled DNA from the most rapidly turning over sub-populations being lost first (early fast decline) and labeled DNA from the other, more slowly turning over, populations being lost later (late slow decline). To study the effect of the shape of the turnover rate distribution on the predicted labeling curve, we plotted the changes in the fraction of labeled DNA as predicted by the model (Figure 3A&B) with different gamma-distributed turnover rates (Figure 3C). When the gamma distribution is highly skewed (i. e.,), the majority of cell sub-populations have very low rates of cell turnover, and the average rate of cell turnover is dominated by a few sub-populations that turn over unrealistically fast. This is best illustrated by calculating the cumulative contribution of a sub-population with a particular rate of turnover to the average turnover rate of the population (Figure 3D): (7) For large values of the shape parameter (e. g.,), the sub-populations with turnover rates that are close to the average turnover rate, are the main contributors to the average rate of cell turnover (Figure 3D). When the gamma distribution is extremely skewed (), the rate of turnover of the sub-populations that contribute significantly to the average turnover rate is as high as per day, which is biologically unrealistic. Therefore, the gamma distribution should be rejected whenever one estimates a high average turnover rate with a highly skewed gamma distribution (i. e., a low value of the shape parameter). As a rule of thumb, should be larger than 0. 1 (Figure 3D); otherwise a relatively large fraction of sub-populations has unrealistically high turnover rates. It is possible, however, that not all cells in the population are turning over. The models above can easily be extended to incorporate this possibility by allowing for the same asymptote as in eqn. (1). An example would be a labeling experiment in which slowly turning over naive T lymphocytes and more rapidly turning over memory lymphocytes are not separated [2]. If only a fraction of cells have turnover rates that are distributed exponentially, and the other cells undergo negligible turnover on the time scale of the experiment, the change of the fraction of labeled nucleotides with time is given by: (8) where is the average of the exponentially distributed turnover rates, and the average rate of cell turnover in the whole population is. It should be noted that the results of this section are applicable both to proliferating and non-proliferating lymphocytes, given the general structure of the cell population in the model (see Figure 1). As a downside of this, the model does not allow to estimate which fraction of labeling of lymphocytes is due to proliferation of precursors (e. g., thymocytes for naive T cells) or due to peripheral proliferation of the lymphocyte population itself. Additional experiments, such as thymectomy in case of studies of naive T-cell turnover, may allow to estimate the separate contribution of peripheral T-cell proliferation [14, Den Braber et al. submitted]. Having analytical expressions for several kinetic heterogeneity models, we analyzed how well these models can recover the (known) average turnover parameter from simulated (artificial) datasets. Three models were used to generate artificial datasets: 1) the kinetic heterogeneity model with gamma-distributed rates of turnover (eqn. (4) ), referred to as the “Gamma model”, 2) the kinetic heterogeneity model in which a fraction of cells have exponentially-distributed rates of turnover (eqn. (8) ), referred to as the “Exponential model”, and 3) a “Two population model” (Eqn. (2) with, turnover rates and, average turnover rate, and). These datasets were subsequently fitted by the same three models as well as by the conventional Asymptote model (eqn. (1) ). Not surprisingly, the models delivered correct estimates for the average turnover parameter if a dataset was fitted with the model that was used to generate the data (Table S1 and Figures 4 and 5). All models described the data sets generated by the other models reasonably well (Figure 4), although some features in the data could not be reproduced. For example, the Asymptote model failed to describe the decreasing rate at which labeled DNA is lost over time, which is observed in the data generated by the Gamma and the Exponential models (see last data points in Figure 4). Some model fits delivered incorrect estimates for the average turnover rate if the data were generated using another model. For example, the Gamma model overestimated the average turnover rate when the data were generated using the Exponential model (up to 2-fold), and underestimated for data generated using the Two populations model (over 2-fold). This is most likely due to the strong constraint of the model that both uplabeling and delabeling curves have to be described with one mechanism, i. e., gamma-distributed turnover rates. On the other hand, the Asymptote model always underestimated the true average turnover rate (up to 2-fold for data generated by the Two-populations model; Table S1). It did perform somewhat better than the Gamma model as judged by the mean square distances, because the rate of uplabeling and downlabeling are relatively independent in the Asymptote model. Given that natural lymphocyte populations are likely to contain resting sub-populations, some extent of saturation in the fraction of deuterium-labeled nucleotides is expected in almost any experimental dataset. In our artificial data, such an asymptote was imposed when using the Exponential model by letting only 50% of all cells to turn over (Figure 4 and Table S1). It is therefore not surprising that the Gamma model, which does not have an explicit asymptote in the uplabeling phase (see eqn. (4) ), did not correctly estimate the average turnover rate for the data generated by the Exponential model (Figure 5). Extending the Gamma model to allow for an explicit asymptote during the labeling phase () indeed improved the estimate of the average turnover rate (with 95% CIs = which includes the true average), even though the estimated fraction of turning over cells was not significantly different from 1 (i. e., an F-test would not reject a model with; results not shown). This exercise illustrates that when fitting experimental data one should check whether allowing for an explicit asymptote in the uplabeling phase leads to different estimates of the average turnover rate. Interestingly, all models underestimated the average rate of cell turnover when the data were generated using the Two populations model. This is because the models did not reproduce the relatively rapid accumulation of the labeled DNA in the first days (Figure 4). Fitting the Two populations model to these data led to better estimates of the average turnover rate (per day with 95% CIs = for 7 days of labeling, and per day with 95% CIs = for 15 days of labeling, where the constant is contained within both ranges, results not shown). Although stable isotope labeling seems to be the best tool at hand to estimate rates of lymphocyte turnover, a recent review [10] pointed out that estimated lymphocyte turnover rates differ consistently, depending on the labeling method used (heavy water or deuterated glucose), and the length of the labeling period. A priori, according to the Asymptote model that is generally used, the estimated average turnover rate should not depend on the length of the labeling period. Using our explicit kinetic heterogeneity models, we analyzed the influence of the length of the labeling period on the estimated average turnover rate. For all models, we found that the duration of labeling had little influence on the estimated average turnover rate (for the chosen labeling periods of 7 and 15 days, Table S1). This suggests that longer labeling periods will not necessarily result in lower estimates of the average cell turnover rate than shorter labeling periods. An overall conclusion of this analysis is that without a good understanding of the underlying model of cell proliferation (i. e., the distribution of turnover rates in the population), one may obtain incorrect estimates of cellular turnover rates, even if the quality of the fit to the data is acceptably good. Therefore, when analyzing experimental data, one should aim at using several alternative models for fitting, and investigate whether estimates of kinetically important parameters, such as the average rate of cell turnover, are independent of the model used. There are two possible outcomes of such an analysis. First, fitting multiple models to data may yield similar estimates of the average rate of cell turnover. This would imply that the average turnover rate can be robustly estimated from the data, even though the precise model for cell kinetics cannot be determined from such an analysis. Second, the estimate of the average turnover rate may depend on the model that was used to fit the data, while the quality of the fit of various models to the data was similar. In this case, the estimate of the average rate of cell turnover is not robust to changes in the model. Additional data on cell kinetics (e. g., the fraction of cells in division or the fraction of dying cells) would then be required to discriminate between the alternative models for cell kinetics, and to obtain more confident estimates of the average rate of cell turnover. We next sought to determine how well the new kinetic heterogeneity models fit experimental data. Using deuterated glucose, Mohri et al. [2] obtained labeling data of T lymphocytes from uninfected healthy human volunteers and from chronically HIV-infected patients. Previously, these data were fitted using an extended 4-parameter source model, to estimate the rates of cell division and death of T lymphocytes in healthy humans, and to obtain insights into how these rates change upon HIV-infection [2]. Lymphocytes were sorted into CD4+ and CD8+ T cells, without distinguishing between their naive and memory subpopulations. Since naive T cells have a much slower rate of turnover than memory T cells [3], it is natural to assume an asymptote in the fraction of labeled nucleotides of unsorted CD4+ and CD8+ T cells. We have refitted the labeling data from the four healthy human volunteers studied by Mohri et al. [2], again using the three models for cell proliferation: the Asymptote model (eqn. (1) ), the Exponential model (with a fraction of cells with exponentially distributed turnover rates, eqn. (8) ) and the Gamma model (with gamma distributed turnover rates, eqn. (4) ). The data were fitted simultaneously for all four healthy volunteers while searching for the minimal number of parameters that describe the data with reasonable quality (using a partial F-test for nested models [15]). Because cells with deuterium-labeled DNA appear in circulation only a few days after the start of labeling [5], we allowed for a time delay in our model. Overall, the models described the data reasonably well (Figure 6 and Table S2 and S3). For CD4+ T cells, the average turnover rate and the delay at which labeled cells appeared in the blood did not differ significantly between patients (Table S2). For all volunteers, the average rate of turnover was about 0. 46% per day with a corresponding estimated half-life of days. There was an average delay of one day before labeled cells appeared in the blood. The average turnover rate of CD4+ T cells from control c1 was always higher than that of the other individuals, irrespective of the model used (Figure 7A), which may be a sign of an immune response to an infection in c1 (see also below). Both the Asymptote model () and the Exponential model () predicted an asymptote in labeling that is smaller than the fraction of memory phenotype CD T cells in humans of that age [3]. The Gamma model could describe these data even better than the other two models with no need for an asymptote. The observation that the estimate of the asymptote can differ dramatically between different models reconfirms our statement that this parameter is of little use for data interpretation [11]. For CD8+ T cells, the parameters differed significantly between different healthy volunteers, with the exception of the asymptote level in the Exponential model which could be fixed between individuals. The estimates of the average turnover rates of CD8+ T cells in healthy volunteers c2–c4 did not strongly depend on the model that was used to fit the data. However, the estimated turnover rate of CD8+ T cells in individual c1, which was much higher than the estimated turnover rate in the other healthy volunteers, depended strongly on the model used and was estimated to be the highest when using the Gamma model. The latter model fitted the labeling data from all four individuals very well and reproduced the non-exponential change in the fraction of labeled DNA in the downlabeling phase (Figure 6F). In all four healthy individuals, CD8+ T cells turned over at a slower rate than CD4 T cells; the average turnover rate of CD8+ T cells was per day with a corresponding half-life of. The fits of the Asymptote model and the Exponential model predicted an asymptote in labeling of (Table S3). Even though the Gamma model lacks an explicit asymptote lower than 1, it fitted these data with equally good quality as the models with explicit asymptotes. Allowing for an explicit asymptote in the Gamma model did not improve the quality of the fit (CD4+ T cells:, CD8+ T cells:, F-test), and the estimated average lymphocyte turnover rates were not affected by the addition of an explicit asymptote (results not shown). It is important to investigate whether the good description of the data of the model with gamma distributed turnover rates is achieved with biologically reasonable parameter values. In all data we estimated the shape parameter of the gamma distribution to be small, i. e., but was estimated to be larger than 0. 1 in seven of the eight fits. Low values of the shape parameter imply that in the population most cells turn over at very slow rates while a few populations turn over very rapidly. To investigate whether such a distribution is biologically reasonable, we calculated the fraction of cells in the population with a turnover rate higher than per day which is the maximal rate of CD8+ T-cell proliferation in rhesus macaques [16]. This fraction is given by for the estimated parameters of the distribution (see Table S2 and S3). For most fits, the fraction of cells with turnover rates higher than 1 per day is, and given the estimated total number of lymphocytes in humans of [17], that would yield only a few cells with unrealistically high rates of turnover. However, for the CD8 T cells of healthy volunteer c1 we found that cells turn over at rates higher than 1 per day, which is unrealistically high. To investigate this further we reanalyzed the CD8+ T-cell labeling data of individual c1 using several extended models. In the first model, a fraction of cells in the population have gamma-distributed turnover rates while the other fraction () of cells turn over at the highest possible rate. This situation may correspond to a scenario where a small fraction of CD8 T cells is responding to an infection. However, this model failed to describe the data with biologically reasonable parameter values (and). In the second extended model, the gamma distribution of turnover rates was truncated at a maximal value (see Text S1 for analytical results). The fit of this model to the labeling data for individual c1 was of similar quality as the fit in which the gamma distribution was not truncated, and it delivered similar estimates for the average turnover rate and the shape parameter (per day and, results not shown). We estimate that in healthy volunteer c1 about 0. 1% of all CD8+ T cells are turning over rapidly at rates between per day, which is reasonable. For example, in mice responding to lymphocytic choriomeningitis virus (LCMV) infection, at the peak of the immune response more than 50% of all CD8+ T cells in the spleen are specific for the virus [18], [19]. Finally, in the third model, we assumed that the CD8+ T-cell population in volunteer c1 consists of naive, memory and effector T-cell subpopulations with 3 different rates of turnover (see Eqn. (2) with). Assuming that the rate of turnover of naive T cells is 0 and that letting for effector cells per day, we could obtain excellent fits of the labeling data with an estimated average turnover rate per day (per day) which is much higher than estimates obtained by other models (Figure 7). Using model selection methods such as the Akaike Information Criterion, we found equal support for the latter model and the model in which the turnover rates follow a truncated gamma distribution [20, results not shown]. We can conclude, therefore, that the average turnover rate of CD8+ T cells in volunteer c1 is at least 0. 62% per day (Gamma model) and could be as high as 1. 4% per day (Three population model). In summary, it seems that the average turnover rate of both CD4+ and CD8+ T cells was increased in individual c1 as compared to other individuals, and this could be explained by a normal immune response in this otherwise healthy volunteer. Differences in CD8+ T cell kinetics between individuals c1 on the one hand and c2–c4 on the other, were in fact to be expected from visual inspection of the labeling data, because those from individual c1 reached a higher peak and had a faster decline in the fraction of labeled DNA after the peak than those of the other volunteers (see Figure 6F). In this paper we have analyzed the models that are commonly used in the literature to estimate the rates of cell turnover from deuterium labeling data (see Table 1 for a summary of our main results). We have shown that the three most commonly used models are mathematically identical and therefore provide identical fits to the data. These models, however, differ in the biological interpretation of the estimated parameters [13]. The simplest summary of labeling data is provided by a model that has two parameters: as the rate of cell death in the population, and as the fraction of cells that undergo turnover, which determines the asymptote of the uplabeling phase (see eqn. (1) ). In this model, gives the estimated average rate of cell turnover. We have extended this model by allowing for multiple sub-populations of size with different turnover rates (see Eqn. (2) ). This extended model can be used to investigate potential heterogeneity of cell populations, by fitting labeling data with a model that has one, two, or more sub-populations with different turnover rates. Using standard techniques of model selection (e. g., the partial F-test or the Akaike Information Criterion), one can investigate which of those models describes the labeling data best, given the number of model parameters [15], [20], or one can study whether the estimated average turnover rate is converging to an invariant value by increasing the number of compartments (work in progress). For the case where the number of sub-populations is large, we derived a model with continuous kinetic heterogeneity. For several continuous distributions such as the exponential and the gamma distribution, the model predicts that the initial rate of loss of labeled DNA after label withdrawal is determined by the duration of the labeling period as has been observed experimentally [9]. Moreover, in the model the average turnover rate, which determines the initial rate of label accumulation in the population, turned out to be independent of the length of the labeling period. However, it should be noted that the average rate of cell turnover that is estimated from experimental data using, for example, the Asymptote model, may in fact depend on the duration of labeling [10, Den Braber et al. (in prepartion) ]. Potential reasons for this discrepancy will be investigated in more detail elsewhere. Previous models had certain artifacts: the asymptote labeling level was dependent on the length of the labeling period, and the accrual of labeled DNA during the uplabeling phase and the loss of labeled DNA during the downlabeling period were always described by single exponential functions. It is, therefore, unclear whether such limited models provide a good description of truly kinetically heterogeneous populations. We have shown that deuterium labeling data could be fitted and parameters estimated reliably, using a model that assumes a large number of kinetically heterogeneous subpopulations. By fitting artificial labeling data, we have validated these new models: they generally give good fits to the data and converge on average turnover rates that are close to the known average turnover rate. Moreover, the new explicit heterogeneity model outperformed the Asymptote model when it came to fitting experimental data, especially when the rates of label accumulation and loss are not exponential (see Figure 6). Importantly, due to its relatively general structure, all results of the kinetic heterogeneity model are applicable to both non-proliferating and proliferating lymphocytes, all having a distribution of turnover values (results not shown). Moreover, because the model naturally incorporates the dependence of the rate of label loss on the length of the labeling period, this is the first model that can be strictly applied to fit labeling data with different labeling periods. We have focused our analysis on a particular type of kinetic heterogeneity in which kinetic properties of cells of a given subpopulation do not change over time and there is no exchange of cells between different sub-populations. Although we have not specified the nature of sub-populations, one possibility would be that cells within a sub-population share the same antigenic specificity (i. e., they are T-cell clones). In that case, within each functional compartment, and averaged over potential temporal heterogeneity, cells expressing the same antigen receptor would be assumed to have similar kinetic properties. It would be interesting to investigate whether T-cell clones or e. g. polyclonal T -cell populations sharing a particular phenotype (like CD44 or CD62L) are indeed kinetically sufficiently homogeneous to qualify as sub-populations of cells with similar kinetic properties. However, during acute immune responses, the assumption of constant kinetic properties of all cells in a sub-population may be violated. Over the course of an infection, lymphocytes do change their kinetic properties over time (e. g., [21]). Under such circumstances one should take such a type of temporal heterogeneity into account. This requires future work to develop sufficiently simple models from earlier examples [22], [23]. Generally, future studies should aim at testing multiple models in how well they describe the labeling data and whether these models deliver similar estimates of important kinetic parameters such as the average rate of cell turnover. When fitting experimental data, the models were extended to allow for the initial delay in the labeling of cells (see also [2]). For example, including a delay in the Asymptote model (given by eqn. (1) ) takes the form (9) To normalize the residuals of the model fits to experimental data, given that the data are expressed as proportions, the data and the model predictions were transformed as where is the frequency of labeled DNA in the population [24]. The models were fitted according to the least squares method by using the FindMinimum routine in Mathematica. Confidence intervals were calculated by bootstrapping the residuals with 1000 simulations.

Understanding of cellular processes is impossible without quantitative estimates of how quickly cells in an organism divide and die. The most widely used approach to measure rates of cell turnover in humans is by labeling dividing cells with deuterium given in the form of deuterated glucose or heavy water. Surprisingly, quantitative estimates of the rates of cell turnover obtained from accumulation and decay of the labeled nucleotides in the cell population varied between different studies. We demonstrate that these differences were not likely to arise because of different mathematical models used in data fitting, since the previously used models have an identical mathematical structure. We extend these previous models to allow for cell populations with different rates of turnover and show how such a new explicit kinetic heterogeneity model can be applied to simulated and experimental data. The new model opens a new way of interpreting data from deuterium labeling experiments and will likely lead to new insights into how infections and/or treatments affect cell turnover in humans.

lay_plos

We previously showed that broadly neutralizing anti-HIV-1 antibody 2G12 (human IgG1) naturally forms dimers that are more potent than monomeric 2G12 in in vitro neutralization of various strains of HIV-1. In this study, we have investigated the protective effects of monomeric versus dimeric 2G12 against HIV-1 infection in vivo using a humanized mouse model. Our results showed that passively transferred, purified 2G12 dimer is more potent than 2G12 monomer at preventing CD4 T cell loss and suppressing the increase of viral load following HIV-1 infection of humanized mice. Using humanized mice bearing IgG “backpack” tumors that provided 2G12 antibodies continuously, we found that a sustained dimer concentration of 5–25 µg/ml during the course of infection provides effective protection against HIV-1. Importantly, 2G12 dimer at this concentration does not favor mutations of the HIV-1 envelope that would cause the virus to completely escape 2G12 neutralization. We have therefore identified dimeric 2G12 as a potent prophylactic reagent against HIV-1 in vivo, which could be used as part of an antibody cocktail to prevent HIV-1 infection. Human efficacy trials of vaccine candidates designed to elicit antibody-based immunity against HIV-1 have mostly failed [1], [2], raising questions as to whether such an approach to HIV-1 vaccination is at all feasible. A recent human vaccine trial in Thailand [3], however, provided a promising signal of efficacy. While there is no direct evidence of which component of the vaccine was effective, it could be antibody-based immunity. In the trial, 98. 6% of vaccinated individuals produced “binding antibodies” against HIV-1 envelope protein gp120 although no broadly neutralizing antibodies. The possibility that antibody-mediated protection was effective has reenergized the search for effective anti-HIV-1 antibodies. Existing broadly neutralizing anti-HIV-1 antibodies are valuable starting points for generating protection against HIV-1. Several broadly neutralizing antibodies have been proposed as the basis for designing protective mechanisms against HIV-1 in recent years [4], [5]. Among them, 2G12 is unique, because it recognizes a constellation of carbohydrates on gp120 [6], [7], [8], [9] and has an unusual structure that involves a domain swap between the two heavy chains [8]. 2G12 is most effective at neutralizing clade B strains of HIV-1 [10]. A series of studies have described the in vivo protective effects of 2G12 against simian/HIV-1 in macaques [11], [12], [13] and against HIV-1 in humans [14], [15], [16], [17]. Interestingly, in the studies where 2G12 was combined with other broadly neutralizing antibodies such as 4E10 and 2F5 [16], [17], 2G12 provided the dominant protective effect against HIV-1. The relatively long in vivo half-life of 2G12 can partially explain this phenomenon [18]. However, albeit protective, 2G12 also selected HIV-1 escape mutants in vivo [16], [19]; therefore, it is important to identify a new reagent or method to minimize the rate of appearance of such escape mutants. We have previously shown that 2G12 IgG1 can form natural dimers that are 50-80–fold more potent than monomeric 2G12 IgG1 in in vitro neutralization of various strains of HIV-1 [20]. 2G12 monomer, in common with typical IgGs, contains two antigen-binding Fabs and one Fc region, but the heavy chain regions of the Fabs are domain-swapped to create a single (Fab) 2 unit [8]. 2G12 dimer contains four Fabs and two Fcs, which form a structure, presumably through inter-molecular domain swapping, that does not interconvert with 2G12 monomer [20]. The present study was designed to investigate the in vivo potency of dimeric 2G12 in controlling HIV-1 infection in a humanized mouse model. We show that dimeric 2G12 is effective at providing protection against HIV-1 without selecting viral mutants that would completely escape 2G12 neutralization, suggesting that the 2G12 dimer is a suitable prophylactic reagent for use against HIV-1. The dimeric form of the monoclonal antibody 2G12 possesses increased in vitro neutralization potency compared to the monomeric form [20]. It is unknown, however, whether dimeric 2G12 would have a long enough half-life to be more effective than the 2G12 monomer at preventing HIV-1 infection in vivo. To address this question, we prepared separate stocks of purified 2G12 monomer and dimer and passively transferred 0. 5 mg/mouse of 2G12 monomer or dimer into Rag2−/−γc−/− mice reconstituted with human immune cells (Supporting Figure S1A). We then challenged the mice intravenously (i. v.) with the CCR5-tropic strain of HIV-1, JR-CSF, at a dose of 400 ng of p24. Using an ELISA targeting a Myc tag fused to the light chain of the purified antibodies, we found that the concentration of 2G12 monomer declined quickly in the mouse plasma whereas the 2G12 dimer was relatively stable (Figure 1A). The elimination (β phase) half-lives of the purified human IgGs in the humanized mouse plasma were estimated as 3. 5±0. 9 days for the 2G12 dimer and 0. 9±0. 2 days for the 2G12 monomer. The 2G12 dimer prevented CD4 T cell loss in the peripheral blood following HIV-1 infection, whereas the 2G12 monomer did not provide protection (Figure 1B). In addition, the 2G12 dimer moderately suppressed the increase of viral load in the blood (Figure 1C), causing an overall reduction of 97. 5% in viral load compared to the control that lacked antibody (Figure 1D). The 2G12 monomer, on the other hand, did not suppress the increase of viral load following HIV-1 infection (Figure 1C). We also analyzed the percentages of T cells and the numbers of p24+ cells in the spleen, thymus, and mesenteric lymph node. As shown in Figure 1E, we found that without 2G12, HIV-1 almost completely depleted CD4+ cells in the spleen. The percentage of splenic CD8+ cells also decreased, presumably because they rely on CD4+ T helper cells for proliferation and survival [21]. Between the two forms of 2G12, the monomer had a minimal effect at preventing the loss of CD4+ and CD8+ splenocytes following HIV-1 infection, whereas the 2G12 dimer was able to rescue nearly half of the CD4+ cells and most of CD8+ cells in the spleen (Figure 1E). A similar effect was observed in the mesenteric lymph node (Figure S1B) but not in the thymus (Figure S1C), presumably because a CCR5-tropic virus was used and there are few CCR5+ T cells in the thymus [22]. Immunohistochemical analysis using an antibody against HIV-1 p24 confirmed that the 2G12 dimer was effective at limiting HIV-1 infection in both the spleen and the mesenteric lymph node (Figure 1F). HIV-1 p24+ cells were hardly found in the thymus (data not shown). These results demonstrated increased protection against HIV-1 of purified 2G12 dimer compared to 2G12 monomer when the antibodies were administered to humanized mice prior to HIV-1 challenge. To investigate whether the higher potency of 2G12 dimer compared to the monomer resulted only from its longer in vivo half-life, we modified the conventional humanized mice [23], [24] to carry antibody-expressing cells as backpacks [25] that produced antibodies continuously throughout the course of HIV-1 infection (Figure 2). This strategy avoided the dramatic fluctuation of antibody concentrations that usually occur when antibodies were administered through multiple administrations [16], [17]. The antibody-expressing cells were injected subcutaneously (s. c.) and formed localized backpacks whose size could be controlled by the administration of ganciclovir, a prodrug that killed backpacked cells co-expressing herpes simplex virus thymidine kinase (TK) along with the antibody [26]. Because the backpack size positively correlated with the concentration of 2G12 in the blood (Figure S2A), we could control the backpack size to limit the antibody concentration within a reasonably small range. We made mice with backpacks that expressed wild-type 2G12 (named “2G12 BP”) and those with backpacks expressing D2, a mutant of 2G12 that is expressed with an increased dimer/monomer ratio [20] (named “D2 BP”). We previously reported that wild-type 2G12 cells produce 78% monomer and 22% dimer whereas the D2 clone produced 60% monomer and 40% dimer; and that the monomers and dimers produced by wild-type 2G12 or D2 2G12 exhibited no significant differences in biophysical and neutralization characteristics [20]. Since the 2G12 monomer and dimer share the same heavy and light chains, an ELISA would not distinguish between the two forms, making it difficult to directly measure the dimer: monomer ratios in the backpacked mice. Size exclusion chromatography, which could be normally used to determine relative levels of monomer and dimer, would require several milliliters of mouse blood for each sample collection, which was not feasible. Instead, we calculated the monomer: dimer ratios based on the production ratios of monomer versus dimer in the two cell lines (3. 5: 1 for 2G12 BP and 1. 5: 1 for D2 BP) and their individual half-lives in the humanized mice (see Materials and Methods for details). We then used the ratios to estimate the concentrations of 2G12 dimer and 2G12 monomer in the blood samples (Table 1; concentrations of total 2G12 and 2G12 dimer are shown; the concentration of 2G12 monomer can be obtained by subtracting the dimer concentration from the concentration of total 2G12). The D2 BP provided an estimated 3-5-fold more dimer than the 2G12 BP during the first 3 weeks of HIV-1 infection (Table 1; p<0. 02 for weeks 0,1, 2 and 3). The concentrations of 2G12 monomer were not significantly different between the D2 BP and 2G12 BP groups at each time point (p>0. 05) although the combined 2G12 concentrations were higher in the D2 BP group due to significantly greater dimer concentrations. Analysis of the peripheral blood lymphocytes showed that 2G12 BP barely had any protective effect on CD4 T cells compared to the control that lacked antibody (Figure 3A, weeks 1,2 and 4). In contrast, D2 BP effectively protected CD4 T cells from being cleared by HIV-1 after one week of infection (Figure 3A, week 1; p<0. 05). D2 BP also appeared to offer some protection for CD4 T cells 2 and 4 weeks after HIV-1 inoculation although the effect was not statistically significant. Analysis of HIV-1 copy numbers in the mouse plasma showed that D2 BP moderately suppressed the viral load at each time point (Figure 3B) and significantly suppressed the overall viral load (Figure S2B; p<0. 01), suggesting that D2 BP is potent at preventing viral entry and/or eliminating HIV-1 from the circulation. The mice with D2 BP also had significantly lower numbers of p24+ cells in the mesenteric lymph node than mice carrying 2G12 BP (Figure 3C), although neither backpack significantly protected the spleen from HIV-1 infection (Figure 3D for the percentage of CD4 T cells and Figure S2C for the number of p24+ cells). Since D2 BP did not completely prevent HIV-1 infection of humanized mice (i. e., HIV-1 viral load was still detectable in the mouse plasma), we asked if increasing the concentration of 2G12 to over 100 µg/ml [11], [12], [13] would provide better protection against HIV-1. Thus, we included a group of mice (named “BP”) that carried large wild-type 2G12 backpacks as a means to maintain both 2G12 monomer and 2G12 dimer at high concentrations in the peripheral blood (Table 1). Our results showed that the large backpacks prevented HIV-1-induced CD4 T cell loss in the peripheral blood (Figure 3A, weeks 1,2, and 4), suppressed HIV-1 viral load in the mouse plasma (Figure 3B and Figure S2B), decreased the number of p24+ cells in the mesenteric lymph node (Figure 3C), and minimized the decrease of CD4 T cell percentage in the spleen (Figure 3D). However, the virus was still detectable in the periphery (Figure 3B). In fact, the overall viral load in BP mice was similar to that of D2 BP mice (Figure S2B), suggesting that the concentration of 2G12 dimer required to neutralize HIV-1 in vivo might be as low as 5–25 µg/ml (Table 1, dimer concentrations in the D2 BP group from week 0 to week 4), a level that led to over 70% neutralization of the virus (Figure 3B, comparing D2 BP to the control group lacking antibody). Providing 10-fold more of the 2G12 dimer could potentially prevent CD4 T cell loss in the peripheral blood for a longer period of time (Figure 3A), but it would not prevent HIV-1 entry or further decrease HIV-1 viral load in the plasma (Figure 3B and Figure S2B) or mesenteric lymph node (Figure 3C). These results showed that a continuous supply of dimeric 2G12 at 5–25 µg/ml during the course of HIV-1 infection is effective at protecting humanized mice against HIV-1 infection. Since 2G12 is known to induce HIV-1 escape mutants [16], [19], we extracted viral RNA from the week-4 plasma of 3 or 4 representative mice per experimental group, cloned the JR-CSF envelope gene from viral cDNA, and sequenced at least 10 clones per mouse sample. Some viral clones had spontaneous mutations at residue N339 regardless of the presence of 2G12 and might represent a background in the inoculum (Table 2 and Figure 4A). In addition, both 2G12 BP and D2 BP selected mutations at residue N386. Surprisingly, we observed an unusually high percentage of mutations at residue N295 when the 2G12 concentration was kept at 100 µg/ml or higher (Table 2 and Figure 4A; BP). This residue, along with N332 that was not significantly mutated in this study, have been suggested as the key anchors of glycans that form the 2G12 epitope [7]. To assess the sensitivity of mouse-derived viruses to 2G12 neutralization, we performed in vitro neutralization assays using pseudoviruses made with JR-CSF envelope genes that we obtained from mouse plasma samples. Both the input virus (the pseudovirus that shared the same JR-CSF envelope as the inoculum) and the virus with mouse-derived envelope that did not encounter any neutralizing antibody in vivo (HIV-1 only; No mutation) were effectively neutralized by 2G12 monomer and 2G12 dimer in vitro (Figure 4B and 4C); but the half maximal inhibitory concentration (IC50) of 2G12 dimer was 33-fold less than the IC50 of the monomer, suggesting that the 2G12 dimer was more potent at neutralizing the JR-CSF strain of HIV-1 than the 2G12 monomer. More importantly, we found that the viral envelope from a BP mouse with the mutation N295S caused the pseudovirus to completely escape the neutralization effect of both the 2G12 monomer (Figure 4B) and the 2G12 dimer (Figure 4C). In contrast, a virus variant with a mutation at residue 386 was partially neutralized by the 2G12 monomer and 2G12 dimer. This suggests that, unlike the >100 µg/ml condition (provided by BP), the presence of 2G12 dimer at 5–25 µg/ml (provided by D2 BP) did not select for complete HIV-1 escape mutants. Therefore, our results showed that although constant administration of 2G12 at high concentrations was potent at protecting humanized mice from HIV-1 infection in vivo, it resulted in HIV-1 envelope mutations that could completely escape 2G12 neutralization. However, at least over the time-course of our experiments, a low level of 2G12 dimer did not specifically select the same mutations, providing an additional benefit to its high potency. In this study, we used a humanized mouse model to investigate the in vivo potency of dimeric 2G12 in controlling HIV-1 infection. This mouse model supports human hematopoietic development, provides human CD4 T cells as natural targets of HIV-1 infection, and allows for possible selection of viral resistance [27]. Using these mice, we first examined the stability and protective effects of monomeric and dimeric forms of 2G12 in HIV-1-challenged humanized mice by passively transferring purified antibodies. We found that the 2G12 dimer had a longer in vivo half-life and was more potent than the 2G12 monomer at controlling HIV-1 infection in vivo. The elimination half-life of the 2G12 dimer was 3. 5 days in humanized mice and comparable to the reported elimination half-life (3. 2 days) of human IgG1 in mice [28]. This is shorter than the half-life of human IgG1 in humans [18] but correlates with the difference in body weight between mice and humans [29]. To investigate whether a continuous supply of the 2G12 monomer would overcome its poor in vivo efficacy, we next used a backpacking approach to provide the antibody continuously. Using wild-type 2G12 as the backpacked gene, we achieved a sustained level of 2G12 monomer and dimer in the mouse plasma. However, constant delivery of 2G12 monomer plus a small amount of 2G12 dimer at a low level (1–4 µg/ml dimer for the first 3 weeks and 16. 6 µg/ml dimer after 4 weeks) did not protect the mice from HIV-1 infection. In contrast, backpacks containing the D2 mutant, which produced increased levels of 2G12 dimer (60% monomer, 40% dimer) provided effective protection against HIV-1 by maintaining a 2G12 dimer concentration of 5–25 µg/ml in the mouse plasma. Thus, our results suggest that, administered either through a single injection or continuously, dimeric 2G12 is a more potent prophylactic anti-HIV-1 antibody than 2G12 monomer. Several in vivo studies have estimated that concentrations of 2G12 of 100 µg/ml or higher exert a protective effect against HIV-1 when the virus is given at a 50% tissue culture infective dose (TCID50) of 500—5,000 [11], [12], [13]. In order to establish a robust and consistent infection in humanized mice, we administered HIV-1 intravenously at a dosage of 400 ng p24, or a TCID50 of 400,000. Although sterilizing immunity was not achieved in this study, we found that, even with high-dose HIV-1 challenge, 2G12 monomer and dimer at combined concentrations of 100 µg/ml or higher could significantly reduce the severity of HIV-1 infection in the humanized mice (Figure 3). More importantly, the D2 BP that delivered 2G12 at a much lower concentration exerted a similar protective effect against HIV-1. In particular, D2 BP provided the 2G12 dimer at 5–25 µg/ml, which was sufficient to prevent peripheral blood CD4 T cell loss (Figure 3A) and suppress the increase of the viral load following HIV-1 infection (Figure 3B and Figure S2B). Therefore, 2G12 dimer represents a promising prophylactic reagent against HIV-1 in vivo because it neutralizes HIV-1 at a relatively low concentration. Having a low effective concentration is not the only advantage of the 2G12 dimer as a protective reagent against HIV-1. 2G12 is known to select HIV-1 escape mutants both in vitro [30], [31] and in vivo [16], [30], [31], with in vivo escape mutants detectable as early as 4 weeks after HIV-1 inoculation [16], [30], [31]. Here we analyzed the diversity of HIV-1 viral RNA isolated from the mouse plasma, focusing on regions of the JR-CSF envelope gene where 2G12 epitope-containing carbohydrates would attach [7], [32]. We found that while low levels of 2G12 dimer induced mutations at residue N386,2G12 at monomer plus dimer concentrations of >100 µg/ml specifically selected mutations at another residue (Table 2). This residue, N295, has been suggested to be one of the two central players in the interaction between 2G12 and its carbohydrate epitope [7]. A mutation at N295 would be more likely to allow HIV-1 to escape 2G12 neutralization than mutations at other sites such as N386 (Figure 4B and Figure 4C). Thus, at least over the time-course of our experiments, dimeric 2G12 provided protection against HIV-1 without selecting for complete HIV-1 escape mutants. In summary, we found in the present study that dimeric 2G12, or the D2 mutant that increases the production of dimeric 2G12, might be potential prophylactic reagents against HIV-1. However, more research is necessary to characterize the tissue distribution of dimeric 2G12 and its in vivo antibody-dependent cellular cytotoxicity activity. It is also important to assess the immunogenicity of 2G12 in its dimeric form since it is twice the size of a typical IgG. In addition, the pharmacokinetics of dimeric 2G12 should be carefully established in human studies, as the half-life of the antibody in humans is likely to be different from that in humanized mice. Furthermore, because the neutralization spectrum of 2G12 is not particular good when tested against a large panel of HIV-1 isolates [10] and neutralizing antibodies have demonstrated synergy when combined together [33], the 2G12 dimer may be more beneficial when used as part of an antibody cocktail to protect people from HIV-1 infection. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of California Institute of Technology (Animal Assurance Number: A3426-01). All animal experiments were conducted under IACUC protocols 1536-09G and 1547-08G. The wild-type 2G12 heavy chain gene (IgG1) and a Myc-tagged 2G12 light chain gene were linked by an F2A sequence and subcloned into a lentivector. The vector is a third-generation, self-inactivating lentiviral vector backbone based on pHRST [34], [35]. Briefly, the StuI fragment of pHRST containing a complete viral genome was ligated into the pUC19 backbone to remove exogenous flanking genomic sequences. PCR-cloning was employed to introduce restriction sites flanking the promoter and transgene to facilitate subsequent cloning. Further modifications were made to pHAGE6 to remove extraneous viral sequences with no effect on virus function (A. B., to be published elsewhere). Lentiviruses were then generated by transient transfection of HEK-293T cells using the Trans-IT reagent (Mirus Bio; Madison, WI) and used to create a 293T stable cell line that produced 2G12. The 2G12-expressing, adherent stable cell line was adapted for growth in suspension for large-scale production of 2G12 at the Caltech Protein Expression Center. Cell culture supernatants were collected and passed over protein A resin (Pierce Biotechnology; Rockford, IL), and eluted using using pH 3. 0 citrate buffer. Protein A eluates were immediately neutralized and then subjected to size exclusion chromatography in 20 mM Tris pH 8. 0,150 mM NaCl using a Superdex 200 16/60 (GE Healthcare). Fractions corresponding to monomer and dimer were collected and then separately passaged over a Superdex 200 10/30 column (GE Healthcare) to remove contaminating amounts of monomer or dimer from the separated purified species. Frozen human cord blood CD34+ cells from single donors were purchased from AllCells (Emeryville, CA) or Lonza (Basel, Switzerland). One-day-old Rag2−/−γc−/− pups were irradiated and intrahepatically (i. h.) injected with 0. 1-0. 2×106 human cord blood CD34+ cells per pup. Mice were then screened for human CD45+ cells at 6 weeks of age and those with good reconstitution were chosen for the study (Figure S1A). For passive transfer experiments, one single dose of 0. 5 mg/mouse of purified 2G12 dimer or 2G12 monomer was injected retro-orbitally (i. v.) into 4-month-old humanized mice 1 day before HIV-1 challenge. The HIV-1 JR-CSF plasmid was obtained from NIH AIDS Research and Reference Reagent Program and transiently transfected into 293T cells to produce infectious HIV-1 particles. The culture medium containing HIV-1 was then harvested and titered using the p24 ELISA kit from PerkinElmer (Waltham, MA). The virus was injected (i. v.) at 400 ng p24/mouse. For non-HIV-1 mice, conditioned medium was injected as the control. All mice involved in this study were age-matched since the CD4: CD8 ratio naturally increased with the age of these mice. Wild-type 2G12 and D2 mutant genes were cloned into lentiviral vectors. Lentiviruses were then generated and used to create stable cell lines that produced wild-type 2G12 and D2, respectively. The parent cell line was a stable 293T cell line that expressed herpes simplex virus thymidine kinase (TK), so the progeny lines were named 293T/TK/2G12 and 293T/TK/D2 cell lines. When well-reconstituted humanized mice were 3-month-old, 1×106 of backpacked cells were injected (s. c.) on the back of the mice at the lower right side. Backpack size (length × width) was measured weekly and controlled by injection (i. p.) of 62. 5 µg or 125 µg (depending on the backpack size) of ganciclovir (Sigma; St. Louis, MO) per mouse after HIV-1 challenge and when the backpack size reached 1. 5 cm2. Weekly blood samples were obtained retro-orbitally and the plasma was immediately separated from blood cells and stored for viral RNA extraction and Myc-specific ELISA (see below for details). The peripheral blood mononuclear cells after antibody staining were analyzed by the FACSCalibur (BD Biosciences; San Jose, CA). Mice were sacrificed 4 weeks after HIV-1 challenge. Blood, spleen, thymus, and mesenteric lymph node were collected for flow cytometry analysis or fixation in formalin. The fixed tissues were then send to University of California, Los Angeles for immunohistochemical analysis. Mouse plasma was diluted 1∶10,1∶100, and 1∶1000 in sample diluent and heat-inactivated at 55°C for 1 h. Myc-tagged 2G12 was captured by anti-human IgG-Fc (Bethyl Laboratories; Montgomery, TX) and detected by anti-Myc conjugated with horseradish peroxidase (Bethyl Laboratories; Montgomery, TX). The plates were read at 450 nm on a SpectroMax Reader (Molecular Devices, Sunnyvale, CA) after the addition of the TMB substrate and the stop solution. In passive transfer experiments, the half-life of the elimination phase (β phase), which took place after the redistribution phase, was determined using a one-phase exponential decay model using data points from week 0 (24 h after the injection of 2G12 monomer or dimer) to week 4. The half-lives were estimated as 3. 5±0. 9 days for the 2G12 dimer and 0. 9±0. 2 days for the 2G12 monomer. In backpacking experiments where both 2G12 monomer and dimer were present in the plasma, we determined their individual concentrations by calculating the monomer: dimer ratios as following: where P = protein (monomer or dimer), β = production rate; α = degradation rate. Assuming that at the time of HIV-1 challenge (4 weeks after backpack injection), the monomer and dimer had reached their individual steady state (i. e.), If the dimer had a production rate of β and a degradation rate of α, then the monomer should have a production rate of 3. 5β (78% monomer versus 22% dimer produced from 2G12 backpacks) and a degradation rate of 3. 9α (dimer: monomer ratio of half-lives 3. 5/0. 9 = 3. 9) for 2G12 backpacks. Thus, Therefore, the 2G12 monomer and dimer concentrations were calculated as: where Ptotal = total 2G12 concentration as measured by Myc-specific ELISA. For D2 backpacks, since the dimer' s production rate was 1. 5β (60% monomer versus 40% dimer produced from D2 backpacks) and the degradation rate stayed the same, Viral RNA was extracted from mouse plasma using QIAamp Viral RNA Mini Kit from Qiagen (Valencia, CA). The RNA (200 ng) was reverse transcribed and quantified using the Taqman RNA-to-CT One-Step Kit (Applied Biosystems; Foster City, CA) and the Eppendorf Realplex real-time PCR system (Hauppauge, NY). The primers were designed to anneal to the pol region of the HIV-1 genome within the first intron, so that only unspliced viral RNA could be detected. The primer sequences were: forward primer, 5′-CAA TGG CAG CAA TTT CAC CA-3′; reversed primer, 5′-GAA TGC CAA ATT CCT GCT TGA-3′. The probe sequence was: 5′-/56-FAM/CCC ACC AAC AGG CGG CCT TAA CTG/36-TAMSp/-3′. HIV-1 RNA standard was generated using the Riboprobe T7/SP6 kit from Promega (Madison, WI) and the pGEM FL2 plasmid was provided by Dr. Dong Sung An at University of California, Los Angeles. The detection limit of the assay was 20,000 HIV-1 copies/ml mouse plasma. Viral RNA was extracted from mouse plasma using QIAamp Viral RNA Mini Kit from Qiagen (Valencia, CA). The RNA (500 ng) was reverse transcribed and amplified using the SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity from Invitrogen (Carlsbad, CA). The primer sequences were: JR-CSF env forward primer, 5′-GGC AAT GAG AGT GAA GGG GAT CAG-3′; JR-CSF env reversed primer, 5′-CAT CTT ATA GCA AAG CCC TTT CCA AGC C-3′. The primers flanked the whole 2. 5-kb envelope gene. The PCR product was then gel-purified and cloned into the TOPO vector using the TOPO XL PCR Cloning Kit from Invitrogen (Carlsbad, CA). More than 10 clones were picked for each RNA sample. The plasmids were then extracted and sent to sequencing at Laragen (Los Angeles, CA) or Sequetech (Mountain View, CA). The sequencing primer was 5′-GTC AGC ACA GTA CAA TGT ACA CAT GGA ATT AG -3′ and annealed upstream of the Asn residues that linked 2G12 epitope-containing carbohydrate chains [7]. Mutations at N295, N332, N339, N386, N392, N448 and adjacent Ser/Thr residues were then analyzed. We used a previously described pseudovirus neutralization assay, which measures the reduction in luciferase reporter gene expression in the presence of 2G12 monomer or dimer following a single round of pseudovirus infection in TZM-bl cells [20]. Pseudoviruses were generated by cotransfection of 293T cells with an envelope expression plasmid and a replication-defective backbone plasmid. (For envelope expression, viral RNA was extracted from mouse plasma 4 weeks after HIV-1 challenge and reverse transcribed. The complete envelope gene was amplified from viral cDNA and the PCR product was then gel-purified and cloned into the pcDNA3 vector.) Each 2G12 protein was tested in triplicate with a 3-fold dilution series, and incubated with the pseudoviruses (250 infectious viral units per well) for 1 h at 37°C. After the incubation, 10,000 TZM-bl cells were added to each well, followed by incubation for 2 days. Cells were then lysed and assayed for luciferase expression by using Bright-Glo (Promega; Madison, WI) and a Victor3 luminometer (Perkin-Elmer; Waltham, MA).

Most successful vaccines function by eliciting antibodies that bind to the surface of pathogens of interest from the host immunologic repertoire. This should also be the case for an HIV-1 vaccine, but broadly neutralizing anti-HIV-1 antibodies have proven hard to elicit with any reagent. Thus, methods to directly administer broadly neutralizing anti-HIV-1 antibodies, such as passive transfusion, become appealing. It is therefore important to find out which antibodies, or antibody cocktails, would provide effective protection against HIV-1 before administering them. Here, we show that the dimeric fraction of 2G12, a unique monoclonal anti-HIV-1 antibody that dimerizes naturally, provides better protection against HIV-1 than its monomeric fraction. As an added bonus, although HIV-1 can evolve to completely escape antibody control, the 2G12 dimer does not favor such evolution. Our study suggests that the 2G12 dimer may be a suitable reagent for direct administration to protect people from HIV-1 infection.

lay_plos

WARRIORS OF THE DEEP BY: JOHNNY BYRNE Part Three First Air Date: 12 January 1984 Running time: 24:02 [SCENE_BREAK] TURLOUGH: Open it! BULIC: The controls are locked. It can now only be opened by the bridge. BULIC: Let him go. BULIC: Stay on guard, Sergeant. GUARD: Yes, sir. BULIC: I must see the Commander. The rest of you follow me. [SCENE_BREAK] TEGAN: That won't do much good. DOCTOR: Get ready. Mind your eyes, Tegan. TEGAN: What did you do to it? DOCTOR: Simple. The charge from the Myrka's body blew up the magazine. TEGAN: It's blinded. DOCTOR: Yes, temporarily. [SCENE_BREAK] TURLOUGH: The master control to bulkhead one, where is it? NILSON: The Commander's orders were to keep that bulkhead closed. TURLOUGH: I know what the Commander's orders were, but now I'm giving you mine. Open that bulkhead. NILSON: The hydraulic valve has blown. TURLOUGH: The bulkhead opened? NILSON: Yes. But I'm not so sure it closed. [SCENE_BREAK] TEGAN: What do we do if the Myrka breaks in, Doctor? DOCTOR: Well, hopefully we can stop it before it does too much damage. TEGAN: You said yourself it's practically indestructible. DOCTOR: Yes, we all have an Achilles' heel, Tegan, including the Myrka. Now we must hurry. I must speak to Vorshak. [SCENE_BREAK] SOLOW: Nilson, I have hidden the woman's body. The escape pod is ready for us. We can leave as soon as Maddox has completed his work. NILSON: Good. [SCENE_BREAK] TARPOK: The Myrka has broken through the bulkhead door. ICHTAR: Command it to make for its objective. [SCENE_BREAK] VORSHAK: Vorshak. NILSON (OOV.): The creature's broken into the Base. Turlough forced us to open the bulkhead door. VORSHAK: Sound full alert, Nilson. Get your best people to Airlock One immediately. They must stop that creature. BULIC: Our weapons have no effect on it. VORSHAK: Have them try! DOCTOR: He's right, Commander. Your weapons are useless against it. VORSHAK: But not against you. You know what Turlough has done? TEGAN: Yes, he saved our lives. VORSHAK: I ordered that bulkhead to be kept closed. DOCTOR: The bulkhead wouldn't have kept the Myrka out for long. PRESTON: Commander, the seals are about to blow. DOCTOR: If it's any consolation, I may just know how to stop it. VORSHAK: When I want your help, I'll ask for it. DOCTOR: You've got nothing to lose. VORSHAK: I should have you shot. DOCTOR: After I've dealt with the Myrka. VORSHAK: All right, but I can't spare you any help. DOCTOR: I need just one person. Someone in authority who can get things done. VORSHAK: Very well. Preston, go with him. PRESTON: Sir. VORSHAK: Kill him if he gives you any trouble. That's an order. The rest of you, get ready. VORSHAK: Get back! Back. [SCENE_BREAK] BULIC: They've got us outgunned, Commander. VORSHAK: I know. It all depends now on whether this bulkhead can hold them. [SCENE_BREAK] SAUVIX: Bring forth the cutting device. [SCENE_BREAK] TURLOUGH: The Doctor and Tegan, where are they? VORSHAK: They're safe, and contributing to the defence of this Base, as you are. TURLOUGH: Sorry? VORSHAK: You're volunteering your services to defend this bulkhead. [SCENE_BREAK] PRESTON: Preston. GUARD (OOV.): The Myrka's in corridor seven. PRESTON: Delay it's progress as much as possible. GUARD (OOV.): We'll do our best. DOCTOR: It can only be heading for one place. PRESTON: The bridge? DOCTOR: Yes, the bridge. PRESTON: I'll inform the Commander. DOCTOR: Do you have ultraviolet converters on the Base? PRESTON: Yes. DOCTOR: Good. Now, will the Myrka have to pass this way to get to the bridge? PRESTON: If it maintains its present course. DOCTOR: Perfect. I need a converter brought here. PRESTON: Tech unit. [SCENE_BREAK] BULIC: It's not going to hold. VORSHAK: We must break radio silence. BULIC: Have every enemy listening post pinpoint our position? VORSHAK: Sea Base Command has to know about these creatures. We have no choice. [SCENE_BREAK] DOCTOR: Ah. Set it up here. PRESTON: That'll be all. Carry on. DOCTOR: Now, what do you think. Will the lighting circuit bear maximum converter load? PRESTON: Just about. TEGAN: What have you got in mind? DOCTOR: I'm planning to bring a little sunshine into the Myrka's life. [SCENE_BREAK] BULIC (OOV.): The Commander is on his way up to signal Sea Base Command. Have Maddox stand by for sync up. NILSON: Understood. SOLOW: What are you going to do? NILSON: Maddox must finish what he has started. [SCENE_BREAK] PRESTON: I suppose you know what you're doing. DOCTOR: The Myrka is a creature of the inkiest depths, or it was until the Silurians tinkered with its biology. Anyway, it has little tolerance to light and hopefully none at all to ultraviolet rays. TEGAN: Can you be sure? DOCTOR: No, Tegan. Perhaps you should ask it nicely to go away. [SCENE_BREAK] NILSON: The work goes well. Wait in the escape pod. I'll join you when I can. SOLOW: What about Maddox's conditioning disc? NILSON: Take it with you. It'll provide essential knowledge. SOLOW: Good luck. [SCENE_BREAK] PRESTON: Ready? TEGAN: Ready now? DOCTOR: Yes, almost. PRESTON: Doctor Solow. DOCTOR: Look! DOCTOR: Perfect. Almost. GUARD: 43Y. [SCENE_BREAK] ICHTAR: How fares the course of battle? SAUVIX: The outcome is certain. The ape primitives are no match for my warriors. They will be crushed. [SCENE_BREAK] VORSHAK: You have everything you need? DOCTOR: Yes, I think so. TEGAN: What happened to Turlough? VORSHAK: He's defending Airlock Five with Bulic. Doctor, I've decided to take your advice. I'm contacting Sea Base Command. DOCTOR: Very wise. GUARD: The Myrka's coming this way, sir. GUARD: It killed Doctor Solow. We found this by the body. DOCTOR: Something wrong? VORSHAK: I pray not, Doctor. Get down to Airlock Five. Preston, come with me. VORSHAK: Vorshak. BULIC (OOV.): They're in, sir. The Sea Devils are everywhere. VORSHAK: You must hold them. [SCENE_BREAK] BULIC: We'll try, Commander. VORSHAK (OOV.): Do what you can. BULIC: Back! Get back! [SCENE_BREAK] NILSON: Don't die on me, Maddox. Not yet. Not till you've served your purpose. NILSON: Now, hurry. [SCENE_BREAK] ICHTAR: Are the ape primitives in full retreat? Do you control all strategic areas? SAUVIX: Yes, Ichtar. ICHTAR: Excellent. We expect the Myrka to take the bridge very soon. SAUVIX: Then the outcome is doubly certain. [SCENE_BREAK] NILSON: Yes? VORSHAK: Maddox's conditioning disc. You were ordered to return it to me. NILSON: Solow was in charge of that disc. I assumed she had. VORSHAK: Where is Maddox? NILSON: Inside, checking the sync circuit. VORSHAK: Get him in here. PRESTON: Commander. [SCENE_BREAK] VORSHAK: Maddox! What are you doing? PRESTON: He's still alive. VORSHAK: Check the damage to the other computers. [SCENE_BREAK] DOCTOR: Here it comes. Now we'll know if my theory was right. TEGAN: Switch it on. DOCTOR: Just a few more feet. TEGAN: Switch it on! DOCTOR: All right, Tegan, close your eyes. Make a wish! TEGAN: Is it dead? DOCTOR: Very. Let's get back to the bridge. TARPOK: The Myrka has been destroyed. ICHTAR: The ape primitives have more cunning than we thought. Sauvix, divert your warriors. We must capture the bridge without delay. SAUVIX: At once, Ichtar. [SCENE_BREAK] VORSHAK: You were in charge of the bridge. How could you not know what was going on in here? PRESTON: Commander! Karina's dead. She's been murdered. VORSHAK: You'll answer to a court martial for this, I promise, but first, you'll revive Maddox and decondition him. I want him ready for sync up. We must signal Sea Base Command. NILSON: I'm afraid we can't do that, Commander. VORSHAK: You'll do it! NILSON: Your weapons, please. [SCENE_BREAK] TEGAN: What do the Silurians want, Doctor? DOCTOR: Obvious. Control the Base and the proton missiles TEGAN: And you control Earth? DOCTOR: Or destroy it. [SCENE_BREAK] VORSHAK: I trusted you, Nilson. NILSON: Don't take it so hard, Commander. Like you, we were only doing our duty. DOCTOR: The Myrka is dead, Commander. NILSON: That's far enough, Doctor. VORSHAK: It seems Nilson is an enemy agent. TEGAN: For the Silurians? VORSHAK: No, our human enemies. NILSON: The power bloc opposed to this Sea Base. DOCTOR: There isn't time for your petty feuds. NILSON: I know what I'm doing, Doctor. DOCTOR: Do you? Before long, the Silurians and the Sea Devils will have control of this Base. VORSHAK: And control of the proton missiles. NILSON: Missiles they can't fire. Maddox has irreversibly rigged the computers to that effect. DOCTOR: The technology of these creatures predates yours by millions of years. If they intend to fire those missiles, they have the means, I assure you. NILSON: They won't have time. As soon as I've left in the escape pod, this Base will be attacked and everything on it, including the creatures and all of you, will perish. MADDOX: Nilson, you made me kill Karina. Now you're going to NILSON: I'm sorry, Maddox, but your usefulness is at an end. NILSON: Leave him! He's dead. NILSON: If any of you try to follow me, she dies. [SCENE_BREAK] SAUVIX: Isolate them. [SCENE_BREAK] PRESTON: The computers have been badly damaged. VORSHAK: Vorshak. GUARD (OOV.): There's not much more we can do, sir. The Sea Devils have just breached the bridge perimeter. VORSHAK: Where are Bulic and Turlough? GUARD (OOV.): Dead, or taken, Commander. VORSHAK: I'm sorry. DOCTOR: I'm going after Nilson. [SCENE_BREAK] SAUVIX: The way to the bridge is cleared. [SCENE_BREAK] TURLOUGH: We've got to get out of here. BULIC: To go where? The Sea Devils are all over the place. TURLOUGH: The TARDIS? At least we'd be safe there. [SCENE_BREAK] NILSON: Be still, woman. Pity all the Doctor's efforts were to no avail. TEGAN: You haven't got away yet. NILSON: Stay where you are. You were very foolish to follow me, Doctor. DOCTOR: Let Tegan go. She's of no use to you now. Killing us won't make your escape any easier. You fire, and every Sea Devil in the area will come running. NILSON: I'll take that risk. DOCTOR: Tegan, make a wish! NILSON: Goodbye, Doctor. DOCTOR: Goodbye. SAUVIX: Your turn.

While the Silurians and Sea Devils attack the bases air locks, the saboteurs take advantage of the distraction to go ahead with their own plans.

summ_screen_fd

Under conditions of stress, such as limited growth factor signaling, translation is inhibited by the action of 4E-BP and PDCD4. These proteins, through inhibition of eIF4E and eIF4A, respectively, impair cap-dependent translation. Under stress conditions FOXO transcription factors activate 4E-BP expression amplifying the repression. Here we show that Drosophila FOXO binds the PDCD4 promoter and stimulates the transcription of PDCD4 in response to stress. We have shown previously that the 5′ UTR of the Drosophila insulin-like receptor (dINR) supports cap-independent translation that is resistant to 4E-BP. Using hippuristanol, an eIF4A inhibitor, we find that translation of dINR UTR containing transcripts are also resistant to eIF4A inhibition. In addition, the murine insulin receptor and insulin-like growth factor receptor 5′ UTRs support cap-independent translation and have a similar resistance to hippuristanol. This resistance to inhibition of eIF4E and eIF4A indicates a conserved strategy to allow translation of growth factor receptors under stress conditions. During times of stress the cell changes its gene expression profile to better manage the cause of the stress. Coordinate changes in both transcription and translation occur (Sengupta et al., 2010; Spriggs et al., 2010). A central pathway that responds to stress stimuli by controlling both protein and RNA synthesis is the insulin and insulin-like receptor-signaling (IIS) pathway. The fundamental molecular architecture of the IIS pathway is conserved from flies to man (Figure 1) (Oldham, 2011). When IIS signaling is high, the protein kinase AKT is activated (Ruggero and Sonenberg, 2005). AKT directly phosphorylates the Foxo family of transcription factors and consequently prevents activated transcription of Foxo target genes (Brunet et al., 1999). AKT also stimulates the activation of the mechanistic target of rapamycin (mTOR) protein (Zoncu et al., 2011). 10. 7554/eLife. 00542. 003Figure 1. Simplified insulin/insulin-like growth factor signaling diagram. (A) When Insulin receptor or Insulin-like growth factor receptor is active signaling through AKT inhibits Foxo transcription factors and activates mTOR. mTOR in turn inhibits 4E-BP and activates S6K. S6K in turn inhibits Pdcd4 and activates eIF4B. When insulin signaling is low inhibition of Foxo is relieved and Foxo activates the transcription of Insulin receptor and 4E-BP. The broken line indicates the proposed activation of Pdcd4 by Foxo. (B) Alignment of human (Hs top) and Drosophila (Dm bottom) Pdcd4 proteins. Conserved Akt and S6K phosphorylation sites are indicated by asterisk. Conserved MA3 domains are indicated by shaded boxes. Arrowheads indicate conserved acidic residues important for eIF4A binding in humans. (C) eIF4A interacts with Pdcd4 in Drosophila cells. Cytoplasmic extracts from a saturated culture of S2 cells were subjected to immunoprecipitation with antisera directed against eIF4A or preimmune serum. Pdcd4 was detected with antisera against Pdcd4. (D) Mutant Pdcd4 binds less efficiently to eIF4A than wildtype. Cytoplasmic extracts from cultures of S2 cells expression wild-type Myc-Pdcd4 or mutant Myc-Pdcd4 (AA) were subjected to immunoprecipitation with antisera directed against eIF4A. Myc-Pdcd4 was detected with mouse monoclonal antibody to the Myc tag. Immunoprecipitated eIF4A was detected with rabbit antisera. (E) Immobilized Drosophila Pdcd4 interacts with Drosophila eIF4A. On the top is a cartoon of approach. On the bottom is an immnoblot of proteins eluted from the affinity columns. Position of the recombinant eIF4A is indicated. DOI: http: //dx. doi. org/10. 7554/eLife. 00542. 003 Activated mTOR stimulates general translation, in part, by influencing the activity of the translation initiation complex eIF4F. The eIF4F complex consists of eIF4E, the 7-methyl-Guanosine-cap (m7G) binding protein, eIF4A, an RNA helicase, and eIF4G, a large scaffolding protein. In addition, the RNA binding protein eIF4B can associate with eIF4F to stimulate the helicase activity of eIF4A (Ma and Blenis, 2009; Sonenberg and Hinnebusch, 2009; Zoncu et al., 2011). mTOR stimulates general translation in part by inactivating translational inhibitors. mTOR phosphorylates and inactivates the translation repressor eIF4E binding protein (4E-BP) (Gingras et al., 1999) allowing efficient formation of the eIF4F complex. In addition, mTOR activates ribosomal protein S6 kinase (S6K) (Sarbassov et al., 2005). S6K stimulates the helicase eIF4A by activating eIF4B and inhibiting programmed cell death protein 4 (Pdcd4), a known eIF4A inhibitor (Figure 1) (Yang et al., 2003; Raught et al., 2004; Dorrello et al., 2006). Thus under conditions of high signaling through AKT and mTOR, cap-dependent translation is stimulated. In times of stress, low levels of signaling through the IIS pathway lead to activated Foxo and 4E-BP in addition to inactive S6K. Foxo moves to the nucleus and controls the transcription of its target genes (Salih and Brunet, 2008). 4E-BP prevents formation of the translation initiation complex eIF4F, thereby inhibiting m7G-dependent translation, and S6K no longer stimulates eIF4A. This in turn leads to lower levels of global protein synthesis. Thus the IIS pathway controls gene expression with two different branches: transcription of Foxo target genes and m7G-cap-dependent translation through 4E-BP and S6K. The IIS pathway in Drosophila contains a mechanism that functionally couples activated transcription to translation. A portion of the system includes a signaling and gene expression feedback loop for direct genetic targets of Drosophila Foxo. The insulin-like receptor (INR) and 4E-BP genes are conserved transcriptional targets of Foxo (Figure 1) (Puig et al., 2003; Puig and Tjian, 2005; Marr et al., 2007; Hu et al., 2008). Paradoxically, the insulin receptor protein, as well as mRNA, is being synthesized and accumulating under the same conditions when 4E-BP activity and expression is induced and S6K is inhibited (Marr et al., 2007). Foxo activates the transcription of the Drosophila insulin receptor gene from three promoters. Each promoter produces a transcript with a distinct 5′ untranslated region (UTR) but identical coding region (Casas-Tinto et al., 2007; Marr et al., 2007). Transcripts derived from promoter 1 are by far the most abundant and ubiquitous form of INR transcript (Casas-Tinto et al., 2007; Marr et al., 2007). The Drosophila INR 5′ UTRs contain an internal ribosome entry site (IRES) that allows the message to escape 4E-BP inhibition of cap-dependent translation. This mechanism provides a functional coupling of transcription and translation in times of stress that allows amplification of insulin receptor expression (Marr et al., 2007). Because IRES containing transcripts can outcompete cap-dependent transcripts under these conditions their translation is actually stimulated (Svitkin et al., 2005; Marr et al., 2007). This leads to an effective switch of the cellular translation machinery to targets of the IIS pathway. Thus, Foxo targets impose a translational program by activation of genes that repress general translation while simultaneously activating targets that are immune to this translational control. This provides these targets with a competitive advantage allowing them to utilize the translation machinery that is freed by the general inhibition. Here we identify Drosophila Pdcd4 as an additional Foxo target further enhancing the coupling of transcription and translation regulation in the IIS pathway. Since the IIS pathway targets translation initiation through control of both eIF4E and eIF4A, we wondered if the most abundant and ubiquitous Drosophila INR 5′ UTR would also provide resistance to inhibition of eIF4A activity. To answer this question we used both an in vitro translation system and a cell based assay to investigate the eIF4A requirements for efficient translation of reporters containing the INR 5′ UTR from Drosophila. Because mammalian systems show the same type of regulation, we also investigated the role of eIF4A inhibition in the murine insulin receptor and insulin-like growth factor receptors. (Giraud et al., 2001; Meng et al., 2008; Spriggs et al., 2009b). We find, in both the Drosophila and mouse systems, that the 5′ UTRs of the mRNAs for these receptors provide resistance to both eIF4E and eIF4A inhibition. Taken together, these results indicate that these cellular messages have some of the lowest requirement for eIF4F activity for translation identified to date. A connection between Foxo activation and translation inhibition was identified when it was discovered that 4E-BP expression is controlled by Foxo in Drosophila and mouse cells (Junger et al., 2003; Puig et al., 2003; Hu et al., 2008). Since the IIS pathway is also known to control eIF4A activity through Pdcd4 (Figure 1A), we tested if this gene is under direct Foxo control in Drosophila. Blast analysis of human Pdcd4 with the Drosophila genome identifies a single homologous protein encoded by CG10990. Alignment of human PDCD4 and CG10990 indicate that important regions of the protein are conserved (Figure 1B) (Cash and Andrews, 2012). The two MA3 domains, including the acidic residues shown to be important for the interaction with eIF4A are conserved (Chang et al., 2009; Waters et al., 2011). In addition, the Akt and S6K phosphorylation sites are conserved (Palamarchuk et al., 2005; Dorrello et al., 2006). To determine if the interaction with eIF4A is conserved, we immunoprecipitated Drosophila eIF4A from cytoplasmic extracts derived from a saturated culture of Drosophila S2 cells. Associated Pdcd4 was detected by immunoblot. Pdcd4 is co-precipitated with antisera against eIF4A but not with preimmune serum indicating that Pdcd4 and eIF4A interact in Drosophila cells (Figure 1C). We next created Myc-tagged expression constructs for Drosophila Pdcd4, one wildtype construct and a construct containing mutations in conserved residues in the first MA3 domain (E282A, D286A). The analogous mutations in human PDCD4 destabilize the interaction with eIF4A (Chang et al., 2009; Waters et al., 2011). The constructs were transfected into growing Drosophila S2 cells. Subsequent immunoprecipitation of eIF4A from these cells reveals a decreased association of the mutant Pdcd4 with eIF4A (Figure 1D). This indicates the mutations induce the same destabilization of the eIF4A–Pdcd4 interaction in Drosophila cells. Interestingly under these conditions we detect multiple forms of Pdcd4 by western blot, most likely phosphorylated forms of Pdcd4, and only the fastest migrating species associates with eIF4A. To determine if Drosophila Pdcd4 can interact directly with eIF4A we used an affinity chromatography assay using recombinant proteins purified from Escherichia coli. A GST fusion to Drosophila Pdcd4 was immobilized on glutathione agarose and recombinant Drosophila eIF4A was passed over the column (Figure 1D). As a control GST was also immobilized on glutathione agarose. The eIF4A bound to the immobilized GST-Pdcd4 but not to GST alone indicating that Drosophila Pdcd4 can interact directly with Drosophila eIF4A. Taken together these data are consistent with the notion that CG10990 is the Drosophila homologue of human PDCD4. There are hints in the literature, based on microarray experiments, indicating this gene is induced in response to nutrient stress and might be controlled by Foxo (Gershman et al., 2007). In an effort to determine if Foxo binds to the Pdcd4 gene in nutrient stressed animals we reanalyzed the only publically available Foxo ChIP (Chromatin immunoprecipitation) dataset (Teleman et al., 2008). These experiments were performed on starved larva. We find Foxo binds the Pdcd4 gene in both the promoter and intronic regions with enrichment values as high as 16-fold over background (Figure 2A). To corroborate this finding, we performed ChIP on genomic DNA from a cell line with an inducible Foxo cDNA gene that has been modified so the Foxo protein produced is constitutively active because it is immune to the negative regulation by insulin signaling (FoxoCA) (Puig et al., 2003; Gershman et al., 2007). This allows us to induce Foxo under conditions of high nutrient signaling and remove possible crosstalk from upstream signaling pathways. We tested the enrichment of genomic sequences by qPCR using primers to the Pdcd4 promoter region (Figure 2A) compared to a region in the first intron of CG15414, a gene just downstream of 4E-BP. We find FoxoCA binds to the promoter region of Pdcd4 at levels comparable to a well-defined direct target, 4E-BP (Junger et al., 2003; Puig et al., 2003; Marr et al., 2007) (Figure 2B). To determine the effect on mRNA production under these conditions we performed quantitative RT-qPCR on induced cells. We find that the steady-state level of Pdcd4 mRNA is increased about threefold in cells expressing active Foxo (Figure 2C). To determine if the effect is due to mRNA stability changes or new transcription we assayed intron-containing pre-mRNAs by RT-qPCR. Since most splicing is co-transcriptional in Drosophila this is a good assay for new RNA synthesis (Khodor et al., 2011). We find that Pdcd4 pre-mRNA is increased, indicating an increase in transcription of the gene. The increased mRNA also leads to increased protein synthesis as determined by immuno-blot with antibodies directed against Drosophila Pdcd4 (Figure 2D). This is likely an underestimate of the effect since these experiments are all done under high serum and insulin conditions that should result in the rapid turnover of Pdcd4 protein (Dorrello et al., 2006). 10. 7554/eLife. 00542. 004Figure 2. Foxo activates Pdcd4 in Drosophila cells. (A) Reanalysis of ChIP-chip data from Teleman et al. (2008). Genomic Browser view of Foxo binding to the Pdcd4 locus in starved larva. The data are plotted as the enrichment (log2) over mock precipitated samples. Primers used for ChIP and qPCR are indicated. (B) ChIP of Foxo at 4E-BP promoter and Pdcd4 locus in Drosophila S2 cells expressing constitutively active Foxo (FoxoCA). The data are plotted as fold enrichment over a background region 1 kb downstream of 4E-BP. Uninduced samples are plotted in white, induced samples in black (error bars indicate SD). (C) RT-qPCR of Pdcd4 mRNA and pre-mRNA in Drosophila S2 cells expressing FoxoCA. Data are plotted as fold-induction (error bars indicate SD). (D) Immunoblot of total protein from Drosophila S2 cells expressing FoxoCA. Positions of Pdcd4 and tubulin are indicated. (E) 4E-BP, GstD1, and Pdcd4 RNA levels in untreated and paraquat-treated animals. The levels of RNA were normalized to RP49 and are plotted as fold-induction relative to untreated animals (error bars indicate SEM). DOI: http: //dx. doi. org/10. 7554/eLife. 00542. 004 We tested if Foxo controls Pdcd4 in adult flies subjected to stress. Adult wildtype or Foxo null flies (Slack et al., 2011) were treated with paraquat to induce oxidative stress and assayed for expression of 4E-BP, Pdcd4, and GstD1 by RT-qPCR. Consistent with previous results (Wang et al., 2005), 4E-BP is induced by paraquat in a Foxo dependent manner. Like 4E-BP, we find that Pdcd4 is induced in response to paraquat in a Foxo dependent manner (Figure 2E). To determine if the effect was due to loss of general oxidative stress response or if it is Foxo specific we examined the induction of GstD1, a gene controlled by Nrf2 in response to oxidative stress (Misra et al., 2011). We find that GstD1 is still responsive indicating that the effects at Pdcd4 and 4E-BP are Foxo-specific and not due to a loss of responsiveness in the mutant flies (Figure 2E). These results are consistent with the idea that in addition to controlling the cap-binding complex through 4E-BP, active Foxo can influence eIF4A through activation of the Pdcd4 gene in response to stress. Given that Foxo is activating transcription of the INR gene while Pdcd4 is also active, we hypothesized that since the INR mRNA is translated efficiently under these conditions (Marr et al., 2007) it must be at least partially immune to diminished eIF4A activity. To determine the effect of increased Pdcd4 on the translation of insulin receptor UTR containing RNAs we modified a dicistronic mRNA assay which we previously used to investigate the effects of 4E-BP on insulin receptor translation in Drosophila (Marr et al., 2007). In this assay a construct is used that produces a RNA in which the open reading frames of renilla luciferase and firefly luciferase are present on the same transcript (Figure 3A). Renilla luciferase levels are an indication of total message produced in the cell and firefly luciferase levels are an indication of IRES dependent translation. As previously reported, insertion of the INR 5′ UTR between the ORFs promotes translation of the second ORF (Marr et al., 2007). The levels of IRES activity of the INR UTR are comparable to the well-characterized IRES from Hepatitis C Virus (Figure 3B) (Tsukiyama-Kohara et al., 1992). The INR UTR and the HCV IRES both produce more firefly signal than the empty vector (Figure 3D). 10. 7554/eLife. 00542. 005Figure 3. Drosophila insulin receptor 5′UTR provides resistance to Pdcd4. (A) Diagram of dicistronic reporters. Translation of the Firefly ORF requires internal ribosome entry. Firefly to renilla activity ratio provides an indication of IRES activity. (B) The Drosophila Insulin receptor UTR provides IRES activity comparable to the activity of HCV. (C) Renilla activity of the reporters. (D) Firefly activity of the reporters. (E) Dicistronic reporter activities in the presence of 4E-BP or Pdcd4 expression. 4E-BP and Pdcd4 stimulate the IRES activity of the insulin receptor UTRs. Mutation of the critical acidic residues of Pdcd4 prevents the stimulation. (F) Renilla activity of the reporters in the presence of expressed proteins. (G) Firefly activity of the reporters in the presence of the expressed proteins (error bars indicate SEM). DOI: http: //dx. doi. org/10. 7554/eLife. 00542. 005 To determine the effect of Pdcd4 on the INR UTR we expressed Pdcd4 in Drosophila S2 cells and measured the activity of the dicistronic reporter. In order to more accurately determine the effects of the protein in question we modified the assay so the production of the dicistronic mRNA is inducible. This allows accumulation of the experimental protein in the cell before the dicistronic mRNA is produced giving a more precise measure of the effects on the activity of the dicistronic message. The levels of expression of the second ORF relative to the first ORF are an indication of the cap-independent translation potential of the insert. To validate this assay we reproduced our previous results with 4E-BP (Marr et al., 2007). As reported previously, expression of 4E-BP stimulates the translation of the second open reading frame in a reporter containing the INR 5′UTR (Figure 3E, G). Expression of Pdcd4 also stimulates translation of the second ORF dependent on the INR 5′UTR similar to the effects seen with the HCV IRES (Figure 3E, G). These effects are specific. Mutation of the key acidic residues (Figure 1B) in Pdcd4 shown to disrupt eIF4A binding in the human system (Waters et al., 2011) prevent the IRES stimulation. To address the role of eIF4A in insulin receptor translation, we used a highly specific small molecule inhibitor of eIF4A, hippuristanol (Bordeleau et al., 2006; Lindqvist et al., 2008). Hippuristanol is a potent translation inhibitor that works in eukaryotes from yeast to human (Lindqvist et al., 2008). Hippuristanol inhibits the ATPase activity and RNA binding of eIF4A (Bordeleau et al., 2006; Lindqvist et al., 2008). The small molecule binds to the protein in conserved regions V and VI in eIF4A homologues (Lindqvist et al., 2008). Importantly, the effects on translation can be rescued by addition of either wild-type or mutant forms of eIF4A that are immune to hippuristanol indicating that the effects are highly specific for eIF4A (Bordeleau et al., 2006; Lindqvist et al., 2008). This small molecule had been used previously in the Drosophila system to investigate eIF4A requirements (Iwasaki et al., 2009). We performed in vitro competitive translation experiments with capped and polyadenylated firefly luciferase reporters (Figure 4A) and a Drosophila embryo extract translation system that has not been treated with micrococcal nuclease (Gebauer et al., 1999; Marr et al., 2007) in the presence of hippuristanol. The RNA reporters contain the 5′ UTR from the Drosophila insulin receptor. In addition we include two control RNAs. One control RNA contains a non-specific UTR derived from plasmid sequences. The other RNA contains the IRES from the Hepatitis C virus (HCV) (Tsukiyama-Kohara et al., 1992). This IRES does not require eIF4A activity and controls for non-specific effects on the extract (Pestova et al., 1998). Under the experimental conditions, translation of the first control RNA is strongly inhibited by hippuristanol while the reporter containing the HCV IRES is completely resistant to eIF4A inhibition (Figure 4A). This small molecule inhibitor exposed a greatly diminished role for eIF4A in the Drosophila INR UTR mediated translation (Figure 4A). At the low and moderate concentrations of hippuristanol, the Drosophila INR UTR reporter retains almost complete activity comparable to the HCV UTR. Even at the highest concentration of hippuristanol tested, the reporter containing the INR UTR retains >50% of the original translation activity. To determine the effect of Pdcd4 in this system we added recombinant Pdcd4 to the translation extract. Consistent with the data using hippuristanol, we find Pdcd4 can inhibit the control RNA but not the Drosophila INR UTR or the HCV IRES (Figure 4B). These finding suggests that translation of the most abundant Drosophila INR transcript can tolerate inhibition of eIF4A. 10. 7554/eLife. 00542. 006Figure 4. Drosophila insulin receptor 5′UTR provides resistance to eIF4a inhibition. (A) Top: Diagram of RNAs used in the in vitro translation assays. Bottom: Titration of hippuristanol in in vitro translation assays. The shade of the bars indicates the final concentration of hippuristanol in the assay. The legend appears above the graph. Data are plotted as the fraction activity of the carrier treated extracts (error bars indicate SEM). (B) Top: Diagram of RNAs used in the in vitro translation assays. Bottom: Activity of these RNAs in in vitro translation assays in the absence (white bars) and presence (black bars) of Drosophila Pdcd4 (error bars indicate SEM). (C) Dicistronic RNA translation in vitro. Top: Diagram of RNAs used in the in vitro translation assays. Bottom: Firefly to renilla ratio in the absence (white bars) and presence (black bars) of hippuristanol. (D) Top: Renilla activity in the dicistronic assay. Bottom: Firefly activity in the dicistronic assay. Shading as in C. Percentage above the bars indicates activity after hippuristanol addition relative to carrier treated samples. DOI: http: //dx. doi. org/10. 7554/eLife. 00542. 006 We used a dicistronic RNA in the in vitro translation assay to directly test the IRES activity under hippuristanol treatment (Figure 4C top). Dicistronic RNAs were synthesized in vitro using T7 RNA polymerase. The RNA was capped and polyadenylated and used to program the same Drosophila embryo translation system described above. The extracts were treated with either hippuristanol or carrier. Consistent with the monocistronic assay, both the INR and the HCV IRES containing transcripts show increased relative translation of the second ORF upon eIF4A inhibition (Figure 4C). The increase is due both to a resistance of the second ORF to the inhibition and a decrease in the cap-dependent translation of the first ORF (Figure 4D). Under these conditions there is a small amount of cryptic translation of the second ORF in the control transcript. However both the renilla and firefly activites respond the same to the hippuristanol treatment. Because the molecular architecture of the IIS signaling pathway is conserved in mammals, we wondered if this level of regulation would be conserved in mammals. To address this, we cloned the 5′ UTR from the longest mRNAs for the mouse insulin receptor (mINR) and the mouse insulin-like growth factor receptor-I (IGFR) and created firefly luciferase reporters under the control of these UTRs (Figure 5A). Previously, it was reported that INR and IGFR UTRs confer cap-independent translation activity in human and rat (Giraud et al., 2001; Spriggs et al., 2009b). To extend this finding to the mouse system and ensure that our competitive rabbit reticulocyte system was capable of supporting cap-independent translation, we assayed translation of the mINR and IGFR reporters in the presence of excess m7G cap along with our control RNA and the HCV IRES reporters described above (Figure 5A). While the control RNA is inhibited by excess cap, translation from the 5′ UTR of mINR and IGFR is not only resistant to excess m7G cap but the activity actually increases in the presence of excess m7G cap. This observation is common with UTRs that contain an IRES (Svitkin et al., 2005). The increase in activity for both the mINR and IGFR UTRs exceeded the IRES activity of the HCV UTR. This indicates that mINR UTR and IGFR UTR are capable of conferring cap-independent translation initiation. 10. 7554/eLife. 00542. 007Figure 5. Mammalian insulin receptor and insulin-like growth factor receptor 5′UTR provide resistance to eIF4a inhibition. (A) Diagram of RNAs used in the in vitro translation assays. (B) In vitro Translation in the absence (white bars) and presence (black bars) of excess m7G analogue. Data are plotted as the fraction activity of the mock treated extracts (error bars indicate SEM). (C) Titration of hippuristanol in in vitro translation assays. Data are plotted as the fraction activity of the mock treated extracts (error bars indicate SEM). DOI: http: //dx. doi. org/10. 7554/eLife. 00542. 007 To directly test the ability of these UTRs to allow internal ribosome entry we performed a dicistronic assay in mammalian cells. The UTRs were subcloned into a plasmid construct between the renilla and firefly open reading frames controlled by the RSV LTR. Both the mINR and the IGFR UTRs supported substantial firefly activity compared to the original vector. This is apparent both in the firefly to renilla ratio and in the raw firefly activity units (Figure 5B). This combined with the in vitro translation assays strongly suggest that the mINR and IGFR UTRs can support cap-independent translation. To test for conservation of resistance to eIF4A inhibition, hippuristanol was titrated into the rabbit reticulocyte translation system. As expected, the activity of the control RNA is reduced to <10% of the mock treated extract, and the HCV IRES is completely resistant to hippuristanol (Figure 5C). In fact, the HCV IRES is stimulated fourfold by addition of hippuristanol under these conditions. Both mINR and IGFR UTRs confer resistance to hippuristanol (Figure 5C). Even at the highest concentrations of the small molecule the mINR and mIGFR UTRs remain roughly 50% active indicating a decreased requirement for eIF4A relative to the control RNA. Stress responses controlled through the IIS pathway result in changes in both mRNA synthesis and protein synthesis. These processes are coordinated to ensure proper expression of the downstream targets. Under the same low IIS signaling conditions that activate 4E-BP, Pdcd4 is stabilized resulting in the inhibition of the DEAD box helicase eIF4A. Thus, under these conditions the eIF4F complex is repressed by two mechanisms. At the same time, the Foxo family of transcription factors is active and increasing the synthesis of certain mRNAs, one of which is the INR transcript itself. Previously we identified a connection between 4E-BP mediated inhibition of the cap-binding complex and INR mRNA translation in Drosophila (Marr et al., 2007). The INR message is immune to the 4E-BP translational repression and thus is preferentially translated under low signaling conditions coupling the increase in mRNA expression to an increase in protein synthesis. In the work presented here we extend this observation of gene expression coordination of INR mRNA to the eIF4A branch of the IIS signaling pathway. First, we show that active Foxo is capable of directly stimulating the transcription of Pdcd4, analogous to the activation of 4E-BP seen previously under these same conditions (Junger et al., 2003; Puig et al., 2003). This provides a mechanism for the pathways controlling Foxo to enhance the inhibition of eIF4A when stressed or when nutrients are low. Second we show that the 5′ UTR of the most abundant Drosophila INR transcript provides resistance to eIF4A inhibition comparable to the resistance seen with the HCV IRES that does not require eIF4A. A similar finding has been seen for the Drosophila reaper 5′ UTR (Hernandez et al., 2004; Iwasaki et al., 2009). The data presented above also support the conservation of the cap-independent mechanism of translation initiation of the insulin receptor and insulin-like growth factor receptor mRNAs in mammals. The mouse transcripts show resistance to hippuristanol under conditions that almost completely inhibit a control RNA indicating that the resistance to eIF4A inhibition is also conserved. There are no easily recognizable conserved sequence elements between the Drosophila and mouse UTRs, but the mode of regulation is conserved suggesting an important role for this type of translational control. This defines a functional characteristic of the insulin receptor and IGF receptor transcripts that is conserved across hundreds of millions of years of evolution (from flies to mammals). Taken together with previous work, these data indicate that the coupling of transcription to translation of insulin receptor mRNA mediated by Foxo targets can culminate in an activated translational response. Our findings highlight a unique characteristic of the insulin receptor and IGF receptor UTRs that differentiates them from other cellular transcripts. In addition to being immune to 4E-BP, these IRESes are resistant to eIF4A inhibition. While viral IRESes are fairly common, cellular IRESes are rare and relatively unexplored. Where it has been explored, most cellular IRESes have a strong requirement for eIF4A (Thoma et al., 2004; Spriggs et al., 2009a). The INR and IGFR UTRs seem to require neither eIF4E nor eIF4A. In fact, these UTRs have the lowest identified requirement for eIF4F activity of any cellular transcript thus far. In addition, they are immune to two of the most important types of translational control, namely 4E-BP control and eIF4A inhibition. Both of these features make sense given the cellular environment when these mRNAs are to be translated. The conserved resistance to eIF4E and eIF4A inhibition of the insulin receptor transcripts should make them capable of out-competing other cellular transcripts with greater need for eIF4A or eIF4E. Using these exceptional characteristics, the insulin receptor mRNA could out-compete more abundant transcripts under times of stress or when nutrients are limiting and 4E-BP and Pdcd4 are active. We focused on the insulin receptor UTRs as a mechanism for continued translation under conditions of general inhibition of protein synthesis as this is one of the initial components of the pathway identified as a direct Foxo target. In more recent work other components of the pathway have been identified as transcriptionally controlled by Foxo. If these targets are to be translated when Foxo is active they should also require mechanisms to escape 4E-BP and Pdcd4 inhibition. It remains to be seen if they will use the same mechanism as the INR mRNA or another mechanism. Wildtype Canton S flies are from the Bloomingtion Stock Center. foxOΔ94 has been described (Slack et al., 2011). Chromatin immunoprecipitation using Foxo antibodies followed by tiling array analysis was performed previously on starved larva (Teleman et al., 2008). The raw. CEL files for Foxo precipitated and mock precipitated arrays were downloaded from the Teleman lab web page (http: //www. dkfz. de/en/signal-transduction-cancer/pages/Data. html). Triplicate samples were combined and the Foxo precipitated samples were compared to mock precipitated samples using using the Affymetrix Tiling analysis (TAS) software. Combined mock arrays were used to set the background signal intensities for the ChIP arrays. The Integrated Genome Browser (Nicol et al., 2009) was used to visualize the resultant profile. Oligonucleotides were synthesized corresponding to annotated transcripts with the longest 5′ for both Insulin receptor (corresponding to EST G430111A11) and IGF-1 receptor (corresponding to ESTs CJ180736 and CJ173921) (Supplementary file 1). The oligos were used to clone the UTRs from cDNA derived from NIH 3T3 cells by PCR. Drosophila eIF4A and Pdcd4 were cloned into pET28 in frame with the 6x HIS tag from cDNA using PCR and standard cloning methods. The plasmid was transformed into BL21* (DE3) cells (Life Technologies, Grand Island, NY) containing pLacIRARE2 (Novagen, EMD Millipore, Billerica, MA). After induction with 1 mM IPTG overnight at 25°C, eIF4A or PDCD-4 was purified using HisPur Ni-NTA Resin (Thermo Fisher Scientific, Rockford, IL) according to manufacturer’s directions. PDCD-4 was eluted from the resin using 500 mM imidazole in PBS. eIF4A was eluted using 50 mM EDTA in 1X PBS. Purified 6His-Pdcd4 and 6His eIF4A was used to make rabbit polyclonal antisera (Cocalico Biologicals, Inc., Reamstown, PA). Full length Drosophila Pdcd4 was cloned into pGEX2TKN in frame with GST. GST-PDCD-4 or GST alone was expressed in BL21* (DE3) cells (Invitrogen, Grand Island, NY) containing pLacIRARE2 (Novagen, EMD Millipore, Billerica, MA). Expression was induced with 1 mM IPTG overnight at 25°C. Cells were resuspended and lysed in 1X PBS with lysozyme. GST or GST-PDCD4 was immobilized on glutathione sepharose 4B (GE Healthcare Biosciences, Pittsburgh, PA). Equal amounts of recombinant eIF4A were applied to 50 µl of resin containing either GST or GST-Pdcd4. The resin was incubated for 1 hr at 4°C. The resin was poured into a small spin column (Pierce, Thermo Fisher Scientific, Rockford, IL) and washed with 100 column volumes wash buffer (20 mM Tris pH 7. 5,100 mM NaCl, 1 mM DTT, 0. 1 mM EDTA, 0. 1 mg/ml BSA). Bound proteins were eluted with wash buffer containing 10 mM reduced glutathione for 1 hr at 4°C. Samples were separated by SDS-PAGE, transferred to nitrocellulose and recombinant eIF4A was detected with a monoclonal antibody directed against the 6x HIS tag on eIF4A (A00186 GenScript, Piscataway, NJ) and a fluorescent secondary antibody against mouse IgG using a Li-Cor Odyssey Infrared imaging system. 15 ml of a saturated culture of Drosophila S2 cells were harvested by centrifugation. The cells were washed once with 1X PBS. The cells were resuspended in two packed cell volumes hypotonic buffer (10 mM HEPES pH 7. 4,10 mM KCl, 5 mM MgCl2,1 mM DTT, 1X protease inhibitors [Sigma-Aldrich Corp., St. Louis, MO]). Triton X-100 was added to 0. 5% and the cells were left on ice 30 min. Nuclei were pelleted 10 min at 6000×g. Supernatant was transferred to a new tube. 10% of the sample was saved for input analysis. Polyclonal rabbit antisera to Drosophila eIF4A was added to the remainder of the sample and the sample was incubated at 4°C overnight with constant mixing. Samples were centrifuged 2 min at 23,000×g. The supernatant was combined with 50 µl protein-A Sepharose (GE healthcare) and incubated at 4°C for 2 hr. The mixture was poured into a small spin column (Pierce, #89869) connected to a needle and washed with 5 ml 1XPBS 0. 1% Triton X-100 followed by 1 ml 1X PBS. Proteins were elute by addition of 2X SDS-PAGE buffer (62. 5 mM Tris-HCl, pH 6. 8,25% glycerol, 2% SDS, 0. 01% Bromophenol Blue, 5% β-mercaptoethanol) incubation at room temperature for 10 min and collected by centrifugation. Samples were separated by SDS-PAGE. The gel was transferred to nitrocellulose and probed with rabbit α-Drosophila Pdcd4 antisera directed against full length 6His-Pdcd4 (1: 1000). To avoid detection of the IgG used for precipitation the immunoblot was developed with biotinylated protein-A (1: 5000) (Lal et al., 2005) followed by fluorescent labeled streptavidin (1: 5000) using a Li-Cor Odyssey Infrared imaging system. For experiments with the mutant Pdcd4,10 ml of Drosophila S2 cells were plated at 1 × 106 cells/ml in Schneider’s media supplemented with 10% FBS in a 10-cm dish. Constructs expressing wild-type myc-Pdcd4 or mutant myc-Pdcd4-282 A286A were transfected using effectene (Qiagen Inc., Valencia, CA). 4 days later the cells were harvested and immunoprecipitation of eIF4A was performed as described above except transfected Pdcd4 was detected with a monoclonal antibody directed against the myc tag (9E10) and a fluorescent secondary antibody against mouse IgG using a Li-Cor Odyssey Infrared imaging system. Protein concentrations were determined using BCA assay (Pierce) and equal amounts of protein were loaded onto a 10% SDS-PAGE gel. The gel was transferred to nitrocellulose and probed with rabbit α-Drosophila Pdcd4 antisera directed against full length 6His-Pdcd4 (1: 500) and mouse α-tubulin antibody (1: 1000). Transcription templates for monocistronic RNAs were created using PCR containing a template-specific forward primer with a T7 promoter incorporated and a vector specific reverse primer. Dicistronic RNA templates were made by digesting the cellular reporter vectors downstream of the firefly luciferase coding sequence and utilizing a T7 promoter incorporated in the vector. Templates were purified using column clean up protocol and eluted in 50 µl 10 mM Tris pH 8. 0 (Epoch Life Science, Missouri City, TX). Templates were transcribed using T7 polymerase and subsequently purified using LiCl precipitation. Transcripts were capped using vaccinia virus capping enzyme (New England Biolabs, Ipswich, MA) as recommended and purified using RNeasy column protocol (Qiagen). Transcripts were tailed using poly (A) polymerase (New England Biolabs, Ipswich, MA) and purified using RNeasy columns (Qiagen). Embryo translation extracts were prepared as described from 0- to 4-hr embryos (Marr et al., 2007). Extracts were left untreated (no Micrococcal nuclease treatment) to allow translation under competitive conditions. Translation assays were performed in 6 µl of Drosophila embryo extract, 0. 1 mM spermidine, 60 µm Amino Acids, 16. 8 mM creatine phosphate, 800 ng of creatine kinase, 24 mM HEPES (pH 7. 4), 0. 4 mM Mg acetate, 30 mM K acetate, 1 µg of calf liver tRNA, and 100 ng of template RNA in a 10-µl reaction. Hippuristanol was added to a final concentration of 2µM or otherwise indicated. For assays containing PDCD-4, protein was added to a final concentration of 320 nM and was preincubated for 15 min with extract before the addition of RNA templates. Translation reactions were incubated at 27°C for 1 hr and luciferase activity was measured using 100 µl of luciferase substrate (Promega Corp., Madison, WI). Firefly luciferase was measured by adding 100 µl of 75 mM HEPES pH 8. 0,5 mM MgSO4,20 mM DTT, 100 µM EDTA, 530 µM ATP, 0. 5 mM coenzyme A, and 0. 5 mM D-luciferin and renilla was measured by adding 100 µl 25 mM Na4PPi, 10 mM NaOAc, 15 mM EDTA, 0. 5 M Na2SO4,1. 0 M NaCl, and 0. 1 mM Coelenterazine, pH 5. 0. All experiments were performed at least twice in triplicate. Translation assays were performed in 6 µl of untreated rabbit reticulocyte extract (no Micrococcal nuclease treatment to allow translation under competitive conditions). (Green Hectares, McFarland, WI), 0. 1 mM spermidine, 60 µm Amino Acids, 16. 8 mM creatine phosphate, 800 ng of creatine kinase, 24 mM HEPES (pH 7. 4), 0. 4 mM Mg acetate, 30 mM K acetate, 1 µg of calf liver tRNA, and 100 ng of template RNA in a 10-µl reaction. Translation reactions were incubated at 37°C for 30 min and luciferase activity was measured using 100 µl of luciferase substrate (Promega). For assays containing excess m7G cap, cap structure analogue (New England Biolabs, #S1407S) was added to a final concentration of 1 mM. All experiments were performed at least twice in triplicate. Drosophila S2 cells with a stable transfection of constitutively active Foxo under the control of the metallothionein A promoter were maintained in Schneider’s Insect Media supplemented with 10% fetal bovine serum (Puig et al., 2003). These cells were plated at 1. 25 × 106 cell/ml, and expression was induced by addition of 500 µm CuSO4 for 16 hr. During induction the media was supplemented with 1 µg/m bovine insulin. For protein samples, cells were lysed in RIPA buffer (PBS containing 10 mM EDTA, 1% Triton X-100,1% SDS, 1% deoxycholate, 1× complete protease inhibitor [Roche, Indianapolis, IN], 10% glycerol). Total RNA was extracted from mock-treated and induced cells using TRI Reagent according to manufacturer’s protocol (Molecular Research Center, Inc., Cincinnati, OH). 5 µg of total RNA were digested by DNaseI (New England Biolabs). First strand cDNA sythesis was done using a mix of oligo-dT and random hexamers with MMLV reverse transcriptase. The final concentrations of the cDNA reaction were 50 mM Tris-HCl (pH 8. 3), 50 mM KCl, 3 mM MgCl2,10 mM DTT, 400 µM dNTPs, 1–2 µg RNA, 500 ng primers, 200 units MMLV RT. cDNAs were diluted 1: 10 in TE pH 8. qPCR was run using 5 µl cDNA, GoTaq qPCR Master Mix (Promega), and primers at a final concentration of 100 nM in a 20-µl reaction. qPCR was done using specific primers against Drosophila Pdcd4, RP49, GstD1, and 4E-BP (Supplementary file 1). Pdcd4 fold-expression was calculated as a fraction of RP49 and normalized to mock-treated expression levels. All experiments were performed at least twice in triplicate. 7-day-old adult male flies were starved for 5 hr and transferred to vials containing 5% sucrose or 5% sucrose+5 mM paraquat. After 24 hr flies were harvested and total RNA was extracted from mock-treated and paraquat-treated flies using TRI Reagent according to manufacturer’s protocol (Molecular Research Center, Inc.). Previously described dicistronic reporter constructs (Marr et al., 2007) were subcloned into a plasmid containing the metallothionein A promoter for metal inducible expression (Marr et al., 2006). The IRES sequence from HCV (Kieft et al., 1999) was subcloned into the inducible expression vector. For expression in S2 cells, Pdcd4 was cloned under a minimal actin promoter. For the Pdcd4 double mutant construct, amino acids 282, and 286 (Figure 1B) were mutated to alanine by site directed mutagenesis. S2 cells were maintained in Schneider’s media with 10% FBS and Penicillin/Streptomycin. For transfection, cells were plated at 1. 25×106 cells/ml in Schneider’s supplemented with 10% FBS and an additional 1 µg/ml bovine insulin. DNA was transfected at a 4: 1 ratio expression plasmid to reporter plasmid using effectene transfection reagent (Qiagen) following instructions for S2 cells. Cells were induced with 500 µM CuSO4 36 hr after transfection. Cells were lysed in passive lysis buffer (Promega) and assayed 36 hr after induction using a dual luciferase assay. Firefly expression was measured in 75 mM HEPES pH 8. 0,5 mM MgSO4,20 mM DTT, 100 µM EDTA, 530 µM ATP, 0. 5 mM coenzyme A, and 0. 5 mM D-luciferin. Renilla expression was measured by addition of an equal volume of 25 mM Na4PPi, 10 mM NaOAc, 15 mM EDTA, 0. 5 M Na2SO4,1. 0 M NaCl, and 0. 1 mM Coelenterazine. All experiments were performed at least twice in triplicate. Drosophila S2 cells expressing constitutively active Foxo were formaldehyde crosslinked. Nuclei were isolated, lysed, and chromatin was sonicated to 500–1000 bp in length. Chromatin was incubated with polyclonal sera against full length Foxo. The chromatin/antibody mix was then incubated with protein A beads to isolate Foxo-bound chromatin from the sample. Purified DNA was assayed by qPCR to determine enrichment for genomic sites bound by Foxo. Enrichment is based on signal increase compared to a region of the genome in the first intron of CG15414 (Supplementary file 1). The mouse insulin receptor and insulin-like growth factor one receptor 5′ UTRs were subcloned into the plasmid pGLRSVRF. This plasmid contains the RSV LTR followed by the renilla luciferase open reading frame, the firefly luciferase open reading frame and the SV40 early polyadenylation signal. The UTRs were cloned between the renilla and firefly open reading frames. For transfection NIH3T3 cells were trypsinized and counted. 350 µl of cells at a concentration of 1 × 105 cells per milliliter were plated in each well of a 24-well plate. Cells were transfected using Effectene (Qiagen) according to manufacturer’s instructions. Cells were lysed in passive lysis buffer (Promega) and assayed 36 hr after induction using a dual luciferase assay (Promega).

Protein synthesis in eukaryotes occurs in two stages: transcription of DNA into messenger RNA (mRNA) in the nucleus, and then translation of that mRNA into a protein by ribosomes in the cytoplasm. These processes are regulated by a complex network of signaling pathways that enables cells to tailor protein synthesis to match current conditions. This involves regulating the expression of the genes that code for these proteins. When cells experience stressful events, such as a shortage of oxygen or nutrients, they reduce the synthesis of most proteins. This response is regulated, in part, by a signaling pathway known as the insulin and insulin-like receptor pathway. In particular, stressful events inhibit a protein complex called eIF4F, which normally initiates the translation of mRNA molecules by binding to a structure on one end of the mRNA called the 5′ cap. Despite this general inhibition, the production of certain other proteins-including the insulin receptor itself-is actually increased in response to stress. Olson et al. have carried out a series of experiments to explore how inhibition of the eIF4F protein complex influences the translation of the mRNA for the insulin receptor. The eIF4F complex is made up of three proteins, including one that binds to the 5′ cap and a helicase that unwinds the RNA. Previous work in the fruit fly Drosophila showed that translation of this mRNA can continue even if formation of the eIF4F complex is inhibited by targeting the cap binding protein. Olsen et al. now show that translation of this mRNA is also independent of the helicase. Instead, translation is maintained under these conditions because the insulin receptor mRNA contains a sequence called an internal ribosome entry site, which allows ribosomes to bind to the mRNA without the influence of the 5′ cap. Olson et al. reveal the details of this regulatory pathway in Drosophila and show that similar mechanisms are at work in mammalian cells, suggesting this pathway represents a crucial regulatory process that has been conserved during evolution. A key question for future research is whether other genes within the insulin and insulin-receptor like signaling pathway use this same trick to evade translational inhibitors.

lay_elife

_29 October._--This is written in the train from Varna to Galatz. Last night we all assembled a little before the time of sunset. Each of us had done his work as well as he could; so far as thought, and endeavour, and opportunity go, we are prepared for the whole of our journey, and for our work when we get to Galatz. When the usual time came round Mrs. Harker prepared herself for her hypnotic effort; and after a longer and more serious effort on the part of Van Helsing than has been usually necessary, she sank into the trance. Usually she speaks on a hint; but this time the Professor had to ask her questions, and to ask them pretty resolutely, before we could learn anything; at last her answer came:-- "I can see nothing; we are still; there are no waves lapping, but only a steady swirl of water softly running against the hawser. I can hear men's voices calling, near and far, and the roll and creak of oars in the rowlocks. A gun is fired somewhere; the echo of it seems far away. There is tramping of feet overhead, and ropes and chains are dragged along. What is this? There is a gleam of light; I can feel the air blowing upon me." Here she stopped. She had risen, as if impulsively, from where she lay on the sofa, and raised both her hands, palms upwards, as if lifting a weight. Van Helsing and I looked at each other with understanding. Quincey raised his eyebrows slightly and looked at her intently, whilst Harker's hand instinctively closed round the hilt of his Kukri. There was a long pause. We all knew that the time when she could speak was passing; but we felt that it was useless to say anything. Suddenly she sat up, and, as she opened her eyes, said sweetly:-- "Would none of you like a cup of tea? You must all be so tired!" We could only make her happy, and so acquiesced. She bustled off to get tea; when she had gone Van Helsing said:-- "You see, my friends. _He_ is close to land: he has left his earth-chest. But he has yet to get on shore. In the night he may lie hidden somewhere; but if he be not carried on shore, or if the ship do not touch it, he cannot achieve the land. In such case he can, if it be in the night, change his form and can jump or fly on shore, as he did at Whitby. But if the day come before he get on shore, then, unless he be carried he cannot escape. And if he be carried, then the customs men may discover what the box contain. Thus, in fine, if he escape not on shore to-night, or before dawn, there will be the whole day lost to him. We may then arrive in time; for if he escape not at night we shall come on him in daytime, boxed up and at our mercy; for he dare not be his true self, awake and visible, lest he be discovered." There was no more to be said, so we waited in patience until the dawn; at which time we might learn more from Mrs. Harker. Early this morning we listened, with breathless anxiety, for her response in her trance. The hypnotic stage was even longer in coming than before; and when it came the time remaining until full sunrise was so short that we began to despair. Van Helsing seemed to throw his whole soul into the effort; at last, in obedience to his will she made reply:-- "All is dark. I hear lapping water, level with me, and some creaking as of wood on wood." She paused, and the red sun shot up. We must wait till to-night. And so it is that we are travelling towards Galatz in an agony of expectation. We are due to arrive between two and three in the morning; but already, at Bucharest, we are three hours late, so we cannot possibly get in till well after sun-up. Thus we shall have two more hypnotic messages from Mrs. Harker; either or both may possibly throw more light on what is happening. * * * * * _Later._--Sunset has come and gone. Fortunately it came at a time when there was no distraction; for had it occurred whilst we were at a station, we might not have secured the necessary calm and isolation. Mrs. Harker yielded to the hypnotic influence even less readily than this morning. I am in fear that her power of reading the Count's sensations may die away, just when we want it most. It seems to me that her imagination is beginning to work. Whilst she has been in the trance hitherto she has confined herself to the simplest of facts. If this goes on it may ultimately mislead us. If I thought that the Count's power over her would die away equally with her power of knowledge it would be a happy thought; but I am afraid that it may not be so. When she did speak, her words were enigmatical:-- "Something is going out; I can feel it pass me like a cold wind. I can hear, far off, confused sounds--as of men talking in strange tongues, fierce-falling water, and the howling of wolves." She stopped and a shudder ran through her, increasing in intensity for a few seconds, till, at the end, she shook as though in a palsy. She said no more, even in answer to the Professor's imperative questioning. When she woke from the trance, she was cold, and exhausted, and languid; but her mind was all alert. She could not remember anything, but asked what she had said; when she was told, she pondered over it deeply for a long time and in silence. * * * * * _30 October, 7 a. m._--We are near Galatz now, and I may not have time to write later. Sunrise this morning was anxiously looked for by us all. Knowing of the increasing difficulty of procuring the hypnotic trance, Van Helsing began his passes earlier than usual. They produced no effect, however, until the regular time, when she yielded with a still greater difficulty, only a minute before the sun rose. The Professor lost no time in his questioning; her answer came with equal quickness:-- "All is dark. I hear water swirling by, level with my ears, and the creaking of wood on wood. Cattle low far off. There is another sound, a queer one like----" She stopped and grew white, and whiter still. "Go on; go on! Speak, I command you!" said Van Helsing in an agonised voice. At the same time there was despair in his eyes, for the risen sun was reddening even Mrs. Harker's pale face. She opened her eyes, and we all started as she said, sweetly and seemingly with the utmost unconcern:-- "Oh, Professor, why ask me to do what you know I can't? I don't remember anything." Then, seeing the look of amazement on our faces, she said, turning from one to the other with a troubled look:-- "What have I said? What have I done? I know nothing, only that I was lying here, half asleep, and heard you say go on! speak, I command you!' It seemed so funny to hear you order me about, as if I were a bad child!" "Oh, Madam Mina," he said, sadly, "it is proof, if proof be needed, of how I love and honour you, when a word for your good, spoken more earnest than ever, can seem so strange because it is to order her whom I am proud to obey!" The whistles are sounding; we are nearing Galatz. We are on fire with anxiety and eagerness. _Mina Harker's Journal._ _30 October._--Mr. Morris took me to the hotel where our rooms had been ordered by telegraph, he being the one who could best be spared, since he does not speak any foreign language. The forces were distributed much as they had been at Varna, except that Lord Godalming went to the Vice-Consul, as his rank might serve as an immediate guarantee of some sort to the official, we being in extreme hurry. Jonathan and the two doctors went to the shipping agent to learn particulars of the arrival of the _Czarina Catherine_. * * * * * _Later._--Lord Godalming has returned. The Consul is away, and the Vice-Consul sick; so the routine work has been attended to by a clerk. He was very obliging, and offered to do anything in his power. _Jonathan Harker's Journal._ _30 October._--At nine o'clock Dr. Van Helsing, Dr. Seward, and I called on Messrs. Mackenzie & Steinkoff, the agents of the London firm of Hapgood. They had received a wire from London, in answer to Lord Godalming's telegraphed request, asking us to show them any civility in their power. They were more than kind and courteous, and took us at once on board the _Czarina Catherine_, which lay at anchor out in the river harbour. There we saw the Captain, Donelson by name, who told us of his voyage. He said that in all his life he had never had so favourable a run. "Man!" he said, "but it made us afeard, for we expeckit that we should have to pay for it wi' some rare piece o' ill luck, so as to keep up the average. It's no canny to run frae London to the Black Sea wi' a wind ahint ye, as though the Deil himself were blawin' on yer sail for his ain purpose. An' a' the time we could no speer a thing. Gin we were nigh a ship, or a port, or a headland, a fog fell on us and travelled wi' us, till when after it had lifted and we looked out, the deil a thing could we see. We ran by Gibraltar wi'oot bein' able to signal; an' till we came to the Dardanelles and had to wait to get our permit to pass, we never were within hail o' aught. At first I inclined to slack off sail and beat about till the fog was lifted; but whiles, I thocht that if the Deil was minded to get us into the Black Sea quick, he was like to do it whether we would or no. If we had a quick voyage it would be no to our miscredit wi' the owners, or no hurt to our traffic; an' the Old Mon who had served his ain purpose wad be decently grateful to us for no hinderin' him." This mixture of simplicity and cunning, of superstition and commercial reasoning, aroused Van Helsing, who said:-- "Mine friend, that Devil is more clever than he is thought by some; and he know when he meet his match!" The skipper was not displeased with the compliment, and went on:-- "When we got past the Bosphorus the men began to grumble; some o' them, the Roumanians, came and asked me to heave overboard a big box which had been put on board by a queer lookin' old man just before we had started frae London. I had seen them speer at the fellow, and put out their twa fingers when they saw him, to guard against the evil eye. Man! but the supersteetion of foreigners is pairfectly rideeculous! I sent them aboot their business pretty quick; but as just after a fog closed in on us I felt a wee bit as they did anent something, though I wouldn't say it was agin the big box. Well, on we went, and as the fog didn't let up for five days I joost let the wind carry us; for if the Deil wanted to get somewheres--well, he would fetch it up a'reet. An' if he didn't, well, we'd keep a sharp lookout anyhow. Sure eneuch, we had a fair way and deep water all the time; and two days ago, when the mornin' sun came through the fog, we found ourselves just in the river opposite Galatz. The Roumanians were wild, and wanted me right or wrong to take out the box and fling it in the river. I had to argy wi' them aboot it wi' a handspike; an' when the last o' them rose off the deck wi' his head in his hand, I had convinced them that, evil eye or no evil eye, the property and the trust of my owners were better in my hands than in the river Danube. They had, mind ye, taken the box on the deck ready to fling in, and as it was marked Galatz _via_ Varna, I thocht I'd let it lie till we discharged in the port an' get rid o't althegither. We didn't do much clearin' that day, an' had to remain the nicht at anchor; but in the mornin', braw an' airly, an hour before sun-up, a man came aboard wi' an order, written to him from England, to receive a box marked for one Count Dracula. Sure eneuch the matter was one ready to his hand. He had his papers a' reet, an' glad I was to be rid o' the dam' thing, for I was beginnin' masel' to feel uneasy at it. If the Deil did have any luggage aboord the ship, I'm thinkin' it was nane ither than that same!" "What was the name of the man who took it?" asked Dr. Van Helsing with restrained eagerness. "I'll be tellin' ye quick!" he answered, and, stepping down to his cabin, produced a receipt signed "Immanuel Hildesheim." Burgen-strasse 16 was the address. We found out that this was all the Captain knew; so with thanks we came away. We found Hildesheim in his office, a Hebrew of rather the Adelphi Theatre type, with a nose like a sheep, and a fez. His arguments were pointed with specie--we doing the punctuation--and with a little bargaining he told us what he knew. This turned out to be simple but important. He had received a letter from Mr. de Ville of London, telling him to receive, if possible before sunrise so as to avoid customs, a box which would arrive at Galatz in the _Czarina Catherine_. This he was to give in charge to a certain Petrof Skinsky, who dealt with the Slovaks who traded down the river to the port. He had been paid for his work by an English bank note, which had been duly cashed for gold at the Danube International Bank. When Skinsky had come to him, he had taken him to the ship and handed over the box, so as to save porterage. That was all he knew. We then sought for Skinsky, but were unable to find him. One of his neighbours, who did not seem to bear him any affection, said that he had gone away two days before, no one knew whither. This was corroborated by his landlord, who had received by messenger the key of the house together with the rent due, in English money. This had been between ten and eleven o'clock last night. We were at a standstill again. Whilst we were talking one came running and breathlessly gasped out that the body of Skinsky had been found inside the wall of the churchyard of St. Peter, and that the throat had been torn open as if by some wild animal. Those we had been speaking with ran off to see the horror, the women crying out "This is the work of a Slovak!" We hurried away lest we should have been in some way drawn into the affair, and so detained. As we came home we could arrive at no definite conclusion. We were all convinced that the box was on its way, by water, to somewhere; but where that might be we would have to discover. With heavy hearts we came home to the hotel to Mina. When we met together, the first thing was to consult as to taking Mina again into our confidence. Things are getting desperate, and it is at least a chance, though a hazardous one. As a preliminary step, I was released from my promise to her. _Mina Harker's Journal._ _30 October, evening._--They were so tired and worn out and dispirited that there was nothing to be done till they had some rest; so I asked them all to lie down for half an hour whilst I should enter everything up to the moment. I feel so grateful to the man who invented the "Traveller's" typewriter, and to Mr. Morris for getting this one for me. I should have felt quite; astray doing the work if I had to write with a pen.... It is all done; poor dear, dear Jonathan, what he must have suffered, what must he be suffering now. He lies on the sofa hardly seeming to breathe, and his whole body appears in collapse. His brows are knit; his face is drawn with pain. Poor fellow, maybe he is thinking, and I can see his face all wrinkled up with the concentration of his thoughts. Oh! if I could only help at all.... I shall do what I can. I have asked Dr. Van Helsing, and he has got me all the papers that I have not yet seen.... Whilst they are resting, I shall go over all carefully, and perhaps I may arrive at some conclusion. I shall try to follow the Professor's example, and think without prejudice on the facts before me.... * * * * * I do believe that under God's providence I have made a discovery. I shall get the maps and look over them.... * * * * * I am more than ever sure that I am right. My new conclusion is ready, so I shall get our party together and read it. They can judge it; it is well to be accurate, and every minute is precious. _Mina Harker's Memorandum._ (Entered in her Journal.) _Ground of inquiry._--Count Dracula's problem is to get back to his own place. (_a_) He must be _brought back_ by some one. This is evident; for had he power to move himself as he wished he could go either as man, or wolf, or bat, or in some other way. He evidently fears discovery or interference, in the state of helplessness in which he must be--confined as he is between dawn and sunset in his wooden box. (_b_) _How is he to be taken?_--Here a process of exclusions may help us. By road, by rail, by water? 1. _By Road._--There are endless difficulties, especially in leaving the city. (_x_) There are people; and people are curious, and investigate. A hint, a surmise, a doubt as to what might be in the box, would destroy him. (_y_) There are, or there may be, customs and octroi officers to pass. (_z_) His pursuers might follow. This is his highest fear; and in order to prevent his being betrayed he has repelled, so far as he can, even his victim--me! 2. _By Rail._--There is no one in charge of the box. It would have to take its chance of being delayed; and delay would be fatal, with enemies on the track. True, he might escape at night; but what would he be, if left in a strange place with no refuge that he could fly to? This is not what he intends; and he does not mean to risk it. 3. _By Water._--Here is the safest way, in one respect, but with most danger in another. On the water he is powerless except at night; even then he can only summon fog and storm and snow and his wolves. But were he wrecked, the living water would engulf him, helpless; and he would indeed be lost. He could have the vessel drive to land; but if it were unfriendly land, wherein he was not free to move, his position would still be desperate. We know from the record that he was on the water; so what we have to do is to ascertain _what_ water. The first thing is to realise exactly what he has done as yet; we may, then, get a light on what his later task is to be. _Firstly._--We must differentiate between what he did in London as part of his general plan of action, when he was pressed for moments and had to arrange as best he could. _Secondly_ we must see, as well as we can surmise it from the facts we know of, what he has done here. As to the first, he evidently intended to arrive at Galatz, and sent invoice to Varna to deceive us lest we should ascertain his means of exit from England; his immediate and sole purpose then was to escape. The proof of this, is the letter of instructions sent to Immanuel Hildesheim to clear and take away the box _before sunrise_. There is also the instruction to Petrof Skinsky. These we must only guess at; but there must have been some letter or message, since Skinsky came to Hildesheim. That, so far, his plans were successful we know. The _Czarina Catherine_ made a phenomenally quick journey--so much so that Captain Donelson's suspicions were aroused; but his superstition united with his canniness played the Count's game for him, and he ran with his favouring wind through fogs and all till he brought up blindfold at Galatz. That the Count's arrangements were well made, has been proved. Hildesheim cleared the box, took it off, and gave it to Skinsky. Skinsky took it--and here we lose the trail. We only know that the box is somewhere on the water, moving along. The customs and the octroi, if there be any, have been avoided. Now we come to what the Count must have done after his arrival--_on land_, at Galatz. The box was given to Skinsky before sunrise. At sunrise the Count could appear in his own form. Here, we ask why Skinsky was chosen at all to aid in the work? In my husband's diary, Skinsky is mentioned as dealing with the Slovaks who trade down the river to the port; and the man's remark, that the murder was the work of a Slovak, showed the general feeling against his class. The Count wanted isolation. My surmise is, this: that in London the Count decided to get back to his castle by water, as the most safe and secret way. He was brought from the castle by Szgany, and probably they delivered their cargo to Slovaks who took the boxes to Varna, for there they were shipped for London. Thus the Count had knowledge of the persons who could arrange this service. When the box was on land, before sunrise or after sunset, he came out from his box, met Skinsky and instructed him what to do as to arranging the carriage of the box up some river. When this was done, and he knew that all was in train, he blotted out his traces, as he thought, by murdering his agent. I have examined the map and find that the river most suitable for the Slovaks to have ascended is either the Pruth or the Sereth. I read in the typescript that in my trance I heard cows low and water swirling level with my ears and the creaking of wood. The Count in his box, then, was on a river in an open boat--propelled probably either by oars or poles, for the banks are near and it is working against stream. There would be no such sound if floating down stream. Of course it may not be either the Sereth or the Pruth, but we may possibly investigate further. Now of these two, the Pruth is the more easily navigated, but the Sereth is, at Fundu, joined by the Bistritza which runs up round the Borgo Pass. The loop it makes is manifestly as close to Dracula's castle as can be got by water. _Mina Harker's Journal--continued._ When I had done reading, Jonathan took me in his arms and kissed me. The others kept shaking me by both hands, and Dr. Van Helsing said:-- "Our dear Madam Mina is once more our teacher. Her eyes have been where we were blinded. Now we are on the track once again, and this time we may succeed. Our enemy is at his most helpless; and if we can come on him by day, on the water, our task will be over. He has a start, but he is powerless to hasten, as he may not leave his box lest those who carry him may suspect; for them to suspect would be to prompt them to throw him in the stream where he perish. This he knows, and will not. Now men, to our Council of War; for, here and now, we must plan what each and all shall do." "I shall get a steam launch and follow him," said Lord Godalming. "And I, horses to follow on the bank lest by chance he land," said Mr. Morris. "Good!" said the Professor, "both good. But neither must go alone. There must be force to overcome force if need be; the Slovak is strong and rough, and he carries rude arms." All the men smiled, for amongst them they carried a small arsenal. Said Mr. Morris:-- "I have brought some Winchesters; they are pretty handy in a crowd, and there may be wolves. The Count, if you remember, took some other precautions; he made some requisitions on others that Mrs. Harker could not quite hear or understand. We must be ready at all points." Dr. Seward said:-- "I think I had better go with Quincey. We have been accustomed to hunt together, and we two, well armed, will be a match for whatever may come along. You must not be alone, Art. It may be necessary to fight the Slovaks, and a chance thrust--for I don't suppose these fellows carry guns--would undo all our plans. There must be no chances, this time; we shall, not rest until the Count's head and body have been separated, and we are sure that he cannot re-incarnate." He looked at Jonathan as he spoke, and Jonathan looked at me. I could see that the poor dear was torn about in his mind. Of course he wanted to be with me; but then the boat service would, most likely, be the one which would destroy the... the... the... Vampire. (Why did I hesitate to write the word?) He was silent awhile, and during his silence Dr. Van Helsing spoke:-- "Friend Jonathan, this is to you for twice reasons. First, because you are young and brave and can fight, and all energies may be needed at the last; and again that it is your right to destroy him--that--which has wrought such woe to you and yours. Be not afraid for Madam Mina; she will be my care, if I may. I am old. My legs are not so quick to run as once; and I am not used to ride so long or to pursue as need be, or to fight with lethal weapons. But I can be of other service; I can fight in other way. And I can die, if need be, as well as younger men. Now let me say that what I would is this: while you, my Lord Godalming and friend Jonathan go in your so swift little steamboat up the river, and whilst John and Quincey guard the bank where perchance he might be landed, I will take Madam Mina right into the heart of the enemy's country. Whilst the old fox is tied in his box, floating on the running stream whence he cannot escape to land--where he dares not raise the lid of his coffin-box lest his Slovak carriers should in fear leave him to perish--we shall go in the track where Jonathan went,--from Bistritz over the Borgo, and find our way to the Castle of Dracula. Here, Madam Mina's hypnotic power will surely help, and we shall find our way--all dark and unknown otherwise--after the first sunrise when we are near that fateful place. There is much to be done, and other places to be made sanctify, so that that nest of vipers be obliterated." Here Jonathan interrupted him hotly:-- "Do you mean to say, Professor Van Helsing, that you would bring Mina, in her sad case and tainted as she is with that devil's illness, right into the jaws of his death-trap? Not for the world! Not for Heaven or Hell!" He became almost speechless for a minute, and then went on:-- "Do you know what the place is? Have you seen that awful den of hellish infamy--with the very moonlight alive with grisly shapes, and every speck of dust that whirls in the wind a devouring monster in embryo? Have you felt the Vampire's lips upon your throat?" Here he turned to me, and as his eyes lit on my forehead he threw up his arms with a cry: "Oh, my God, what have we done to have this terror upon us!" and he sank down on the sofa in a collapse of misery. The Professor's voice, as he spoke in clear, sweet tones, which seemed to vibrate in the air, calmed us all:-- "Oh, my friend, it is because I would save Madam Mina from that awful place that I would go. God forbid that I should take her into that place. There is work--wild work--to be done there, that her eyes may not see. We men here, all save Jonathan, have seen with their own eyes what is to be done before that place can be purify. Remember that we are in terrible straits. If the Count escape us this time--and he is strong and subtle and cunning--he may choose to sleep him for a century, and then in time our dear one"--he took my hand--"would come to him to keep him company, and would be as those others that you, Jonathan, saw. You have told us of their gloating lips; you heard their ribald laugh as they clutched the moving bag that the Count threw to them. You shudder; and well may it be. Forgive me that I make you so much pain, but it is necessary. My friend, is it not a dire need for the which I am giving, possibly my life? If it were that any one went into that place to stay, it is I who would have to go to keep them company." "Do as you will," said Jonathan, with a sob that shook him all over, "we are in the hands of God!" * * * * * _Later._--Oh, it did me good to see the way that these brave men worked. How can women help loving men when they are so earnest, and so true, and so brave! And, too, it made me think of the wonderful power of money! What can it not do when it is properly applied; and what might it do when basely used. I felt so thankful that Lord Godalming is rich, and that both he and Mr. Morris, who also has plenty of money, are willing to spend it so freely. For if they did not, our little expedition could not start, either so promptly or so well equipped, as it will within another hour. It is not three hours since it was arranged what part each of us was to do; and now Lord Godalming and Jonathan have a lovely steam launch, with steam up ready to start at a moment's notice. Dr. Seward and Mr. Morris have half a dozen good horses, well appointed. We have all the maps and appliances of various kinds that can be had. Professor Van Helsing and I are to leave by the 11:40 train to-night for Veresti, where we are to get a carriage to drive to the Borgo Pass. We are bringing a good deal of ready money, as we are to buy a carriage and horses. We shall drive ourselves, for we have no one whom we can trust in the matter. The Professor knows something of a great many languages, so we shall get on all right. We have all got arms, even for me a large-bore revolver; Jonathan would not be happy unless I was armed like the rest. Alas! I cannot carry one arm that the rest do; the scar on my forehead forbids that. Dear Dr. Van Helsing comforts me by telling me that I am fully armed as there may be wolves; the weather is getting colder every hour, and there are snow-flurries which come and go as warnings. * * * * * _Later._--It took all my courage to say good-bye to my darling. We may never meet again. Courage, Mina! the Professor is looking at you keenly; his look is a warning. There must be no tears now--unless it may be that God will let them fall in gladness. _Jonathan Harker's Journal._ _October 30. Night._--I am writing this in the light from the furnace door of the steam launch: Lord Godalming is firing up. He is an experienced hand at the work, as he has had for years a launch of his own on the Thames, and another on the Norfolk Broads. Regarding our plans, we finally decided that Mina's guess was correct, and that if any waterway was chosen for the Count's escape back to his Castle, the Sereth and then the Bistritza at its junction, would be the one. We took it, that somewhere about the 47th degree, north latitude, would be the place chosen for the crossing the country between the river and the Carpathians. We have no fear in running at good speed up the river at night; there is plenty of water, and the banks are wide enough apart to make steaming, even in the dark, easy enough. Lord Godalming tells me to sleep for a while, as it is enough for the present for one to be on watch. But I cannot sleep--how can I with the terrible danger hanging over my darling, and her going out into that awful place.... My only comfort is that we are in the hands of God. Only for that faith it would be easier to die than to live, and so be quit of all the trouble. Mr. Morris and Dr. Seward were off on their long ride before we started; they are to keep up the right bank, far enough off to get on higher lands where they can see a good stretch of river and avoid the following of its curves. They have, for the first stages, two men to ride and lead their spare horses--four in all, so as not to excite curiosity. When they dismiss the men, which shall be shortly, they shall themselves look after the horses. It may be necessary for us to join forces; if so they can mount our whole party. One of the saddles has a movable horn, and can be easily adapted for Mina, if required. It is a wild adventure we are on. Here, as we are rushing along through the darkness, with the cold from the river seeming to rise up and strike us; with all the mysterious voices of the night around us, it all comes home. We seem to be drifting into unknown places and unknown ways; into a whole world of dark and dreadful things. Godalming is shutting the furnace door.... * * * * * _31 October._--Still hurrying along. The day has come, and Godalming is sleeping. I am on watch. The morning is bitterly cold; the furnace heat is grateful, though we have heavy fur coats. As yet we have passed only a few open boats, but none of them had on board any box or package of anything like the size of the one we seek. The men were scared every time we turned our electric lamp on them, and fell on their knees and prayed. * * * * * _1 November, evening._--No news all day; we have found nothing of the kind we seek. We have now passed into the Bistritza; and if we are wrong in our surmise our chance is gone. We have over-hauled every boat, big and little. Early this morning, one crew took us for a Government boat, and treated us accordingly. We saw in this a way of smoothing matters, so at Fundu, where the Bistritza runs into the Sereth, we got a Roumanian flag which we now fly conspicuously. With every boat which we have over-hauled since then this trick has succeeded; we have had every deference shown to us, and not once any objection to whatever we chose to ask or do. Some of the Slovaks tell us that a big boat passed them, going at more than usual speed as she had a double crew on board. This was before they came to Fundu, so they could not tell us whether the boat turned into the Bistritza or continued on up the Sereth. At Fundu we could not hear of any such boat, so she must have passed there in the night. I am feeling very sleepy; the cold is perhaps beginning to tell upon me, and nature must have rest some time. Godalming insists that he shall keep the first watch. God bless him for all his goodness to poor dear Mina and me. * * * * * _2 November, morning._--It is broad daylight. That good fellow would not wake me. He says it would have been a sin to, for I slept peacefully and was forgetting my trouble. It seems brutally selfish to me to have slept so long, and let him watch all night; but he was quite right. I am a new man this morning; and, as I sit here and watch him sleeping, I can do all that is necessary both as to minding the engine, steering, and keeping watch. I can feel that my strength and energy are coming back to me. I wonder where Mina is now, and Van Helsing. They should have got to Veresti about noon on Wednesday. It would take them some time to get the carriage and horses; so if they had started and travelled hard, they would be about now at the Borgo Pass. God guide and help them! I am afraid to think what may happen. If we could only go faster! but we cannot; the engines are throbbing and doing their utmost. I wonder how Dr. Seward and Mr. Morris are getting on. There seem to be endless streams running down the mountains into this river, but as none of them are very large--at present, at all events, though they are terrible doubtless in winter and when the snow melts--the horsemen may not have met much obstruction. I hope that before we get to Strasba we may see them; for if by that time we have not overtaken the Count, it may be necessary to take counsel together what to do next. _Dr. Seward's Diary._ _2 November._--Three days on the road. No news, and no time to write it if there had been, for every moment is precious. We have had only the rest needful for the horses; but we are both bearing it wonderfully. Those adventurous days of ours are turning up useful. We must push on; we shall never feel happy till we get the launch in sight again. * * * * * _3 November._--We heard at Fundu that the launch had gone up the Bistritza. I wish it wasn't so cold. There are signs of snow coming; and if it falls heavy it will stop us. In such case we must get a sledge and go on, Russian fashion. * * * * * _4 November._--To-day we heard of the launch having been detained by an accident when trying to force a way up the rapids. The Slovak boats get up all right, by aid of a rope and steering with knowledge. Some went up only a few hours before. Godalming is an amateur fitter himself, and evidently it was he who put the launch in trim again. Finally, they got up the rapids all right, with local help, and are off on the chase afresh. I fear that the boat is not any better for the accident; the peasantry tell us that after she got upon smooth water again, she kept stopping every now and again so long as she was in sight. We must push on harder than ever; our help may be wanted soon. _Mina Harker's Journal._ _31 October._--Arrived at Veresti at noon. The Professor tells me that this morning at dawn he could hardly hypnotise me at all, and that all I could say was: "dark and quiet." He is off now buying a carriage and horses. He says that he will later on try to buy additional horses, so that we may be able to change them on the way. We have something more than 70 miles before us. The country is lovely, and most interesting; if only we were under different conditions, how delightful it would be to see it all. If Jonathan and I were driving through it alone what a pleasure it would be. To stop and see people, and learn something of their life, and to fill our minds and memories with all the colour and picturesqueness of the whole wild, beautiful country and the quaint people! But, alas!-- * * * * * _Later._--Dr. Van Helsing has returned. He has got the carriage and horses; we are to have some dinner, and to start in an hour. The landlady is putting us up a huge basket of provisions; it seems enough for a company of soldiers. The Professor encourages her, and whispers to me that it may be a week before we can get any good food again. He has been shopping too, and has sent home such a wonderful lot of fur coats and wraps, and all sorts of warm things. There will not be any chance of our being cold. * * * * * We shall soon be off. I am afraid to think what may happen to us. We are truly in the hands of God. He alone knows what may be, and I pray Him, with all the strength of my sad and humble soul, that He will watch over my beloved husband; that whatever may happen, Jonathan may know that I loved him and honoured him more than I can say, and that my latest and truest thought will be always for him.

Includes the October 29th and October 30th entries of Dr. Seward's diary; the Ocotber 30th entries of Mina Harker's journal; the October 30th entry of Jonathan Harker's journal; the October 30th entries of Mina Harker's journal; Mina Harker's memorandum ; the October 30th, October 31st, November 1st, and November 2nd entries of Jonathan Harker's journal; the November 2nd, November 3rd, and November 4th entries of Dr. Seward's diary; and the October 31st entries of Mina Harker's journal. Mina's latest trance allows the group to learn that Dracula is still near the waterfrom the signs of it, river water. Van Helsing believes that Dracula may have great difficulty being transported back to his castle, as running water and daylight are both dangerous for him, and an inspection of his coffin's contents will undo him. The old professor hopes that the group can reach Galatz before the box departs. Seward notes that it is growing more difficult for Van Helsing to hypnotize Mina. When they arrive in Galatz, they learn that the box was passed off to Immanuel Hildesheim. The ship's skipper informs them that his "superstitious" Romanian crew wanted to destroy the box instead. The friends find the businessman and question him, to learn that he passed the box on to Petrof Skinsky, a trader who deals with the Slovaks who trade down the river to the port. Petrof is found shortly afterward in a cemetery, his throat ripped open. At a loss over their next step, the men retire to the hotel and try to figure out what to do. Mina takes the maps and all of the facts at her disposal and makes a thorough analysis of the situation, until she correctly reasons the most likely route for the Count's escape. He is going to take the river Sereth up to the river Bistritza, which makes a loop up near the Borgo Pass. The men plan: Arthur and Jonathan will take a steamboat upriver, Quincey and Dr. Seward will take horses and follow the river, and Van Helsing and Mina will cut straight through to the castle, where the professor will sterilize Dracula's home. Jonathan protests at being separated from Mina, and he hates the idea of Mina going to the castle. But Van Helsing needs to use hypnotism to track down the places that he will need to sterilize; otherwise, if Dracula escapes, he can sleep for centuries. Funded by Quincey and Arthur, the group arm themselves to the teeth and part ways. From Jonathan, Dr. Seward, and Mina, we read accounts of the early part of the journey into the country. Van Helsing buys a carriage and horses, so that he and Mina can make the seventy-mile trek. Arthur and Jonathan are detained by an accident when they try to make it up a very turbulent part of the river. No sign of the box yet.

booksum

Background GPRAMA requires OMB to coordinate with agencies to develop long- term, outcome-oriented federal government priority goals for a limited number of crosscutting policy areas and management improvement areas every 4 years. Furthermore, with the submission of the fiscal year 2013 budget, GPRAMA required OMB to identify a set of interim priority goals.interim CAP goals, 9 of which were related to crosscutting policy areas and 5 of which were management improvement goals. The President’s 2013 budget submission included a list of 14 The CAP Goal Leader. As required by GPRAMA, each of the interim CAP goals had a goal leader responsible for coordinating efforts to achieve each goal. CAP goal leaders were given flexibility in how to manage these efforts, and were encouraged by OMB to engage officials from contributing agencies by leveraging existing inter-agency working groups, policy committees, and councils. For information on the position of the goal leader and the interagency groups used to engage officials from agencies contributing to each interim CAP goal, see figure 1. For more information on the interagency groups used to engage agency officials in efforts related to each goal, see appendix III. According to OMB and PIC staff, because CAP goal leaders were responsible for managing efforts related to the achievement of the goals as part of a larger portfolio of responsibilities, staff from the PIC, OMB, and—in some cases—from agencies with project management responsibilities, provided additional capacity for coordinating interagency efforts and overseeing the process of collecting, analyzing, and reporting data. Specifically, PIC staff provided logistical support, assisting with the regular collection of data, updates to Performance.gov, and the development of CAP goal governance structures and working groups. They also provided support in the area of performance measurement and analysis. For example, PIC staff supported the Exports goal leader by informing discussions of how to measure the success and impact of export promotion efforts, providing expertise in the development and selection of appropriate performance measures, and assisting in the collection and analysis of relevant data. Progress Reviews. GPRAMA also requires that the Director of OMB, with the support of the PIC, review progress towards each CAP goal with the appropriate lead government official at least quarterly. Specifically, the law requires that these should include a review of progress during the most recent quarter, overall trends, and the likelihood of meeting the planned level of performance. As part of these reviews, OMB is to assess whether relevant agencies, organizations, program activities, regulations, tax expenditures, policies, and other activities are contributing as planned to the goal. The law also requires that OMB categorize the goals by risk of not achieving the planned level of performance and, for those at greatest risk of not meeting the planned level of performance, identify strategies for performance improvement. In an earlier evaluation of the implementation of quarterly performance reviews at the agency level, we found that regular, in-person review meetings provide a critical opportunity for leaders to use current data and information to analyze performance, provide feedback to managers and staff, follow up on previous decisions or commitments, learn from efforts to improve performance, and identify and solve performance problems. As part of this work we also identified nine leading practices that can be used to promote successful performance reviews at the federal level. To identify these practices, we conducted a review of relevant academic and policy literature, including our previous reports. We refined these practices with additional information obtained from practitioners at the local, state, and federal level who shared their experiences and lessons learned. Nine Leading Practices That Can Be Used to Promote Successful Performance Reviews Leaders use data-driven reviews as a leadership strategy to drive performance improvement. Key players attend reviews to facilitate problem solving. Reviews ensure alignment between goals, program activities, and resources. Leaders hold managers accountable for diagnosing performance problems and identifying strategies for improvement. There is capacity to collect accurate, useful, and timely performance data. Staff have skills to analyze and clearly communicate complex data for decision making. Rigorous preparations enable meaningful performance discussions. Reviews are conducted on a frequent and regularly scheduled basis. Participants engage in rigorous and sustained follow-up on issues identified during reviews. Reporting Requirements. In addition to requiring quarterly reviews, GPRAMA requires that OMB make information available on “a single website” (now known as Performance.gov) for each CAP goal on the results achieved during the most recent quarter, and overall trend data compared to the planned level of performance. In addition, information on Performance.gov is to include an assessment of whether relevant federal organizations, programs, and activities are contributing as planned, and, for those CAP goals at risk of not achieving the planned level of performance, information on strategies for performance improvement. New CAP Goals. As required by GPRAMA, in March 2014, OMB announced the creation of a new set of CAP goals in the fiscal year 2015 budget. It then identified 15 CAP goals with 4-year time frames on Performance.gov—7 mission-oriented goals and 8 management-focused goals. Five goal areas—Cybersecurity; Open Data; Science, Technology, Engineering, and Mathematics (STEM) Education; Strategic Sourcing; and Sustainability (renamed Climate Change (Federal Actions))—were carried over from the set of interim CAP goals, while the other 10 are new goal areas. OMB stated on Performance.gov that more detailed action plans for each of the goals, including specific metrics and milestones that will be used to gauge progress, will subsequently be released. The new CAP goals will also have co-leaders; one from an office within the Executive Office of the President (EOP) and one or more from federal agencies. According to OMB staff, this change was made to ensure that CAP goal leaders can leverage the convening authority of officials from the EOP while also drawing upon expertise and resources from the agency level. CAP Goal Leaders Reported Performance on Goals, but Many Lacked Key Information to Demonstrate Progress GPRAMA Requirements for Establishing Planned Performance for CAP Goals GPRAMA requires the Director of OMB to establish, in the annual federal government performance plan, a planned level of performance for each CAP goal for the year in which the plan is submitted and the next fiscal year, as well as quarterly performance targets for the goals. GPRAMA Requirements for Reporting CAP Goal Performance Information GPRAMA requires the Director of OMB to publish on Performance.gov information about the results achieved during the most recent quarter and trend data compared to the planned level of performance for each CAP goal. OMB released the federal government performance plan on Performance.gov concurrently with the fiscal year 2013 budget submission that identified the 14 interim CAP goals. The information on Performance.gov included a goal statement for each of the interim goals that established an overall planned level of performance. During the two- year interim goal period, OMB addressed the requirement to report on results achieved during the most recent quarter for each of the CAP goals by publishing 5 sets of quarterly updates to the interim CAP goals on Performance.gov. The first set of updates, for the fourth quarter of fiscal year 2012, was published in December 2012 and the final set of updates, for the fourth quarter of fiscal year 2013, was published in February 2014. These documents described general accomplishments made to date, specific actions completed, or both. The updates to the Broadband CAP goal, for instance, included short descriptions of general progress made towards each of the five strategies identified for achieving the goal, as well as specific milestones accomplished. The quarterly updates did not, however, consistently identify required interim planned levels of performance and data necessary to indicate progress being made toward the CAP goals. Updates to eight of the goals included quarterly, biannual, or annual data that indicated performance achieved to date toward the target identified in the goal statement. Three of the eight goals (Cybersecurity, Energy Efficiency, and Strategic Sourcing) also contained the required annual or quarterly targets that defined planned levels of performance, which allowed for an assessment of interim progress. For example, the Cybersecurity goal’s updates stated that the goal would not be met within its established time frame, and provided quarterly performance data compared to quarterly targets for the entirety of the goal period to support the statement. In contrast, the updates for the other five goals did not contain annual or quarterly targets, which made it difficult to determine whether interim progress towards the goals’ overall planned levels of performance was being made. For example, updates to the Exports goal included data on the total amount of U.S. exports by quarter for calendar years 2012 and 2013 but did not include a target level of performance for those years or quarters. Therefore, it was unclear whether the goal’s overall planned level of performance of doubling U.S. exports by the end of 2014 is on track to be met. Furthermore, updates to six interim CAP goals did not include trend data to indicate progress being made towards the goals’ overall planned levels of performance. Figure 2 below identifies the frequency with which data on CAP goal performance were reported, as well as the overall performance CAP goal leaders reported making compared to the goal’s planned level of performance through the fourth quarter of fiscal year 2013. Through our review of information on Performance.gov and interviews with managers of the six interim CAP goals that did not report any data on progress towards the stated goal, we identified reasons that included: Lack of quantitative planned level of performance (targets). The Entrepreneurship and Small Business CAP goal lacked a quantitative performance target. The quarterly updates to the goal explained that efforts were focused on the goal’s 10 sub-goals. Most of these sub- goals, however, also lacked quantitative performance targets. The deputy goal leader told us that some of the sub-goals did not have quantitative targets by design, as goal managers thought it more appropriate to use qualitative milestones to track progress towards them. The quarterly updates to the “Streamline immigration pathways for immigrant entrepreneurs” sub-goal lacked a quantitative target but had a range of qualitative milestones. For example, the Department of Homeland Security and the Department of State established a milestone to identify reforms needed to ease the application and adjudication processes for visas available to certain immigrant entrepreneurs. Unavailable data. Some CAP goal managers told us that the data needed to assess and report progress toward their goals’ performance targets were unavailable or not yet being collected. For example, a manager of the Job Training CAP goal told us that staff had not established a baseline number of participants served by federal job training programs against which progress towards the goal could be tracked. In addition, managers of the Real Property CAP goal told us that they did not have data available for tracking progress toward the goal of holding the federal real property footprint at its fiscal year 2012 baseline level. Where key data were not reported, some goal managers took actions to obtain previously unavailable data or developed an alternative approach for assessing progress. Job Training CAP Goal. The first quarterly update for the Job Training CAP goal, published on Performance.gov in December 2012, stated that federal agencies were surveyed to compile a list of all job training programs in the federal government, including the number of participants served by those programs, and that a working group was developing a baseline for measuring progress towards the goal of preparing 2 million workers with skills training by 2015. A goal manager told us that the deputy goal leader and staff from the PIC gathered baseline information for most of the programs within the scope of the CAP goal, but that they were unable to complete the efforts by the end of the goal period. Real Property CAP Goal. Managers of the Real Property CAP goal told us that they worked to establish a baseline and metrics for measuring future performance and would be able to report on progress after the goal period ended. Closing Skills Gaps CAP Goal. A manager of the Closing Skills Gaps goal told us that the goal’s managers decided early on that it did not make sense for each of the goal’s identified mission-critical occupations to have the same skills gaps reduction target. Instead, managers of the goal’s sub-goals identified efforts to reduce skills gaps in their specific occupations. They identified an individual targeted level of performance for that effort and collected and reported data on progress made towards the target. For instance, managers of the Acquisitions sub-goal established a target for increasing the certification rate of GS-1102 contract specialists to 80 percent. The final quarterly status update to the Closing Skills Gaps CAP goal reported that the target was met and the certification rate increased to 81 percent. Veteran Career Readiness CAP Goal. The leader of the Veteran Career Readiness CAP goal told us that efforts were made to collect data to assess the veteran employment situation. For instance, she said that an interagency data-gathering working group reviewed sources of available data, integrated those data – such as the unemployment rate for various sub-populations of veterans – into dashboards for senior leadership review, and made proposals to improve data availability. In addition, the Army led a working group to develop a more complete picture of veterans receiving unemployment compensation. She said that these and other efforts led to a concerted effort to improve the availability of data, and to develop and implement metrics measuring career readiness and attendance in a veteran career transition assistance program. However, no data to track progress towards the overall goal were reported during the interim goal period. As we have previously reported, no picture of what the federal government is accomplishing can be complete without adequate performance information. However, OMB and CAP goal leaders did not identify interim planned levels of performance or targets for most of the interim CAP goals. Furthermore, they established a number of CAP goals for which data necessary to indicate progress towards the goal could not be reported. In so doing, they limited their ability to demonstrate progress being made towards most of the CAP goals and ensure accountability for results from those who helped to manage the goals. CAP Goal Leaders Described What Contributed to Goal Achievement, but in Some Cases Information Was Incomplete GPRAMA Requirement for Establishing Milestones GPRAMA requires the Director of OMB to establish, in the federal government performance plan, clearly defined quarterly milestones for the CAP goals. GPRAMA Requirement for Reporting on Contributions towards Cross-Agency Priority Goals GPRAMA requires that OMB identify the agencies, organizations, program activities, regulations, tax expenditures, policies, and other activities that contribute to each CAP goal on Performance.gov. It also requires OMB to make available on the website an assessment of whether relevant agencies, organizations, program activities, regulations, tax expenditures, policies, and other activities are contributing as planned. In the status updates that were published on Performance.gov, managers of each of the CAP goals reported the general approaches, strategies, or specific initiatives being employed to make progress towards the achievement of the goal, as well as the departments, agencies, and programs that were expected to contribute to goal achievement. For example, the leader of the Science, Technology, Engineering, and Mathematics (STEM) Education CAP goal identified a number of general strategies for making progress towards the achievement of its goal of increasing the number of graduates in STEM subjects by 1 million over the next 10 years, such as “Address the mathematics preparation gap that students face when they arrive at college” and “Identifying and supporting the role of technology and innovation in higher education.” In addition, the goal leader identified a number of programs and goals within four departments and agencies that were likely to contribute in part or in whole to the goal. Figure 3 below illustrates how this information was presented in the update to the STEM Education CAP goal for the fourth quarter of fiscal year 2013. In a May 2012 report on our work related to the CAP goals, we noted that information on Performance.gov indicated additional programs with the potential to contribute to each of the CAP goals may be identified over time. We then recommended that OMB review and consider adding to the list of CAP goal contributors the additional departments, agencies, and programs that we identified, as appropriate. OMB agreed with the recommendation, and in the quarterly updates to the CAP goals published in December 2012 and March 2013, OMB added some of the departments, agencies, and programs we identified in our work to some CAP goals’ lists of contributors. For example, we had noted that 12 member agencies of the Trade Promotion Coordinating Committee had not been identified as contributors to the Exports CAP goal. OMB added additional information about contributors to the Exports goal in the update published in December 2012. During our review, in some cases CAP goal managers told us about additional organizations and program types that contributed to their goals, but which were not identified on Performance.gov or in our previous report. For example, the leader of the STEM Education CAP goal told us that representatives from the Smithsonian Institution led an interagency working group that contributed to key efforts towards achieving the goal. Although the CAP goal updates indicate that the Smithsonian Institution is involved in federal STEM education efforts, it was not identified in a dedicated list of contributors to the goal. We have previously found that federal STEM education programs are fragmented across a number of agencies. We continue to believe that the federal government’s efforts to ensure STEM education programs are effectively coordinated must include all relevant efforts. Furthermore, the leader of the Broadband CAP goal told us that he is aware that tax deductions available to businesses making capital investments contributed to the goal by incentivizing investments in broadband. We have long referred to such deductions, along with other reductions in a taxpayer’s liability that result from special exemptions and exclusions from taxation, credits, deferrals of tax liability, or preferential tax rates, as tax expenditures. As we have previously reported, as with spending programs, tax expenditures represent a substantial federal commitment to a wide range of mission areas. We have recommended greater scrutiny of tax expenditures. Periodic reviews could help determine how well specific tax expenditures work to achieve their goals and how their benefits and costs compare to those of programs with similar goals. As previously mentioned, GPRAMA also requires OMB to identify tax expenditures that contribute to CAP goals. However, tax expenditures were not reported as contributors to the Broadband CAP goal in the quarterly status updates published on Performance.gov. CAP Goal Leaders Identified Milestones for Tracking Progress, but in Some Cases Milestones Were Missing Key Information Leading practices state that a clear connection between goals and day-to- day activities can help organizations better articulate how they plan to accomplish their goals. In addition, a clear connection between goals and the programs that contribute to them helps to reinforce accountability and ensure that managers keep in mind the results their organizations are striving to achieve. Milestones—scheduled events signifying the completion of a major deliverable or a set of related deliverables or a phase of work—can help organizations demonstrate the connection between their goals and day-to-day activities and that they are tracking progress to accomplish their goals. Organizations, by describing the strategies to be used to achieve results, including clearly defined milestones, can provide information that would help key stakeholders better understand the relationship between resources and results. GAO-13-174; GAO-13-228; and GAO, Managing for Results: Critical Issues for Improving Federal Agencies’ Strategic Plans, GAO/GGD-97-180 (Washington, D.C.: Sept. 16, 1997). actions, however, lacked clear time frames for completion. Figure 4 below illustrates the “next steps” identified for the Strategic Sourcing CAP goal in the update for the third quarter of fiscal year 2013. Completion status: The Real Property CAP goal update for the second quarter of fiscal year 2013 identified two planned actions as “next steps.” “After agencies submit their Revised Cost Savings and Innovation Plans to OMB, OMB will evaluate agency plans to maintain their square footage baselines, while balancing mission requirements,” and “Updates on agency square footage baselines and projects are forthcoming and will be posted on Performance.gov.” These two actions were again identified as “next steps” in the update for the third quarter of fiscal year 2013, but no update was provided on the status of the actions. By establishing planned activities that, in many of the CAP goal updates, did not have information about their alignment with the strategies they supported, their time frames for completion, or their completion status, CAP goal leaders did not fully demonstrate that they had effectively planned to support goal achievement or were tracking progress toward the goal or identified milestones. OMB did not issue formal guidance to CAP goal leaders on the types of information that were to be included in the CAP goal updates, including information about contributors and milestones. Standards for internal control in the federal government emphasize the importance of documenting policies and procedures to provide a reasonable assurance that activities comply with applicable laws and regulations, and that managers review performance and compare actual performance to planned or expected results and analyze significant differences.staff told us they provided an implementation plan template to goal leaders, which outlined the data elements to be reported in the quarterly status updates. The template was also used to collect information for internal and public reporting. Some CAP goal managers told us that OMB or PIC staff, in their role supporting the collection, analysis, and presentation of data on CAP goal performance, occasionally provided feedback on the information that the individuals submitted in draft updates that OMB reviewed before they were published on Performance.gov. For example, one CAP goal manager told us that during a review of an update submission PIC staff told him that he should develop additional milestones to be completed during a specific future fiscal year quarter. This is in contrast to the detailed guidance that OMB issued on the types of information that agencies must provide for the updates for agency priority goals (APG), which are also published quarterly on Performance.gov. The APG guidance includes explicit instructions for agencies to identify, as appropriate, the organizations, regulations, tax expenditures, policies, and other activities within and external to the agency that contribute to each APG, as well as key milestones with planned completion dates for the remainder of the goal period. Because guidance for the types of information that should have been included in the CAP goal updates was never formally established, CAP goal leaders were at a heightened risk of failing to take into account important contributors to the goal and providing incomplete information about milestones that could help demonstrate progress being made. OMB and Goal Leaders Established Processes for Reviewing Cross- Agency Priority Goal Progress, but Not All Review Processes Were Consistent with Requirements and Leading Practices OMB Established a Quarterly Process for Reviewing Progress on CAP Goals, but Did Not Consistently Outline Improvement Strategies Where Goal Achievement Was at Risk GPRAMA Requirement for OMB Progress Reviews GPRAMA requires that, not less than quarterly, the Director of OMB, with the support of the PIC, shall review progress on the CAP goals, including progress during the most recent quarter, overall trends, and the likelihood of meeting the planned level of performance. GPRAMA also requires that, as part of these reviews, OMB categorize goals by their risk of not achieving the planned level of performance and, for those goals most at risk of not meeting the planned level of performance, identify strategies for performance improvement. As required by GPRAMA, OMB reviewed progress on CAP goals each quarter, beginning with the quarter ending June 30, 2012. This review process consisted of the collection of updated information for each CAP goal by OMB or PIC staff, and the development of a memorandum for the Director of OMB with information on the status of the CAP goals. To develop these memorandums, OMB staff told us that approximately 6 weeks after the end of each quarter, OMB and PIC staff worked with CAP goal leaders to collect updated data and information on goal metrics and milestones, and to update the narratives supporting the data. CAP goal leaders, or staff assisting leaders with the management of efforts related to the goal, would provide this information to OMB using a template for the status updates ultimately published on Performance.gov. In addition to the memorandums developed for the Director of OMB, OMB published more detailed information through the quarterly status updates available on Performance.gov. OMB and PIC staff told us that to support OMB’s quarterly review efforts, PIC staff were to conduct assessments rating the overall health of implementation efforts and goal leader engagement. They were also to assess the execution status of each goal, including the quality and trend of performance indicators. One purpose of these assessments was to identify areas where risks, such as goal leader turnover, could affect the ability to achieve the planned level of performance. Consistent with this intent, several of the quarterly OMB review memorandums we examined highlighted turnover in goal leader or deputy goal leader positions as risks, and suggested the need to find or approve replacements. Although PIC staff have been tasked with assessing these elements of CAP goal implementation, and said that there was a shared understanding between involved staff as to how these assessments would be carried out, the PIC has not documented its procedures or criteria for conducting these assessments. Standards for internal control in the federal government emphasize the importance of documenting procedures, including those for assessing performance. Without clearly established criteria and procedures, PIC staff lack a means to: consistently assess implementation efforts and execution across all goals; bring any deficiencies, risks, and recommended improvements identified to the attention of leadership; and ensure consistent application of criteria over time. While these quarterly review memorandums identified one goal as being at risk of not achieving the planned level of performance, and identified other instances where progress on goals had been slower than planned, the memorandums did not consistently outline the strategies that were being used to improve performance or address identified risks. For example, the Cybersecurity CAP goal was the one goal specifically described as being at risk of not achieving the planned level of performance, both in these memorandums and in the status updates on Performance.gov. Specifically, the memorandum for the third quarter of fiscal year 2012 identified the risk of not achieving the planned level of performance, and outlined seven specific risks facing the goal and the steps being taken to mitigate them. Similarly, the memorandum for the second quarter of fiscal year 2013 also acknowledged that some agencies were at risk of not meeting their Cybersecurity CAP goal targets. However, in contrast to the earlier memorandums, no information was included about the specific steps that were being taken to mitigate these risks, although information on planned and ongoing actions to improve government-wide implementation was included in the milestones section of the status update for that quarter on Performance.gov. The memorandum for the fourth quarter of fiscal year 2012 also acknowledged that the pace of progress on the STEM Education and Closing Skills Gaps goals had been slower than expected. While the memorandum stated that additional OMB attention was needed to support implementation and assure sufficient progress, no information on the specific strategies being employed to improve performance was mentioned. According to OMB staff, however, these memorandums were used to inform subsequent conversations with OMB leadership, which would build on the information presented in the memorandums. Furthermore, because the data necessary to track progress for some goals were unavailable, the Director of OMB would not have been able to consistently review progress for all CAP goals, or make a determination about whether some CAP goals were at risk of meeting their planned levels of performance. This fact was acknowledged in the quarterly review memorandums for quarters one and two of fiscal year 2013, which acknowledged that progress on three goals (Entrepreneurship and Small Business, Job Training, and STEM Education) was difficult to track, and that additional work was needed on data collection. However, no information on the specific steps that were being taken to address these shortcomings was included. A lack of specific information about the steps being taken to mitigate identified risk areas and improve performance could hinder the ability of OMB leadership—and others—to adequately track the status of efforts to address identified deficiencies or risks and to hold officials accountable for taking necessary actions. CAP Goal Leaders Established Processes to Review Progress, but Their Consistency with Leading Practices and Their Effects on Performance and Collaboration Varied GPRAMA Requirement for Goal Leader and Agency Involvement in Progress Reviews As part of the quarterly review process, GPRAMA requires that the Director of OMB review each priority goal with the appropriate lead government official, and include in these reviews officials from the agencies, organizations, and program activities that contribute to the achievement of the goal. According to OMB staff, to encourage goal leaders and contributing agencies to take ownership of efforts to achieve the goals, OMB gave goal leaders flexibility to use different approaches to engage agency officials and review progress at the CAP-goal level. While guidance released by OMB in August 2012 encouraged goal leaders to leverage existing interagency working groups, committees, and councils in the management of the goals as much as practicable, it did not include information on the purpose of reviews, expectations for how reviews should be conducted to maximize their effectiveness as a tool for performance management and accountability, or the roles that CAP goal leaders and agency officials should play in the review process. Again, standards for internal control in the federal government emphasize the importance of documenting procedures for reviewing performance against established goals and objectives. This is in contrast to the detailed guidance that OMB released for agency priority goal and agency strategic objective reviews, which outlined the specific purposes of the reviews, how frequently they should be conducted, the roles and responsibilities of agency leaders involved in the review process, and how the reviews should be conducted. We also found that this guidance for reviews at the agency level was broadly consistent with the leading practices for performance reviews that we previously identified. While no official guidance was published to guide how reviews involving goal leaders and staff from contributing agencies could be conducted for the CAP goals, OMB staff said the principles of the guidance released for agency reviews, which reflected many of the leading practices, was referenced in conversations with CAP goal leaders and teams. OMB has emphasized that flexibility is needed to ensure that goal leaders can use review processes that are appropriate given the scope of interagency efforts, the number of people involved, and the maturity of existing reporting and review processes. The guidance for agency reviews gave agencies flexibility to design their performance review processes in a way that would fit the agency’s mission, leadership preferences, organizational structure, culture, and existing decision- making processes. In our previous work, we detailed how several federal agencies had implemented quarterly performance reviews in a manner consistent with leading practices, but which were also tailored to the structures, processes, and needs of each agency. In this way, flexible implementation of review processes is possible within a framework that encourages the application of leading practices. A lack of clear expectations for how progress should be reviewed at the CAP-goal level resulted in a number of different approaches being used by goal leaders to engage officials from contributing agencies to review progress on identified goals and milestones, ranging from regular in- person review meetings led by the CAP goal leader to the review of written updates provided to the goal leader by officials from contributing agencies. See appendix IV for more detailed information on the various processes used by goal leaders to collect data on, and review progress towards, identified goals. Some Goal Leaders Used Review Processes Broadly Consistent with Leading Practices, and Noted Their Positive Effects on Performance, Accountability, and Collaboration Instituting review processes consistent with the leading practices we previously identified can help ensure that reviews include meaningful performance discussions, provide opportunities for oversight and accountability, and drive performance improvement. Taken together, these leading practices emphasize the importance of leadership involvement in the review process, data collection and review meeting preparation, participation by key officials, and rigorous follow-up. Through our evaluation of how goal leaders and contributing agency officials reviewed progress towards the interim goals, we identified two CAP goals—Cybersecurity and Closing Skills Gaps—and one sub-goal— the Entrepreneurship and Small Business sub-goal on improving access to government services and information (BusinessUSA sub-goal)—where goal managers instituted in-person review processes with officials from contributing agencies that were broadly consistent with the full range of leading practices for reviews, which we have summarized in four categories below. The processes used by other CAP goal leaders to engage agency officials in the review of progress did not reflect the full range of leading practices. Leadership Involvement. Leading practices indicate that leaders should use frequent and regular progress reviews as a leadership strategy to drive performance improvement and as an opportunity to hold people accountable for diagnosing performance problems and identifying strategies for improvement. The direct and visible engagement of leadership is vital to the success of such reviews. Leadership involvement helps ensure that participants take the review process seriously and that decisions and commitments can be made. The goal leaders managing the Cybersecurity and Closing Skills Gaps goals, as well as the BusinessUSA sub-goal, were directly involved in leading in-person reviews for these goals, and in using them as opportunities to review progress, identify and address performance problems, and hold agency officials accountable for progress on identified goals and milestones, as detailed in table 1. Data Collection and Review Meeting Preparation. Leading practices also indicate that those managing review processes should have the capacity to collect, analyze, and communicate accurate, useful, and timely performance data, and should rigorously prepare for reviews to enable meaningful performance discussions. The collection of current, reliable data on the status of activities and progress towards goals and milestones is critical so that those involved can determine whether performance is improving, identify performance problems, ensure accountability for fulfilling commitments, and learn from efforts to improve performance. The ability to assess data to identify key trends and areas of strong or weak performance, and to communicate this to managers and staff effectively through materials prepared for reviews, is also critical. As detailed in table 2, those supporting the Cybersecurity and Skills Gap goals, and the BusinessUSA sub-goal, instituted processes to regularly collect and analyze data on progress towards identified goals and milestones, and to ensure these data would be communicated through materials prepared for review meetings. Participation by Key Officials. Leading practices indicate that key players involved in efforts to achieve a goal should attend reviews to facilitate problem solving. This is critical as their participation enables those involved to break down information silos, and to use the forum provided by the review to communicate with each other, identify improvement strategies, and agree on specific next steps. Reviews for both the Cybersecurity and Closing Skills Gaps CAP goals, and the BusinessUSA sub-goal, were structured so that relevant agency officials playing a key role in efforts to carry out the goal were included, as detailed in table 3. Review Follow-Up. Leading practices indicate that participants should engage in sustained follow-up on issues identified during reviews, which is critical to ensure the success of the reviews as a performance improvement tool. Important follow-up activities include identifying and documenting specific follow-up actions stemming from reviews, those responsible for each action item, as well as who will be responsible for monitoring and follow-up. Follow-up actions should also be included as agenda items for subsequent reviews to hold responsible officials accountable for addressing issues raised and communicating what was done. Goal managers for the Cybersecurity and Closing Skills Gap CAP goals, as well as the BusinessUSA sub-goal, took steps to follow up on action items identified in these meetings, and to ensure that steps were taken towards their completion, as detailed in table 4. Review Effects. Goal leaders and managers we interviewed said that these review processes were valuable in driving improved performance, establishing a greater sense of accountability for progress on the part of contributors, and in providing a forum for interagency communication and collaboration. For example, according to DHS staff involved in the management of the Cybersecurity CAP goal, implementation of Personal Identity Verification (PIV) requirements across the federal government had been stagnant for several years prior to the introduction of cybersecurity as a CAP goal. The review process was used to hold agencies accountable for improved PIV implementation, which helped bring an increased focus on the issue and drive recent progress. Since the reviews were instituted in 2012, DHS has reported improved PIV adoption in civilian agencies, which has increased from 1.24 percent in fiscal year 2010, to 7.45 percent in fiscal year 2012, to 19.67 percent in the fourth quarter of fiscal year 2013. According to data from DHS, while still falling short of the target, this has contributed to the overall increase in PIV adoption across the federal government—including both civilian agencies and the Department of Defense—from 57.26 percent in fiscal DHS year 2012 to 66.61 percent in fourth quarter of fiscal year 2013.staff also added that agencies generally had not previously collaborated on cybersecurity issues or worked to identify best practices. According to DHS staff, the reviews have created an important point of collaboration between DHS, OMB, National Security Staff, and agencies, and provided an opportunity to inform agencies of best practices and connect them with other agencies that are meeting their targets to learn from them. Similarly, OPM officials and sub-goal leaders involved in the management of the Closing Skills Gap CAP goal said that the quarterly review meetings were a critical means to ensure sub-goal leaders and staff were demonstrating progress. Having sub-goal leaders report out on progress, and hear about the progress made in other sub-goal areas, provided additional pressure for continuous improvement and the need to remain focused on driving progress towards their goals. Having the goal leader lead the review was also a way to demonstrate leadership commitment to the achievement of each sub-goal. According to OPM officials and sub- goal leaders, the review meetings also served as an important forum for discussing innovative approaches being taken to address skills gaps in different areas, opportunities for collaboration to address challenges shared by different sub-goals, and how leaders could leverage the efforts of other sub-goals to drive progress on their own. The BusinessUSA sub-goal leader said that having it as the basis for a CAP sub-goal elevated the cross cutting nature of the initiative. In addition to reviewing performance information and the status of deliverables, discussions at inter-agency Steering Committee meetings were used to discuss how contributors could work together to meet the initiative’s performance goals. This communication and coordination led to connections between agencies and to discussions about how programs could be working in a more integrated way. For example, these discussions were used to identify ways that programs could more effectively integrate program information on the BusinessUSA website to increase customer satisfaction. Other Goal Leaders Did Not Use Review Processes Consistent with the Full Range of Leading Practices for Reviews We found that the processes used by other CAP goal leaders to engage agency officials in the review of progress, which are summarized in appendix IV, did not reflect the full range of leading practices. For example, the process for reviewing progress on the Job Training CAP goal involved staff from the PIC collecting updates on recent milestones from agencies, which were then compiled in the quarterly status update and reviewed by the goal leader. This approach was used by the goal leaders for the Broadband and STEM Education CAP goals to review progress as well. While goal leaders and managers for these goals indicated that they used the collection and review of information as an opportunity to communicate with officials from contributing agencies, this approach contrasts with OMB guidance for reviews of agency priority goals, which states explicitly that performance reviews should not be conducted solely through the sharing of written communications. As OMB noted in its guidance, in-person engagement of leaders in performance reviews greatly accelerates learning and performance improvement, and personal engagement can demonstrate commitment to improvement, ensure coordination across agency silos, and enable rapid decision making. While not employing the full range, goal leaders for a number of goals did use processes that reflected one or more leading practices. For example, many CAP goal leaders led or participated in interagency meetings with representatives of contributing agencies. While these were used to facilitate interagency communication and collaboration on the development of plans and policies, it was unclear whether many of these meetings were consistently used to review progress on identified CAP goals and milestones. The goal leader for the Strategic Sourcing CAP goal used processes that reflected leadership involvement, participation by key officials, and the collection and analysis of relevant data. Specifically, according to goal managers, the goal leader led regular meetings of the Strategic Sourcing Leadership Council (SSLC), which were attended by senior procurement officials from eight agencies that combine to make up almost all of the federal government’s total procurement spending. To prepare for each SSLC meeting, staff from OMB’s Office of Federal Procurement Policy (OFPP) held a meeting for supporting staff from each agency, who would then prepare the SSLC member from their agency for the issues to be discussed in the SSLC meeting. OFPP also established a regular data collection process where each agency would report on its adoption and spending rates for two strategic sourcing options, which would then be used for the purposes of reporting on the CAP goal. However, it was unclear how regularly, if at all, SSLC meetings were used to engage agency officials in the review of data on agency adoption of, and spending on, strategic sourcing options, or how regularly meetings were used to review progress that was being made towards the CAP goal. It was also unclear what mechanisms, if any, were used to ensure rigorous follow-up on issues raised in these meetings, a key leading practice, as there were no official meeting minutes maintained. The lack of an official record could hinder follow-up and accountability for any identified actions that need to be taken. Some Goal Leaders Reported Minimal Effects on Performance and Collaboration Representatives of some goals stated that it was difficult to isolate the impact of the CAP goal designation, and its associated reporting and review requirements, on performance and collaboration. According to some goal managers, because their interim goals were based on initiatives that had been previously established in executive orders or Presidential memorandums, much of the interagency activity supporting their efforts would have happened without the CAP goal designation and its reporting and review requirements. For example, a manager for the Data Center Consolidation CAP goal told us that the previously established Federal Data Center Consolidation Initiative was used to drive progress and that the CAP goal designation and quarterly reporting and review requirements had little impact. Similarly, Job Training CAP goal managers said that interagency collaboration on job training issues had been established prior to the creation of the CAP goal, that the goal’s reporting and review requirements were incidental to the contributors’ ongoing work, and that it did not add an additional level of accountability for the completion of job training initiatives. However, this is a goal where no data were reported to demonstrate its impact on federal job training programs, and which was identified in multiple OMB reviews as having slower than anticipated progress due, in part, to extended periods of time in which there was no deputy CAP goal leader to provide support necessary to improve coordination and collaboration. While many CAP goal leaders and staff we interviewed noted the progress they had made with their existing interagency meetings and approaches, a lack of clear expectations or guidance for how review processes at the CAP goal level should be carried out can lead to a situation where reviews are implemented in a manner that is not informed by, or fully consistent with, leading practices. This could result in missed opportunities to realize the positive effects on performance and accountability that can stem from the implementation of review processes that regularly and consistently involve leaders and agency officials in the analysis of performance data to identify and address performance deficiencies, and use rigorous follow-up to ensure accountability for commitments. Conclusions Many of the meaningful results that the federal government seeks to achieve require the coordinated efforts of more than one federal agency. GPRAMA’s requirement that OMB establish CAP goals offers a unique opportunity to coordinate cross-agency efforts to drive progress in priority areas. That opportunity will not be realized, however, if the CAP goal reporting and review requirements and leading review practices are not followed. The reporting and review requirements for the CAP goals, and leading practices for the reviews, are designed to ensure that relevant performance information is used to improve performance and results, and that OMB and goal leaders actively lead efforts to engage all relevant participants in collaborative performance improvement initiatives and hold them accountable for progress on identified goals and milestones. OMB reported performance information in the quarterly CAP goal status updates it published on Performance.gov. While updates for most goals reported data on performance towards the identified planned level of performance, the information in the updates did not always present a complete picture of progress towards identified goals and milestones. For example, while updates for 8 of the 14 goals included data that indicated performance towards the identified overall planned level of performance, only 3 also contained annual or quarterly targets that allowed for an assessment of interim progress. Updates for the other 6 of the 14 goals did not report on performance towards the goal’s primary performance target because the goal was established without a quantitative target or because goal managers were unable to collect the data needed to track performance. In other cases, planned activities that were identified as contributing to the goal were sometimes missing important elements, including alignment with the strategies for goal achievement they supported, a time frame for completion, or information on their implementation status. The incomplete picture of progress that many of the updates gave limited the ability of goal leaders and others to ensure accountability for the achievement of targets and milestones. Holding regular progress reviews that are consistent with GPRAMA requirements and the full range of leading practices can produce positive effects on performance and collaboration. Engaging contributors in regular reviews of data on performance can help ensure interagency efforts are informed by information on progress towards identified goals and milestones, which can be used to identify and address areas where goal or milestone achievement is at risk. Reviews can also be used to reinforce agency and collective accountability for the achievement of individual and shared outcomes, helping to ensure that efforts to improve performance or address identified risks are implemented. Lastly, reviews can be used to foster greater collaboration, ensuring opportunities for communication and coordination between officials involved in efforts to achieve shared outcomes. While OMB and CAP goal leaders instituted processes for reviewing progress on the interim CAP goals, if GPRAMA requirements and leading practices for reviews are not consistently followed, it may result in missed opportunities to improve performance, hold officials accountable for achieving identified goals and milestones, and ensure agency officials are coordinating their activities in a way that is directed towards the achievement of shared goals and milestones. Recommendations for Executive Action We recommend that the Director of OMB take the following three actions: Include the following in the quarterly reviews of CAP goal progress, as required by GPRAMA: a consistent set of information on progress made during the most recent quarter, overall trends, and the likelihood of meeting the planned level of performance; goals at risk of not achieving the planned level of performance; and the strategies being employed to improve performance. Work with the PIC to establish and document procedures and criteria to assess CAP goal implementation efforts and the status of goal execution, to ensure that the PIC can conduct these assessments consistently across all goals and over time. Develop guidance similar to what exists for agency priority goal and strategic objective reviews, outlining the purposes of CAP goal progress reviews, expectations for how the reviews should be carried out, and the roles and responsibilities of CAP goal leaders, agency officials, and OMB and PIC staff in the review process. To ensure that OMB and CAP goal leaders include all key contributors and can track and report fully on progress being made towards CAP goals overall and each quarter, we recommend that the Director of OMB direct CAP goal leaders to take the following four actions: Identify all key contributors to the achievement of their goals; Identify annual planned levels of performance and quarterly targets for each CAP goal; Develop plans to identify, collect, and report data necessary to demonstrate progress being made towards each CAP goal or develop an alternative approach for tracking and reporting on progress quarterly; and Report the time frames for the completion of milestones; the status of milestones; and how milestones are aligned with strategies or initiatives that support the achievement of the goal. Agency Comments We provided a draft of this report for review and comment to the Director of OMB, the Secretaries of Commerce and Homeland Security, the Director of the Office of Personnel Management, the Administrator of the Small Business Administration, as well as the officials we interviewed to collect information on the interim CAP goals from the Council on Environmental Quality, Department of Education, Department of Labor, Department of Veterans Affairs, National Science Foundation, and the Office of Science and Technology Policy. OMB and PIC staff provided oral comments on the draft, and we made technical changes as appropriate. OMB staff generally agreed to consider our recommendations. For example, while they said that OMB and PIC staff will continue to work directly with CAP goal leaders to convey suggested practices for reviewing performance, they will consider referencing principles and practices for data-driven performance reviews in future Circular A-11 guidance related to the management of CAP goals. Furthermore, while they noted that quantitative performance data for some key measures may not available on a quarterly basis, they said that they will continue to work to develop more robust quarterly targets. Officials or staff from the Departments of Commerce and Veterans Affairs, and the Office of Science and Technology Policy provided technical comments, which we incorporated as appropriate. We are sending copies of this report to the Director of OMB as well as appropriate congressional committees and other interested parties. The report is also available at no charge on the GAO website at http://www.gao.gov. If you or your staff have any questions concerning this report, please contact me at (202) 512-6806 or mihmj@gao.gov. Contact points for our Offices of Congressional Relations and Public Affairs may be found on the last page of this report. Key contributors to this report are listed in appendix VI. Appendix I: Objectives, Scope, and Methodology This report is part of our response to a mandate that we evaluate the implementation of the federal government priority goals under the GPRA Modernization Act of 2010 (GPRAMA). Due to the timing of our work, we focused on the implementation of the reporting and review requirements for the 14 interim cross-agency priority (CAP) goals established in February 2012.about progress made towards the interim CAP goals; and (2) how, if at all, quarterly progress reflected GPRAMA requirements and leading practices for data-driven reviews, as well as how they contributed to improved cross-agency performance and collaboration. Specifically, this report assesses (1) what is known To address these objectives, we interviewed representatives of 13 of the 14 interim goals. For 8 of the 13 goals we spoke directly with the goal leader or deputy goal leader, along with, in some cases, staff from Office of Management and Budget (OMB) and agencies involved in supporting efforts related to the goals. For the other five goals (Closing Skills Gaps, Cybersecurity, Data Center Consolidation, Exports, and Job Training) we met with agency officials or OMB staff playing a key role in the management of interagency efforts related to the CAP goal. During these interviews, we asked officials questions concerning how the goal leader and officials from contributing agencies reviewed progress on the goal; the interagency groups used to engage agency officials and manage efforts related to the goal; the role that staff from OMB and the Performance Improvement Council (PIC) played in the review process; and any impact the CAP goal designation and review processes had on performance, collaboration, and accountability. We also participated in interviews with the goal leaders of 11 agency priority goals that were aligned with, or identified as a contributor to, a CAP goal. To further address the first objective, and assess what is known about progress made toward the interim CAP goals, we analyzed information on identified performance metrics and milestones included in the quarterly status updates for each CAP goal published on Performance.gov. We also analyzed relevant information collected through our interviews with CAP goal leaders, deputies, and supporting staff. We compared the data and information made available through the quarterly status updates with requirements in GPRAMA that Performance.gov include information for each goal on results achieved during the most recent quarter and overall trend data. To assess the reliability of performance data and information available through Performance.gov we collected information from OMB and PIC staff, and CAP goal representatives, about data quality control procedures. We determined that the data and information were sufficiently reliable for our analysis of what was reported on Performance.gov about progress towards identified goals and milestones. To address the second objective, we reviewed quarterly review memorandums developed for OMB leadership for five quarters, from the third quarter of fiscal year 2012 to the third quarter of fiscal year 2013.We compared the contents of these review memorandums with requirements for the OMB quarterly reviews established in GPRAMA. We also interviewed staff from OMB and the PIC to discuss the various approaches being used to review progress at the CAP-goal level, the data collection and review process, and the role of the PIC in supporting the quarterly review process. To further address the second objective we reviewed (where available) documents created for interagency meetings, such as meeting agendas, presentation materials, meeting notes, and attendee lists. We also observed one quarterly review meeting held for the Closing Skills Gap goals, and conducted interviews with sub-goal leaders from the Closing Skills Gaps and Entrepreneurship and Small Business CAP goals. These interviews were used to learn more about the involvement of officials from contributing agencies in the quarterly review process for each CAP goal, the processes that had been established to review progress at the sub- goal level, and to gain a more complete picture of participating agency officials’ perceptions of the impact of the CAP goals and review processes. We selected these sub-goals through a two-part process. Of the eight CAP goals for which we had completed interviews through the end of 2013, the team selected one goal for which the goal leader held quarterly meetings dedicated to reviewing progress toward the CAP goal with the goal’s contributors (Closing Skills Gaps). The team also selected a second goal for which the goal leader used a review process that did not rely on quarterly meetings between the goal leader and contributing agencies (Entrepreneurship and Small Business). To ensure that the team would have at least one goal representing each type of goal, the team also ensured that one goal would be an outcome-oriented policy goal and one goal would be a management goal. For both the Closing Skills Gaps and Entrepreneurship and Small Business CAP goals the team then selected four sub-goals for interviews. For the Closing Skills Gaps CAP goal the team interviewed the sub-goal leaders for the Economist; Information Technology/Cybersecurity; Science, Technology, Engineering, and Mathematics (STEM) Education; and Human Resources sub-goals. For the Entrepreneurship and Small Business CAP goal the team held interviews with the sub-goal leaders for the sub-goals to “Accelerate commercialization of Federal research grants,” “Advance federal small business procurement goals,” “Improve access to government services and information,” and “Streamline immigration pathways for immigrant entrepreneurs.” These were selected to ensure that the team would capture sub-goals in which a range of approaches for measuring and reviewing progress were being used. Specifically, sub- goals were selected to ensure the team would have some that did, and did not, hold regular meetings, and some that did, and did not, track quantitative measures of performance or milestones with time frames. Our selection of these sub-goals was nonstatistical and therefore our findings from these interviews are not generalizable to the other CAP goals. We compared what we learned about review processes at the CAP goal and sub-goal levels, through interviews and the collection of documentation, used by leaders from each goal against leading practices for performance reviews previously identified by GAO. Because the scope of our review was to examine the implementation of quarterly progress reviews, we did not evaluate whether these goals were appropriate indicators of performance, sufficiently ambitious, or met other dimensions of quality. We conducted our work from May 2013 to June 2014 in accordance with generally accepted government auditing standards. Those standards require that we plan and perform the audit to obtain sufficient, appropriate evidence to provide a reasonable basis for our findings and conclusions based on our audit objectives. We believe that the evidence obtained provides a reasonable basis for our findings and conclusions based on our audit objectives. Appendix II: Interim Cross-Agency Priority Goals and Goal Statements Goal Broadband Goal Statement As part of expanding all broadband capabilities, ensure 4G wireless broadband coverage for 98 percent of Americans by 2016. Closing Skills Gaps Close critical skills gaps in the federal workforce to improve mission performance. By September 30, 2013, close the skills gaps by 50 percent for three to five critical federal government occupations or competencies, and close additional agency-specific high risk occupation and competency gaps. Cybersecurity Executive branch departments and agencies will achieve 95 percent implementation of the administration’s priority cybersecurity capabilities by the end of FY 2014. These capabilities include strong authentication, trusted Internet connections, and continuous monitoring. Data Center Consolidation Improve information technology service delivery, reduce waste, and save $3 billion in taxpayer dollars by closing at least 2,500 data centers by fiscal year 2015. Energy Efficiency Increase energy productivity (amount of real gross domestic product in dollars/energy demand) 50 percent by 2030. Entrepreneurship and Small Business Increase federal services to entrepreneurs and small businesses with an emphasis on 1) startups and growing firms and 2) underserved markets. Exports Improper Payments Double U.S. exports by the end of 2014. The federal government will achieve a payment accuracy rate of 97 percent by the end of 2016. Job Training Ensure our country has one of the most skilled workforces in the world by preparing 2 million workers with skills training by 2015 and improving the coordination and delivery of job training services. Real Property The federal government will maintain the fiscal year 2012 square footage baseline of its office and warehouse inventory. Science, Technology, Engineering, and Mathematics (STEM) education In support of the president’s goal that the U.S. have the highest proportion of college graduates in the world by 2020, the federal government will work with education partners to improve the quality of STEM education at all levels to help increase the number of well- prepared graduates with STEM degrees by one-third over the next 10 years, resulting in an additional 1 million graduates with degrees in STEM subjects. Strategic sourcing Reduce the costs of acquiring common products and services by agencies’ strategic sourcing of at least two new commodities or services in both 2013 and 2014, that yield at least a 10 percent savings. In addition, agencies must increase their use of Federal Strategic Sourcing Initiative vehicles by at least 10 percent in both fiscal years 2013 and 2014. Sustainability By 2020, the federal government will reduce its direct greenhouse gas emissions by 28 percent and will reduce its indirect greenhouse gas emissions by 13 percent by 2020 (from 2008 baseline). Veteran Career Readiness By September 30, 2013, increase the percent of eligible service members who will be served by career readiness and preparedness programs from 50 percent to 90 percent in order to improve their competitiveness in the job market. Appendix III: Interagency Group Membership and Meeting Frequency and Purpose Goal leaders for 13 of 14 cross-agency priority (CAP) goals leveraged interagency groups for the purposes of coordinating efforts designed to contribute to progress on the cross-agency priority goal. This appendix includes information on the membership of these interagency groups, the frequency with which they met, and the purposes of those meetings. Membership Fourteen agencies with federal property management or transportation funding responsibilities, and broadband or other related expertise. To discuss best practices on broadband-related land management issues, and actions to implement an executive order on accelerating broadband infrastructure deployment. Senior officials from agencies considered major spectrum stakeholders and users of spectrum, including the Departments of Defense, Justice, Homeland Security (DHS), Commerce, and the National Aeronautics and Space Administration (NASA). To provide advice on spectrum policy and strategic plans, discuss commercial transfer of federal agency spectrum, and resolve issues affecting federal/non-federal users. Closing Skills Gaps To review progress on performance metrics and actions taken to close skills gaps in each of the six sub- goal areas. Cybersecurity Officials from National Institute of Standards and Technology, General Services Administration (GSA), DHS, National Security Staff, Office of Management and Budget (OMB) and Performance Improvement Council. Twice each quarter Beginning in 2013, a meeting was held each quarter prior to the collection of data on agency progress on cybersecurity metrics. Another was held after data had been collected and analyzed to review and discuss agency progress. Data Center Consolidation Data center consolidation program managers from 24 federal agencies. To identify and disseminate key information about solutions and processes to help agencies make progress towards data center consolidation goals. Goal or sub-goal Energy Efficiency Entrepreneurship and Small Business Improve Access to Government Information and Services Sub-Goal Interagency group No interagency groups were used to manage efforts related to this goal. Interagency groups were used to manage efforts at the sub-goal level. Senior-level representatives from 24 participating agencies. To oversee strategy, resources and timetables for the development of the BusinessUSA website, resolve interagency issues and ensure department/agency viewpoints are represented. Mid-to-senior level program, technology and customer service managers from 24 participating agencies. To assist the BusinessUSA program management office coordinate the design, development, and operation of the BusinessUSA website, and to track and monitor performance metrics on customer service and outcomes. Commercialization of Federal Research Grants Sub-Goal SBIR/STTR program managers from11 agencies, and coordinating officials from Small Business Administration (SBA) and Office of Science and Technology Policy (OSTP) To discuss the development of SBIR/STTR program policy directives, the implementation of requirements, outreach and access to the programs, and program best practices. Streamlining Pathways for Immigrant Entrepreneurs Sub- Goal To provide updates on relevant agency activities and identify opportunities for interagency collaboration. Small Business Procurement Sub-Goal To share best practices for expanding contracting to small and disadvantaged businesses, and reviewing progress on agency simplified-acquisition threshold goals. To provide officials from the White House, SBA, Commerce, and OMB with an opportunity to meet with senior agency leaders and discuss the steps agencies are taking to increase small business contracting. Goal or sub-goal Exports Membership Principals (cabinet secretaries and deputies) and staff from 20 agencies involved in export policy, service, finance, and oversight. To review progress on deliverables supporting the National Export Strategy, communications, and the status of individual export promotion initiatives. Improper Payments Bi-weekly to monthly To review the status of agency implementation of Do Not Pay requirements and milestones, and guidance for implementation. Officials from agencies with “high-priority” programs, as designated by OMB. To discuss the government- wide improper payment initiative and overall strategy. Job Training To discuss expanding access to job training performance data, and opportunities to promote its use at the local, state, and federal levels. Real Property Among other policy discussions, to discuss the development of agency “Freeze the Footprint” plans. Sustainability To discuss policy to guide the federal government on sustainability issues, and to discuss sustainability goals. Science, Technology, Engineering, and Mathematics (STEM) Education Every 4-6 weeks, during the development of the 5-year strategic plan. To develop a 5-year strategic plan for federal support for STEM education. Strategic Sourcing Representatives from Departments of Defense, Energy, and Veterans Affairs (VA), DHS, HHS, GSA, NASA, and SBA. To discuss the development and adoption of strategic sourcing options. Goal or sub-goal Veteran Career Readiness To review ongoing policy initiatives and opportunities for collaboration between agencies. To develop and implement a redesigned veterans transition program. Appendix IV: Description of Interim Cross- Agency Priority Goal Review Processes Goal Broadband Energy Efficiency meetings with officials from each agency. According to OMB staff, during these reviews participants reviewed metrics from across the agency’s information technology portfolio, which included, in some cases, those related to data center consolidation. Each quarter staff supporting the goal leader would collect updated information on contributing agency priority goals for the purposes of updating the quarterly status update. Each quarter the deputy goal leader would collect updated information on goals and milestones from the leaders of each of 10 sub-goals for the purposes of developing the quarterly status update. The deputy goal leader would follow-up with sub-goal leaders or agency officials, as necessary, to address issues or questions about the status of efforts. The goal leader would then review and approve the quarterly status update. Some sub-goal leaders would hold in-person meetings with officials from contributing agencies to, among other things, review progress on identified goals and milestones. See appendix III for information on interagency groups that were used to manage efforts for four of the sub-goals. Each quarter the goal leader, with the assistance of staff from Commerce and the PIC, would collect updated information on relevant agency metrics and activities for the purposes of updating the quarterly status update. Periodic meetings of the Export Promotion Cabinet/Trade Promotion Coordinating Committee, and its Small Business and other working groups, were also used to discuss the status of export promotion efforts and progress on specific deliverables. Each year OMB would collect and report data on agency improper payment rates. Staff from the OMB Office of Federal Financial Management led monthly meetings with agency representatives to discuss the implementation of the Do Not Pay initiative, which was designed to contribute to the reduction of improper payments. The Department of Treasury, as the agency leading implementation of the Do Not Pay initiative, would track agency progress on implementation milestones. Science, Technology, Engineering, and Mathematics (STEM) Education Each quarter staff from the PIC would collect updated information on progress towards agency milestones, and work with the goal leader on the development of the quarterly status update. After this goal was revised in the second quarter of 2013, a new review process to track agency adherence to the goal was under development by OMB. Twice a year the Council on Environmental Quality (CEQ) would collect and review quantitative and qualitative data on agency progress towards established sustainability goals, including the reduction of agency greenhouse gas emissions. Following the collection of these data, the goal leader hosted meetings of the Steering Committee on Federal Sustainability, which were used to discuss federal sustainability policy and progress on sustainability goals. According to CEQ staff, the goal leader and CEQ staff would meet with representatives from agencies about sustainability issues on an ad hoc basis. In instances where there was a gap between an agency’s actual performance and the target established in that area, the goal leader, or other staff from CEQ, would meet with officials from that agency to discuss ways to address the performance gap. Each quarter the goal leader would collect updated information on agency milestones for inclusion in the quarterly status updates. Progress on some identified strategies to achieve the goal, such as the National Science Foundation’s efforts to improve undergraduate STEM education, were reviewed at the agency level. After progress was reviewed at the agency level the information was passed onto the goal leader and reported publicly in the quarterly status update. Each quarter the General Services Administration would collect data on agency adoption and spending rates for the Federal Strategic Sourcing Initiative (FSSI) solutions for domestic delivery and office supplies. The Strategic Sourcing Leadership Council met bi-monthly to guide the creation and adoption of new FSSI options, and, as part of that effort, might review quarterly data on agency adoption and spending rates. According to the goal leader, each month staff from the Departments of Defense and Veterans Affairs, and the PIC, would provide data for “one-pagers” and other status update documents with key pieces of relevant information, such as the veterans’ unemployment rate and the number of active employers on the Veteran’s Job Bank. These one-pagers would be used to inform regular Interagency Policy Council (IPC) discussions, along with more specific briefing memorandums, which were used to cover the latest issues, keep stakeholders focused on overall outcomes, and to inform discussion around specific outliers. Some of the data in these one-pagers would also be incorporated into the quarterly status updates. More frequently, issue papers and data analysis were provided to Veterans Employment Initiative (VEI) Task Force and IPC members as needed to address topical issues. Ongoing milestone reviews held by the VEI Task Force and its associated working groups on Education, Employment, Transition, and Entrepreneurship, provided an opportunity to discuss strategies being employed to improve performance. Appendix V: Full Text for Interactive Figure 2 on Frequency of Data Reporting for Cross- Agency Priority Goals’ Overall Planned Levels of Performance This appendix includes the print version of the text and rollover graphics contained in interactive figure 2. Overall Planned Level of Performance …achieve 95 percent implementation of the Administration’s priority cybersecurity capabilities by the end of fiscal year 2014. Data reported for primary performance goal …save $3 billion in taxpayer dollars by closing at least 2500 data centers by fiscal year 2015. Data reported for primary performance goal “Agencies have already closed 640 data centers…” Cross-Agency Priority Goal Exports Data reported for primary performance goal Overall Planned Level of Performance Double U.S. exports by the end of 2014. Frequency of Data Reporting for Overall Goal Quarterly …agencies’ strategic sourcing of at least two new commodities or services in both 2013 and 2014, that yield at least a 10 percent savings… In addition, agencies must increase their use of Federal Strategic Sourcing Initiative vehicles by at least 10 percent in both fiscal years 2013 and 2014. Broadband …ensure 4G wireless broadband coverage for 98 percent of Americans by 2016. Cross-Agency Priority Goal Improper Payments Overall Planned Level of Performance …achieve a payment accuracy rate of 97 percent by the end of 2016. Data reported for primary performance goal “Data Not Reported” Entrepreneurship and Small Businesses Increase federal services to entrepreneurs and small businesses with an emphasis on 1) startups and growing firms and 2) underserved markets. Data reported for primary performance goal “Data Not Reported” Job Training Data reported for primary performance goal “Data Not Reported” Real Property The Federal Government will maintain the fiscal year 2012 square footage baseline of its office and warehouse inventory. Data reported for primary performance goal “Data Not Reported” Science, Technology, Engineering, and Math (STEM) Education …increase the number of well-prepared graduates with STEM degrees by one-third over the next 10 years, resulting in an additional 1 million graduates with degrees in STEM subjects. Cross-Agency Priority Goal Data reported for primary performance goal Frequency of Data Reporting for Overall Goal “Data Not Reported” Veteran Career Readiness Data reported for primary performance goal “Data Not Reported” Appendix VI: GAO Contact and Staff Acknowledgments GAO Contact Staff Acknowledgments In addition to the contact named above, Elizabeth Curda (Assistant Director) and Adam Miles supervised the development of this report. Virginia Chanley, Jehan Chase, Steven Putansu, Stacy Ann Spence, and Dan Webb made significant contributions to this report. Deirdre Duffy and Robert Robinson also made key contributions.

The federal government faces complex, high-risk challenges, such as protecting our nation's critical information systems. Effectively managing these challenges is essential for national and economic security and public health and safety. However, responsibility for addressing these challenges often rests with multiple agencies. To effectively address them, shared goals and cross-agency collaboration are fundamental. This report responds to GAO's mandate to evaluate the implementation of GPRAMA. It assesses (1) what is known about progress made towards the interim CAP goals; and (2) how, if at all, quarterly progress reviews reflected GPRAMA requirements and leading practices for reviews, as well as how reviews contributed to improved cross-agency performance and collaboration. To address these objectives, GAO analyzed CAP goal status updates and other documents from OMB and CAP goal progress-review meetings, and interviewed OMB staff and CAP goal representatives. GAO compared this information to GPRAMA requirements and to leading practices for performance reviews previously reported on by GAO. CAP Goal Progress. The GPRA Modernization Act of 2010 (GPRAMA) requires the Office of Management and Budget (OMB) to coordinate with agencies to: (1) establish outcome-oriented, federal government priority goals (known as cross-agency priority, or CAP, goals) with annual and quarterly performance targets and milestones; and (2) report quarterly on a single website now known as Performance.gov the results achieved for each CAP goal compared to the targets. In February 2012, OMB identified 14 interim CAP goals and subsequently published five quarterly updates on the status of the interim CAP goals on Performance.gov. While updates for eight of the goals included data that indicated performance towards an overall planned level of performance, only three also contained annual or quarterly targets that allowed for an assessment of interim progress. Updates for the other six goals did not report on progress towards a planned level of performance because the goals lacked either a quantitative target or the data needed to track progress. The updates on Performance.gov also listed planned activities and milestones contributing to each goal, but some did not include relevant information, including time frames for the completion of specific actions and the status of ongoing efforts. The incomplete information in the updates provided a limited basis for ensuring accountability for the achievement of targets and milestones. OMB Quarterly Progress Reviews. GPRAMA also requires that OMB-with the support of the Performance Improvement Council (PIC)-review CAP goal progress quarterly with goal leaders. OMB instituted processes for reviewing progress on the goals each quarter, which involved the collection of data from goal leaders and the development of a memorandum for the OMB Director. However, the information included in these memorandums was not fully consistent with GPRAMA requirements. For example, GPRAMA requires OMB to identify strategies for improving the performance of goals at risk of not being met, but this was not consistently done. Without this information, OMB leadership and others may not be able to adequately track whether corrective actions are being taken, thereby limiting their ability to hold officials accountable for addressing identified risks and improving performance. Leading Practices for Reviews. At the CAP-goal level, goal leaders for two CAP goals and one sub-goal instituted in-person progress reviews with officials from contributing agencies that were broadly consistent with the full range of leading practices for reviews, such as leadership involvement in reviews of progress on identified goals and milestones, and rigorous follow-up on issues identified through these reviews. In these cases, goal managers reported there were positive effects on performance, accountability, and collaboration. In contrast, review processes used by other goal leaders did not consistently reflect the full range of leading practices. Effective review processes consistently engage leaders and agency officials in efforts to identify and address performance deficiencies, and to ensure accountability for commitments. Thus, not using them may result in missed opportunities to hold meaningful performance discussions, ensure accountability and oversight, and drive performance improvement.

gov_report

Tweet with a location You can add location information to your Tweets, such as your city or precise location, from the web and via third-party applications. You always have the option to delete your Tweet location history. Learn more Image copyright Reuters Image caption Canadian Prime Minister Justin Trudeau called the attempted suicides "heartbreaking" An indigenous community in northern Canada has declared a state of emergency after 11 people attempted to take their own lives in one day. The Attawapiskat First Nation in Ontario saw 28 suicide attempts in March and more than 100 since last September, Canadian media said, with one person reported to have died. Prime Minister Justin Trudeau called the news "heartbreaking". Canada's 1.4 million indigenous people have high levels of poverty. Their life expectancy is also below the Canadian average. Bruce Shisheesh, the chief of the Attawapiskat First Nation community, said 11 people attempted to take their own lives on Saturday, prompting him to declare a state of emergency. He tweeted that the regional Weeneebayko Health Authority was flying in a crisis team, mental health nurses and social workers, and that he was waiting for Ontario's minister of health to deliver more emergency workers to the area. The First Nations government was sending a crisis response unit to the community following the declaration on Saturday, Canadian media said. Image copyright Getty Images Image caption Attawapiskat Chief Theresa Spence held a hunger strike in 2013 over living standards Image copyright Getty Images Image caption The plight of indigenous people has sparked protests in Ottawa Attawapiskat First Nation isolated First Nation tribe in Kenora District, northern Ontario, Canada population around 2,000 on the reserve former chief Theresa Spence had a hunger strike in 2013 to protest over the Canadian government not providing enough money, education and health care for the tribe had a state of emergency in 2011, the third in three years, due to low temperatures and insufficient housing in 2013 accused Stephen Harper's Conservative government of being right-wing and racist The Health Canada federal agency said in a statement it had sent two mental health counsellors as part of that unit. Charlie Angus, the local MP, said: "This is a systemic crisis affecting the communities." "There's just not been a serious response from any level of government until now," he said. "We'll continue to work to improve living conditions for all Indigenous peoples," Prime Minister Trudeau said. Another Canadian aboriginal community in the western province of Manitoba appealed for federal aid last month, citing six suicides in two months and 140 suicide attempts in two weeks. Suicide and self-inflicted injuries are among the top causes of death for First Nations, Métis and Inuit people, according to studies from Health Canada. National Chief of the Assembly of First Nations Perry Bellegarde called for a national strategy to combat indigenous suicide last month. "Immediate support to communities is essential & a #FirstNations driven national strategy on mental health," he tweeted on Monday. More from BBC Magazine BBC reporter Joanna Jolly went on the trail of the murdered and missing to find out why so many of Winnipeg's Aboriginal women and girls have been killed. Read full article A group of First Nations adults and youth finish a spiritual journey from Attawapiskat First Nation to Parliament Hill on Feb. 24, 2014. SEAN KILPATRICK/THE CANADIAN PRESS Where is Attawapiskat? The Attawapiskat First Nation is located at the mouth of the Attawapiskat River on James Bay in Northern Ontario and is home to about 2,000 people. It has been plagued by suicides for decades. How did it start? The suicide epidemic in Attawapiskat started in the fall, when a number of people tried to kill themselves, said Jackie Hookimaw, a member of the community. She said the latest round of youth suicide attempts started with the death of her great-niece Sheridan, who took her life in October. She was 13 years old. What is being done? Chief Bruce Shisheesh and council declared a state of emergency on Saturday. The designation has meant that a Nishnawbe Aski Nation crisis response unit is being sent to the community. The federal and Ontario health ministers said the Weeneebayko Area Health Authority hospital was immediately flying in a crisis team, including mental health nurses and social workers. Meanwhile, Perry Bellegarde, national chief of the Assembly of First Nations, said he has offered Chief Shisheesh his support and is pressing for long-term help for the community in addition to immediate action, adding that the crisis is a "national tragedy." Story continues below advertisement The news from Attawapiskat is heartbreaking. We'll continue to work to improve living conditions for all Indigenous peoples. — Justin Trudeau (@JustinTrudeau) April 10, 2016 The crisis in context 1. Suicide is not just an Attawapiskat problem Previous studies have suggested suicide and self-inflicted injuries are among the leading causes of death among First Nations, Métis and Inuit people. Waves of suicides like the one in Attawapiskat are not new, but several others in recent months have gained renewed attention – such as a string of deaths in northern Manitoba's Pimicikamak Cree Nation, also known as Cross Lake, which has also declared a state of emergency. A grave site for Finola Muswaggon, who died at Cross Lake in March, 2016. She was 14. ANN MARY FOR THE GLOBE AND MAIL Mr. Bellegarde said last month that there should be a national strategy to combat indigenous suicide. "Our young people need hope and inspiration," Mr. Bellegarde said. "They don't see that right now. We've got to make those key strategic interventions now. It's a life-and-death situation." 2. Suicide is not just an on-reserve problem The renewed attention on suicide crises on reserve had led some critics to propose urban resettlement as a solution. Indigenous suicide problems do not disappear in cities, however. A Statistics Canada report released in January found that more than one in five off-reserve First Nations, Métis and Inuit adults reported having suicidal thoughts at some point in their lives. Problems with suicide on reserve can be more acute, though, due to challenges of distance and availability of mental-health services. Charlie Angus, the MP for the Attawapiskat area and NDP indigenous affairs critic, said northern communities aren't given the resources to deal with complicated grief. "When a young person tries to commit suicide in any suburban school, they send in the resources, they send in the emergency team. There's a standard protocol for response. The northern communities are left on their own," he said. "We don't have the mental health service dollars. We don't have the resources." 3. Indigenous suicide is not just a mental-health problem Conditions on reserves lag behind those in the rest of Canada in more respects than just suicide and health: Unemployment, lack of access to education and substandard infrastructure are factors too. Attawapiskat declared a state of emergency five years ago over a housing crisis that James Anaya, then UN Special Rapporteur on indigenous peoples, said "seems to represent the condition of many First Nation communities living on reserves throughout Canada, which is allegedly akin to Third World conditions." Studies have pointed to economic hardships and the legacy of colonialism as key factors in high indigenous suicide rates. The January Statscan study, for instance – which reviewed findings from the 2012 Aboriginal Peoples Survey and the 2012 Canadian Community Health Survey – found a strong connection between residential-school experience and suicidal thoughts. Doing more to prevent suicide was one of the recommendations of last June's report by the Truth and Reconciliation Commission on residential schools. Federal Health Minister Jane Philpott said in a statement regarding the Attawapiskat suicide crisis that improving the wellness of indigenous peoples will require a focus on improving the socioeconomic conditions they face. "I have worked with the province to set up a joint action table so that federal and provincial governments can work together, hand-in-hand with First Nations leadership, to find concrete solutions," Ms. Philpott said. Story continues below advertisement 4. Attawapiskat is more than just a community with problems During some of Attawapiskat's previous moments in the national spotlight – the housing crisis, former chief Theresa Spence's contentious role in the Idle No More movement – Attawapiskat residents have sometimes criticized outside observers for only paying attention during times of crisis. Residents tried to challenge that narrative in 2014 with a 1,000-kilometre trek to Ottawa to urge politicians and First Nations chiefs to renew discussion of treaty rights. Danny Metatawabin, middle, walks with fellow Omushkegowuk Walkers and supporters while marching towards Parliament Hill in Ottawa on Feb. 24, 2014. CHRIS WATTIE/REUTERS Youth from Attawapiskat and neighbouring communities held a healing walk last week to create awareness of the current suicide crisis, Jackie Hookimaw told The Canadian Press. "They said, 'We will not give up, because our youth are killing themselves … We will not be defeated.'" Graphics by Murat Yükselir With report from Evan Annett and Gloria Galloway MORE READING Edit video ‘Something horribly wrong’ with First Nations health: Chief 0:32 The suicide epidemic of Cross Lake: Consider urban resettlement Suicide is at crisis levels in First Nations communities, reflecting the grim realities their young people face, Gary Mason writes. After an increasing number of young people attempted to take their lives in a remote northern Ontario First Nation last week, the chief and council have declared a state of emergency. The suicide epidemic in Attawapiskat started in the fall, when a number of people tried to kill themselves, said Jackie Hookimaw, a resident of First Nation on James Bay. Hookimaw’s great niece Sheridan took her own life in October. She was 13 years old. Hookimaw said Sheridan had a big heart, but she was plagued with multiple health conditions and was bullied at school. More recently, Hookimaw said, she was at the community’s hospital where she saw a number of teenage girls being treated after purposely overdosing on drugs. As she was leaving, a man came in for treatment. Later, she would learn that he, too, had tried to take his own life. There’s different layers of grief The Attawapiskat First Nation, which has been plagued by suicides for decades, is home to about 2,000 people, and Hookimaw said the community needs more resources. She said the latest round of youth suicide attempts started with Sheridan’s death. Sheridan’s peers were grieving, Hookimaw said. They didn’t have the support they needed to manage their grief, so they tried to end their lives, she said. “There’s different layers of grief,” she said. “There’s normal grief, when somebody dies from illness or old age. And there’s complicated grief, where there’s severe trauma, like when somebody commits suicide.” Charlie Angus, the MP for the area and NDP indigenous affairs critic, said northern communities aren’t given the resources to deal with complicated grief. “When a young person tries to commit suicide in any suburban school, they send in the resources, they send in the emergency team. There’s a standard protocol for response. The northern communities are left on their own,” he said. “We don’t have the mental health service dollars. We don’t have the resources.” He said it’s been a “rolling nightmare” of more and more suicide attempts among young people throughout the winter. They didn’t think it could get any worse than it was in March, he said. But April brought even more attempts. We don’t have the mental health service dollars. We don’t have the resources On Saturday alone, 11 people tried to commit suicide, Chief Bruce Shisheesh told the CBC. It’s that situation that led the Attawapiskat chief and council to declare a state of emergency on Saturday. The designation has meant that a Nishnawabe Aski Nation crisis response unit is being sent to the community. On Twitter, Prime Minister Justin Trudeau called the news from Attawapiskat “heartbreaking.” “We’ll continue to work to improve living conditions for all indigenous peoples,” Trudeau tweeted. The federal and Ontario health ministers said the Weeneebayko Area Health Authority hospital was immediately flying in a crisis team, including mental health nurses and social workers. “We will be providing additional health-care experts as needed and we have contacted the ministry of children and youth services about providing emergency life-promotion supports,” Ontario Health Minister Eric Hoskins said in a release. Federal Health Minister Jane Philpott said in a statement that improving the wellness of indigenous peoples will require a focus on improving the socio-economic conditions they face. “I have worked with the province to set up a joint action table so that federal and provincial governments can work together, hand-in-hand with First Nations leadership, to find concrete solutions,” Philpott said. Hoskins said Ontario has asked Ottawa to accelerate the work of the action table and will be identifying additional community programming that can help “give people hope in this terrible time.” Angus said that the work of grief counselling often gets left to untrained community members who are dealing with their own grief. “It’s the local cops, it’s the local teachers, it’s parents,” he said. And now, Hookimaw said, some of the youth are taking healing into their own hands. Youth from Attawapiskat and neighbouring communities held a healing walk last week to create awareness, she said. “They said, ’We will not give up, because our youth are killing themselves … We will not be defeated.”’ Feel like you need help and want to speak with someone? Call the Canadian Association for Suicide Prevention at 204-784-4073, contact a local crisis centre or call Kids Help Phone at 1-800-668-6868 or reach out online at The chief and council for the Attawapiskat First Nation on remote James Bay have declared a state of emergency, saying they're overwhelmed by the number of attempted suicides in the community. On Saturday night alone, 11 people attempted to take their own lives, Chief Bruce Shisheesh said. Shisheesh and the council met Saturday night and unanimously voted to declare the state of emergency. That compels such agencies as the Weeneebayko Health Authority in Moose Factory, Ont., and Health Canada to bring in additional resources. Including Saturday's spate of suicide attempts, a total of 101 people of all ages have tried to kill themselves since September, Shisheesh said, with one person dying. The youngest was 11, the oldest 71. The Cree community — home to about 2,000 residents — saw 28 attempts in March alone. Last September, a group of five girls overdosed and had to be medevaced out of the community, Shisheesh said. I have relatives that have attempted to take their own lives... cousins, friends. - Attawapiskat Chief Bruce Shisheesh Four health-care workers without specialized mental health training are trying to keep up with the attempts and to avert future ones, he said. "I'm asking friends, government, that we need help in our community," Shisheesh said. "I have relatives that have attempted to take their own lives... cousins, friends." The council has hired security to keep an eye on patients currently in hospital. The Mushkegowuk Council, which represents eight northern Ontario First Nations, is also trying to help. "These four workers, crisis workers, are burned out. They can't continue working daily because of the amount of suicides [that] have happened. They're backlogged," said the council's Deputy Grand Chief Rebecca Friday. "There are no services at the moment, no counsellors in the community." A few support workers have been brought in by the Nishnawbe Aski Nation, an association of northern Ontario First Nations, she said, but that's not enough to stem the number of attempted suicides. Government response The federal and Ontario health ministers announced support for Attawapiskat on Sunday evening, saying that the Weeneebayko Area Health Authority hospital was immediately flying in a crisis team, including mental-health nurses and social workers. Ontario also said it would deploy its Health Ministry's Emergency Medical Assistance Team to "get to work immediately" as well as to "determine what further supports are needed for the medium term." "Hearing about the loss of life to suicide and the feelings of despair felt by the community of Attawapiskat reminds us of how important it is to work with First Nations and indigenous peoples across the country to address the very real challenges facing their communities," federal Health Minister Jane Philpott said in a statement. Prime Minister Justin Trudeau also weighed in on the situation Sunday, calling the news from Attawapiskat "heartbreaking." The news from Attawapiskat is heartbreaking. We'll continue to work to improve living conditions for all Indigenous peoples. —@JustinTrudeau Charlie Angus, the region's MP and NDP indigenous affairs critic, echoed others' sentiments that northern communities aren't given the resources to deal with complicated grief. "When a young person tries to commit suicide in any suburban school, they send in the resources, they send in the emergency team. There's a standard protocol for response. The northern communities are left on their own," he said. "We don't have the mental health service dollars. We don't have the resources." Angus said it's been a "rolling nightmare" of more and more suicide attempts among young people throughout the winter. Triggers include overcrowding, abuse As for the triggers, there are many, Shisheesh said. Overcrowding with 14 to 15 people living in one home is difficult, he said. Bullying at school is another trigger. Emotional damage caused by abuse during enrolment at residential schools is having a ripple effect through the generations, he said. (Erik White/CBC ) Drug abuse is another factor, Shisheesh said. People try to numb themselves after physical and sexual abuse. "We have people that are on prescriptions. We have people that are selling pills. And I believe that's how some of them have withdrawals and they feel unwanted, or they don't know how to express their feelings and they have to use a drug to drown their problems or their pains," Shisheesh said. "And when you don't have money to buy drugs, that's when they turn to suicide." As for the near-daily attempts in March, Shisheesh said he had no explanation. He just said he worried every time his phone rang in case it was more bad news. The chief and council are planning to meet Monday to formulate a strategic plan. "We need help in Attawapiskat," Shisheesh said.

A "suicide epidemic" that started last fall in a northern Ontario community-with 11 suicide attempts this past Saturday alone, per the CBC-has led the Attawapiskat First Nation to declare a state of emergency, per the National Post. The remote enclave of 2,000 people has reportedly experienced suicides for decades, but the latest string is so worrisome that the Attawapiskat community is pleading for help. Resident Jackie Hookimaw says the latest string of deaths was spurred by the fall suicide of her 13-year-old great-niece. Chief Bruce Shisheesh tells the CBC that 101 people between the ages of 11 and 71 have attempted suicide since September; one has died. "I'm asking friends, government, that we need help in our community," Shisheesh says. "I have relatives that have attempted to take their own lives." Members of the Attawapiskat community say citizens suffering from drug abuse, overcrowding, and bullying-as well as intense poverty, per the BBC-don't receive enough government help. "When a young person tries to commit suicide in any suburban school, they send in the resources, they send in the emergency team," the area's MP tells the Post, calling the problem a "rolling nightmare" often left to untrained teachers, cops, and parents to handle. "The northern communities are left on their own." There are four health-care workers, but they lack training and are "burned out" and "backlogged," says the deputy grand chief of the Mushkegowuk Council, representing eight Ontario First Nations. On Sunday, federal and Ontario health officials said a crisis team of mental health nurses and social workers, as well as an emergency medical team, was being sent ASAP. Canadian PM Justin Trudeau lent his own moral support Sunday, tweeting, "The news from Attawapiskat is heartbreaking. We'll continue to work to improve living conditions for all Indigenous peoples."

multi_news

In bacteria, laterally acquired genes are often concentrated within chromosomal regions known as genomic islands. Using a recently developed zebrafish infection model, we set out to identify unique factors encoded within genomic islands that contribute to the fitness and virulence of a reference urosepsis isolate—extraintestinal pathogenic Escherichia coli strain CFT073. By screening a series of deletion mutants, we discovered a previously uncharacterized gene, neaT, that is conditionally required by the pathogen during systemic infections. In vitro assays indicate that neaT can limit bacterial interactions with host phagocytes and alter the aggregative properties of CFT073. The neaT gene is localized within an integrated P2-like bacteriophage in CFT073, but was rarely found within other proteobacterial genomes. Sequence-based analyses revealed that neaT homologues are present, but discordantly conserved, within a phyletically diverse set of bacterial species. In CFT073, neaT appears to be unameliorated, having an exceptionally A+T-rich composition along with a notably altered codon bias. These data suggest that neaT was recently brought into the proteobacterial pan-genome from an extra-phyletic source. Interestingly, even in G+C-poor genomes, as found within the Firmicutes lineage, neaT-like genes are often unameliorated. Sequence-level features of neaT homologues challenge the common supposition that the A+T-rich nature of many recently acquired genes reflects the nucleotide composition of their genomes of origin. In total, these findings highlight the complexity of the evolutionary forces that can affect the acquisition, utilization, and assimilation of rare genes that promote the niche-dependent fitness and virulence of a bacterial pathogen. As a species, Escherichia coli is best known for colonizing the lower intestine of humans and other warm-blooded vertebrates [1], [2]. The contingent exit from the intestinal tract presents strains of E. coli with a multitude of secondary habitats, including host-associated and free-living niches [2], [3], [4], [5], [6]. A subset of E. coli designated extraintestinal pathogenic E. coli (ExPEC) excels at colonizing host-associated extraintestinal environments, resulting in an array of human diseases including urinary tract infections, bacteremia, and meningitis [7]. ExPEC strains also exhibit an impressive zoonotic capacity, being able to persist and cause disease in a variety of domesticated animals [8], [9], [10], [11]. Collectively, ExPEC-related diseases represent daunting medical, agricultural, and economic burdens that threaten to worsen as antibiotic-resistant strains become more prevalent [8], [12], [13]. The evolutionary forces that underlie the emergence and niche tropisms of ExPEC have yet to be completely defined. Considering gene content, substantial intra-specific variation often exists between bacterial isolates, particularly among strains of pathogenic E. coli. Key questions regarding the origin of this heterogeneity and its impact on the fitness of virulent strains remain unanswered. Bacteria are proficient at rapidly developing innovative, selectable traits to maintain fitness within complex environments—a property known as ‘evolvability’ [14], [15], [16], [17], [18]. Despite being largely asexual organisms that multiply by binary fission, bacteria engage in a genetically promiscuous behavior known as ‘lateral gene transfer’ (LGT). Laterally acquired genes can provide context-specific functions, such as the ability to metabolize atypical substrates [19], adhere to a variety of surfaces [7], neutralize antibiotics and other toxic compounds [5], or participate in niche construction [20]. Bacteria have several means of obtaining potentially beneficial elements through LGT: direct acquisition from the environment (transformation), transfer through cell-to-cell mating (conjugation), and acquisition from bacterial viruses known as bacteriophages (transduction) [14], [21], [22], [23], [24]. It has been estimated that ∼81% of all genes within a bacterial chromosome have been involved in LGT at some point, suggesting that this behavior is not just an anomalous event, but that over time it is a foundational component of bacterial evolution [25]. The genomes of E. coli are laden with the signatures of past LGT events. Since the first genome sequencing projects it has been apparent that E. coli chromosomes are highly mosaic [26], [27], [28]. In part, this chromosomal architecture results from the presence of ‘genomic islands’ (GI) that intermittently disrupt synteny [29], [30], [31], [32], [33], [34]. Many GIs exhibit clear signs of having been involved in past LGT events as they are often in proximity to mobile elements, such as transposons, or are themselves integrated phages or plasmids [35]. Accompanying this interchangeable chromosomal arrangement is a vast superset of genes defined as the pan-genome [32], [36], [37]. Whereas an average E. coli genome contains about 4,700 genes, the pan-genome of this species is estimated to be over 17,000 genes. Most E. coli strains share a subset of the pan-genome, which encodes vertically inherited genes that dictate the fundamental cellular properties of the lineage. This core genome surprisingly accounts for only 40–50% of the genetic makeup of any particular isolate. The rest of the chromosome contains strain-specific combinations of genes that are infused throughout the core genome and encode a variety of accessory functions that can provide unique selective advantages [32], [37], [38]. With this information in mind, we systematically screened GIs of a urosepsis ExPEC isolate for laterally acquired genes that affect virulence in a surrogate zebrafish host model. We identified a novel gene—designated neaT (nomadically evolved acyltransferase) —that is required during blood-borne, but not localized, infections in both zebrafish and mice. The neaT locus was unexpectedly rare in the genomes of closely related E. coli strains and other Proteobacteria, suggesting that it was obtained from outside the contemporary E. coli pan-genome. Proteobacterial neaT homologues, in general, exhibit a high degree of allelic variance, have reduced guanine and cytosine (G+C) content, and are often localized within the integrated genomes of unrelated bacteriophages. These observations indicate that neaT-like alleles may have been recently acquired on multiple occasions by the proteobacterial supraspecies pan-genome. Together, our results provide molecular and bioinformatic evidence that the acquisition of unique genes like neaT during the evolution of particular ExPEC isolates can significantly impact bacterial fitness and virulence within specific host environments. Possible evolutionary forces that generate the observed sequence-level features of neaT and the role that bacterial individuality plays in pathogenesis are considered. The ExPEC strain CFT073 was isolated from the blood of a human patient with acute pyelonephritis (kidney infection) [26], [39]. This urosepsis isolate is versatile, with the apparent ability to traverse several host microenvironments to reach the bloodstream, and has a relatively large genome of 5,369 protein-coding genes and several GIs. In previous work, we found that CFT073 is exceptionally lethal in an infection model that uses zebrafish embryos as surrogate hosts for the high-throughput analysis of ExPEC virulence [40]. At 48 h post-fertilization (hpf), zebrafish possess an innate immune system composed primarily of phagocytic cells and antimicrobial peptides [41], [42], [43], [44]. These defenses mirror those employed by mammalian hosts to combat ExPEC. To identify GI-associated virulence factors carried by CFT073, we screened 11 previously described deletion mutants that each lack a specific GI (Table 1 and Figure 1A) [45]. In blinded assays, 48 hpf zebrafish embryos were infected with 1,000 to 2,000 colony-forming units (CFU) of either wild type CFT073 or one of the 11 GI mutants. Bacteria were delivered into one of two injection sites: the fluid-filled sac surrounding the heart referred to as the pericardial cavity (PC), which mimics a localized tissue infection, and the circulation valley (CV), which facilitates rapid dispersal of bacteria into the bloodstream [40]. Each of these sites likely challenges the pathogen with different nutrient limitations, receptor availability, and host defenses. In this infection model, increased growth of ExPEC is associated with decreased survival of the host [40]. All 11 GI mutants, with the exception of ΔGI-aspV, grew equally well in broth culture at 28. 5°C and 37°C (data not shown). Following inoculation into the PC, only deletion of the 123 kb GI PAI-pheV [I] resulted in a significant decrease in virulence relative to wild type CFT073 (Figure 1B, top). This was not surprising as PAI-pheV [I] encodes the notable ExPEC-associated virulence factors α-hemolysin (pore-forming toxin), SAT (vacuolating toxin), P pili (adhesive organelles), aerobactin (iron acquisition system), and K2 capsule (immune evasion). The ability of the ΔPAI-pheV mutant to still kill approximately half of the embryos suggests that additional factors with overlapping roles in virulence within the PC are encoded outside of PAI-pheV and the 10 other GIs tested. The ΔPAI-pheV mutant was also attenuated following inoculation of the CV to initiate systemic infection, as were the GI mutants ΔGI-selC, ΔGI-cobU, and Δφb0847 (Figure 1B, bottom). In addition to several hypothetical genes, the selC and cobU islands harbor genes that appear to be components of polyamine and iron transport systems, respectively. Both polyamines and iron acquisition systems are known to be important mediators of ExPEC fitness in mouse models of infection [46], [47], [48]. Although the ΔGI-cobU mutant exhibited only a modest reduction in virulence in these assays using inoculation doses of 1,000–2,000 CFU/embryo, with slightly higher doses between 2,000 to 3,000 CFU/embryo this mutant displayed more dramatic and significant (p<0. 05) attenuation (Table S1). This observation supports previous findings indicating that the inoculation dose can markedly influence the discernibility of some mutant phenotypes in the zebrafish host [40]. The remaining GI showing a phenotype in our screen is composed of an intact integrated phage genome (prophage) named ‘φb0847’ (Figure 1B) [45]. This prophage is 33 kb in length and contains 48 predicted open reading frames, most of which encode recognizable phage proteins that share homology with genes of tailed phages belonging to the order Caudovirales. More specifically, φb0847 carries genes involved in regulation, replication, and virion assembly that are related to and syntenic with the genes of phage P2 and its relatives (Figure 2). From this analysis, it is clear that the φb0847 prophage is a member of the P2-like phage group and likely represents a fully functional phage genome complete with all the essential genes associated with P2-like phages [49]. Aside from the ΔpheV mutant, with its fairly well characterized assortment of virulence genes, Δφb0847 displayed the most pronounced defect of the island mutants examined. Therefore, the φb0847 GI became the primary focus of our investigation. To further define the contribution of φb0847 to the virulence and fitness of CFT073, we carried out competitive assays in which a one-to-one mixture of wild type and mutant bacteria were injected into the CV of zebrafish embryos (Figure 1C). At the indicated time points, the infected embryos were homogenized and bacteria present were enumerated by dilution plating on selective agar. Δφb0847 carries a kanamycin resistance cassette that was used to distinguish wild type and mutant strains. No differences between wild type CFT073 and the Δφb0847 mutant were observed until 6 h post-inoculation (hpi), when Δφb0847 titers began to decline (Figure 1C). These results indicate that the φb0847 island is dispensable during initial stages of a systemic infection, but enhances bacterial fitness at later time points, coordinate with the upregulation of host inflammatory responses engage. The Δφb0847 mutant displayed more modest, though still significant, decreases in fitness during competitive assays against wild type CFT073 within the PC and yolk sac at 18 hpi (Figure 1D). Phagocytes are recruited into the PC en masse in response to infection with ExPEC [40], possibly contributing to the competitive disadvantage of the Δφb0847 mutant within this niche. On the other hand, the yolk is a rich source of nutrients for bacteria and is mostly free of phagocytes and other immunosurveillance mechanisms. However, the yolk does contain maternally inherited antimicrobial compounds that could account for the slight reduction in fitness of Δφb0847 within this host environment [50]. Competitive experiments in broth culture did not reveal appreciable differences between the wild type and mutant strains (data not shown). To identify genes within φb0847 that, when deleted, recapitulate the attenuated phenotypes of Δφb0847, we constructed partial deletion mutants lacking one of three nearly equal-sized regions of the prophage island (designated Δ1–2, Δ2–3, and Δ3–4, as indicated along the φb0847 genome in Figure 2). In competitive assays, the Δ1–2 and Δ2–3 mutants were significantly more fit than the full Δφb0847 mutant at 12 hpi (Figure 3A). Analysis at 12 hpi allowed time for selection to take place, while limiting artifacts due to bacterial replication at later time points in dead and dying hosts where selective pressures are presumably weaker. In these assays, only the Δ3–4 mutant phenocopied the complete φb0847 island deletion mutant (Figure 3A). Lethality of this mutant variant was also significantly reduced in comparison to wild type CFT073 and the Δ1–2 and Δ2–3 mutants in independent challenges (Figure 3B). These results indicate that one or more genes within the terminal 3–4 region of the φb0847 prophage enhances both the fitness and virulence of CFT073 during systemic infections within the zebrafish host. Temperate prophage genomes like φb0847 can carry ‘lysogenic conversion’ genes that affect the bacterial host but are not essential for lytic phage growth. To avoid disruption of critical phage processes, the integration of this genetic material is generally tolerated only in certain regions of the prophage genome. These added sequences are known as ‘morons’, because bacteriophages with such insertions have more DNA [51], [52]. Moron genes typically contain their own regulatory elements and vary among individual phage genomes. They often alter the surface structure or physiology of the bacterial host and can benefit the phage by making its host refractory to competing parasites or otherwise promoting bacterial survival and growth [52], [53]. The P2-like phages appear to have at least two variable moron positions (Figure 2). Using phage P2 as a reference, the location of moron position 1 is between the DNA replication gene A and head assembly gene Q, and moron position 2 is between the tail fiber gene G and tail sheath gene FI (Figure 2) [49]. In φb0847 within CFT073, the second moron site, which is absent from the Δ3–4 mutant, contains one open reading frame that is oriented in the opposite transcriptional direction to the flanking tail genes. This gene, which we named neaT for reasons described later, encodes a putative acyltransferase (Pfam: PF01757). This gene is not conserved among P2-like phages and is likely not critical for lytic replication of φb0847. In light of this information, neaT, the immediately proximal gene yfdK (homologous to P2 phage tail gene G), and the collection of distal tail genes (FI through D) were individually deleted from the φb0847 prophage in CFT073. All three mutant derivatives—ΔyfdK, ΔneaT, and ΔFI-D—were attenuated in their ability to kill zebrafish embryos after injection into the blood via the CV (Figure 3C). Despite the significantly reduced virulence of these mutants, no defects in fitness were observed in competitive assays with wild type CFT073 (data not shown). The lack of any discernable fitness defects in competition assays may 1) reflect the ability of wild type CFT073 to trans-complement the mutant strains in vivo and/or 2) indicate that there is cooperative interplay among the yfdK, neaT, and FI-D loci. Of note, disruption of loci flanking neaT did not appreciably alter its expression in broth culture (Figure S1). Furthermore, we found no evidence that the neaT mutant could be complemented in vivo during competition assays by acquiring φb0847 sequences from the wild type strain (Figure S2). Interestingly, a yfdK homologue was recently shown to aid the survival of a K-12 laboratory strain of E. coli in acidic environments [54], but no mechanism for this effect is known, and to the authors' knowledge, yfdK homologues have not been implicated in pathogenesis. The in vivo assays presented in Figure 3C and bioinformatic analyses described below highlight neaT as a gene of potential importance to the fitness and virulence of CFT073. To test this possibility, the neaT locus, including an upstream promoter region of 211 bp, was amplified from the CFT073 chromosome and cloned into the high-retention plasmid pGEN-mcs, yielding pGEN-neaTPnative. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) indicated that neaT transcript levels made from the pGEN-neaTPnative vector in broth culture were about 1. 7-fold higher than those observed in wild type CFT073 (Figure S3). Complementation experiments were performed comparing the lethality of wild type CFT073/pGEN-mcs, Δφb0847/pGEN-mcs, and Δφb0847/pGEN-neaTPnative in zebrafish embryos after inoculation of the CV (Figure 3D). The complete prophage deletion mutant Δφb0847 carrying the empty vector pGEN-mcs exhibited a significant delay in killing relative to either the wild type strain CFT073/pGEN-mcs or the complemented mutant Δφb0847/pGEN-neaTPnative. In total, these experiments identify neaT as a virulence determinant contained within the φb0847 island of CFT073; therefore, the uncharacterized neaT gene became the focal point for the remainder of our investigation. To extend our observations made using zebrafish, we employed a murine model to further define the requirement for neaT during localized and systemic infections. For localized challenges, we took advantage of a well-characterized mouse model of urinary tract infection. Wild type CFT073 and the ΔneaT mutant were mixed at a one-to-one ratio and inoculated via transurethral catheterization into adult female CBA/J mice. After 3 days, animals were sacrificed and bacterial titers within the bladders and kidneys were enumerated, revealing no outright competitive advantage for wild type CFT073 over the ΔneaT mutant in either organ (Figure 4A). To appraise the requirement for neaT during systemic infections, we utilized a recently described sub-lethal bacteremia model in which CBA/J mice were injected with a one-to-one mixture of wild type and mutant bacteria via the tail vein [55]. At 24 hpi the ΔneaT mutant was recovered at significantly reduced levels from the spleen and liver compared to wild type CFT073 (Figure 4B). These results confirm and extend our findings in the zebrafish host, demonstrating that neaT provides niche-specific advantages to CFT073 during systemic infections. There are no closely related homologues of NeaT in E. coli. Only four matches were found in the current NCBI collection of 170 RefSeq E. coli genomes (as of June 2012) that produce an alignment E value<10−6 with similarity over >50% of the NeaT protein length. A PCR-based survey for the presence of neaT within various clinical E. coli isolates corroborated our in silico observation that neaT is rare among this taxon (Figure S4). Out of 21 randomly chosen isolates, none carried the CFT073 neaT allele. Homologues of neaT are also rarely detected in P2-like phage genomes; among 45 randomly chosen P2-like phages and prophages in E. coli, Salmonella, Shigella, and Enterobacter that we examined, only φb0847 carries a neaT-like gene. The closest match to NeaT in the NCBI database is encoded by locus Ent638_2581 of Enterobacter sp. 638, whose protein product is only about 33% identical to NeaT. We note that several more distantly related neaT homologues are present in the genomes of other temperate phages and prophages of the bacterial family Enterobacteriaceae (Table 2). They are found, for example, in the Shigella flexneri phage Sf6 genome and several uncharacterized prophages of S. flexneri, in E. coli phage φV10 and a nearly identical prophage in the Shiga toxin-producing E. coli isolate DEC4D, and in a putative prophage within Citrobacter rodentium strain ICC168. The above Enterobacter homologue Ent638_2581 is also carried within a putative prophage that is similar to Shigella phage SfV. Each of these phage-associated neaT homologues is un-ameliorated with respect to its bacterial host genome (see below), and each lies within a known moron position in its phage genome. Because neaT homologues differ substantially in sequence conservation and are found in a variety of tailed-phages, neaT-like genes may have been laterally acquired by Enterobacteriaceae lineages on several occasions, possibly via phage. Multiple neaT acquisition events would indicate that this gene has an underlying evolutionary importance to either the phages themselves or their hosts. In considering its putative function (see Figures S5, S6, and Text S1), its apparent lateral acquisition, and its allelic variance within the proteobacterial lineage, this gene was named ‘neaT’—nomadically evolved acyltransferase. In the following sections we explore the evolutionary history of this gene by analyzing the diversity and distribution of neaT-like genes in more detail. To investigate the evolutionary source of E. coli neaT genes, we assessed the phyletic distribution of its homologues. BLASTp alignments were performed on the publically available NCBI database using NeaT from CFT073 as a probe for the search sets of Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, Spirochaetes, and Fusobacteria [56]. Sequences were declared to be homologous if they had an alignment significance (E value) of <10−6 over >50% of their lengths [57]. These searches retrieved a total of 317 non-paralogous NeaT-like sequences. The distribution of phyla containing these sequences is depicted in Figure 5A (left), revealing that the majority of neaT homologues are from the Firmicutes and Bacteroidetes rather than Proteobacteria. In an attempt to control for the inherent bias of NCBI databases, we plotted the number of available gene sequences for each phylum represented in Figure 5A (right). This plot demonstrates that the high number of neaT homologues identified among non-proteobacterial phyla is not due to a skew in sequence abundances. To the contrary, total proteobacterial gene sequences overshadow those from other phyla and therefore underscore the relative rarity of neaT alleles in this taxon. To quantify the phyletic distribution of NeaT homologues with greater statistical confidence, we performed bi-directional alignments of NeaT using BLASTp with a manually assembled database of open reading frames from a representative, yet broad, assortment of 165 phylogenetically classified bacterial genomes and associated plasmids obtained from NCBI (Table S3). This analysis confirmed that, compared to random chance, neaT homologues are significantly enriched among species belonging to the phyla Firmicutes and Bacteroidetes (Figure 5B). Moreover, many of the neaT homologues were detected in notable plant and animal pathogens, including Erwinia spp., Bacillus spp., Staphylococcus aureus, Streptococcus oralis, Clostridium botulinum, and Porphyromonas spp. Results from similar alignments of neaT and all other φb0847-encoded genes are presented graphically in Figure 5C. For each prophage gene, the number of non-paralogous matches found in the custom database are represented as bars (upper axis) and the percent of those hits that are harbored within proteobacterial genomes (lower axis) are plotted against the position of the gene within φb0847 (x-axis). Given the host range of known P2-like phages, it is not unexpected that the majority of genes within φb0847 were exclusive to the proteobacterial phylum. Exceptions, in addition to neaT, include homologues of φb0847 genes encoding the phage integrase and a Dam methylase. However, neaT is unique among the φb0847 prophage genes in that over 75% of its matches (16 of 21) were from outside the Proteobacteria (Figure 5C and Table S4). The discordant conservation of neaT highlights its likely extra-phyletic origin. If a gene has origins outside its immediate genome, it would carry sequence-level vestiges of its previous host until it adopts the characteristics of the current host—a process known as ‘amelioration’ [27], [58]. Commonly used parameters that distinguish laterally transferred and unameliorated genes are atypical codon usage and guanine-cytosine (G+C) content [57], [59], [60]. We analyzed these features of neaT in the context of the CFT073 genome and φb0847 prophage. Using all 5,369 protein-coding genes of CFT073, the frequency with which specific codons are used for each amino acid was calculated (Table S5). Each gene was then assigned a ‘codon deviation score’ representing how often it uses atypical codons (Methods and Table S6). Scoring correlates with conformity; genes scoring low have a more typical codon usage, whereas poorly conformed genes score high. This analysis shows that neaT possesses a significantly abnormal codon usage compared to the rest of the CFT073 genome (p = 0. 0260) (Figure 6A, left panel). The neaT gene was also observed to be G+C-poor (29. 84%), making it a significant outlier from the CFT073 genome-wide median of 51. 5% (p = 0. 0001) (Figure 6A, right panel). We also analyzed the codon deviation score (Figure 6B, upper axis) and nucleotide composition (Figure 6B, lower axis) of neaT with respect to the genome of φb0847. Most genes withinφb0847 conform to the codon usage and G+C content of CFT073. This is expected for a parasite that has been co-evolving with proteobacterial hosts over an extensive period of evolutionary time [59]. Thus, the aberrant codon usage and nucleotide composition of neaT is not simply an inherited trait of φb0847. Because of its relatively low G+C content and poorly conformed codon usage, we conclude that neaT is a relatively recent acquisition by both φb0847 and the genome of CFT073. To determine if the apparently unameliorated state of neaT in CFT073 is unique or if it is hinting at a more widespread phenomenon, we plotted the G+C content of a representative subset of neaT homologues identified in Figure 5 against the G+C content of their respective genomes (Figure 6C). As a control, we also plotted the G+C content of poxB, which encodes the metabolic enzyme pyruvate oxidase and exists in an ameliorated state within several phyla (Figure 6C, inset). Most proteobacterial neaT genes were significantly less ameliorated than those found in the genomes of Bacteroidetes and many Firmicutes (Figure 6C and D). Interestingly, even though Firmicutes genomes generally have a low G+C content, neaT-like genes within this lineage are still relatively G+C-poor, at least in a major fraction of Firmicute species (Figure 6C). Cumulatively, these results indicate that, at least among the three phyla compared here, neaT-like genes have likely been associated with Bacteroidetes the longest, whereas acquisition by the Proteobacteria was a more recent event. Presented here are the results from a screen conducted using the ExPEC isolate CFT073 and a high-throughput zebrafish surrogate host model of infection. We screened GIs for novel virulence genes, which were expected to have a history of lateral gene transfer. Three loci within the P2-like prophage φb0847 were found to contribute to the virulence of CFT073 during systemic infection. A previously uncharacterized gene—designated here as neaT—was discovered to augment the virulence capacity of CFT073, independent of other prophage components (Figure 3D). We demonstrated that neaT is conditionally required for maximal fitness during bacteremic infections of both zebrafish and mice, suggesting that CFT073 has potentially co-opted this phage-borne gene for specific virulence behaviors. By tracing the evolutionary history of the neaT gene, we found that it is relatively rare and has sequence-based features suggesting that it was recently absorbed into the proteobacterial supraspecies pan-genome. Signs of its novelty are typified by high allelic variance—possibly a result of multiple entries into the Proteobacteria lineage via phage—and its mostly unameliorated state within proteobacterial genomes. We also investigated the putative function (s) of NeaT in vitro. The NeaT protein shares homology with several characterized acyltransferases encoded within a variety of non-E. coli genomes. These putative membrane-localized enzymes can modify components of the bacterial cell wall, particularly peptidoglycan [61], [62], [63], [64]. Alteration of this macromolecule can often provide bacterial pathogens with protection from host antimicrobial peptides and enzymes such as lysozyme. However, deletion of neaT had no effect on the sensitivity of CFT073 to lysozyme, the antimicrobial cationic peptide polymyxin B, or antibacterial factors present in human serum (see accompanying supplemental Text S1). Interestingly, expression of neaT did alter the behavior of CFT073 in swarming assays and induced bacterial aggregation on swim plates (Figure S5A–C, Text S1) —phenotypes that may be attributable to NeaT-mediated modification of components within the bacterial envelope. We also found that expression of recombinant NeaT can inhibit production of surface structures like curli and cellulose in some strain backgrounds (Fig. S5D–E, Text S1), supporting the notion that NeaT can affect salient properties of the bacterial surface and thereby alter bacterial group behavior. The apparent capacity of NeaT to modulate bacterial aggregation (Fig. S5C) is especially intriguing in light of a recent work demonstrating that aggregate formation can promote bacterial survival within the bloodstream of infected mice [65]. Building on these observations, we found that expression of the neaT gene from a low copy number plasmid significantly decreased the capacity of CFT073 to associate with murine macrophages, suggesting that NeaT serves as an immune evasion factor (Fig. S6). The specific mechanism (s) by which NeaT promotes bacterial fitness during systemic infections, as well as the environmental cues that control neaT expression, require further investigation. As it stands, this work contributes to the idea that ExPEC isolates do not all share the same set of virulence factors, which are likely dictated by the distinct evolutionary trajectory and particular niche tropism of each strain. Our analysis defines neaT as a recently acquired locus of the Proteobacteria. Evidence for this is drawn from its discordant conservation, abnormal codon usage, and low G+C content. In large part, the unameliorated state of neaT-like genes in Proteobacteria and Firmicutes genomes suggests that there is a general phenomenon accounting for its relative A+T-rich composition beyond having originated in an A+T-rich genome, as previously suggested [59]. We posit that the observed A+T-richness of laterally transferred genes can be, to some extent, accounted for by an ‘exploratory mechanism’ [16]. Upon introgression of a foreign gene, its retention depends on its adaption to the host' s genetic and cellular machinery, a process that can take several millions of years [66]. During this time the gene may fall under relaxed selection whereby mutations accrue until a beneficial allele is ‘discovered’ and acted upon by selection. Connecting relaxed selection to reduced G+C content is the observation that there is a universal mutation bias for G/C to A/T transitions in bacterial genomes [67], [68], [69]. It then follows that immediately after a gene is acquired, it will initially accumulate A+T-rich character until a selectable version is ameliorated. From the findings presented here, we speculate that the neaT variant in CFT073 is an example of a newfound allele that is being used to promote bacterial fitness in pathogenic contexts. Arguably, neaT may represent an ancient gene that has simply failed to fix within the proteobacterial lineage. Therefore, an alternative hypothesis is that the conditional requirement for neaT by CFT073 within different environments may have driven its current evolved state. We observed in two vertebrate model systems that neaT contributes significantly to pathogen fitness primarily during systemic infections. Considering the ecology of many bacterial pathogens, a question often left unaddressed is: what are the evolutionary forces that act on niche-specific genes in the absence of selective pressure? Particularly for E. coli, which has a complex multi-niche life cycle, the evolutionary consequences resulting from time outside selective environments on genes like neaT are not clear. Work directly addressing this question is scarce. However, insight into this issue is provided by findings that genes under relaxed constraint have increased variance at the sequence level [17], [70], [71], [72], [73]. In contrast to relaxed selection, which occurs when purifying selection is alleviated, as discussed above, ‘relaxed constraint’ refers to a limitation in the exposure of a particular gene to selection. For example, eukaryotic genes with expression patterns that are sex-restricted are effectively ‘hidden’ from selection in half of the population. This is the case for the Drosophila spp. maternal-effect gene bicoid, which is maternally-restricted and critical for the embryonic development of fruit flies [70]. The bicoid gene was found to have a 2-fold higher heterozygosity compared to zygotically-expressed genes. Similarly, genes with caste-biased expression (i. e., queen versus worker) in the social insects Solenopsis invicta (fire ant) and Apis mellifera (honey bee) were shown to be evolving more rapidly than genes expressed among all castes [71]. For both of these situations, the higher mutation rate observed for contextually expressed genes was concluded to be due to relaxed constraint. Further investigation into the exploratory mechanism and relaxed constraint hypotheses of neaT evolution is required and must be considered in parallel with other processes and factors, including, for example, the susceptibility of laterally transferred genes to endogenous restriction enzymes [59]. There exists an enormous amount of genetic heterogeneity among Eubacteria lineages. Genome sequencing and bioinformatic analyses have underscored this extensively. Perhaps the most intriguing aspect of this diversity is that even closely related members of the same species can differ greatly with respect to their gene contents. Strikingly, any two E. coli genomes can differ by up to 20–30% of their respective gene contents—in sharp contrast to the relatively minor difference of 1% that exists between, for example, the mouse and human species [32], [74]. Decades worth of epidemiological and experimental studies have focused on the identification of genes that define the pathogenic behavior of ExPEC [7], [32]. However, it appears that a single, ubiquitous genetic identifier of ExPEC, such as a gene encoding a particular toxin or adhesin, does not exist and, rather, what actually binds these pathogens is more qualitative and multigenic in nature [32], [75]. In support of this view, we recently demonstrated that the toxin α-hemolysin, shared among many ExPEC isolates, is differentially required for virulence depending on strain background [40]. Similarly, we found that the pathogenicity of particular ExPEC isolates depends on another toxin, cytotoxic necrotizing factor, while other equally virulent strains naturally lack this gene. Coupled with the work presented here, these observations suggest that there exists a spectrum of only partially overlapping virulence gene requirements among ExPEC, reflecting the idea that these pathogens have emerged from distinct evolutionary trajectories driven by LGT [76], [77]. Accordingly, we found that the expression of NeaT from plasmid pGEN-neaTPnative in other E. coli strains, including Nissle 1917 (gut isolate), F11 (cystitis isolate), and S88 (meningitis isolate), did not augment virulence in the zebrafish infection model (data not shown). These findings suggest that the ability of a rare gene like neaT to affect fitness and virulence is dependent upon the genetic background of individual bacterial strains. The beneficial effects of neaT, and its potential to sweep through bacterial populations, is therefore likely linked to the presence, or coordinate acquisition, of other as-yet undefined bacterial factor (s). The identification, characterization, and continued monitoring of rare genes like neaT will be important to our understanding of ExPEC evolution. As a case in point, we note that the sasX gene, originally defined as rare among strains of methicillin resistant Staphylococcus aureus (MRSA), increased in prevalence among MRSA isolates between 2003 and 2011 and is now considered an emerging virulence determinant [78]. Interestingly, like neaT, sasX is also maintained within a prophage and can affect bacterial interactions with phagocytes. At this point, it is difficult to predict if neaT will sweep ExPEC populations in the future, but work presented here along with recent findings concerning sasX underscore how laterally acquired genes can alter the virulence potential of bacterial pathogens, continually challenging the development of broad spectrum therapeutics. Going forward, as we continue to characterize the composition of pan-genomic elements of ExPEC and other pathogens, it will be important to consider the evolutionary context of their virulence genes. Identifying the spatial and temporal parameters that govern the lateral acquisition of virulence genes from distant lineages will need to be reconciled. Genome compatibility (codon and tRNA usage) and ecology are thought to be influential in the success of LGT events between bacteria [79], [80], [81]. In light of this, several interesting questions arise. How did neaT come to be in the proteobacterial gene pool? How does residence of neaT within a prophage impact its evolution? What conditions fostered the assimilation of neaT into the virulence regulon of its host? Using neaT as a stepping-stone, it will be informative to resolve the amount of strain-specific innovation that goes into producing and fine-tuning pathogen genomes. By understanding the mechanisms of chromosome assembly and the sources of individual genetic components, unrealized patterns may emerge that could prove useful for future diagnostics and disease mitigation. Animals used in this study were handled in accordance with IACUC protocols approved at either the University of Utah or the University of Michigan Medical School following standard guidelines as described at www. zfin. org and in the Guide for the Care and Use of Laboratory Animals, 8th Edition [55], [82]. All bacterial strains and plasmids used in this study are listed in Table 3. Unless specified otherwise, bacteria were cultured statically at 37°C for 24 h in 20 ml of a defined M9 minimal medium (6 g/l Na2HPO4,3 g/l KH2PO4,1 g/l NH4Cl, 0. 5 g/l NaCl, 1 mM MgSO4,0. 1 mM CaCl2,0. 1% glucose, 0. 0025% nicotinic acid, 0. 2% casein amino acids, and 16. 5 mg/ml thiamine in H2O). Antibiotics (kanamycin or ampicillin) were added to the growth medium when necessary to maintain recombinant plasmids or select for mutants. Targeted gene knockouts were generated in the ExPEC isolate CFT073 using the lambda Red-mediated linear transformation system [83], [84]. Briefly, a kanamycin resistance cassette was amplified using polymerase chain reaction (PCR) from pKD4 with 40-base pair overhangs specific to the 5′ and 3′ ends of each targeted locus. PCR products were introduced via electroporation into CFT073 carrying pKM208, which encodes an IPTG (isopropyl-β-D-thiogalactopyranoside) -inducible lambda red recombinase. Knockouts were confirmed by PCR. Primer sets used are listed in Table S8. Cloning and construction of neaT expression constructs were done using standard molecular techniques employing the high-retention plasmid pGEN-mcs [85]. For native regulation, neaT (locus tag: c0970), plus 211 bp of upstream sequences, were amplified from the chromosome of CFT073 and TA-cloned into pCR2. 1-TOPO vector per manufacture' s protocol (Invitrogen). Subsequently, the cloned fragment was isolated using BamHI and NotI restriction enzymes (New England Biosciences) and ligated into pGEN-mcs using the same sites, yielding pGEN-neaTPnative. For construction of pGEN-neaTPlac, a synthetic ribosome binding sequence was introduced upstream of neaT within the 5′ PCR primer, and the resulting PCR product was ligated via an engineered NdeI restriction site with the lac promoter amplified from pGFPmut3. 1 (Clonetech). The ligated Plac-neaT product was amplified and TA-cloned into the pCR2. 1-TOPO vector. Using BamHI and NcoI restrictions sites, the Plac controlled neaT variant was then sub-cloned into pGEN-mcs. All experiments involving pGEN-neaTPlac were performed without IPTG induction. Primer sequences used to generate these plasmids are listed in Table S8. *AB wild-type zebrafish embryos were collected from a laboratory-breeding colony that was maintained on a 14-h/10-h light/dark cycle. Embryos were grown at 28. 5°C in E3 medium (5 mM NaCl, 0. 17 mM KCl, 0. 4 mM CaCl2,0. 16 mM MgSO4) containing 0. 000016% methylene blue as an anti-fungal agent. One ml from each 24 h bacterial culture was pelleted, washed once with 1 ml sterile PBS (Hyclone) and re-suspended in 1 ml PBS to obtain appropriate bacterial densities for microinjection. Prior to injection, 48 hpf embryos were manually dechorionated, briefly anesthetized using 0. 77 mM ethyl 3- aminobenzoate methanesulfonate salt (tricaine) (Sigma-Aldrich), and embedded in 0. 8% low-melt agarose (MO BIO Laboratories) without tricaine. Approximately 1 nl of bacteria was injected directly into the pericardial cavity or the blood via the circulation valley located ventral to the yolk sac using a YOU-1 micromanipulator (Narishige), a Narishige IM-200 microinjector, and a JUN-AIR model 3-compressor setup. For each experiment, average CFU introduced per injection were determined by adding 10 drops of each inoculum into 1 ml 0. 7% NaCl, which was then serially diluted and plated on Luria-Bertani (LB) agar plates. For co-challenge experiments, input doses were plated on LB agar+/−kanamycin (50 µg/ml) to determine relative numbers of the wild type and mutant strains present. After injection, embryos were carefully extracted from the agar and placed individually into wells of a 96-well microtiter plate (Nunc) containing E3 medium lacking both tricaine and methylene blue. For lethality assays, fish were examined at indicated times over the course of a 48 or 72 h period and scored for “death”, defined here as the complete absence of heart rhythm and blood flow. Survival graphs depict total pooled results from two or more independent experiments in which groups of 10 to 20 embryos were injected. To quantify bacterial numbers during the course of co-challenge experiments, embryos were homogenized at the indicated time points in 500 µL PBS containing 0. 5% Triton X-100 using a mechanical PRO 250 homogenizer (PRO Scientific). Homogenates were then serially diluted and plated on LB agar+/−kanamycin (50 µg/ml) to determine relative numbers of wild type and mutant bacteria. For co-challenge during urinary tract infection, seven- to nine-week old female CBA/J mice (Jackson Labs) mice were anesthetized using isoflurane inhalation and inoculated via transurethral catheterization with 50 µl of a 1∶1 wild type to mutant bacterial suspension containing a total of 107 bacteria suspended in PBS. Bladders and kidneys were recovered 3 days later and each was weighed and homogenized in 1 ml containing 0. 025% Triton X-100. Homogenates were serially diluted and plated on LB agar+/−kanamycin (50 µg/ml) to determine number of both wild type and mutant bacteria. Mouse experiments were repeated at least twice. For systemic infections, female CBA/J mice (Jackson Labs) aged 6 to 8 weeks were restrained using a Universal Restrainer (Braintree Scientific, Braintree, MA) and inoculated via the tail vein over a 30 s period with a 100 µl bacterial suspension, delivering 106 CFU/mouse. The inoculum was prepared by re-suspending overnight cultures in PBS and diluting them to 1×107 CFU/ml. For co-challenges, wild type and mutant suspensions were mixed 1∶1 before inoculation. Perfusion was performed on euthanized animals by cutting a small hole in the right cardiac ventricle and infusing the left ventricle slowly with 40 ml 0. 9% sterile saline before organ removal. Blanching of the organs occurred with the first 20 ml of sterile saline. Excised spleens and livers were homogenized in 3 ml PBS using a mechanical homogenizer (Omni International, Marietta, GA), and homogenates were plated using an Autoplate 4000 (Spiral Biotech, Norwood, MA) onto LB agar+/−kanamycin (50 µg/ml) to differentiate wild type and mutant strains. Kaplan-Meier survival and scatter plots were generated using GraphPad Prism 5. For Kaplan-Meier survival plots (independent challenges), the log-rank (Mantel-Cox) test was used to determine statistical differences between datasets. For competitive assays (co-challenges), numbers of wild type and mutant bacteria present in the inoculum and recovered from host tissues were determined as described above and a competitive index was calculated using the following equation where wt represents numbers wild type bacteria: Negative values obtained using the competitive index equation indicate a reduction in mutant fitness. To determine statistical significance, the Wilcoxon signed-rank test (with a hypothetical value of 0) on log-transformed competitive index values was used for co-challenges and the two-tailed Mann-Whitney statistical analysis was performed to determine significant differences between samples in non-competitive assays.

Bacterial pathogens, even those belonging to the same species, can be incredibly diverse with regard to the genes they carry. However, the design of vaccines and antibiotics typically relies upon identification of general molecular features shared by the targeted organisms. Thus, we have traditionally focused on broadly conserved characteristics of pathogenic bacteria, often ignoring the genes that account for their individuality. In this article we report the discovery of a unique gene, neaT, that promotes the fitness of a pathogenic Escherichia coli isolate in zebrafish and mouse models of systemic blood infections. Surprisingly, neaT is rarely found in other related strains of E. coli and appears to have been recently acquired from distant lineages of bacteria via a process known as 'lateral gene transfer' that is used by microbes to swap genetic material. Expression of the neaT gene appears to help pathogens avoid interactions with host immune cells, possibly by altering bacterial surface structures. This work provides an interesting example of how the lateral acquisition of a rare gene can impact the niche-specific virulence properties of a pathogen, shedding light on the mechanisms that drive pathogen evolution and diversity.

lay_plos

Cody Roman Dial disappeared on a trip through Costa Rica's Corcovado National Park. His parents realized something was amiss a week after he was supposed to have left the jungle. Photo: Jonathan Hey/Flickr On July 10, Cody Dial, the son of legendary Alaskan adventurer Roman Dial, embarked on what was supposed to be a five-day trekking trip in Costa Rica. Thirty-one days later, nobody has heard from him. The 27-year-old Cody Roman Dial— “R2” to friends and family—was traveling through Corcovado National Park, Costa Rica’s largest and most ecologically diverse landscape, when he went missing. Tourists have been required to hike with a guide since February, but Cody was alone and told his parents that he intended to trek illegally off the permitted trails. Details about Cody’s disappearance remain contradictory, but several facts are accepted: Officials have no record of his entrance into the park, he was reported missing by his parents on July 22, the search mission began on July 24, and this was Cody’s second time visiting Corcovado. He’d accompanied his father to the park as a teenager. That’s about all we know. The Tico Times, a Costa Rican English-language paper, reports Cody was last seen entering the southeast portion of the park on July 22. But later reporting casts doubts on the sighting. Authorities were also told that Cody was spotted outside the park, but none of the tips have been substantiated. In one case, a local man claimed that Cody hired him as a guide and paid him with cash withdrawn from an ATM. His parents, Roman and Peggy, did say that claim can’t be true. They were able to obtain withdrawal records from Cody’s bank in Alaska, and found that no such transfers were made from Cody’s account. “He’s very cautious, extremely well-educated, and he knows the dangers," Peggy said. "I'm confident." The Corcovado National Park is an eco-tourism and research hot-spot (Roman has led teaching and research trips there to study the ecology of the jungle canopy). It sits on Costa Rica’s Pacific coast, just north of the Panamanian border, and is partially isolated from mainland development on the Osa Peninsula. It’s 160-square miles of mountains, beaches, and rain forests are biologically diverse. But the park can be a dangerous place, even for experienced travelers. Canyons and rivers, hidden by thick foliage, can trap hikers, and the terrain makes even simple navigation dangerous. There are also the venomous snakes and roughly 400 illegal gold mines. Complicating matters further, officials have been unable to distinguish between illegal miners’ campsites and any traces Cody may have left. In late July, rescuers began patrolling the sites with the help of miners, but have not turned up any sign of the American. Last week, the Red Cross and Costa Rican government suspended their search operations. After several weeks of delays, Roman received permission to search for his son on his own, Peggy said. He is focusing his efforts on remote waterfalls and canyons. Cody may have been trying to follow a river out of the wilderness—a sound strategy in less densely vegetated Alaska—and inadvertently become stuck in a deep canyon. Both Cody’s family and friends in Alaska believe a happy outcome to the search is still possible. Cody grew up exploring the Alaskan wilderness, and, like his father, is a backcountry expert. He was studying for a Masters in Environmental Science at Alaska Pacific University, but put the program on hold in January to spend the winter and spring trekking through every major national park in Central America. Roman has been an Alaskan legend since arriving in the state in the late 1970s. A National Geographic Explorer, he pioneered the use of packrafts—inflatable kayak-style boats made of industrial-grade fabric. The boats revolutionized backcountry travel in Alaska and launched modern adventure racing, a sport in which Roman was a dominant figure for two decades. He’s also a four-time winner of the grueling Alaska Mountain Wilderness Classic, first in 1982 and most recently in 2002. The race predates, and many Alaskans claim was the inspiration for, the later crop of more famous and more commercial adventure races such as the Raid Gauloises and Eco-Challenge. The Classic has no checkpoints, no prize money, and no race officials. Cody became the youngest person ever to finish the race when he completed the Eureka Summit to Talkeetna course in 2004* at age 17. Just last weekend at the 2014 event, 10-year veteran of the race Rob Kehrer died while attempting to raft the Tana River. “He’s very competent and strong and smart and that’s probably why there’s hope in this situation,” said Paul Twardock, who, like Roman, is both a long-time professor at APU in Anchorage and an accomplished backcountry guide and outdoorsman. The Dials are asking that checks be sent to Margaret Dial at Alaska USA Federal Credit Union, PO Box 196613, Anchorage, AK, 99519. All checks should say “Cody Roman Dial Donation Account” on the memo line. All donations will go toward defraying costs associated with the Dials' search. Matt White (@PJMatt) was a Pararescueman in the U.S. Air Force and Alaska Air National Guard. He has written about Alaska for Outside, SBNation Longform, Los Angeles Magazine, and other outlets. *An earlier version of this story stated that Cody completed the course in 2014. We regret the error. Still reeling from the end of The Jinx? Not ready to let go of Making a Murder and The People v. O.J. Simpson just yet? The National Geographic Channel is filling the true crime void with Missing Dial, and EW has an exclusive first look at the six-part docuseries. Missing Dial traces the events surrounding the mysterious disappearance of Cody Dial, 27-year old son of National Geographic explorer Roman Dial. Following in his father’s footsteps, Cody set out on a journey through Corcovado National Park in Costa Rica in 2014… and he hasn’t been seen since. After searching the Costa Rican rainforest, which is rife with illegal gold-mining and other criminal activity, Roman enlisted the help of two private investigators — Kenneth Fornier (a retired Pararescue jumper) and Carson Ulrich (a former DEA special agent) — and a film crew, hoping the presence of cameras would increase the urgency needed to discover the truth about what happened to Cody. “I was right,” Roman said of his decision to involve the network. “Within a week, we received a significant break in the case.” Roman previously called for public donations via a GoFundMe campaign, raising $23,610 to help with his search for the truth shortly after Cody’s disappearance in July 2014. For Missing Dial, Roman and his investigators spent eight months combing the jungle for clues, interviewing locals, and scouting for new witnesses. According to the National Geographic Channel, Missing Dial “pushes further and digs deeper into the mystery, retracing Cody’s last-known whereabouts, using hidden cameras and secret identities to uncover a web of lies, surprising clues and a shocking plot twist that rivals a blockbuster movie.” Though he and his wife, Peggy, spent months contacting the U.S. Embassy regarding the case, Roman said the painstaking search yielded few results. “You don’t know how bad agony can be until your son has gone missing and no one can tell you what’s happened,” Roman said. “It’s a pain that keeps me awake every night, and it’s the same pain that drives me on my search for answers.” Watch an exclusive sneak peek of the series in the video above. Missing Dial premieres Sunday, May 22 at 10 p.m. ET on the National Geographic Channel. View Images Roman Dial, a biologist and veteran explorer, has been searching for his son Cody, who disappeared during a solo trip into the Costa Rican rain forest in 2014. Photograph by National Geographic Channel In early 2014, a 27-year-old Alaskan named Cody Roman Dial embarked on a grand and rambling overland journey through Mexico and Central America. He was the son of National Geographic Explorer Roman Dial, a biologist and multisport virtuoso renowned for audacious feats in mountaineering, ice climbing, rafting, and grueling backcountry endurance races. In Anchorage's adventure circles, Roman is a mythic figure. Cody's odyssey seemed to be driven at least in part by an attempt to carve out his own space in the wilderness. That spring, after a rafting trip in Mexico with Roman, Cody set out alone and began threading his way southward. He climbed Guatemala's 14,000-foot Tajumulco, the highest peak in Central America. He slept in a gold miners' camp and scuba dived with whale sharks. After a surfing idyll in Nicaragua, he crossed into Costa Rica on July 3. Several days later, Cody emailed his father from an Internet café in a small town called Puerto Jiménez. He planned to hike through Corcovado National Park—a notoriously challenging and dynamic rain forest—avoiding the main tourist trails and flouting the government mandate to hire a guide. On July 10, Cody entered Corcovado carrying a backpack and a crude map, which he had printed off the Internet. Then he disappeared. For the next 12 days, Costa Rican search-and-rescue teams scoured Corcovado's labyrinth of ravines and fast-flooding rivers. Roman himself flew to Puerto Jiménez to assist the effort, at one point sneaking into the jungle to conduct his own illegal search. Costa Rican authorities suspended their operation in early August but, believing Cody might still be alive, Roman soon called in a team from an Anchorage-based wilderness survival school. The group searched for five days around an area where a gold miner reported seeing Cody—an effort I joined and wrote about for Men's Journal. Ultimately, they found no trace of Cody or his equipment. Roman has since repeatedly returned to Costa Rica to continue the search. His efforts have been documented in the six-part series Missing Dial, airing on the National Geographic Channel, which premieres May 22. View Images Roman Dial with his son Cody in 1987. Photograph courtesy the Dial family When we last spoke, you had traveled to Panama to search for Cody, thinking he might have tried to cross the Darién Gap, a notoriously wild stretch of jungle, after leaving Costa Rica. What’s happened since then? We went down there and there was no record of him crossing into Panama, according to the authorities, no passport record of him going across. But we thought the border is really porous, and it would have been really simple for him to sneak across. And so I flew to Panama City and drove all the way to the last town you can drive to before you can get into the Darién Gap. It was just really apparent it would’ve been extremely difficult for him to get all the way down there. There’s a bunch of checkpoints all the way and so I just decided he didn’t go to Panama. Later that spring, Paul Lima [director of development at This Is Just a Test Productions] approached [my wife] Peggy and said there’s a possibility of doing a TV show about our search for Cody, and this guy had lost his father in Honduras. Using cameras and the media had really helped him get a conviction for his father’s killer. Peggy thought that sounded like a good approach for us to use to figure out what had happened to Cody Roman down in Costa Rica. Do you think media attention might help shed light on his case? When there’s a media spotlight, it calls attention to everyone’s behavior. It makes people more aware of what they’re doing. If you don’t go down [to Costa Rica] and rattle their cage, they’re not going to really do anything. It doesn’t seem like there’s a lot of urgency to figure out what’s going on. But I’m the father, so I do want things to move forward. You brought in two investigators—a former DEA agent and a retired Air Force pararescue jumper (PJ). Was this to continue searching in Corcovado National Park, the last place Cody was seen alive? I came to the conclusion that Cody wasn’t lost in the jungle. He had been killed or kidnapped. I needed someone who could go around and ask questions and know what kind of questions to ask. We went back in July 2015, spent six weeks, and then I went back in November and January [2016] and then again in March. Missing Dial Trailer National Geographic launches a covert investigation into the disappearance of adventurer Cody Dial. Missing Dial premieres May 22 at 10/9c on the National Geographic Channel. Walk me through what happened once you were on the ground. Carson Ulrich is a retired DEA agent who worked in Latin America for 25 years and speaks Spanish. He's a big, intimidating guy. Ken Fornier is a retired PJ and easygoing. When we got there, they started snooping around and interviewing people I had talked to before—Jenkins for example [the gold miner who reported seeing Cody]. And then we spent some time with the OIJ, the Costa Rican FBI, and at the American embassy. What did you find out? We were able to piece together that Cody had gone into the jungle and then come back out. Cody and this character, Pata de Lora [which translates to "parrot foot"], traveled back to Dos Brazos together. A couple of people had seen them. Carson and Ken retraced that path by engaging some informants who traveled through the mining camps to piece together what else happened. It all seemed to center around this trip that Cody had taken after he had come out of the jungle. Pata de Lora's name figured in the case early on. He told police he guided Cody into Corcovado, but you initially dismissed him as a suspect, partly because locals reported seeing him smoking pot with a gringo, which wasn’t characteristic of Cody. What changed? Cody consistently said I don’t want a guide. The second thing was the timing was off—all the dates that people gave about when they saw Pata de Lora were a week late. Those things made me think it was a different person. I dismissed it given what I'd known. As for the pot smoking, I’d be surprised. Soon after you arrived, there was a big development in the case—the biggest since Cody disappeared. When [Cody and I] were together in Mexico, we had a rental car that got broken into and Cody’s pack got stolen. So he bought himself another pack. Apparently, it had been at the hostel [in Puerto Jiménez]. The pack was given to the OIJ and then they gave it to this official at the embassy in March 2015. The embassy held on to it until the end of June and then sent me an email: “By the way, we’ve got Cody’s backpack.” They had it for two months and didn’t tell us. It proved he had come out of the jungle. Did you develop a new theory about Cody's disappearance? The only evidence we have is what people say. There’s no hard physical evidence other than a foam pad I found in a miner’s tent in January that I'd given to Cody. I found that pad in January with the OIJ and a member of the embassy and some cadaver dogs. They asked the miner, but I don’t know what to say about that. Why, because there’s a pending case? Or because that’s the limit of the information? It's a pending case. It really is the only piece of physical evidence that we’ve got. Have the Costa Rican authorities arrested or charged anyone? No. We know that Cody was murdered, and we know there is a suspect. Once the documentary starts, it’s going to be on TV for six weeks. They have six weeks to move on it. We hope they use this time to build their case. How do you know Cody was murdered? I'd prefer to wait until the show airs or better yet an arrest has been made. View Images Roman (right) enlisted the aid of Carson Ulrich, a former DEA special agent, to help him investigate Cody's disappearance. Photograph by National Geographic Channel It sounds like a promising investigative path. Has that helped bring some measure of closure for you and Peggy? When you and I were there, almost two years ago, I was just looking for my son. I called out his name and blew a whistle and listened for him and thought I might find him broken but alive. I’d be able to take him out. At the end of that, I realized he wasn’t there, and he probably was dead, and I wanted to find out what had happened. Now I want justice. One thing that Jon Krakauer, your friend and climbing partner, told me was that “to live an overly cautious life in many ways is as dangerous as its opposite.” Has this experience altered your own feelings about the nature of risk? Yes, certainly it has. When you’re doing active things, you’re aware there’s a certain amount of risk. There’s a self-focus, it’s about you. I guess I never realized how painful the loss is to the people who love you. It’s made me think the adventure stuff we do is pretty selfish. When you die, it’s over. The people who are going to be suffering are the people who loved you and lost you. This interview has been edited for length and clarity. Early last April, a 27-year-old biology student named Cody Roman Dial set out into the remote jungle of northern Guatemala. Equipped with a crude map and a compass, he planned to traverse the Petén, a lowland rain forest teeming with snakes, illegal gold miners, and cocaine traffickers. His biggest concern, though, was dehydration — save for jeep-track mud puddles, the area lacks ready sources of freshwater. Cody had spent the previous week preparing for the trip, talking to locals and poring over maps. He had bought a machete and commissioned a local tailor to stitch together a tent of his own design. His plan was to spend about 10 days in the Petén, bushwhacking through the jungle to a sprawling Mayan ruin called El Mirador. But, he emailed his parents shortly before beginning the trip, "I expect I'll spend a couple days out there, eat a snake, get scared, and turn around." A bright, quiet introvert with a sharp wit and passing resemblance to Harry Potter, Cody had taken a hiatus from his graduate studies in environmental science at Alaska Pacific University to boot around Mexico and Central America. Having grown up exploring Alaska's wilderness, he was an experienced outdoorsman and a competent navigator. He was also the son of adventure royalty. His father, 53-year-old Roman Dial, a National Geographic explorer and a legendary figure in Alaska, had pioneered dozens of first ascents in the state's mountain ranges. Dial was considered the father of packrafting, a mode of river travel using inflatable kayak-like boats, and in the 1980s had helped found a grueling backcountry footrace called the Alaska Mountain Wilderness Classic. The race, which usually covers several hundred miles and prohibits outside support, is called the most difficult wilderness challenge in the world — and, more simply, life-threatening. Nearly every year, several participants require helicopter evacuation. In 2004, 17-year-old Cody, with his father as teammate, became the second youngest person ever to cross the finish line. After a week of gathering supplies, Cody hiked into the Petén. Though he'd often accompanied his father into tropical rain forests as a child, this was his first real trip alone into truly wild jungle. He picked his way through bullhorn acacia and green spiny palm and, as dusk settled, camped beside a jeep track. In the morning, he encountered a wild pig crashing through the brush; not long after, a cougar loped down the trail. After the jeep track ended, Cody began threading thin jungle trails and soon became lost. For the next two days, he wandered in the wilderness. On the third, he backtracked by following blazes that he'd hacked into tree trunks and, eventually, made his way to a Mayan ruin called Naachtun — where he encountered an archaeologist named Carlos Morales-Aguilar. "I thought he was crazy," Morales-Aguilar says. Navigating by compass, Cody then pushed through to El Mirador. After five days, he limped out of the jungle, filthy, heat-exhausted, elated. Behind him was a trail of gobsmacked Guatemalan park rangers, astonished to see a lone gringo hacking his way through the Petén. Photograph Courtesy Roman Dial As he had done throughout his journey, Cody soon emailed his father to let him know he was safe. The trip "sounds not only superbadass but kind of foolhardy," Cody wrote. "It wasn't really either of those things, just walking for eight days and asking people where to go." Dial was relieved that Cody was alive. For the past couple of decades, he had raised his son to survive in the wild — navigating the Chugach Mountains south of Anchorage, enduring freezing temperatures on backpacking trips through Alaska's 700 square-mile Harding Ice Field, and once hiking 18 miles in 35 hours through the Alaska Range. Just a few weeks earlier, Cody had gotten disoriented coming down a fog-shrouded volcano at night. "Luckily," he wrote his father, "I've followed Roman Dial around the wilderness all my life, so my instincts led me to the right place." Dial had encouraged his son to undertake his first, grand, solo adventure, but after Cody initially spoke to him about the Petén trek, he had considered telling him not to go. "It just sounded crazy," Dial says. "He was going to walk for 10 days in the wildest place in Central America. But I didn't send an email. I was like, 'Who am I?' The reason he's doing it is because I want him there. So I said, 'Be careful with your machete and watch out for snakes.' " Through May and June, Cody ticked off an enviable list of adventures: diving with whale sharks in Honduras, riding a dugout canoe into the remote Mosquito Coast. In Nicaragua, he spent two weeks surfing and began researching a trip through the Darién Gap, a notoriously lawless patch of jungle between Panama and Colombia. Costa Rica was next on Cody's itinerary, and, on June 7, he emailed his father to ask about a trip the two had taken there in 2001, when Cody was 14 and Dial was conducting research on the jungle canopy. "You and I went to Guanacaste and Santa Rosa National Parks," Dial replied. "Also Corcovado National Park." Over the past two decades, much of Costa Rica has been transformed into a placid eco-tourism paradise, but the Osa Peninsula — a genie-slipper-shaped protuberance jutting into the Pacific Ocean — remains wild and remote. Corcovado occupies much of the Osa and contains the only remaining old-growth rain forest in Central America. Its terrain is complex and dynamic: a jungle labyrinth of steep ridges, deep ravines, and gorges that fill with astonishing speed during the rainy season's torrential downpours. Jaguars, crocodiles, and snakes — such as the deadly fer-de-lance — prowl the interior, along with a sizable population of illegal gold miners. Warnings about entering the park alone are abundant. Every year, according to Lonely Planet, insufficiently prepared visitors "become injured, sick, or even dead." Corcovado challenges even the experienced. In 2006, Costa Rica's minister of the environment, an accomplished outdoorsman, spent three days lost in his own park after being attacked by a bovine-like animal called a tapir. On July 3, Cody crossed the border from Nicaragua into Costa Rica. At some point — it remains unclear exactly when — he arrived in Puerto Jiménez, a fishing town on the Osa's east coast. Five days later, he checked into a budget hostel called the Corner. The following morning, he emailed Dial and his mother, Peggy, an outline of his intended route through Corcovado. A few hours later, having settled on a new course, he sent another message: He planned to enter from the park's eastern perimeter near a river called the Rio Rincon. Navigating through the dense jungle, he would pick up another river, the Rio Davíd, then follow that to the Rio Claro, which led to the Osa's photogenic Pacific shore. From there, he merely had to walk to any village along the coast and catch a bus back to Puerto Jiménez. On July 10, Cody set out for Corcovado. He carried a backpack containing food, his tent, and a cooking stove. He also had the machete and a compass, along with an imprecise map of the park he'd printed off the internet. His proposed route would skirt a popular tourist trail in favor of an off-limits path used mainly by illegal gold miners. Several months before, MINAE, the ministry that manages Costa Rica's parks, had begun requiring all tourists entering Corcovado to hire a professional guide, a decree intended to limit environmental damage as well as the number of costly rescues every year. It was a rule that Cody planned to flout. Fiercely independent, emboldened by his trip in the Petén, and having been tutored by his father — one of the U.S.'s most skilled backcountry navigators — he almost certainly believed such assistance was unnecessary. "I am not sure how long it will take me, but I'm planning on doing four days in the jungle and a day to walk out," Cody wrote in his last email. "I'll be bounded by a trail to the west and the coast everywhere else, so it should be difficult to get lost forever." On the day Cody entered Corcovado, Dial was on summer break from Alaska Pacific University, where he teaches biology and math, and packrafting with several friends on a river in the Talkeetna Mountains. On July 15, five days after Cody had started his trip, Dial returned to his home in Anchorage and, scrolling through scores of emails, saw Cody's first message from Costa Rica. His son, Dial thought, was still only planning his trip, and so he and Peggy spent the next three days dipnetting for salmon on the Kenai River. Cody's second email, tucked beneath the first, went unnoticed. By the time Roman and Peggy returned from their trip on July 18, Cody had been gone for eight days. He typically checked in every 10 to 12 days, so they were aware, but not yet alarmed, that they hadn't heard from him. Five days later, while driving to Home Depot, according to Dial, Peggy began feeling inexplicably nauseous. "She said, 'I feel like I'm going to throw up,' " Dial remembers. "We went home, and I finally read that second email underneath and realized, 'Holy shit, he was supposed to be back' " almost 10 days earlier. As with his Petén traverse, Cody would have emailed his parents as soon as he was safely out of Corcovado. Photograph Courtesy Roman Dial Immediately, on July 22, Dial alerted the American embassy in Costa Rica that his son was missing. Twenty-four hours later, he was on a flight to San José, the capital. The next day, the Costa Rican Red Cross dispatched two search-and-rescue teams: one to an entrance on the park's eastern perimeter and another to Cody's planned exit point at the Rio Claro. Several more teams — aided by park rangers and officers from the national Fuerza Pública police force — began scouring Corcovado over the next few days. The strategy of the searchers, led by veteran Red Cross official Gilbert Dondi, was to begin at the park's perimeter and work their way toward the center. If Cody was still fighting his way out, then, in theory, their paths would intersect. The search team faced a daunting challenge: Corcovado encompasses an area larger than 120 Central Parks. Its jungle canopy is sufficiently thick to thwart aerial infrared cameras, and since Cody had avoided primary hiking trails, ground teams would have to scour the park's rugged hinterland — a treacherous patchwork of ridges, ravines, and gorges whose topography Dial likened to "taking a piece of paper and wadding it up in your hand." In addition to deadly snakes, a species of wild pig called a white-lipped peccary also inhabits the Osa. Peccaries travel in packs up to 200 strong, and many locals consider them the most dangerous animal in the park. Their presence is said to be preceded by a strong smell of onion. As the Red Cross' teams continued searching, Dial was conducting his own investigation. A mix of rumor and strange reports about Cody swirled through the Osa. The owner of the Corner, where Cody had checked in on July 7, told authorities that Cody had returned on the 21st, then caught a bus the next morning to Dos Brazos, a small town on the southeastern edge of the park. Residents there reported seeing a gringo backpacker matching Cody's description on July 22. In Puerto Jiménez, there were also dark whispers that a local criminal known as Pata de Loro, or Parrot Foot, was somehow involved in Cody's disappearance. Questioned by police, Parrot Foot, whose real name is José Fallas, claimed Cody had hired him as a guide in mid-July. After hiking into Corcovado from Dos Brazos, Fallas said, he and Cody spent two nights in the jungle and then parted ways in Puerto Jiménez. Many locals' stories were conflicting — there were reports of Cody in two locations simultaneously — or they appeared to describe someone else entirely. (Parrot Foot, according to several residents in Dos Brazos, was accompanied by a backpacker wearing an earring, which Cody lacked, and smoking marijuana, which, according to friends, Cody avoided.) Based on reports from the hostel owner and other locals, however, the Red Cross developed a new theory: Cody had likely entered and exited the park on at least two occasions. According to Dondi, the search coordinator, he was possibly still hiking another trail — or had left Corcovado altogether. Dial quickly dismissed the idea, as well as the locals' stories. His son would have contacted him if he were out of Corcovado — and he never would have hired a guide. Photograph Courtesy Roman Dial Then, on July 30, the Red Cross search team in Dos Brazos received what seemed like a credible lead. A gold miner named Jenkins Segura reported encountering someone resembling Dial's son deep inside the park several weeks before. The young man had been eating breakfast by a stream and, Segura said, introduced himself as a biologist from Alaska. He also seemed disoriented. According to the miner, Cody had asked whether they were in the Golfo Dulce forest, a national reserve adjacent to Corcovado. In some respects, Segura's story was as suspect as the others — he was hazy on the dates and initially also described the backpacker as wearing an earring — but Dial met him the next day in Dos Brazos and found his story believable. "He spoke good English," Dial says. "We quizzed him for a long time." That afternoon, the pair snuck into Corcovado and hiked for several hours up a river called the Rio Tigre, then walked a trail that led to an old gold-mining camp. Nearby was the spot where Segura claimed to have encountered Cody. To Dial, it seemed like an ideal place for his son to rest: a small clearing, surrounded by ridges, with a stream burbling up from the ground. "There's no water on the ridges," Dial says. "It'd make perfect sense that he stopped at the first place where there's water and made breakfast." But according to Segura, he had never seen a gringo in that area before and was sufficiently concerned to have told Cody that Dos Brazos was only two hours away — he simply had to follow another short trail that led to the Rio Tigre and then walk the river back to town. Not far from the stream, however, Dial discovered a massive landslide that had recently rumbled down the south wall of a canyon. It had obliterated part of the trail Segura had recommended with a bewildering mass of earth, trees, and tangled vines. Along one stretch, a towering tree — wet, slippery, snake infested — had come to rest some 100 feet over a creek. Searching the area, Dial became increasingly convinced that his son had encountered the landslide and attempted to navigate around it. "The forest is really disorienting. If you get off-trail, it's real easy to get lost," he says. In the process, he believed, Cody could have been bitten by a snake, slipped and fallen into a gully, or "gotten injured and couldn't climb up to where the best trails are, then got caught between some waterfalls." On August 2, the Red Cross dispatched patrols to search the area where Segura reported seeing Cody, as well as a gold-mining tunnel where they suspected he could have gotten trapped. A helicopter soon flew several aerial searches up and down the Rio Tigre. But two days later, the organization's senior staff convened a meeting with Dial at MINAE headquarters in Puerto Jiménez. Dondi showed him a Google Earth display marking where its teams had searched: a Pollock painting of red pins and blue lines overlaying Corcovado's green expanse. Ten teams had spent 12 days combing the park; they were exhausted and had found no trace of Cody or his equipment. Without new evidence, they told Dial, the search was over. In Anchorage, news of Cody's disappearance spread quickly. Well-wishers inundated social media with hopes of a speedy resolution. Organizers of the Alaska Mountain Wilderness Classic, slated to begin on August 3, considered calling off the race but instead donated the $200 entrants' fees — normally pooled for helicopter rescues — into a fund for the family's search effort. Over the years, Dial, a "Lower 48er" in his youth who had fallen hard for Alaska, had staked out a lonely position in the state defending and often lecturing about Chris McCandless, the young, idealistic adventurer whose untimely death is chronicled in Jon Krakauer's bestselling book Into the Wild. Many Alaskans, including himself, Dial liked to argue, had undertaken similarly risky adventures as young men — and survived simply by luck. Perhaps inevitably, Cody's vanishing invited comparison to McCandless' — a notion that Dial, believing his son both more competent and less rash than the "dreamy half-cocked greenhorn" Krakauer described, entirely rejected. "The only thing that's similar," Dial told me, "is that it's a young guy on a quest." A former colleague at APU also wrote an op-ed in the Alaska Dispatch News to dispel any such comparisons. "There is no debate this time about competence or experience (both of which Cody Roman has in spades) or even about the young man's good judgment," she wrote. "We didn't know Chris McCandless, but a lot of us know Cody Roman (or 'R2,' as we call him) and his family. I don't care why R2 went into the jungle by himself or whether or not it was a good idea and neither does anyone else. We just want him to come home." Photograph Courtesy Roman Dial Although Dial was raised in Falls Church, Virginia, a suburb of Washington, D.C., his connection to Alaska was profound. When he was nine, his parents — entering a turbulent period in their marriage — packed him off to spend the summer with three uncles in Usibelli, a rugged coal-mining town on the edge of Denali National Park. Dial was largely left to fend for himself. He acquired a.22 rifle and a dirt bike on which to roam the backcountry. For company, he had a wolf-dog named Moose. "It was life-changing," he says. "I had all this freedom to do what I wanted. After that, I went back to the suburbs, and it was really boring." Dial returned to Alaska when he was 14 and undertook a series of "really foolish" solo backpacking trips — including one excursion in which he nearly drowned crossing the Resurrection River, near Seward. As a child, Dial was fascinated by turtles and lizards and dreamed of being a scientist. As a young teenager, he began reading John Muir and became enamored of alpine climbing. In 1977, Dial graduated from high school two years early, partly so he could head west. His parents pushed for the Ivy League, but Dial chose the University of Alaska-Fairbanks — largely because the school's catalog featured one of the state's most majestic peaks on its cover. After his freshman year, Dial moved to a cabin in the woods without running water and, in the winter, skied to class. One day in 1980, a pretty, plainspoken freshman looked out of her dorm window and saw a wild-haired young man climbing the exterior of the student center. "I thought, 'Oh, he looks interesting. I've got to meet this guy,' " Peggy remembers. She soon introduced herself. "I was never into drinking and partying," she says. "When I met him, we went for a walk, and he immediately starts talking about the plants and the grouse that are mating. I thought, 'This guy's cool.' " Dial by then had become a formidable alpine mountaineer and soon pulled off a string of audacious routes in Alaska's Hayes Range. About a week before he met Peggy, Dial — braving whiteout conditions and persistent avalanches — completed the first ascent of the vertiginous east face of a 10,000-foot peak in the backcountry. Years later, Dial attempted what renowned mountaineer David Roberts described as his "masterpiece." Along with fellow climber Chuck Comstock, Dial set out to ascend the east face of 11,400-foot McGinnis Peak. After several pitches up a 3,000-foot gully of ice, the pair made the summit in four days. As they started to down-climb, Comstock broke through a cornice and plummeted off one side of a ridge. Dial, belaying him, threw himself over the other side as a human counterweight — dropping some 200 feet before the ropes caught him, saving Comstock from a fatal fall. Though he was at the peak of his climbing career, Dial's already notoriously thin margin of error had become intolerably fine. He soon quit alpine mountaineering. "We rappelled down, and I was like, I'm done and I'm getting married," he says. After he and Peggy married in 1985, Dial dreamed up a 900-mile, four-season traverse of the entire Brooks Range. He skied one section in the early spring, then returned with his new wife to complete another leg in May, but the couple quickly became tent bound. "The snow went rotten, so we got stuck on a mountain," he says. "I found out later that the name of the creek we were above meant 'place to make love many times [in Nunamiut].' " Nine months later, Peggy gave birth to Cody Roman. Dial still completed the traverse, by packraft and kayak, and set a record. "My first trip to Alaska was in 1974, and you just heard stories about him," says Jon Krakauer, who later became a frequent climbing partner of Dial's. "He set a new standard for ice climbing. Then he got into the wilderness stuff. In a day, he'd see how fast he could ski the length of the Alaska Range. With this whole packrafting thing, he's changed the way people move in the wilderness. He made people rethink what you could do, how far you could travel." Years later, Dial would successfully complete an 800-mile bicycle traverse of the entire Alaska Range. To save weight, he slept in a floorless tent and ate with bike tools. In 2006, he pulled off a 620-mile-long march through the Arctic to the remotest point in the U.S. carrying only a single bag of food — mostly just to see if he could. Both trips embodied the Dial ethos: grueling, audacious, pushing as far and as fast with as little equipment as possible. According to friends and competitors, Dial is blessed with an almost otherworldly ability to navigate terrain. His endurance is also legendary, as is his risk tolerance. "He cut it as fine as a person could and emerged unscathed," Krakauer says. Dial is, by many accounts, also brilliant. After earning a double master's degree at APU, he and Peggy moved to Palo Alto, California, so he could pursue a Ph.D. in biology at Stanford. The couple had their second child, Cody's sister, Jazz, and then resettled in Anchorage in 1992. (Following in the family tradition, Jazz would later become a competitive rock climber and a fearless downhill skier.) Dial and his young son soon embarked on their first grand adventure together: a long ramble across Umnak, a barren and mountainous island in the Aleutian archipelago, which extends like a long comma from mainland Alaska. Dial bought a cheap ticket to a neighboring island, then talked his way onto a plane flying a Japanese film crew to Umnak. Photograph by Frank Marley "It was 60 miles across the island," Dial says. "He was six, and I didn't make him carry a pack because I didn't want to spoil it for him. Sometimes I would carry him on my back. That's actually where he started calling himself Roman. There's only people on both ends of the island, but nothing in between. There's a family on one end, and when we landed, they go, 'Well, what's your name? What are you doing here with your son?' I said, 'My name's Roman, and I'm going out on a nice walk.' They look at Cody and go, 'Well, what's your name, little guy?' and he said, 'Well, I'm Roman, too.' " After the Red Cross called off its search in Corcovado, Dial began working back channels to get the U.S. military involved in a rescue effort. As days passed, though, his request seemed to vanish in the murky depths of American bureaucracy. On August 11, frustrated and increasingly desperate, Dial visited a psychic who gave him a set of map coordinates in Corcovado. He hired a plane and flew to the location — a ranger station near the Pacific coast that was along Cody's intended route — but found nothing after searching the area on foot. Days later, Alaska senator Lisa Murkowski, who had been shepherding Dial's appeal, told him the Defense Department had concluded it lacked legal authority to mount a search-and-rescue operation in Costa Rica. Dial immediately began calling friends in Anchorage to put together a covert search-and-rescue mission. "I was tired of waiting for the cavalry to arrive," he says. "He's stuck somewhere, and I've got to get him." In Alaska, Peggy got in touch with Brian Horner — an affable former instructor with the Air Force's notoriously punishing SERE (Survival, Evasion, Resistance, and Escape) school and the owner of an Anchorage-based survival company called Learn to Return (LTR) — who agreed to send an LTR team to Corcovado, free of charge. Hours later, Horner and two employees, a square-jawed former Special Forces soldier named Clint Homestead and Frank Marley, a search-and-rescue expert, met at LTR's hangar-like training facility to assemble gear: climbing rope, rope ascenders, machetes, radios, trauma kits. Twenty-four hours later, they boarded a plane bound for Costa Rica. Two other Alaskans — Todd Tumolo, a 29-year-old Denali mountain guide, and an aristocratic bird-watcher named Brad Meiklejohn — also flew in to join the effort. Dial, meanwhile, was in San José trying to wrangle permits for the search — though, if the attempt failed, he intended to enter the park illegally. At a pretrip briefing, Horner told his team that if they got into legal trouble, the emergency code words were Warren Zevon: Send lawyers, guns, and money. On August 21, Dial and the Alaska group drove to the Rio Tigre. Their plan was to search an approximately one-square-mile area around where Segura reported seeing Cody. Lugging packs loaded with ropes and rappelling gear, they labored upriver in the sweltering jungle heat. Around 12:30 pm, the group reached a fork in the river, walked up the left branch, and then scrambled over the landslide. While setting up camp nearby, they were inundated by a torrential downpour. Over the next four days, the rains would begin every afternoon with near mechanical precision, turning the park's gullies into fast-filling traps and its ridges into mud-slicked high wires. In the morning, Dial began searching a gully near where he suspected Cody might have gotten trapped. "I just used my intuition about what he would have done if he had run into this landslide," Dial says. He and Tumolo then found a trail that led up to a ridge. They looked for bent branches, machete marks, a burst of color signaling a piece of clothing in the brush — anything to indicate Cody might have passed through. The LTR team, meanwhile, concentrated on the landslide. Homestead, who would earn the nickname "Chop Chop" for his zeal with the machete, cleared a path through brush trapped in the wall of debris. After combing the area, he, Horner, and Marley hiked down to the fork in the Rio Tigre and then walked back up the right branch — a high-walled canyon filled with water deep enough to reach their chests. Soon the sky darkened. Not wanting to get trapped in a flood, the Alaskans turned back for their camp. Homestead led the team on its return down the river. Then he suddenly stopped. Two figures were coming toward him. One day before, I had hopped a 12-seat turboprop from San José and landed in Puerto Jiménez. After making contact with Dial, my plan had been to hire a local guide, link up with his team, and then accompany them during the search. Corcovado's topography and thick jungle canopy, however, had prevented my calls from reaching Dial's sat phone. That afternoon, I hastily hired Reyes Morales, a gold miner with a roguish grin and an Errol Flynn mustache who knew Corcovado as well as perhaps anyone in the Osa. He was also a skilled tracker. Morales would, I hoped, pick up a sign from one of Dial's teams, then take me to their base camp. At the last minute, Dial had managed to secure legal permits for his search. At MINAE headquarters in Puerto Jiménez, however, an officious park ranger with gelled hair had informed me that I would not receive similar permission. Early the next morning, Morales and I waded into the Rio Tigre and began hiking upriver. Steep canyon walls rose high above, trees clinging to their sides at oblique angles. Morales walked fast, hopping over rocks and clambering up the river's steep banks to catch shortcuts through the forest. I moved cautiously, wary of a shattered ankle and snakes. The river soon grew deeper, the terrain rockier. All around, Corcovado pressed in — dense, choking, stiflingly hot. Navigating its immensity with a compass and a rudimentary map seemed a heroic undertaking. Three hours later, Morales stopped at a fork in the river. "Roman aquí," he said, pointing at boot prints leading up the right branch. Twenty minutes later, he stopped again. Several hundred yards away, Homestead was walking downstream — along with a park ranger and a police officer, part of a government contingent accompanying Dial's search. Alarmed by a strange gringo and an unknown local in an off-limits part of the park, the officer drew his pistol. "Where is your permission to be here?" the park ranger, a short man in a dirty green T-shirt, asked me. Dial's team had just encountered a group of poachers, Horner explained, and the Costa Ricans were jumpy. He then clicked on a GoPro camera. "In case you disappear," Horner said. After a protracted discussion, the park ranger ordered Morales and me to leave Corcovado. As we slogged back down the river, the police officer stayed close behind me, while the park ranger trailed Morales. Two hours later, at a ranger station in Dos Brazos, I was told to wait. The official with gelled hair from Puerto Jiménez soon arrived. Looking displeased, he waved me over to the back of his pickup. "If you go in again, then I will have you arrested," he said. The Costa Ricans, it seemed, were unenthusiastic about the possibility of another gringo going missing in Corcovado. Raising Cody, Dial envisioned a fellow comrade in adventure and forged him in what might be described as the Alaska mold: independent, intrepid, self-sufficient. "I always wanted him to be a companion," Dial says. "My dad left home when I was nine or 10, and I don't feel like he ever shared anything with me except mathematics." According to Krakauer and other friends, Dial could be brash and even combative — he once punched a climbing partner in the gut — but, unlike many type A parents, Dial says he avoided pressuring his son. "I went on a trip with him every year pretty much since he was three," he told me. "I'd say, 'What do you think? Do you want to do this?' I didn't push him into it." After Dial started teaching at APU, his research soon took him to far-flung jungles around the world, with Cody as his frequent companion. In 1998, Dial traveled with a class from APU to Corcovado. Father and son, along with Dial's students, spent nearly a week hiking the main tourist trail from Los Patos — the same area where Cody likely entered in July — to La Sirena, then walking north along the coast. At night, Dial gave lectures about tropical ecology. "He was fascinated," Dial recalled of his son. "He would sit up close and take better notes than anyone else." Over the next several years, Dial and Cody traveled to jungles together around the world: Corcovado again in 2001, Borneo, and Bhutan. By virtually all accounts, Cody was more risk-averse than his father. "A deliberate, thoughtful kid," Dial says. "Not bold — more like his mom that way." But Cody also embraced some of the same challenges that captivated his father. When he was 17, he and Dial competed in the Alaska Mountain Wilderness Classic as a team. That year's race called for a sprint from Eureka to Talkeetna — a distance of about 150 miles and one of the Classic's most technically challenging routes. They rode mountain bikes through tundra hills, lugged them over a high pass, then lashed the bikes to packrafts and paddled miles of whitewater on the Talkeetna River. They came in sixth, but beat the previous year's winning time. "I felt like a bad parent, teaching my kid about sleep deprivation," Dial says. "But he knew he could go without sleep. He knew he could get cold and wet. We trained a lot for the trip. I was very proud of him." After attending William & Mary, in Virginia, Cody returned to Anchorage and enrolled in APU's environmental sciences master's program. In 2012, he met Katelyn Barnett, a junior with a mordant sense of humor and an irresistible laugh. She and Cody were soon spending weekends backpacking or skiing to her cabin in Talkeetna. "He liked being cold and wet and miserable — I can only assume because he grew up doing it with his dad," she says. But, Barnett believed, Cody was also not immune to the pressure of being the son of one of Alaska's most famed outdoorsmen. "It's hard to live in the shadow of Dial," she told me. "Little Roman's not his dad, but he wants to be his dad." Photograph by Peter Bohler As Cody worked on his master's thesis — a study of a small, omnivorous ocean crustacean — he began to encounter problems with some of his data. He had planned to have his paper finished by the spring of 2013, but months passed and, as the project lagged, he grew frustrated. By later that summer, it had stalled completely. "He was trying to finish in August, and he didn't," Barnett says. "And then he was like, 'Fuck this, I'm going to go travel for a year.' " After spending a few months bumming around the Lower 48, Cody took a flight on New Year's Eve from Washington, D.C., to Mexico City. By chance, his father was also flying in with several friends for a packrafting trip. Cody hopped a bus to Veracruz, and he and Dial spent 10 days paddling miles of whitewater on the state's famously turbulent and dangerous Alseseca River. "It has all these waterfalls — we counted like 15 or 20 in two miles," Dial says. "You have to use special techniques to go off." According to his father, Cody had mostly stopped paddling after injuring his back in 2008 and was out of practice. "He swam a bunch — no one else was swimming that much — but he'd get back in his boat. We walked around the Class V waterfalls, but other sections were solid Class IV. On the last day, we ran it again and he ran every waterfall without swimming at all. It was beautiful — the sun was shining straight down and the mist was capturing these rays of light. I went first, and I'd be looking back, watching to see how he was doing, and he was just styling all the moves, doing perfect." It was the last time Dial would see his son. By the evening of his third day in Corcovado, Dial was growing dispirited. Cody had now been missing for 45 days. It was possible, though increasingly unlikely, that Cody was still alive. Dial had been in Costa Rica for a month, much of that time spent in the jungle. He was gaunt and worn down, older-looking. He also feared for the safety of his fellow searchers. Earlier, Horner had slid down the side of a ridge, crashing through trees before a vine arrested his fall. There had been several close calls with snakes — including an eyelash viper Tumolo chopped in half with his machete. That night, a 130-foot tree, waterlogged from the daily rains, toppled over and nearly crushed the LTR team while they slept. Despite the dangers, Dial wanted to check one last location: a patch of jungle by the fork in the Rio Tigre where he had seen vultures circling. In the morning, he, Tumolo, and Meiklejohn hiked down to the area. After hours of bushwhacking through thick brush and humping over corrugated terrain, they still had found nothing. "We were walking up and down these hills and cutting our way through vines and finding nothing. Finding nothing at all," Dial told me. At the same time, the LTR team explored a ridge above the landslide. Homestead took point, moving slowly to watch for snakes. The ridgeline gradually narrowed to a foot-wide path, dropped downhill, and delivered them to a cliff. After rigging ropes to a tree, the three men rappelled off the top as the afternoon downpour began. Fifty feet down, they found themselves in a flooding canyon. They charged downstream, then scurried over the side of a 12-foot waterfall. "The water was roaring," according to Horner, "and we're doing that, 'OK, don't panic, come on.' " Six more waterfalls lay ahead. Using each other as human ladders, they scrambled down one after the next as the canyon filled. The next morning, having found no evidence of Cody — and by decree of the Costa Rican government, which had limited Dial's search to five days — the two teams broke camp and hiked back out to Dos Brazos. "I kept thinking I was going to find him," Dial told me the next day. "But we really searched the place. I'm convinced he isn't there — at this point, all of us are. There's not that many probable places that a person can go, but there's an infinite number of improbable places. And you just can't search the improbable ones." A logician by temperament, Dial had spent long hours turning over the possibilities in his mind. It was possible that Cody — propelled by stubbornness or perhaps still disoriented — had continued searching for his original, intended route. Following his compass and fighting through the dense jungle, any number of potentially lethal dangers awaited: snakes, peccaries, treacherously slick ravines. Dial vigorously rejected this notion, however, and, after days searching the park, had become increasingly convinced his son had followed the Rio Tigre toward Dos Brazos. The route is arduous, but not lethal. It is also well traveled. That Cody had not been found along the river, Dial suspected, indicated his son could have been murdered before he made it out of the park. "I don't feel like he's in the jungle hurt or dead of natural causes," Dial told me. "That leaves the other two possibilities: He decided to disappear, go have his own life. That would be really out of character for him, but I don't leave it out of the realm of possibility. The third thing is he was murdered. That's what I'm left with." Such a theory is not far-fetched. Over the past decade, at least a dozen expats and tourists have either mysteriously vanished or been murdered in Costa Rica, with nearly half of the confirmed killings taking place in the Osa. In an eerily similar case to Cody's, a 28-year-old graduate student from Chicago named David Gimelfarb disappeared in another national park in mainland Costa Rica in 2009. His parents, at one point, suspected Gimelfarb was the victim of foul play. It is entirely plausible that Cody Roman Dial met some similarly grim fate. It is also true, though, that for a parent confronting the death of a child, a son who may have died engaged in a pursuit passed down to him by his father, the prospect of outside agency offered some glimmer of relief. "I don't know if he's alive or dead, but I'm convinced that my son didn't disappear in the jungle," Dial told me. "That feels good to me that he didn't. I'd rather he not disappear that way because then it's like all of this training all of his life — and he did something wrong." A few hours before, we had driven to meet another local who reported seeing someone resembling Cody. But the lead hadn't panned out, and Dial, who had appeared invigorated by the familiar comfort of forward momentum, now seemed drained. He stared out the truck window as we crossed a rickety bridge over the Rio Rincon. In a few hours, Peggy and Cody's sister, Jazz, would be flying in from Anchorage so the family could regroup. "There's nothing worse than losing your kid," he said after a while. "It's like losing a part of yourself — like being alive and dying at the same time." Late last September, I sat with Dial and Peggy in their kitchen in the quiet suburbs of Anchorage. The couple's one-story house is messy and comfortable, with a coil of climbing rope hanging from a birch in the front yard. From the kitchen window, the Chugach Mountains are visible in the distance. It had been nearly a month since Dial returned from Costa Rica — and nearly three months since Cody disappeared — and he and his wife were trapped in a kind of agonizing holding pattern, tethered to the jungle thousands of miles away. They had hired a private investigator to dig for new information. He had tracked down Pata de Loro, who admitted to having guided a different backpacker, according to Peggy, but found little else. A few weeks before, Dial also announced that they would return to Corcovado in late December — to continue the search. Dial had now largely given up hope that Cody had been abducted or disappeared to start a new life. Kidnappers would have contacted the family with a ransom demand; the latter notion, he felt, was wishful thinking. Instead, Dial had come to increasingly believe his son was the victim of foul play. Once he and Peggy returned to the Osa, he said, "we're going to look around and go places we didn't go before. We have some crazy ideas — meet with Catholic priests holding mass and see if anyone knows anything. I hope something comes from it." Peggy planned to meet Segura, whom she found suspicious, and go door-to-door in Dos Brazos and other towns. "Somebody knows something," she told me. A few hours before, Katelyn Barnett had dropped off a plastic bag filled with some of Cody's belongings. Peggy, an energetic and purposeful woman, had placed the bag in another room and then resumed cooking a plate of hamburgers. Now, sitting at the table, she rested her head in her hands. "It's like you just want an answer — a good one," she said. "I don't know. There's too many possibilities, and they keep running through your head. I don't sleep, because all I dream about is finding him. I'm in the jungle looking. I'm always searching for him." The most devastating aspect of Cody's disappearance was, perhaps, the lack of conclusive evidence. A body, though terrible to behold, might provide some measure of closure. Haunted by the unknown, the parents of David Gimelfarb had been returning to the country every year to search for their son. Before I left Costa Rica, Brian Horner had pulled me aside to talk for a few minutes. In 2008, he said, his 19-year-old daughter had died of an aberrant heart attack — an experience that had compelled him to join the search in Corcovado, but a fact he had not divulged to Dial until that night. "I told him, 'I want you to realize this is going to hurt you. It's going to hurt really bad.' " Horner paused. "All the things he's done," he said, "he's never had to confront losing." The death of a child is a violent unraveling of the natural order. The experience is irreparable, utterly shattering, and without reference point in human experience. Books on the subject sometimes compare the trauma to an arduous pilgrimage or a mountain which, in time, can be summited. But the pain can neither be navigated nor overcome with indomitable will. There is no route through such grief. That Cody had come from a family that deeply embraced adventure — as well as its risks — would bring its own tormenting questions. Dial had cheated death on many occasions and survived; his son, very likely, had not. "A lot of parents would be appalled to think of letting their kids take those kinds of chances," Krakauer told me. "But I got it with Roman — his kids were the same way he was. The whole family had this philosophy that is really admirable. To live an overly cautious life in many ways is as dangerous as its opposite. What I worry most is there'll be this guilt they feel or criticism, 'R2's death was your fault.' I don't buy that. It was a terrible thing, but I applaud Roman for the way he's raised his kids, the way he's lived his life. I hope people see him as a role model and not a cautionary tale." A few days later, I met Dial at his house once more. He answered the door unshaven and bleary-eyed. The night before, he said, he'd begun reading The Cloud Garden, a book written by two Brits who'd been kidnapped by guerrillas while attempting to cross the Darién Gap in Panama. We sat down in the kitchen. "I think, 'I may not see him again,' " Dial said. "I get upset a couple times a day. I miss him." Dial had set up his computer on the kitchen table. He played a video of his 2012 trip through Bhutan, Cody flickering in and out of the frame as they drove along the small mountain kingdom's roads. Opening another folder, Dial played a slide show he'd made of his search in Corcovado: the small creek in the clearing, the landslide, the bamboo forest. After several minutes, I asked him about something that had puzzled me since I left Costa Rica: Corcovado was crisscrossed by a dizzying labyrinth of hundreds, probably thousands, of small trails. How was he sure Cody hadn't taken one of them, hoping to push through the jungle, as he had in the Petén, but then become lost or hurt attempting to reach the coast? The question upset him, perhaps because he resented its implication of error, or simply because he had tolerated prying for long enough. "My son wouldn't go on those little trails," Dial said, leaning forward and thumping the table. "I've spent 25 years walking trails with him. He wouldn't blindly fuck off — that's what Chris McCandless would do. That's what you would do. That's what someone who has no experience would do. If you have experience, you backtrack and get back on a good trail. How do I know? Because he's my son."

In early 2014, the son of a biologist and legendary Alaskan adventurer took time off of school for an an epic trip into the depths of Central America. In July, after scuba diving with whale sharks, climbing Guatemala's 14,000-foot Tajumulco, and surfing in Nicaragua, 27-year-old Cody Roman Dial disappeared into a Costa Rican jungle the size of 120 Central Parks and hasn't been seen-at least by his family-since. His father, Roman Dial, has spent the better part of the past two years searching for his son in and around the Corcovado National Park, and National Geographic is airing a six-part series documenting his travails, Missing Dial, on the National Geographic Channel beginning Sunday. Entertainment Weekly reports that the elder Dial and his investigators spent eight months interviewing locals and combing the jungle, and that one week in they had, as Dial puts it, a "significant break" in the case. The team manages to "uncover a web of lies, surprising clues and a shocking plot twist that rivals a blockbuster movie," as the National Geographic Channel puts it, but Dial himself won't reveal any more than that "we know that Cody was murdered, and we know there is a suspect." Dial had raised his son to "survive in the wild," Men's Journal reported soon after Cody's disappearance, with the two of them enduring harsh climates and vast wilderness throughout Alaska. Cody was up against canyons, rivers, thick foliage, venomous snakes, and roughly 400 illegal gold mines, reports Outside, the last of which may have proved most dangerous. "I'll be bounded by a trail to the west and the coast everywhere else, so it should be difficult to get lost forever," Cody wrote. But in the end, the elder Dial says of the reality of living so adventurously, "The people who are going to be suffering are the people who loved you and lost you."

multi_news

Host colonization by lymphotropic γ-herpesviruses depends critically on expansion of viral genomes in germinal center (GC) B-cells. Myc is essential for the formation and maintenance of GCs. Yet, the role of Myc in the pathogenesis of γ-herpesviruses is still largely unknown. In this study, Myc was shown to be essential for the lymphotropic γ-herpesvirus MuHV-4 biology as infected cells exhibited increased expression of Myc signature genes and the virus was unable to expand in Myc defficient GC B-cells. We describe a novel strategy of a viral protein activating Myc through increased protein stability resulting in increased progression through the cell cycle. This is acomplished by modulating a physiological post-translational regulatory pathway of Myc. The molecular mechanism involves Myc heterotypic poly-ubiquitination mediated via the viral E3 ubiquitin-ligase mLANA protein. EC5SmLANA modulates cellular control of Myc turnover by antagonizing SCFFbw7 mediated proteasomal degradation of Myc, mimicking SCFβ-TrCP. The findings here reported reveal that modulation of Myc is essential for γ-herpesvirus persistent infection, establishing a link between virus induced lymphoproliferation and disease. Myc is a transcription factor that enhances the expression of genes involved in cellular growth and proliferation. Hence, it is not surprising that viruses have evolved mechanisms to modulate Myc to promote their own life cycle. Myc heterodimerizes with Max, through a basic region/helix-loop-helix/leucine-zipper domain, to regulate the transcription of specific E-box-containing genes in response to mitogenic stimuli. Myc functions as a universal amplifier of gene expression by promoting the transcriptional elongation of RNA polymerase II driving biomass accumulation and enhanced cellular bioenergetic pathways [1], [2], [3]. The expression of c-myc is tightly regulated with extremely short half-lives for mRNA and protein. In non-transformed cells, Myc is continuously subjected to ubiquitination and proteasomal-degradation, resulting in a highly unstable protein with a half-life of about 15–20 minutes [4]. Several mechanisms of Myc regulation have been identified that operate at the level of protein stability. The best characterized mechanism involves the interplay between phosphorylation at two specific residues and ubiquitination. Phosphorylation at serine (S) 62 by extracellular signal-regulated kinase (ERK) stabilizes Myc resulting in enhancement of its transcription activity. In contrast, phosphorylation of Myc at threonine (T) 58 by glycogen synthase kinase 3 (Gsk-3), which is dependent on previous phosphorylation of Myc at S62, leads to proteasomal degradation of Myc [5]. The mechanism involves the assembly of homotypic poly-ubiquitin chains on Myc specifically dependent on lysine (K) 48 linkage by SCF (Skp1/Cul/Fbox) Fbw7 [6], [7]. Myc turnover by SCFFbw7 is antagonized by polymerization of mixed heterotypic poly-ubiquitination chains via SCFβ-TrCP on the N-terminus of Myc [8]. Thus, SCFFbw7 and SCFβ-TrCP assemble different K-linkage poly-ubiquitin chains with functionally distinct outcomes on Myc stability, i. e., degradation versus stability. The physiological relevance of regulating Myc activity through protein stability is underscored by observations that point mutations at or near T58, which render Myc resistant to proteasomal degradation, occur with high frequency in B-cell lymphomas [9]. Examples of viruses that modulate Myc activity include Kaposi' s sarcoma associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Infection by these γ-herpesviruses is characterized by the establishment of latent infection in memory B-cells. Access to this cell type is gained by virus-driven proliferation of germinal centre (GC) B-cells [10], where virus genomes replicate and segregate in step with normal cell division. This process is mediated by episomal maintenance proteins, which include EBV nuclear antigen-1 (EBNA-1) [11] and latency associated nuclear antigen (LANA) encoded by ORF73 of γ-2-herpesviruses [12]. Given the essential role of Myc for the initiation and maintenance of GCs [13], [14], it is not surprising that γ-herpesviruses have evolved mechanisms to modulate Myc activity. In the case of KSHV-associated primary effusion lymphoma, Myc was shown to be abnormally stabilized [15], [16]. The mechanism involves the direct interaction of the viral protein LANA with Gsk-3 resulting on reduced levels of Myc T58 phosphorylation [17]. Another strategy appears to be employed by EBV encoded EBNA-3C protein. This viral protein was shown to increase the transcriptional activity of Myc through an interaction with both Myc and SCFSkp2. Surprisingly the mechanism proposed does not involve poly-ubiquitination but rather the action of SCFSkp2 functioning as a transcription co-factor for Myc [18]. Here, we utilized murid herpesvirus-4 (HuHV-4) infection of mice as a model system to address the role of Myc in the pathogenesis of γ-herpesviruses. We show that Myc expression is required for the expansion of MuHV-4 infection in GC B-cells. The mechanism involves heterotypic poly-ubiquitination of Myc mediated through the ElonginC/Cullin5/SOCS (supressors of cytokine signaling) (EC5S) E3 ubiquitin-ligase activity of mLANA encoded by ORF73 of MuHV-4. EC5SmLANA mimics SCFβ-TrCP by antagonizing SCFFbw7-mediated proteasomal turnover of Myc but unlike the cellular E3 ubiquitin-ligases its activity is not dependent on the phosphorylation status of Myc. Our results underscore the importance of modulating Myc activity during γ-herpesvirus driven lymphoproliferation providing a link between persistent infection and lymphoproliferative disease. Experiments were designed to investigate the role of Myc expression on gammaherpesvirus pathogenesis. We utilized MuHV-4 infection of laboratory mice as the model, which is characterized by the expansion of latently infected B-cells in GCs and virus persistence in memory B-cells [19]. We analysed the transcription of several Myc target genes in infected versus non-infected GC B-cells, purified from the same pool of splenocytes. We utilized a recombinant MuHV-4 expressing a yellow fluorescent protein (YFP) [20] to segregate infected (CD19+CD95hiGL7hiYFP+) from non-infected (CD19+CD95hiGL7hiYFP−) GC B-cells derived from C57BL/6 mice, at day 13 post-infection. We analysed the transcription of Myc signature genes involved in cell cycle entry (encoding for cyclins B1, D1, D2 and E, and cyclin-dependent kinase 4) and GC B-cell activation (encoding for IL-10, B-ATF, MIF and CD70). Cyclin D3 was included as a gene non-regulated by Myc. The transcription of Myc target genes was significantly increased in infected GC B-cells when compared with their non-infected counterparts (Figure 1A). These data show that during the expansion of latent infection in GC B-cells, MuHV-4 induces a transcription profile compatible with increased Myc transcriptional activity. To define the impact of Myc expression on gamma-herpesvirus pathogenesis, we generated mice with conditional deletion of c-myc in B-cells undergoing GC reaction. This was achieved by breeding homozygous c-mycfl/fl mice, where second and third exons of the c-myc locus are flanked by two loxP sites [21] to heterozygous Cγ1-cre mice, in which expression of Cre recombinase is induced by transcription of the Ig-γ1 constant region gene segment early in GC development during immunoglobulin class-switch recombination [22]. Resulting progeny Cγ1-creKI/WT; c-mycfl/fl, hereafter designated GC Myc KO, is expected to have specific deletion of c-myc in class-switched GC B-cells, after immunization with T-dependent antigens. Thus, we next investigated GC responses in the absence of Myc expression. GC Myc KO mice were immunized with the Th-2 cell-dependent antigen 4-hydroxy-3-nitrophenylacetyl (NP) –chicken γ-globulin (CGG) adsorbed to alum. Frequencies of GC B-cells (CD19+CD95hiGL7hi) were analysed at day 10 post-immunization. Compared to control litter mates Cγ1-creWT/WT; c-mycfl/fl, hereafter designated control mice, GC Myc KO mice showed a marked impairment in GC development (Figure 1B), accompanied by a strong reduction in IgG1+ B-cell numbers (CD19+IgD−IgM−IgG1+) (Figure 1C). Immunization with ovalbumin (OVA) emulsified in complete Freund' s adjuvant (CFA) further confirmed that GC Myc KO mice were defective to mount a normal GC response, though less deficient than upon immunization with NP-CGG, and presented reduced levels of IgG1 expressing B-cells (Figure 1D and 1E, respectively). However frequencies of IgG2a/2b+ B-cells (CD19+IgD−IgM−IgG2a/2b+), whose class-switching is not strictly dependent on Ig-γ1 promoter, revealed that GC Myc KO mice are competent in developing IgG2 class-switched B-cells (Figure 1F). These data demonstrate a clear requirement for Myc expression in order to generate GC reactions to T cell dependent antigens. Our results are in direct agreement with those recently published that demonstrate the lack of GCs in mice in which c-myc is ablated early during GC induction [13]. Since we obtained the two transgenic mice lines carrying the alleles c-mycfl/fl and Cγ1-cre from these authors, and independently generated GC Myc KO mice, our data are directly comparable. The requirement of Myc for gammaherpesvirus pathogenesis was next investigated by infecting GC Myc KO mice with MuHV-4. Analysis of the percentages of GC B-cells revealed no significant differences between control and GC Myc KO mice (Figure 2A), with the majority of infected GC B-cells falling into dark zone (Figure 2B) as previously described for infection of wild type mice [23]. To determine if MuHV-4 infected GC B-cells had been or not exposed to Cre-mediated c-myc deletion, we FACS purified infected cells from GC Myc KO mice, at day 14 post-infection. A PCR assay was employed to detect floxed and deleted c-myc alleles in DNA from infected GC B-cells, compared to DNA from uninfected total B-cells (CD19+YFP−), purified from the same pool of splenocytes. Infected cells were found to have undeleted (floxed) c-myc allele and no c-myc rearrangement could be detected (Figure 2C). This contrasted with total non-infected B-cell population where c-myc deletion could be readily detected (Figure 2C). To further confirm the integrity of the c-myc locus in infected GC B-cells in GC Myc KO mice, we quantified Myc mRNA levels. Comparison of GC B-cells from GC Myc KO with wild type infected mice revealed no significant differences in Myc transcription (Figure 2D). These data imply that MuHV-4 is expanding exclusively in GC B-cells where the c-myc locus did not undergo Cre-mediated deletion. Accordingly, GC Myc KO mice infected with MuHV-4 were unable to class-switch to IgG1, which is consistent with Myc deficiency in these cells (Figure 2E). However, these infected mice showed high numbers of IgG2a/2b positive B-cells, equivalent to infected control mice counterparts (Figure 2F). Quantification of the frequency of viral DNA positive cells in GCs, assessed by limiting dilution combined with real time PCR, showed an approximately 10-fold deficit of latent infection at 14 days post-infection in GC Myc KO mice (Figure 2G). This deficit was likely a reflection of infection of B-cells that did not result in the expansion in GC reactions due to Ig-γ1 Cre mediated c-myc deletion. However, at day 21 post-infection latent viral loads in control and conditional KO mice were equivalent (Figure 2G), indicating a recovery from the early deficit in expansion. Collectively data obtained with GC Myc KO mice demonstrated that infection with MuHV-4 does not compensate for Myc loss in GC B-cells and virus is found to amplify exclusively in c-myc intact cells. Therefore, Myc is essential for the expansion of latently infected GC B-cells, thus critical for the establishment of persistent infection. We have shown before that the ORF73 protein encoded by MuHV-4, designated mLANA by homology with the latency associated nuclear antigen encoded by KSHV, is selectively transcribed in GC B-cells [24]. Thus mLANA was a strong candidate to be responsible for the observed increased transcription of Myc target genes in MuHV-4 infected GC B-cells. To address this hypothesis, we analysed the transcription of Myc target genes in mLANA expressing cells. When compared to control transfected cells, mLANA expression induced the transcription of all Myc target genes analysed (Figure 3A). Expression of mLANA had no effect on c-myc mRNA levels indicating that its putative modulatory effect on Myc was post-transcriptional (Figure 3B). We have also shown before that mLANA acts as the substrate recognition factor of an ElonginC-Cullin5-SOCS (suppressor of cytokine signalling) (EC5S) E3 ubiquitin-ligase towards the p65/RelA cellular transcription factor NF-κB [25]. The mechanism involves the assembly of an EC5S -like complex, mediated by a viral unconventional SOCS-box motif present in mLANA. Hence, we analysed if mLANA-mediated modulation of transcription of Myc target genes could be attributed to its function as an E3 ubiquitin-ligase. To this end, we utilized a previously characterized mLANA mutant, designated mLANA-SOCS where residues V199, L202, P203 and P206 were substituted by alanines abrogating E3 ubiquitin-ligase function [25]. This mutant was no longer able to modulate the expression of Myc target genes when compared with intact mLANA (Figure 3A). To define if the observed mLANA modulatory effect on cellular transcription was Myc specific, we carried out gene reporter assays using a synthetic Myc reporter plasmid containing three copies of E-box sequences driving the expression of luciferase. As expected, overexpression of Myc was translated into a significant increase on luciferase activity (Figure 3C). Cells expressing mLANA exhibited comparable levels of luciferase activity, which increased further when Myc was concomitantly expressed. In contrast, mLANA-SOCS expression showed no effect on luciferase levels. We next proceeded to analyse the modulatory effect of mLANA on Myc in B-cells, which are physiological more relevant given the tropism of MuHV-4. When compared to control and mLANA-SOCS transfected cells, mLANA expression induced the transcription of all Myc target genes analysed (Figure 3D). As before, expression of mLANA in A20 B-cells had no effect on c-myc mRNA levels confirming that its modulatory effect on Myc was post-transcriptional (Figure 3E). Myc transcriptional activation of genes encoding proteins involved in cell cycle entry results in transition from G0-G1 to S phase. Thus, we analysed cell cycle profiles in B-cells expressing mLANA in comparison to control or mLANA-SOCS. These experiments showed a clear decrease in the number of mLANA expressing cells in G1 phase, with a concomitant increase in the number of cells in S and G2-M phases (Figure 3F). Combined these data indicate that mLANA is modulating Myc-dependent transcription and progression through cell cycle in B-cells through its activity as an EC5S E3 ubiquitin-ligase. Co-immunoprecipitation experiments also showed that mLANA and Myc exist in the same heteromolecular complex in a context of virus infection. This was demonstrated using a murine B-cell lymphoma-derived cell line latently infected with MuHV-4, designated S11 cells (Figure 3G). We also showed that this interaction was reduced for mLANA-SOCS (Figure 3H, compare lanes 3 and 5). This reduction could be due to lower levels of Myc in mLANA-SOCS expressing cells, when compared to mLANA, indicating that the interaction is independent of the SOCS-box motif. Alternatively it is plausible that the SOCS-box is participating in mLANA-Myc interaction. We next set out experiments to investigate if EC5SmLANA was able to mediate poly-ubiquitination of Myc. We started by performing a nickel-nitrilotriacetic acid (Ni-NTA) pull-down in the presence of histidine-tagged ubiquitin. Upon culture and cell lysis, ubiquitinated proteins were extracted from total cellular lysates with Ni-NTA beads and resolved by SDS-PAGE. The levels of ubiquitinated Myc present in each condition were analysed by immunoblotting. We observed that when mLANA was expressed, the levels of ubiquitinated Myc were significantly enhanced (Figure 3I, compare lanes 3 and 4). To confirm this activity in a more relevant biological context, we evaluated the ability of mLANA to promote the ubiquitination of endogenously expressed Myc. In comparison with control transfected and mLANA-SOCS transfected cells, higher levels of ubiquitinated Myc were detected in the presence of intact mLANA (Figure 3J, compare lane 2 with 1 and 3). We have previously demonstrated that mLANA E3 ubiquitin-ligase activity towards p65/RelA required the E2 ubiquitin-conjugating enzyme UbcH5 [25]. Here we showed that EC5SmLANA requires UbcH5 as its E2 conjugating partner to mediate poly-ubiquitination of Myc (Figure S1). Myc protein has 25 lysine residues that can be potentially ubiquitinated. To define if the ubiquitination ladder observed was due to the ability of mLANA to mediate poly-ubiquitination or multiple mono-ubiquitination of Myc in different lysine residues, we made use of a previously described lysine-free (K−) version of Myc, which can only be ubiquitinated on its N-terminal residue [8]. By performing an in vivo ubiquitination assay with K−Myc, we observed the following. First, mLANA was able to mediate poly-ubiquitination of the N-terminal residue of Myc (Figure 3K, compare lanes 3 and 4). Secondly, K−Myc in the presence of mLANA exhibited a ladder of ubiquitination indicating the assembly of poly-ubiquitin chains (Figure 3K, compare lanes 3 and 4). Finally, the ubiquitination pattern of wild type Myc and K−Myc are distinctive, implicating that other lysine residue (s) are targets for mLANA-mediated poly-ubiquitination of Myc. The addition of ubiquitin chains to a target protein can occur with different moieties that are emerging as determinants of biological outcome. These include, mono-ubiquitination and poly-ubiquitination. Ubiquin chains can be assembled using seven internal lysine residues (K) and thus define homotypic poly-ubiquitination (same K-linkage) or heterotypic poly-ubiquitination (mixed K-linkages) [26]. To investigate the type of lysine (K) -linkage involved in mLANA-mediated poly-ubiquitination of Myc we utilized of a series of ubiquitin mutants with every single lysine residue, of the possible seven, substituted by an arginine. By performing in vivo ubiquitination assays in mLANA expressing cells we observed that in the absence of K33, K48 or K63 residues the ability of mLANA to promote Myc poly-ubiquitination was suppressed (Figure 4A). Next, Myc transcription reporter assays were carried out in cells expressing the same ubiquitin mutants. Consistent with the poly-ubiquitination pattern, replacement of K33, K48 and K63 in ubiquitin rendered mLANA unable to positively modulate Myc transcriptional activity (Figure 4B). Measurement of Myc cellular levels in extracts from mock transfected control or mLANA-transfected cells co-expressing each ubiquitin mutant, revealed that in the presence of wild type ubiquitin, mLANA-expressing cells exhibit augmented Myc levels (Figure 4C, upper panel, compare lanes 1 and 2). The same result is observed when residues K6, K11, K27 and K29 of ubiquitin were substituted by arginines (Figure 4C, upper and lower panels). However, in good agreement with the previous data, substitution of K33, K48 and K63, diminished or abolished the ability of mLANA to promote the increase in Myc cellular levels (Figure 4C). Our observations demonstrate the involvement of different lysine-linkages in EC5SmLANA Myc poly-ubiquitination. However, these data do not distinguish between homotypic poly-ubiquitination at different K residues in Myc from mixed K-linkage poly-ubiquitination. Hence, we next utilized K−Myc where ubiquitination is only possible at the first metionine. In vivo ubiquitination assays and transcription reporter assays with K−Myc, in combination with each of the K ubiquitin mutants, essentially recapitulated the above observed results with wild type Myc (Figure 4D and E). Hence, heterotypic poly-ubiquitination of the N-terminus of Myc is sufficient for mLANA modulatory activity. Collectively, these data show that EC5SmLANA requires different K-linkages to ubiquitinate Myc. Moreover, they demonstrate a direct correlation between the ability of mLANA to mediate Myc poly-ubiquitination, to increase Myc cellular levels, and to promote its transcriptional activity, supporting that all three activities are directly linked. Although classically associated with protein degradation, ubiquitination is now emerging as regulator of a wide variety of non proteolytic cellular signalling functions [26]. Therefore, and in agreement with a positive effect on Myc activity, we further confirmed that mLANA-mediated poly-ubiquitination of Myc was non-degradative. To that end, we performed an in vivo ubiquitination assay in the presence of the proteasome inhibitor MG132. In control-transfected cells, the presence of MG132 favoured the accumulation of Myc ubiquitinated species (Figure 5A, top panel, compare lanes 1 and 2), as well as the cellular levels of Myc protein (Figure 5A, bottom panel, compare lanes 1 and 2). In contrast, in cells expressing mLANA, inhibition of proteasomal degradation had a negligible influence on both Myc poly-ubiquitination and Myc protein levels (Figure 5A, compare lanes 3 and 4). The importance of these results is threefold. Firstly, they confirm that under physiological conditions Myc turnover is highly regulated by the proteasome. Secondly, they show that treatment with the proteasome inhibitor MG132 has no effect on mLANA-mediated poly-ubiquitination of Myc, thus mLANA in not directing Myc for proteasomal degradation. Thirdly, the increase in Myc levels in response to mLANA expression was not altered under conditions of proteasomal inhibition, suggesting that expression of mLANA is preventing the proteasomal degradation of Myc. To assess this hypothesis, we next compared the half-life of Myc in control and mLANA expressing cells. Cells were treated with the protein synthesis inhibitor cycloheximide (CHX), and Myc protein levels were analyzed by immunoblotting at different time-points post-treatment. Under these experimental conditions, expression of mLANA led to a pronounced raise in Myc stability with an increase of half-life from ≈20 minutes to over 4 hours (Figure 5B). Collectively these results demonstrate that mLANA expression has a positive effect on Myc cellular levels by preventing its proteasomal turnover, thus prolonging its half-life, which is associated with increased transcriptional activity. Under physiological conditions Myc half-life is tightly regulated through poly-ubiquitination by two distinct Skp1/Cul1/F-box (SCF) E3 ubiquitin-ligases. That is, poly-ubiquitination of Myc by SCFβ-TrCP antagonizes SCFFbw7-mediated proteasomal dependent turnover [6], [8]. Therefore, we hypothesised if mLANA could be modulating Myc stability by counteracting degradation by Fbw7 and mimicking the activity of β-TrCP. We overexpressed Fbw7 and analysed Myc cellular levels in the presence of co-expressed mLANA. As expected, overexpression of Fbw7 led to a decrease in Myc levels (Figure 5C, compare lanes 1 and 2). Overexpression of Fbw7 had no effect on Myc levels when mLANA was concomitantly expressed (Figure 5C, compare lanes 3 and 4). Notably, the antagonizing activity of mLANA towards Fbw7 was more pronounced when compared with that afforded by β-TrCP (Figure 5C, compare lanes 4 and 6). We further characterized the mLANA effect on Fbw7 and β-TrCP interplay by depletion of the expression of the two cellular E3 ubiquitin-ligases and analysis of Myc levels. When Fbw7 was depleted Myc levels increased further by the presence of mLANA (Figure 5D, compare lanes 3 and 4). In agreement, the turnover effect on Myc levels caused by depletion of β-TrCP was counteracted by concomitant expression of mLANA (Figure 5E, compare lanes 3 and 4). Together these results demonstrate that mLANA mimics SCFβ-TrCP by antagonizing SCFFbw7-mediated proteasomal turnover of Myc. Fbw7 control of Myc turnover is dependent on the interaction between the ubiquitin-ligase and its substrate. Fbw7 recognizes Myc when phosphorylated on threonine (T) 58 and catalyses its poly-ubiquitination resulting in Myc proteasomal degradation [6], [7]. Phosphorylation of Myc on T58 is sequentially preceded by phosphorylation on serine (S) 62, which activates and promotes Myc stability [5]. Thus T58 and S62 are key phospho-residues that regulate Myc activity at the protein level. Therefore, we set to investigate the influence of Myc phosphorylation on the modulatory activity of mLANA. Using phospho-specific antibodies we observed that under mLANA expression Myc is phosphorylated on both S62 and T58 (Figure 6A, lane 2, first and second panels, respectively). To analyse if sequential phosphorylation of Myc was intact on mLANA expressing cells we proceed to substitute S62 or T58 to alanines (A) on Myc and assess the phosphorylation status of those Myc mutants. Compatible with the model of sequential phosphorylation, in which phosphorylation of S62 precedes phosphorylation of T58, MycT58A was readily phosphorylated on S62, whereas MycS62A was not phosphorylated (Figure 6A, lanes 3–6, first and second panels, respectively). Remarkably, analysis of total Myc cellular levels revealed that, regardless of Myc phosphorylation on either S62 or T58, co-expression of mLANA led to increased Myc levels (Figure 6A, third panel). These data not only demonstrate that mLANA is not interfering with Myc phosphorylation, but also indicates that mLANA modulatory functions override cellular pathways that control Myc activity. Consistent with this hypothesis, mLANA is co-immunoprecipitated by both Myc mutants (Figure 6B), and it is able to mediate their poly-ubiquitination (Figure 6C). Transcriptional activities of MycT58A and MycS62A were also increased by expression of mLANA (Figure 6D), further supporting that mLANA targets Myc independently of cellular mechanisms of Myc regulation. In this study, we describe the first example of a viral protein activating Myc transcriptional activity through increased protein stability by mimicking a physiological post-translational regulatory pathway. Modulation of Myc function was shown to be essential for the lymphotropic γ-herpesvirus MuHV-4 biology as infected cells exhibit increased expression of known Myc target-genes, and using a genetic approach, virus was found to amplify exclusively in intact Myc GC B-cells. The molecular mechanism involved heterotypic poly-ubiquitination of Myc mediated via the mLANA protein encoded by ORF73. This was reminiscent of a newly described pathway of Myc regulation through poly-ubiquitination. Popov et al. showed that the cellular E3 ubiquitin-ligase SCFβ-TrCP uses UbcH5 ubiquitin-conjugating enzyme to form heterotypic poly-ubiquitin chains on the N-terminus of Myc. Poly-ubiquitination of Myc by SCFβ-TrCP leads to Myc stabilization and was shown to antagonize SCFFbw7-mediated proteasomal turnover of Myc [8]. Like SCFβ-TrCP, EC5SmLANA uses UbcH5 and antagonizes SCFFbw7. Furthermore, as previously demonstrated for SCFβ-TrCP, single substitutions of K33, K48 or K63 of ubiquitin reduced or eliminated the ability of EC5SmLANA to poly-ubiquitinate, stabilize or increase the transcriptional activity of Myc. However, our results suggest that the molecular mechanism of mLANA modulation of Myc activity is not limited to Fbw7 antagonism. This is supported by the fact that mLANA protective effects on Myc stability, in conditions of Fbw7 over expression, are significantly more pronounced than observed for β-TrCP. Moreover, mLANA was able to increase the stability and activity of MycT58A, a Myc version that is not recognized by Fbw7. This contrasts with what has been reported for β-TrCP that albeit being able to poly-ubiquitinate MycT58A it did not impact on its turnover [8]. Thus, the ability of mLANA to increase Myc transcriptional activity through poly-ubiquitination, independently of the phosphorylation status of Myc on S58 and T62, indicates that this novel viral modulatory mechanism does not rely on post-translation cellular regulation. Modulation of host B-cell biology is of vital importance for γ- herpesviruses, as they depend critically on the expansion of latently infected B-cell in GC reactions for host colonization. GC reactions exhibit two distinct morphological areas. These include the dark zone (DZ), where centroblasts are rapidly dividing, and the light zone (LZ), where B-cells exit the cell cycle to differentiate into plasma cells or memory B-cells or reenter the DZ for additional rounds of cell division. Recently two studies have established the importance of Myc for GC biology [13], [14]. These studies show that Myc expression is restricted to minute clusters of B-cells that initiate GCs and a small fraction of LZ B-cells. Furthermore, genetic interference with Myc expression or activity blocks GC formation and results in the collapse of mature GCs. Combined these studies demonstrate that expression of Myc is essential for the initiation and maintenance of GCs preceding B-cell proliferation in the DZ. It has been proposed that the lack of Myc expression in DZ B-cells, in conjunction with its short half-life, settles strict limits on the number of cell divisions afforded by centroblasts [27]. Therefore, by overriding the post-translational physiological control of Myc and significantly prolonging its half-life, mLANA favors an increase in the number of cell divisions during the expansion of MuHV-4 infected B-cells in the GC competitive niche. This modulation is likely to operate in infected B-cells at the initiation of a GC reaction and re-entry into the DZ. This interpretation is consistent with the observation that around 70% of MuHV-4 infected GC B-cells are rapidly dividing centroblasts versus approximately 20% infection in centrocytes. However, we show here that MuHV-4 is not able to latently expand in B-cells depleted of Myc expression. Hence, it is dependent on previous expression of Myc in B-cells. This is supported by our observation that MuHV-4 infected cells do not exhibit increased levels of Myc mRNA. Combined, our data is in good agreement with a post-translational mechanism of mLANA-mediated Myc stabilization in latently infected B-cells to increase their proliferative potential within GC reactions. This modulation is compatible with the selective expression of mLANA within GC B-cells [24] and its viral episomal maintenance properties [28]. By increasing the proliferative potential of latently infected GC B-cells through the modulation of Myc, γ-herpesviruses are effectively promoting host colonization. However, different γ-herpesviruses have evolved distinct mechanisms to accomplish Myc modulation. KSHV achieves this via LANA through a mechanism that involves targeting of Gsk-3 [17] whereas EBV appears to increase the transcription activity of Myc via interaction of EBNA3C with SCFSkp2 [18]. However, how vital is Myc modulation for γ-herpesvirus host colonization? We have previously reported that EC5SmLANA mediates poly-ubiquitination-dependent proteosomal degradation of the NF-κB family member p65/RelA [25]. In that study we demonstrate that a recombinant MuHV-4 with a disrupted SOCS-box motif in mLANA loses the ability of the virus to expand in GC B-cells and persist in the mouse. Given that this recombinant lacks ubiquitin-ligase activity we cannot ascribe its phenotype to NF-κB or Myc modulatory effects since both activities depend on an intact SOCS-box motif. We show here that mLANA interacts with Myc through a motif independent of the SOCS-box and have shown before that this also applies to interaction with p65/RelA [25]. We have mapped both interactions to the N-terminal half of mLANA but have been unable to identify any discrete binding motif to both cellular targets (unpublished observations). Since structural modeling of the N-terminal half of mLANA predicts it to be unstructured we envisage that conformational rather than linear binding motifs may be required for interaction of mLANA with p65/RelA and Myc. Current mLANA structural studies in our laboratory are addressing this question. However, the property of mLANA to modulate both Myc and p56/RelA supports that maintenance of a proliferative GC reaction through Myc stabilization requires simultaneous inhibition of NF-κB signaling. The molecular basis for EC5SmLANA-mediated poly-ubiquitination of Myc and p65/RelA resulting in opposed outcomes is not known. Effector proteins with ubiquitin binding domains (UBDs) trigger specific cellular responses by recognizing different types of ubiquitin topologies [26]. Hence, the decoration of Myc and p65/RelA with distinct K-linkages and lengths by EC5SmLANA may determine distinct ubiquitin-mediated cellular functions. It is interesting to note that in this respect a parallel exists between EC5SmLANA and SCFβ-TrCP, as the latter also targets IκBα for proteasomal-mediated degradation [29] whereas it promotes Myc stabilization. The identification of UBD containing proteins that discriminate poly-ubiquitin chains topologies, which determine different biological outcomes is a field under intensive investigation. mLANA, therefore, provides as a good in vivo model for future studies. Herein, we described a novel viral mechanism of stabilization of Myc through heterotypic poly-ubiquitination mediated by mLANA. The findings presented sustain the interpretation that increasing Myc stability is critical for the amplification of γ-herpesviruses in GC B-cells, thus persistence in the host. Therefore, this study provides a pathogenesis link between Myc and γ-herpesviruses associated lymphoproliferative disease. This study was carried out in strict accordance with the recommendations of the Portuguese official Veterinary Directorate (Portaria 1005/92). The Portuguese Experiments on Animal Act strictly comply with the European Guideline 86/609/EEC and follow the FELASA. Animal experiments were approved by the Portuguese official veterinary department for welfare licensing under the protocol number AEC_2010_017_PS_Rdt_General and the IMM Animal Ethics Committee. For reporter gene assays, cells were transiently transfected with 500 ng of reporter vector, 1 µg of Myc and mLANA/mLANA–SOCS expression plasmids. In all transfections, a Renilla luciferase plasmid (10 ng) was used to normalise luciferase activity. Firefly and Renilla luciferase activities were assayed using Dual-Luciferase (Promega). Results are shown as fold induction relative to firefly luciferase activity measured in control-transfected cells. Cell cycle distribution profiles were analysed using Vybrant DyeCycle Violet Stain (Invitrogen) according to the manufacter' s instructions. Briefly, 24 h post-tranfection with GFP, GFP-mLANA or GFP-mLANA-SOCS expressing plasmids, 1×106 A20 cells were incubated with 1 µl of Vybrant DyeCycle in complete RPMI for 30 minutes, at 37°C. The percentage of cells in the various phases of cell cycle was determined using FlowJo software (Tree Star, Inc), implementing the Dean-Jett-Fox model. Three independent experiments were performed for each experimental condition and a representative experiment is shown. Cells were transiently transfected with plasmids encoding Myc or Myc phospho- mutants (2 µg), ubiquitin or ubiquitin with specific lysines mutated to arginines (4 µg), UbcH5 (2 µg) and/or mLANA or mLANA-SOCS (2 µg). Cells were disrupted in 10 mM Tris–HCl (pH 7. 5), 150 mM NaCl, 1% Triton X-100,1 mM NaF, 100 mM Na3VO4 and protease inhibitors (Complete; Roche). Supernatants were processed for immunoprecipitation as described [30]. Analysis of Myc or K− Myc ubiquitination was performed under denaturing conditions (20 mM Tris–HCl (pH 7. 5), 5 mM EDTA, 1% SDS, 10 mM dithiothreitol and Complete). Lysates were boiled for 10 minutes at 100°C, diluted 1/10 in lysis buffer and proceeded to immunoprecipitation. Levels of in vivo ubiquitinated Myc were determined by pull-down using Ni-NTA agarose beads. Cells were transfected with plasmids carrying His6-ubiquitin (4 µg), Myc (2 µg), and/or mLANA (2 µg). When indicated, cells were incubated for 8 hr in 10 µM MG132 (Calbiochem). Transfected cells were lysed in urea buffer (8M urea, 50 mM Tris-HCl (pH 7. 5), 300 mM NaCl, 1% Triton X-100,10 mM imidazole, 1 mM Na3VO4 and Complete), incubated for 2 hr at 4°C with Ni-NTA beads that were collected and washed with urea buffer. Proteins were eluted, denatured by boiling in Laemmli' s buffer and analyzed by immunoblotting. Total RNA from FACS-purified uninfected or infected GC B-cells, or transfected HEK 293T cells or A20 B cells was extracted with Trizol (Invitrogen). RNA (500 ng) was used for cDNA synthesis (DyNAmo, Finnzymes). qPCR was performed using DyNAmo Flash SYBR Green (Finnzymes). Primer sequences are available in Table S1. All reactions were run in duplicates. Amplification efficiencies and threshold cycle values were defined by the fractional cycle number at which fluorescence crosses the fixed threshold. Relative mRNA values, normalized to GAPDH, were calculated by the Pfaffl method [31]. Cγ1-Cre mice were provided by Dr. Kurosaki (Japan), with the agreement of Dr. Rajewsky and Dr. Casola. c-myc floxed mice were a gift from Dr. Moreno de Alborán (Spain). Cγ1-creKI/WT; c-mycfl/fl mice were generated by breeding heterozygous Cγ1-cre mice [22], with homozygous c-mycfl/fl mice [21]. Cγ1-creWT/WT; c-mycfl/fl mice littermates were used as controls. C57BL/6 mice were obtained from Charles River Laboratories International Inc. Mice were bred and housed at IMM. All experimental protocols were performed in animals with 6–8 weeks of age. Immunizations were performed via intraperitoneal injection with 100 µg NP-CGG (Biosearch Technologies, Inc.) adsorbed to 3 mg of aluminium hydroxide (SERVA Electrophoresis GmbH), or 100 µg ovalbumin (OVA) grade V in CFA (Sigma). OVA immunized mice were challenged 7 days post-primary immunization with the same antigen/adjuvant combination. Inoculation of MuHV-4 was performed intranasally with 104 p. f. u. in 20 µl of PBS under halothane. Frequencies of MuHV-4 genome-positive cells in GC B-cells were determined by limiting dilution combined with real-time PCR as previously described [32]. GC B cells were FACS purified from pools of five spleens using a BD FACSAria Flow Cytometer (BD Biosciences) and serially two-fold diluted. Eight replicates of each dilution were analysed by real time PCR (Rotor Gene 6000, Corbett Life Science). The primer/probe sets were specific for the MuHV-4 M9 gene (5′ primer: GCCACGGTGGCCCTCTA; 3′ primer: CAGGCCTCCCTCCCTTTG; probe: 6-FAM-CTTCTGTTGATCTTCC–MGB). Samples were subjected to a melting step of 95°C for 10 min followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. Real-time PCR data was analyzed on the Rotor Gene 6000 software. The purity of sorted cells was always greater than 97%, as analyzed by flow cytometry. Information provided in protocols S1.

Being obligatory intracellular parasites, it is not surprising that viruses have evolved mechanisms to induce cellular proliferation to promote their own life cycle. This is notorious in the case of γ-herpesviruses, such as Epstein-Barr virus (EBV) and Kaposi' s sarcoma virus (KSHV), which are human pathogens associated with lymphoproliferative disease and several tumors. Host colonization by γ-herpesviruses is critically dependent on the ability to expand latent infection in proliferating B-cells. Virus-induced cellular proliferation is a process mediated by the expression of specific viral proteins. One of such proteins is the latency-associated protein (LANA) of KSHV. In this study, we use murid herpesvirus-4 (MuHV-4) as a mouse model of γ-herpesvirus pathogenesis. We show that the MuHV-4 LANA (mLANA) stabilizes the cellular oncogene Myc, increasing its half-life, thus promoting its activity as a potent inducer of cellular proliferation. The molecular mechanism involves heterotypic poly-ubiquitination of Myc mediated via mLANA. The findings here reported demonstrate that modulation of Myc is essential for γ-herpesvirus persistent infection, establishing a link between virus induced lymphoproliferation and disease. The implication is that revealing a critical function of a viral protein possibly allows the development of small molecule probes to disrupt mLANA-Myc interaction, therefore inhibit virus induced lyhophoproliferative disease.

lay_plos

The first images of Joseph Fiennes as Michael Jackson that you can't unsee CLOSE Skip in Skip x Embed x Share A British TV production hired white actor Joseph Fiennes to portray Michael Jackson. The recently-released trailer, gives viewers the first glimpse of the controversial casting choice in action. USA TODAY Bad news, 2017: The Joseph Fiennes-as-Michael Jackson comedy has arrived, and it's not pretty. The Internet, and Jackson's family members, were aghast when the first trailer for the UK show Urban Myths dropped Tuesday night, with Fiennes' prosthetics-heavy visage barely resembling like the singer he's supposed to be playing. Featuring a host of actors playing figures from Bob Dylan to Adolf Hitler, Urban Myths takes "a slightly tongue in cheek, mischievous – and deliberately ambiguous – look at what might have happened" in famous stories of celebrity folklore. Joseph Fiennes as Michael Jackson (Photo: Sky Arts) In Fiennes' episode, titled "Elizabeth, Michael & Marlon," the trio of Elizabeth Taylor, Michael Jackson and Marlon Brando flee post-9/11 New York in a rental car together, "reflecting on their lives, loves, careers and egos as they go." The 30 minute-long clip also features a cameo from the late Carrie Fisher as a "diner burger flipper," according to a release. But Fiennes' casting, announced in the thick of the #OscarsSoWhite controversy last year, drew jeers from social media users, criticizing the series' choice of a white actor playing the African-American singer. Abigail Tye, a spokeswoman for Urban Myths broadcaster Sky Arts, called the program a "lighthearted look at a reportedly true event," about a supposed car ride that Jackson took with Elizabeth Taylor and Marlon Brando. In another segment, the program features Harry Potter's Rupert Grint playing "Hitler's friend," alongside actor Iwan Rheon's portrayal of the young Fuhrer. "At least if it was Meryl Streep, we'd be like, 'Yeah I mean, but she's dope.' But Joseph Fiennes? The guy who played Shakespeare? That's like the whitest guy in history!" Trevor Noah joked about the casting decision last year. Fiennes, who rose to fame in 1998's Shakespeare in Love, told ET he's " as shocked as you may be" that he's playing the late music icon. "I'm a white, middle-class guy from London," he said. The actor defended Urban Myths against claims that it whitewashed Jackson, who suffered from vitiligo. "[Jackson] definitely had an issue, a pigmentation issue, and that's something I do believe," he said. "He was probably closer to my color than his original color." Read or Share this story: http://usat.ly/2jvshwu Perhaps you have a few questions upon viewing this image, I know I certainly did. Namely: Why? Also: What? Lastly: Will it go away? Advertisement What fresh horror 2017 hath wrought: here is Joseph Fiennes, brother of Ralph, also known as “the hotter one,” as Michael Jackson in the forthcoming Urban Myths from our friends across the pond at Sky Arts. It’s a comedy series featuring dramatic retellings of fictionalized stories about celebrities that have become “urban folklore.” The Michael Jackson story in question is a since-disputed report about the King of Pop, Elizabeth Taylor and Marlon Brando fleeing in a car after 9/11. Who knows if that actually happened; all I know is that looking at Joseph Fiennes, a white man, as late-in-life Michael Jackson, a man who was most certainly black, is a nightmare. Advertisement Joseph Fiennes is well aware of the rightful outrage of being cast as a black man. and responded to the backlash last year when his role was announced, saying to The Hollywood Reporter in 2016 “The decision with the casting and the producers — I wrangled with it, I was confused and shocked at what might come my way and I knew the sensitivity, especially to Michael’s fans and to Michael’s family. It doesn’t negate who he was.” The director of the series, Ben Palmer, defended his casting decision in the Guardian, noting that casting someone to play Michael Jackson at that stage in his life “obviously is a challenge in terms of his physical resemblance,” but said that what he was really looking for was someone who could “unlock the spirit” of the deceased star. “We were really looking for the performance that could unlock the spirit, and we really think Joe Fiennes has done that. He’s given a really sweet, nuanced, characterful performance,” he said. Ben, my dude, I’m sure you were, but holy guacamole, this is frankly insane. Advertisement Sponsored What works about this series? Stockard Channing as Elizabeth Taylor, for one –those brows, that fur! Also, Iwan Rheon (that’s Ramsey Bolton, guys) as Adolf Hitler and Rupert Grint as “Hitler’s friend.” What does not work, however, is Joseph Fiennes playing a waxen, powdery Michael Jackson with a nose that looks like two hot dog halves stitched together and covered in concealer. Please. This year will be long enough without this. The series premieres on January 19. Home TV News Sky Arts Urban Myths Sky Arts reveals Urban Myths comedy season Sky Arts is to broadcast eight one-off comedy shows featuring fictional stories about cultural figures Starting later this January, Urban Myths features stars including Katherine Parkinson and David Threlfall Stories include Hitler at art school, Muhammad Ali helping a suicidal stranger, and Cary Grant on LSD Sky Arts is to broadcast a season of comedies under the title Urban Myths. The eight one-off shows will "look at remarkable stories from well known historical, artistic and cultural figures, which may or may not have happened in real life..." The channel will begin broadcasting the 30 minute shows from later this January, and have signed up a host of stars to play the famous faces. Katherine Parkinson, David Threlfall, Paul Ritter, Iwan Rheon and Rupert Grint are amongst the actors involved. Carrie Fisher, who died last month, also appears in one of the episodes. The channel explains: "Each of the eight original titles in the series has its own unique take on an Urban Myth; some well-known and some that will delight viewers with their obscurity. "The films include Bob Dylan and Dave Stewart hanging out in Crouch End; an 18 year-old Adolf Hitler attempting to get into art school; Cary Grant and Timothy Leary taking LSD; Salvador Dali summoning Alice Cooper to sit for him; Elizabeth Taylor, Michael Jackson and Marlon Brando going on a road-trip; Samuel Beckett driving Andre The Giant to school; Muhammad Ali saving a suicidal man; and The Sex Pistols and the story behind the infamous expletive-filled TV interview which announced the arrival of punk rock. "Using a generous dose of artistic license, the comedies are designed to mischievously spark viewers' imaginations and answer the question; 'if these events really did happen, how might they have played out?'" Phil Edgar-Jones, Director of Sky Arts, says: "We are always looking at new ways of shining a light on the wonderfully bizarre and endlessly fascinating worlds you find across the Arts, and our brand new series of Urban Myths does just that, exploring those tales we've kind of heard of and hope are true. We are delighted that some of Britain's most talented and loved writers, directors and performers have had such a lot of fun with these tales and let their imagination and creative brilliance beam out from every frame." The individual comedies making up the season are as follows: Urban Myths: Bob Dylan Music legend Bob Dylan decides to play an impromptu visit to his friend and fellow musician Dave Stewart at home. Unfortunately Dave isn't home but his wife Ange reveals he will be back shortly, so 'would he like to wait over a cuppa?'. Sure enough Dave returns, but far from Dave Stewart, this is Dave the plumber - and massive fan of Bob Dylan. Bob sticks around to offer relationship advice. Bob Dylan is played by Eddie Marsan, Dave by Paul Ritter and Ange by Katherine Parkinson. The episode is written and produced by Neil Webster and directed by Ben Palmer. It will be shown on Sky Arts at 10pm on Thursday 19th January. Guide Urban Myths: Samuel Beckett And Andre The Giant David Threlfall portrays playwright Samuel Beckett in this comedy. The premise of the programme suggests to viewers that, long before he went on to become a famous wrestler and film star, Andre The Giant was chauffeured to school by Beckett. Samuel steps in to help Andre, who is his builder's son, as his size means he is already too big to be able to get the school bus. Along the journey a formative lesson in love and life ensues. The show, also written by Webster and directed by Palmer, will be on Sky Arts on the 26th January. Guide Urban Myths: Hitler The Artist A comedy drama observing as an 18 year-old Adolf Hitler pursues his passion for art, as he and his best friend August, a talented musician, live in Vienna hoping to study at the revered Arts Academy. The only problem is that young Adolf is 'crap' at art and is rejected, incurring a wrath that would have far-reaching consequences. Hitler is played by Misfits actor Iwan Rheon and Gustl by Harry Potter star Rupert Grint. The episode was written by Ben Edwards and has been directed by Dan Zeff. It will be shown on TV on the 2nd February. Guide Urban Myths: Cary Grant And Timothy Leary Focused on the set of Alfred Hitchcock's North by NorthWest, this comedy drama suggests Hollywood heartthrob Cary Grant introduced the psychologist and soon to be drugs advocate Timothy Leary to LSD, resulting in an unforgettable kaleidoscopic trip. Ben Chaplin plays Cary Grant, with Aiden Gillen as Timothy Leary. Written by Ed Dyson and Roger Drew, Sky Arts is due to broadcast this programme on the 9th February. Guide Season 2 The second batch of programmes is expected to be shown on Sky Arts in April. Most are still in production, so some details are still to follow. However, the information that has been revealed about them is as follows... Urban Myths: Elizabeth, Michael & Marlon A comedy drama written by Neil Forsyth that imagines what would have happened if long-time friends Elizabeth Taylor, Michael Jackson and Marlon Brando had hired a Budget rental car together to escape Manhattan during the September 11th attacks. Elizabeth Taylor is played by Stockard Channing, Michael Jackson by Joseph Fiennes and Marlon Brando by Brian Cox. This programme also features one of the final performances of Star Wars actor Carrie Fisher. She plays a burger flipper in a diner that the trio visit. Guide Story update, 13th January: This programme has proved to be highly controversial and Sky Arts has now dropped it. Full story Urban Myths: Muhammad Ali In the early 1980s, after his recent and high profile defeat to Larry Holmes, boxing legend Muhammad Ali takes it upon himself to talk a suicidal stranger down from a ledge. Using his unique brand of wit and wisdom, he succeeds in persuading the man that life is worth living, and learns a huge amount about himself in the process. This offering has been written and produced by Neil Webster, and features Noel Clarke as Muhammad Ali. Guide Urban Myths: The Sex Pistols A comedy about The Sex Pistols' infamous 1976 appearance on the early evening Bill Grundy show, when they outraged a nation with their four-letter tirade, heralding the arrival of punk rock. This comedy by Simon Nye takes a wry look at the stories surrounding the appearance, from Johnny Rotten answering viewers' complaints to Freddie Mercury's role in giving the Sex Pistols notoriety. Set to be filmed by Sacha Baron Cohen's company, this production will draw its cast from the National Youth Theatre, the first product of a new partnership between Sky Arts and the NYT. Guide Urban Myths: Salvador Dali and Alice Cooper In this programme, which has yet to be cast, Salvador Dali will summon Alice Cooper to sit for him whilst he creates a hologram. It is described as "a comedic exploration of the moment when these two egos - and Dali's muse Gala - collide." Guide Here is the trailer for the first four episodes: Introducing Urban Myths, a brand new #comedy series based on (mostly) true stories... Coming to your screens from 19th January, 10pm. pic.twitter.com/fwmtBqXnNX-- Sky Arts (@SkyArts) January 8, 2017 Share this page Sky Arts It’s been nearly a year since the announcement that Joseph Fiennes would play Michael Jackson in a TV episode based on a possibly apocryphal story about him, Elizabeth Taylor, and Marlon Brando going on a road trip after 9/11, a year during which the incandescent insanity of that idea has gradually faded away. But now it’s back and burning bright, thanks to the trailer for Sky Arts’ Urban Myths, an eight-part TV series that includes the already infamous Jacko/Liz/Brando episode. The trailer, which draws from several different episodes, only gives us a short glimpse of Fiennes’ M.J., but that’s enough to show that he’s been made up with skin the color and texture of uncooked pizza dough and a nose that looks like an unintentional tribute to Jackson’s character from The Wiz. In short, it’s a nightmare, even before you take the implications of having a white actor play a man who, whatever his skin tone, was never not black, into account. Advertisement The first footage of Joseph Fiennes as Michael Jackson has emerged – and people aren't quite sure how to feel. The predominant emotion appears to be horror and dismay, although, if Twitter is anything to go by, some observers are more amused than upset. The casting of Fiennes as Jackson, for an episode of the new Sky Arts series Urban Myths, attracted controversy when it was first announced almost a year ago. At the time, however, the Shakespeare in Love star said that he did not believe it was inappropriate for a white man to play the music star, due to the fact that the pigmentation of Jackson's skin had significantly changed by the time of his death. “[Jackson] definitely had an issue – a pigmentation issue – and that’s something I do believe,” he told Entertainment Tonight. “He was probably closer to my color than his original color.” UPDATE: The episode has been pulled indefinitely after outcry from Jackson's fans and daughter Paris. Joseph Fiennes (controversially) portrays Michael Jackson in the first trailer for a new comedy show that reimagines the urban legend about the King of Pop's post-9/11 road trip with Marlon Brando and Elizabeth Taylor. Related Joseph Fiennes Addresses 'Sensitive' Michael Jackson Casting "I deal in imagination, so I don't think imagination should have rules stamped on them," actor says of playing black singer Originally called Elizabeth, Michael & Marlon, the film has since been retitled and integrated into Urban Myths: A Brand New Collection of Comedies, an anthology that revolves around other "true…ish stories" that have circulated as urban legends. In the trailer, Fiennes' Jackson appears as a largely silent, wild-eyed passenger as Taylor (played by Stockard Channing) and Brando (Brian Cox) drive cross-country after the airports have been shut down in the aftermath of 9/11. At one point, the famous trio get pulled over by a highway patrolman who is shocked to encounter the three megastars in a rented red sedan. "This is territory that is sensitive," Fiennes previously said of the role after he was accused of "whitewashing" the singer. "One must determine if this portrayal is one that is going to be positive entertainment, and one that will not bring about division and put anyone's noses out of joint, so I went with the mind that this was a positive light-hearted comedy." Sky Arts, which will broadcast Urban Myths in the United Kingdom, told Rolling Stone in a statement, "Joseph Fiennes is cast as Michael Jackson. It is part of a series of comedies about unlikely stories from arts and cultural history. Sky Arts gives producers the creative freedom to cast roles as they wish, within the diversity framework which we have set." Other Urban Myths stories include Cary Grant's alleged love of LSD, a story about Adolf Hitler and "Hitler's friend" and another episode involving Samuel Beckett. Eddie Marsan plays Bob Dylan in an episode about the singer's search for someone named "Dave." Paris Jackson vehemently objected to Joseph Fiennes' portrayal of her late pop-icon father Michael Jackson in an 'Urban Myths' trailer. Watch here. Throughout a lifetime, there will come occasions in which a person has to show restraint. Take the high road. Do absolutely everything in their power not to say what is on their minds. And not because whatever those thoughts are, are not completely true and justified. Going high when they go low is never easy, chief among those reasons being because one must live with the knowledge that they are choosing to withhold very, very valid information. This is one of those times.

Behold, white British actor Joseph Fiennes playing black American King of Pop Michael Jackson. You can do just that now that the first trailer is out for the TV special in which Fiennes as Jackson, Stockard Channing as Elizabeth Taylor, and Brian Cox as Marlon Brando depict a road trip the odd trio supposedly once took from New York to Ohio after the 9/11 terror attacks. The story may not actually be true; it will be featured on new series Urban Myths on the arts-oriented channel Sky Arts, E! reports. The show, whose tagline is "True...ish Stories," will run eight 30-minute episodes telling similarly strange may-or-may-not-have-actually-happened stories, per the British Comedy Guide. It debuts in the UK Jan. 19, but the Michael Jackson episode isn't slated until April. Needless to say, the casting of Fiennes as Jackson has caused some controversy: E!'s take on it: "Throughout a lifetime, there will come occasions in which a person has to show restraint. Take the high road. Do absolutely everything in their power not to say what is on their minds.... This is one of those times." Slate does not take that road. Fiennes has "been made up with skin the color and texture of uncooked pizza dough and a nose that looks like an unintentional tribute to Jackson's character from The Wiz," writes Sam Adams. "In short, it's a nightmare." "People aren't quite sure how to feel," notes Rebecca Hawkes at the Telegraph. "The predominant emotion appears to be horror and dismay." She rounds up some of the best tweets on the matter. USA Today simply calls the images of Fiennes as Jackson "unsettling." Rolling Stone chooses "bizarre" to describe the trailer as a whole. And Jezebel goes with "horrifying" and "insane."

multi_news

Precise patterns of spatial and temporal gene expression are central to metazoan complexity and act as a driving force for embryonic development. While there has been substantial progress in dissecting and predicting cis-regulatory activity, our understanding of how information from multiple enhancer elements converge to regulate a gene' s expression remains elusive. This is in large part due to the number of different biological processes involved in mediating regulation as well as limited availability of experimental measurements for many of them. Here, we used a Bayesian approach to model diverse experimental regulatory data, leading to accurate predictions of both spatial and temporal aspects of gene expression. We integrated whole-embryo information on transcription factor recruitment to multiple cis-regulatory modules, insulator binding and histone modification status in the vicinity of individual gene loci, at a genome-wide scale during Drosophila development. The model uses Bayesian networks to represent the relation between transcription factor occupancy and enhancer activity in specific tissues and stages. All parameters are optimized in an Expectation Maximization procedure providing a model capable of predicting tissue- and stage-specific activity of new, previously unassayed genes. Performing the optimization with subsets of input data demonstrated that neither enhancer occupancy nor chromatin state alone can explain all gene expression patterns, but taken together allow for accurate predictions of spatio-temporal activity. Model predictions were validated using the expression patterns of more than 600 genes recently made available by the BDGP consortium, demonstrating an average 15-fold enrichment of genes expressed in the predicted tissue over a naïve model. We further validated the model by experimentally testing the expression of 20 predicted target genes of unknown expression, resulting in an accuracy of 95% for temporal predictions and 50% for spatial. While this is, to our knowledge, the first genome-wide approach to predict tissue-specific gene expression in metazoan development, our results suggest that integrative models of this type will become more prevalent in the future. Gene expression is regulated through the interplay of transcription factors binding to cis-regulatory modules (CRMs), chromatin modifications and the basal transcriptional machinery recruited to promoter elements. CRMs function as discrete regulatory elements [1], [2], that can act at varying genomic distances from their target genes [3]. Despite recent advances in our understanding of the regulatory steps of transcription, the ability to predict both spatial and temporal aspects of gene expression remains surprisingly limited. Efforts in this direction can be broadly divided into two groups: (1) Predicting cis-regulatory or enhancer activity, where recent studies in yeast [4] Drosophila [5]–[7] and C. elegans [4] have made substantial progress. In one such study the tissue specificity of the neighboring gene' s expression was used to guide the search for specific TF combinations [7], while in another the combination of sequence motif matches was used to predict gene expression [4]. Although, these are important steps, integrating the activity of multiple cis-regulatory elements that regulate overlapping or distinct aspects of a gene' s spatio-temporal expression remains a key challenge (Fig. 1a, Fig. S1). (2) Using chromatin state dynamics to predict gene expression [8]–[12]), with or without information on transcription factor (TF) and insulator data. For example, in Drosophila a logistic regression was used to predict temporal (not tissue-specific) gene expression in embryogenesis [11], showing a performance better than random for 23. 6% genes, with a 2. 5 fold enrichment over control experiments where the connectivity between TFs and their targets was reshuffled. In c. elegans an SVM classifier was used for a similar task of discerning highly and lowly expressed transcripts based on measured chromatin marks [13], although tissue specificity was not examined. This approach, based on transcripts and chromatin marks in their immediate vicinity (+/−4 kb) achieves high accuracy (average AUC for all stages = 0. 82), reflecting the strong correlation between transcription and chromatin marks on the gene body, such as the H3K79 methylation and Pol II occupancy consistent with the results by Karlic et al [14]. However, while virtually all regulatory elements appear to reside within 5 kb of the transcriptional start site (TSS) in C. elegans, this is not the case in other species. In Drosophila, mouse and humans there are many examples of remote CRMs acting at large distances from the TSS [15]–[18] spanning many intervening genes [19], [20], where large chromatin loops are thought to bring the enhancers and the target gene' s promoter in close physical proximity [21]. In addition, genes, especially developmental regulators, are controlled by multiple CRMs, giving rise to partially overlapping patterns of activity [22], [23]. In order to capture, and thereby predict, the full spectrum of a gene' s spatial expression, two key issues need to be addressed directly: (1) accurately linking CRMs to their target genes and (2) integrating the activity from multiple CRM, as is done naturally for most developmental genes in multicellular species. There is currently very little biological information or understanding of how the activity of multiple elements is integrated at the promoter level. While some studies have suggested that each CRM acts in an additive manner so that the gene' s expression pattern is the simply sum of all elements, other studies have shown that the gene can be expressed in a broader [8], [24]) or more restricted [9], [10] spatial domain than the sum of its individual regulatory elements. It is therefore currently not clear how best to integrate separate computational models of cis-regulatory elements to accurately reflect this convergence of regulatory information controlling a gene' s expression in vivo. These difficulties have limited spatial predictions of gene expression to a small number of very well characterized genes [6], [25], or more globally to focusing on predicting on-off states in single cell systems [26], [27], thereby circumventing the inherent complexity of spatial expression within a multicellular organism. High-resolution ChIP-chip or ChIP-seq approaches facilitate the mapping of distant regulatory elements based on transcription factor occupancy [26]–[32], co-factor binding [33] or chromatin marks [34], providing new possibilities to develop better predictive models of global gene expression patterns. However, there are still several levels of information missing, including a complete catalog of all enhancers active during specific stages of development, information on the identity and timing of the TFs recruited to each enhancer, cell-type specific information on chromatin status, the activity state of the associated target gene and a general lack of information on the physical association of CRMs with promoter elements. Despite this incomplete knowledge, we asked if the current level of information is sufficient to accurately predict spatio-temporal gene expression within the context of a multicellular embryo, reasoning that the predictive power of the model should only improve as more information becomes available. We developed a probabilistic model, integrating diverse types of data generated from whole embryos and thereby containing mixed signals from many tissues, to predict both spatial and temporal aspects of gene expression, with particular emphasis on the mesoderm and derived muscle types. More specifically, using Drosophila embryogenesis as a model system, we integrated six types of data relevant to transcriptional regulation: (i) 19,000 TF binding peaks derived from ChIP-chip experiments for mesoderm specific TFs, clustered into 8008 non-overlapping cis-regulatory modules (ChIP-CRMs), (ii) spatio-temporal activity data for 343 CRMs from in vivo transgenic reporter assays, (iii) the genomic distance of CRMs in relation to transcriptional start sites, (iv) 37,923 occupancy peaks for 6 insulator binding proteins, (v) H3K4me3 enrichment measured for promoter regions of 14689 genes, and (vi) spatio-temporal expression of 5,995 genes derived from in-situ hybridization (see Table S1 for a detailed data description). Note, as chromatin modifying enzymes for canonical histones and insulator binding proteins are ubiquitously expressed, the whole embryo data from (iv) and (v) does not contain any inherent cell-type specific (spatial) information, and (v) represents merged temporal signal over the entire period of embryogenesis, which is 24 hr in Drosophila. TF occupancy (i) and gene expression (vi) data provide information on potential regulatory input and the final spatio-temporal output, respectively, but little means to connect the two, highlighting the need to integrate diverse layers of information. Previous studies suggest that cis regulatory elements function, to a large extent, independently of each other [35]. Assuming that this is correct, there are two natural levels to model gene expression based on: (i) the relationship between TF occupancy and CRM activity and (ii) the relationship between models of multiple CRMs' activity and a gene' s expression (Fig. 1a). This first step was recently addressed using support vector machine (SVM) models, which demonstrated that TF occupancy alone is sufficient to predict spatio-temporal CRM activity during mesoderm development [5]. It was postulated [36] that the same method could in principle be adapted to model gene expression prediction, although this would require linking CRMs to their appropriate target genes and integrating inputs from multiple CRM models to reflect a target gene' s expression. Taking advantage of the wealth of data on TF occupancy at mesodermal CRMs [5], we tested this assumption by building a simple additive model that assigns each CRM to the nearest gene and then sums the SVM prediction scores for all assigned CRMs to obtain a spatio-temporal expression prediction at the gene level. Overall, the predictions were of poor quality (Fig. S2), indicating that a model based on these simple assumptions does not reflect the biological complexity of the system. Using well-characterized gene loci to examine why the model failed revealed that enhancers do not always regulate the nearest gene, but often a more distant gene (Fig. 1a twist locus) or can even act across an intervening inactive gene to reach its appropriate target (Fig. S1a bagpipe locus). Such inactive ‘bystander’ genes [37] can be located within the intron of a target gene (Fig. S1c Fas3 locus) or vice versa (CG6981), further confounding the problem of appropriate target gene assignment. This demonstrates the need to move to a more integrative model that includes information on promoter activity (H3K4me3 enrichment) and insulator occupancy within a gene locus. As insulator binding proteins mediate long-range regulatory interactions between enhancers and their target genes [3], [38], we reasoned that insulator occupancy could improve the ability to recognize ‘bystander genes’, while the presence of H3K4me3 at promoters will identify active genes within the vicinity of active CRMs. To deal with this complexity, we applied a Bayesian model to probabilistically integrate diverse types of data in an iterative manner, which has the advantage of being able to cope with uncertainty and incompleteness within each dataset using conditional probabilities. The model consists of three components (Fig. 1b): (i) a Bayesian Network (BN) that describes the probability of a CRM being active in a tissue or time-point as a function of its occupancy by different TFs, (ii) a custom probabilistic model that describes the probability of a gene being expressed at a given stage and tissue depending on the activity of surrounding CRMs, the location of CRMs and insulators relative to the promoter, and the activity state of the promoter (Fig. 1), and (iii) an expectation maximization (EM) procedure [39] that functions to find an optimal set of parameters within the overall Bayesian model, iterating between the BN and custom model until convergence. To accurately predict gene expression, the model must be able to cope with dynamic changes in the regulatory context of genes, which determines their activity state at different stages of development and in different tissues. To account for this, we trained the model using spatio-temporal expression information of 5,082 non-ubiquitous genes generated from large-scale in-situ hybridization experiments [40], describing when and where genes are expressed during embryogenesis. As a proof-of-principle we focused on five temporal windows of development and five tissue classes (10 prediction classes; Supplementary text S1.). In more detail, the first component, modeling CRM activity as a function of TF binding events, was achieved using a BN, allowing for accurate representation of conditional probability (Fig. 2a, described in detail in Supplementary text S1 – in “Layer 1-TF binding” and “Layer 2-CRM activity”). The model uses measured TF binding events on CRMs as input (from ChIP-chip data) and spatio-temporal CRM activity data as output (from in vivo transgenic-reporter assays) (depicted in Fig. 1a). The nodes within the BN are of two types: specific TF binding events (factor-F at time-point-T, representing 15 variables) and activity classes (tissue or time-point, representing 10 variables). Each edge between nodes represents the probability of a CRM being active in a given activity class as a function of a particular binding event (e. g. CRM activity in tissue-A depends on the binding of factor-F at time-point-T). The correct topology of connections was reconstructed using the Bayesian Dirichlet equivalence score as implemented in the BNfinder software [41]. Once the most likely topology was known, the conditional probabilities of CRM activity in different classes (temporal and spatial) were calculated from the training data using the maximum likelihood principle. The trained BN and the conditional probability distributions were then used to provide probability estimates for the spatio-temporal activity of all 8008 CRMs, not only the 147 used in the training dataset. Based on these probability estimates, we compared the BN model with the previously published SVM approach [5]. Overall, our model gives slightly better predictions of previously unseen CRM activity (Fig. S3b), even though it was not explicitly optimizing the accuracy at the CRM level. In addition, unlike ‘black box’ type models such as SVMs, the learned BN network topology provides interpretable insights into the most important TF binding events for each spatio-temporal activity. For example, the BN revealed that Biniou (a FoxF TF) enhancer occupancy is the key predictive signal for visceral muscle activity (Fig. 2a), which matches the known essential role of this TFs for visceral muscle development from genetic studies [2]. The second component of the Bayesian model addresses how genes integrate probabilistic signals from one or many CRMs by relating this information to known gene expression patterns within the training set (described in detail in Supplementary text S1 – “Layer 3-gene activity”). For each gene, we consider the location of its transcriptional start site (TSS) and the CRMs present in its broad environment (+/−100 kbp, where there is one gene per ∼8 kb in the Drosophila genome). As the majority of known Drosophila enhancers are located within +/−20 kb of their target gene' s promoter, the probability of activation decreases linearly with respect to the distance from the TSS. The only parameter that the model fits is the maximal distance between a CRM and the TSS within a +/−100 kbp window. To facilitate linking CRMs to their appropriate target gene, the model integrates information on the occupancy of six insulator binding proteins [42] relative to the location of CRMs and surrounding genes (Fig. 1a). As insulator proteins can block enhancers from inappropriately activating nearby promoters [43], CRM-promoter interactions are considered blocked if they operate across an insulator boundary (see Methods). To obtain a probability for a promoter being in an active or inactive state, we used the presence of H3K4-trimethylation ChIP-seq signal at promoters as an indicator of promoter activity [44] (Fig. 1a, Fig. S1, methods). The model requires both an active promoter and at least one active CRM to activate a gene in a given spatio-temporal context. The classifier accuracy was determined using the area under a receiver-operator curve (AUC) for varying posterior probabilities of gene activation. To train the model for tissue specific or developmental stage specific chromatin context, we used in-situ hybridization data of 5082 genes [40] to identify genes in specific spatio-temporal classes. For simplicity, expression patterns were divided into a number of binary classes: focusing on 5 tissue classes (mesoderm, somatic muscle, visceral muscle, mesoderm+somatic muscle and visceral+somatic muscle) and 5 time-windows (stages 4–6,7–8,9–10,11–12,13–16, spanning ∼85% of embryonic development). Separate variables were incorporated for a gene or CRM activity in each class, allowing each class to be evaluated individually using the probability of a gene to be expressed in a particular spatial or temporal domain. The coupling between the two mentioned components of the model is through the intermediate layer representing the activity of the CRMs (Supplementary text S1 “Integrating the different layers of the model using iterative optimization”). Since the activity of the vast majority of the ChIP-defined CRMs is unknown, the variables in the intermediate layer are latent. Under this setting, an iterative Expectation maximization (EM) [39] procedure was used to facilitate using data of varying degrees of completeness at different levels of the model. The TF binding data is very extensive for all 8008 CRMs (at least within the scope of the five TFs), as is the insulator occupancy and promoter activity data, although the later two represent merged signals from mixed tissue types and have very low temporal resolution. Spatio-temporal expression data is available for a substantial number of genes (∼33% of predicted Drosophila genes), which contrasts with the scarcity of knowledge on CRM activity, which is available for only ∼4% of CRMs. This level of CRM activity data is sufficient to train a predictive model of CRM activity, using a BN (Fig. 2a) or SVM [5] approach. However, there is not a single gene in the Drosophila genome where the activity of all ChIP-defined CRMs in its vicinity are known. As such, there are no complete examples that could be used to fit a model representing convergence of multiple CRM activities to a single gene' s expression. To address this, the activity of CRMs was consistently treated as a hidden variable in the model, and the CRM activity information was only used for model initialization. EM was used to iteratively improve both the CRM activity predictions and gene expression predictions (see methods and Fig. 1b), resulting in an effective model with local maximal likelihood. By performing the EM procedure in a 10-fold cross-validation framework, we assessed the ability of the model to predict gene expression for genes not used for training. The average AUC value for all 10 prediction classes exceeds 0. 8 (Fig. S4), a significant improvement over the simple additive SVM method (p-value<10−7; Fig. S3a). Importantly, the cross-validation estimated performance is comparable to that of the model trained on the full dataset (Fig. S5), indicating that the model is not over-fitted. The difference in AUC slightly underestimates the improvement of the model as it is based on predictions made for all genes, while only a minority of Drosophila genes are expected to be specifically expressed in each activity class and the majority of genes are correctly predicted not to be regulated by mesodermal CRMs. For example, from all 5082 Drosophila genes with characterized non-ubiquitous expression, only 137 have annotated expression in the activity class somatic muscle, 135 in mesoderm and 60 in VM [40]. Extrapolating these numbers to the entire genome estimates that the percentage of genes expressed in each activity class is in the range of 1–2%, excluding ubiquitously expressed genes. With this in mind, we examined the top 2% of predictions from the trained Bayesian model, which identified on average a 15-fold enrichment in gene expression in the predicted tissue compared to a random classifier, for all activity classes, with the best class having a 45-fold enrichments (Fig. 2b). To investigate the most important aspects of the model' s predictions, we compared the results to simpler approaches that do not use either chromatin state (insulator binding data or H3K4me3) or an EM procedure, all of which obtained inferior results (Fig. 2b, Fig. S6). Adding H3K4me3 promoter activity signal to TF binding, for example, reduces the number of false-positive predictions by 1. 5 fold, thereby increasing the enrichment of correct predictions (Fig. 2b). The method also demonstrates improved performance over a simpler two-layer model predicting gene expression directly from ChIP peaks, skipping the intermediate CRM layer [45] (, Methods). Although this 2-layer model is not accurate enough to make reliable predictions, the approach can be very valuable for initiation of the EM algorithm in cases where there is no CRM activity database available. In many organisms obtaining information on CRM activity for a large number of regulator elements is difficult. We therefore tested whether our approach could provide comparable results without providing the measured activity of selected CRMs. To avoid random fluctuations we have used the gene expression data for genes with very closely (<500 bp) associated CRMs as a proxy for enhancer activity. While this is certainly introducing some erroneous information by both erroneous target assignment and by assigning total gene activity to only one selected enhancer, it seems to give only slightly worse results for classes with multiple genes associated to it (VM, SM, MESO, see Fig. S10). To validate the true performance of the model we took advantage of spatio-temporal expression data for more than 600 genes not included in our training set that was part of the third release of the Berkeley Drosophila Genome Project (BDGP) in-situ database [40]. We used models trained on the whole training dataset and assessed their performance on the genes present only in the new dataset by calculating the AUC for each activity class (Fig. S8). The performance was comparable to the cross-validated-based estimates, with the average AUC of 0. 78 (compared to 0. 82; Fig. 2c). To further validate the quality of the trained model, we chose a tissue with a relatively restricted spatial expression, the visceral muscle (Fig. 3a, AUC 0. 87), and manually curated the top 100 genes predicted to be expressed in this tissue (VM). Examining the literature and BDGP, we identified spatio-temporal expression for 46 of the 100 genes, 67% of which are expressed in visceral muscle, while the expression of the remaining 33% did not fit with our prediction (Dataset S8). We randomly selected 22 genes for which there was either no expression data available, or were apparent prediction errors from the model (within the 33%). Double fluorescent in-situ hybridization using a visceral muscle specific marker revealed that the timing of expression of 21 out of 22 genes match their temporal prediction (95%), while the expression of 50% match their spatial prediction (Fig. 3c, Fig. S9), representing a 42-fold enrichment in gene expression in visceral muscle compared to the 1. 2% of genes annotated in the BDGP database (Fig. 3b). The high success rate of the model, despite the presence of inaccurate expression annotations within the training dataset, demonstrates the general robustness of this iterative approach. This study represents a first attempt to build an integrative probabilistic genome-wide model that predicts both the spatial and temporal aspects of gene expression, within the context of a multicellular embryo. The Bayesian model integrates diverse types of genomics data, including transcription factor occupancy, chromatin modification and insulator binding information, using in vivo CRM activity information and gene expression data to train the model. In addition to predicting gene expression, introspection of the model' s parameters reveals a number of additional insights. First, the iterative trained Bayesian network improved the accuracy of the previously published SVM approach for CRM and gene activity prediction [5] (Fig. S3), and recovered, without any prior information, the known dependencies between specific TFs and respective tissues. Second, through expectation-maximization, the model learns the optimal distance of a CRM to its target gene. This revealed extensive long-range enhancer activity, which may be much more widespread in Drosophila than previously anticipated. Although there are a handful of known enhancers acting >30 kb from their target gene [19], [20], the majority of CRMs identified to date are <+/−20 Kb of their target gene. This apparent close proximity, however, most likely reflects the current biases in how CRMs are identified in single gene studies, starting from the gene moving out, or in global studies where CRMs are typically linked to the closest proximal gene. The iterative Bayesian model revealed that CRMs as far as 50 kbp from the transcriptional start site have important contributions to accurately predict a gene' s activity. Third, the model suggests that enhancer sharing between genes may be an inherent property of developmental enhancers where a CRM contributes to the predictions of 3 genes on-average. This observation, which came directly from the trained model, has recently been observed using an experimental technique to link CRMs to genes (chromatin conformation capture) [12], and has exciting implications for how transcriptional networks are regulated during development. Taken together, this approach provides a method to move from descriptive ‘omics’ type data to predictive models of gene expression. Given the exponential increase in measurements of chromatin state and TF occupancy in the coming years, we expect this type of iterative analyses to become increasingly useful as a complement to ongoing attempts to map global gene expression patterns by experimental approaches and as a tool to uncover novel properties of transcriptional regulation. CRM occupancy data and CAD database were used as published by Zinzen et al. [5]. Gene expression patterns were obtained from the BDGP in-situ hybridization database [46] - release 2 served as the training data, while release 3 (beta release downloaded on May 27th 2010) was used as the testing dataset. Only genes with tissue specific expression (excluding ubiquitous and maternal expression) were analyzed. Anatomical terms from BDGP were grouped into more general classes (mesoderm, somatic muscle, visceral mesoderm), similarly to the procedure used by Zinzen et al. [5]. Temporal classes were based on the staged groups used by the BDGP in their annotations (st. 4–6,7–8,9–10,11–12,13–16). Whole embryo ChIP-seq data of histone H3K4 tri-methylation was from ModEncode [47] for three time-points: 4–8 h, 8–12 h and 12–16 h (ModEncode sample IDs: 790,791,792). Averaged processed signal was calculated for a region surrounding all transcriptional start sites (−100, +400 bp from TSS) and then discretized into low and high values (threshold 0. 3) for training the Bayesian network. Whole embryo ChIP-chip data for the six insulator proteins was obtained from Negre et al. [42] (using a 1% FDR). The Bayesian model is composed of three main layers of different nature. The first layer represents variables corresponding to Transcription Factor binding to CRMs; second represents the CRMs activity under different conditions and the third is concerned with gene activity under the same set of conditions. We made the assumption that the only True causal connections are either coming from the first to second layer (TF binding causing CRM activity) or from the second to third layer (CRM to gene activity). No direct dependencies from first to third layer are allowed. A Bayesian Network was used to model the dependency between binding and CRM activity, while gene expression was assumed to be independently initiated by any active CRM within an acceptable range. Bipartite Bayesian network was used to describe dependencies between TF binding and CRM activity. For each CRM, a quantitative binding score was computed for each of 15 TF/stage combinations (as previously described [48]) representing quantitative measurements for actual binary events of TF binding (Dataset S1). Each expression class (temporal or spatial) was represented by a separate binary variable. There were 5 temporal classes, representing stages 4–6,7–8,9–10,11–12 and 13–16, following the BDGP nomenclature and 5 tissue-specific classes mesoderm (MESO), somatic muscle (SM), visceral muscle (VM), mesoderm and SM (MESO+SM), somatic and visceral muscle (SM+VM). Edges in the network represent dependencies of the conditional probability function of the variable corresponding to the CRM being active in a given condition on any variables representing TF binding events. Measured binding and activity for each CRM were considered to be a single observation from the same underlying joint distribution and they were used to find an optimal network. The network structure was constrained to only contain edges of this kind and probability distributions were optimized using BNfinder [24] software using Bayesian Dirichlet equivalence (BDe) score. No constraints on the resulting cpd function were set, however the binding signal was converted by the BNfinder software to probabilistic readouts of binary variable using a Gaussian mixture model. For detailed parameters used see Supplementary Text S1. All distances between a CRM and a transcriptional start site of a gene that were lower than 100 kb were tabulated based on FlyBase genome annotations, version 5. 17 [49] (Dataset S2). For each CRM-promoter pair, the total number of insulator peaks was calculated in between them. Each gene is assumed to be able to respond to the activation signal from any of the paired CRMs, depending on the distance and the number of insulator peaks between them. It is assumed that the probability of activation by a CRM over a given distance d is linearly decreasing with d until it reaches 0 at the distance dmax or when the predefined limit of insulator peaks have been exceeded. Each promoter is assigned a probability of being activated in development based on the histone modification (H3K4me3) level measured within the 500 bp around the TSS, using non tissue-specific data (Dataset S4). For details see Supplementary text S1. The majority of CRMs (>95%) have unknown activity, so we treat all variables corresponding to CRM activity as latent and use a maximum likelihood principle to estimate them. We define the likelihood function Lwhere G represents gene activity, i indexes CRMs, j indexes genes, Ai represents activity of the i-th CRM, Wij represents the weight of CRM-promoter interaction (depending on distance and insulators, as described earlier) and Rj representing the probability of a given promoter responding under specific conditions. Given this likelihood function we aim to find the most likely parameters of the model, i. e. the Bayesian Network and the optimal dmax. We use the Expectation Maximization (EM) strategy, by iteratively improving our current estimate of the parameters. Since the EM is a local optimization strategy, the result is highly dependent on the starting model. Normally this can be solved by starting from multiple randomized models, however in our case properly sampling a space of all Bayesian Networks would be difficult and likely to produce non-biological models. Instead we begin by initializing the BN parameters based on known CRM activity data (CAD [5]) by making the first inference not on the full training dataset but on the subset of the training set with experimentally measured tissue-specific activity. The dmax parameter could also have a strong impact in the initial stage of EM if it is set too low and therefore excludes some CRM data from the whole learning process. For this reason we initially set dmax to the maximum possible value and allow it to change freely from then on. The EM procedure is composed of alternating iteration of the expectation (E-step) and maximization (M-step) steps until convergence (improvement in the likelihood below 2%). In the E-step, we calculate estimated probability of each CRM being active in each condition based on our current model parameters (BN and dmax). Since the model has three layers and we are interested in the estimation of the hidden variables from the middle one, we use an approach based on the forward-backward algorithm frequently used to infer the probabilities of the hidden variables in Hidden Markov Models [7]. In our case, the forward probability is the probability of the CRM being active given the TF binding data, and can be easily computed using the BN for all CRMs. The backward probability is the probability of the CRM being active given the gene expression data. We can ignore all genes j such that wij = 0 as the change of the i-th CRM activity will not affect the total likelihood. For each CRM we need to consider all genes with wij>0 and the CRM can only be inactive if each of the genes in its range is turned on by another CRM, which, by assumption of independent action of CRMs, can be computed using Bayes theorem and total probability. The overall activity of each CRM is determined by a smoothing step as the product of the respective forward and backward probabilities. In the M-step, current estimates of CRM activity (the latent variables) are used for finding the model parameters (BN and dmax) maximizing the likelihood function L. For the BN, we are using the Bayesian Dirichlet equivalence optimization implemented in the BNfinder [24] library. Due to the constrained structure of the BN, it is possible to find a globally optimal network representing observed combinations of binding and activity very efficiently. As the likelihood function is not monotonous with respect to dmax we employed an exhaustive strategy to find the optimal dmax giving the maximum likelihood under assumed CRM activity and gene expression. This can be done quite efficiently with a step size of 200 bp, equal to the minimal size of the CRM. The process was repeated until convergence; in the tested cases ∼10 iterations were required to reach improvement in one step below 2%. For a more detailed description see Supplementary text S1. For each expression class (temporal or spatial) the posterior probability calculated from the model was used as the ranking criteria to calculate the area under the curve (AUC) for the receiver operator characteristic (ROC) curve. The AUC value can be interpreted as the probability of a random positive example to have a higher posterior probability of expression than a random negative example. To assess the significance of the achieved AUC measures in comparison to random classifier or in comparison between different models we used the procedure proposed by Hanley and McNeil [50]. To avoid over-fitting, all models were trained in a 10-fold cross-validation scheme based on BDGP gene expression database release 2. Then the entire BDGP release 2 dataset was used for training the final models (Dataset S5), which were then tested on the gene expression patterns from BDGP release 3 (Dataset S6), excluding those from the training set. The same models were used to select genes from the visceral muscle activity class for validation by in-situ hybridization experiments. All training sets are available in Dataset S7. The EM algorithm was implemented in Python using the BNfinder [24] library for estimating Bayesian networks, ROC curves were plotted with ROCR [51] package for R. All the scripts are available at https: //code. launchpad. net/bnfinder/GEpredict In-situ hybridizations in Drosophila embryos were carried out using standard protocols as described previously [52]. The following ESTs from the Drosophila Gene Collection (DGC) were used to generate Digoxigenin-labeled probes: GM02640 (Eip75B), LD09907 (Hex-A), RE05370 (CG9194), GM10074 (bt), AT24194 (Rya-r44f), LP05734 (Hsp22), GH06348 (CG1516), RH17388 (CG10654), GH24653 (A3-3), SD01953 (by), LP03829 (CG6981), GH27027 (Ncc69), SD11716 (CG14709), HL01392 (fau), LP06027 (Cpr78E), GH06222 (CG13124), LD02379 (nrv1), RE70568 (Lim3), LD44720 (CG7530), GH23506 (CG14655), LP04481 (CG6770), GH19382 (CG4945). biniou cDNAs (from M. Frasch) was used to generate Biotin-labeled probe. Double in-situs hybridizations were performed by using anti-Digoxigenin-POD and anti-Biotin-POD antibodies (Roche) and detected sequentially with FITC and Cy3 (Perkin-Elmer TSA kit). A Zeiss LSM 510 confocal microscope was used for imaging.

Development is a complex process in which a single cell gives rise to a multi-cellular organism comprised of diverse cell types and well-organized tissues. This transformation requires tightly coordinated expression, both spatially and temporally, of hundreds to thousands of genes specific to any given tissue. To orchestrate these patterns, gene expression is regulated at multiple steps, from TF binding to cis-regulatory modules, general transcription factor and RNA polymerase II recruitment to promoters, chromatin remodeling, and three-dimensional looping interactions. Despite this level of complexity, the regulation of gene expression is typically modeled in the context of transcription factor binding and a single enhancer' s activity as this is where the majority of experimental data is available. Recent advances in the measurement of chromatin modifications and insulator binding during embryogenesis provide new datasets that can be used for modeling gene expression. Here we use a Bayesian approach to integrate all three levels of information to combine the activity of multiple regulatory elements into a single model of a gene' s expression, implementing an expectation maximization strategy to overcome the problem of missing data. Importantly, while the data for histone modifications and insulator binding represents merged signals from all cells in the embryo, the model can extract cell type specific and stage-specific predictions on gene expression for hundreds of genes of unknown expression.

lay_plos

Aneuploidy causes birth defects and miscarriages, occurs in nearly all cancers and is a hallmark of aging. Individual aneuploid cells can be eliminated from developing tissues by unknown mechanisms. Cells with ribosomal protein (Rp) gene mutations are also eliminated, by cell competition with normal cells. Because Rp genes are spread across the genome, their copy number is a potential marker for aneuploidy. We found that elimination of imaginal disc cells with irradiation-induced genome damage often required cell competition genes. Segmentally aneuploid cells derived from targeted chromosome excisions were eliminated by the RpS12-Xrp1 cell competition pathway if they differed from neighboring cells in Rp gene dose, whereas cells with normal doses of the Rp and eIF2γ genes survived and differentiated adult tissues. Thus, cell competition, triggered by differences in Rp gene dose between cells, is a significant mechanism for the elimination of aneuploid somatic cells, likely to contribute to preventing cancer. Aneuploidy (gain or loss of whole chromosomes resulting in an abnormal karyotype) is a hallmark of spontaneous abortions and birth defects and observed in virtually every human tumor (Hassold and Hunt, 2001; Hanahan and Weinberg, 2011; López-Otín et al., 2013). It was suggested over 100 years ago that aneuploidy contributes to cancer development (Boveri, 1914). Aneuploidy can change the copy number of important oncogenes and tumor suppressors, cause stress due to gene expression imbalance, and promote further genetic instability (Naylor and van Deursen, 2016; Rutledge and Cimini, 2016; Chunduri and Storchová, 2019; Ben-David and Amon, 2020; Zhu et al., 2018). Mouse models of chromosome instability that result in aneuploidy are oncogenic (Foijer et al., 2008; Baker et al., 2009; Mukherjee et al., 2014). Drosophila cells with chromosome instability undergo a p53-independent death, but can form tumors if their apoptosis is prevented (Dekanty et al., 2012; Gerlach and Herranz, 2020; Morais da Silva et al., 2013). Because aneuploidy is thought to be detrimental to normal cells, aneuploid cells arising sporadically in vivo should, as a rule, grow poorly (Sheltzer and Amon, 2011). Studies of yeast carrying extra chromosomes reveal a stress response in these cells, thought to result from the cumulative mismatch in levels of many proteins that interact in the cell, which inhibits growth (Torres et al., 2007; Zhu et al., 2018; Terhorst et al., 2020). Increasing evidence points to the capacity of normal tissues to recognize and eliminate aneuploid cells (Hook, 1981; van Echten-Arends et al., 2011; Bazrgar et al., 2013; Pfau et al., 2016; Santaguida et al., 2017). Array Comparative Genome Hybridization detects mosaic aneuploidy in as many as 60% of normal human embryos, which can nonetheless develop into healthy babies without birth defects or evidence of aneuploid cells, suggesting their elimination (Greco et al., 2015). In mice, chimeric embryos can be constructed using both normal diploid cells and cells with a high rate of aneuploidy due to treatment with reversine, an inhibitor of the spindle assembly checkpoint. The reversine-treated cells are actively eliminated from the chimeric embryos, which can develop into morphologically normal adult mice from which reversine-treated cells have been eliminated (Bolton et al., 2016). Other observations point to the loss of aneuploid cells in other biological processes. For example, the cortex of normal mouse embryos contains as many as 30% aneuploid cells, but only ~1% are detected by 4 months post-partum, suggesting selective loss of the aneuploid fraction (Andriani et al., 2016). The mechanisms of recognition and removal of aneuploid cells are still poorly understood. In mouse tissues, cells with complex karyotypes may be recognized by the immune system (Santaguida et al., 2017). In Drosophila, clones of segmentally aneuploid cells (cells with loss or gain of chromosome segments) can survive development and differentiate in the adult abdomen, but their representation decreases as more genetic material is lost, whereas cells carrying extra genetic material are less affected (Ripoll, 1980). Clonal loss of heterozygosity is also tolerated in the abdomens of DNA repair pathway mutants, and studies with genetic markers indicate that this frequently represents loss of substantial chromosome segments (Baker et al., 1978). The Drosophila adult abdomen derives from larval histoblasts. In the head and thorax, which develop instead from the larval imaginal discs, there is evidence that aneuploid cells undergo apoptosis. DNA damage following ionizing irradiation rapidly leads to apoptosis but is followed by a smaller amount of delayed apoptosis that is independent of p53 and Chk2 and therefore unlikely to reflect unrepaired DNA damage (Brodsky et al., 2004; Wichmann et al., 2006). A similar biphasic response is seen after mitotic breakage of dicentric chromosomes, and in this case, the delayed, p53-independent cell death only occurs in genotypes likely to lead to aneuploid products (Titen and Golic, 2008). Accordingly, it is suggested that post-irradiation apoptosis independent from p53 also represents removal of aneuploid cells that arise following DNA repair, and that ‘cell competition’ may provide the p53-independent mechanism (McNamee and Brodsky, 2009). The term ‘cell competition’ was originally coined to describe the elimination of Drosophila cells heterozygous for mutant alleles of ribosomal protein genes (Rp genes) (Morata and Ripoll, 1975). Most Rp’s are essential, even to the individual cell, so that homozygosity for Rp- mutations is rapidly lethal, whereas Rp+/- heterozygotes are viable and fertile, although slow growing with minor morphological defects such as thin adult bristles (Marygold et al., 2007). By contrast to their whole animal viability, individual Rp+/- heterozygous cells or clones are actively eliminated from mosaic Drosophila tissues (Morata and Ripoll, 1975; Simpson, 1979). This involves apoptosis specific to Rp+/- heterozygous cells near to Rp+/+ cells (Morata and Ripoll, 1975; Simpson, 1979; Moreno et al., 2002; Li and Baker, 2007). The defining feature of cell competition is therefore the elimination of cells based on their difference from other neighboring cells rather than based on their intrinsic properties (Morata and Ripoll, 1975; Baker, 2020). The 80 eukaryotic Rp’s are mostly encoded by single copy genes transcribed by RNA polymerase II, and are dispersed throughout the genome in both humans and in Drosophila (Uechi et al., 2001; Marygold et al., 2007). Accordingly, aneuploidy and other large-scale genetic changes will usually affect Rp gene dose. Since Rp proteins are required stoichiometrically for ribosome assembly, which generally stalls when any one Rp is limiting, imbalanced Rp gene dose can perturb ribosome biogenesis (de la Cruz et al., 2015). This provides an almost perfectly suited mechanism to serve as an indicator of unbalanced chromosome content (McNamee and Brodsky, 2009). Cell competition may thus have evolved to recognize and remove cells with large-scale genetic changes such as aneuploidy, recognized on the basis of their mis-matched Ribosomal protein (Rp) gene complements. In this view, cells heterozygous for point mutations in Rp genes are eliminated because they mimic larger genetic changes. A stress response pathway that is activated by Rp mutations in Drosophila has recently been described, and is required for Rp point mutated cells to undergo cell competition (Baillon et al., 2018; Kale et al., 2018; Lee et al., 2018; Ji et al., 2019; Blanco et al., 2020). In Rp+/-genotypes, RpS12, an essential, eukaryote-specific component of the ribosomal Small Subunit, is required to activate expression of Xrp1, a rapidly evolving AT-hook, bZip domain transcription factor (Lee et al., 2018; Ji et al., 2019; Blanco et al., 2020). Although rpS12 null mutations are homozygously lethal, a particular point mutation, rpS12G97D, appears defective only for the cell competition aspect of RpS12 function. Homozygotes for the rpS12G97D mutation are viable, showing only minor effects on morphology and longevity, yet rpS12G97D prevents elimination of Rp+/- cells by cell competition (Kale et al., 2018; Ji et al., 2019). A key target of RpS12 appears to be the putative transcription factor Xrp1, because Xrp1 protein is barely detected in wild type cells but significantly elevated in Rp+/- wing discs. Xrp1 controls most of the phenotype of Rp+/- cells, including their reduced translation (Lee et al., 2018; Ji et al., 2019; Blanco et al., 2020). Xrp1 mutants have negligible effect in wild-type backgrounds, and normal lifespan (Baillon et al., 2018; Lee et al., 2018; Mallik et al., 2018). In response to RpS12 and Xrp1 activities, Rp+/- cells both grow more slowly than surrounding Rp+/+ cells and are also actively eliminated by apoptosis that occurs where Rp+/- cells and Rp+/+ cells meet (Baillon et al., 2018; Lee et al., 2018; Ji et al., 2019). Why apoptosis occurs at these interfaces in particular is not certain. A role for innate immune pathway components has been proposed (Meyer et al., 2014), and different levels of oxidative stress response in Rp+/- cells and Rp+/+ cells (Kucinski et al., 2017) or local induction of autophagy have also been suggested (Nagata et al., 2019). Here, we test the hypothesis that cell competition specifically removes cells with aneuploidies that result in loss of Rp genes in Drosophila imaginal discs. We show that, as hypothesized previously (McNamee and Brodsky, 2009), most of the p53-independent cell death that follows irradiation resembles cell competition genetically. We then use a targeted recombination method to investigate the fate of somatic cells that acquire large-scale genetic changes directly, and confirm that it is cell competition that removes cells heterozygous for large deletions, when they include ribosomal protein genes. By contrast, when ribosomal protein genes are unaffected, or when the cell competition pathway is inactivated genetically, cells carrying large deletions remain largely un-competed, proliferate, and contribute to adult structures. Thus, cell competition is a highly significant mechanism for elimination of aneuploid somatic cells. We discuss how cell competition to remove aneuploid cells could play a role preventing tumor development in humans. The idea that cell competition removes aneuploid cells was suggested by studies of p53-independent cell death following chromosome breakage or ionizing irradiation (Titen and Golic, 2008; McNamee and Brodsky, 2009). Importantly, in Drosophila cell competition of Rp+/- cells does not depend on p53 (Kale et al., 2015). If the model was correct, it would be expected that the p53-independent apoptosis that follows irradiation would depend on the genes recently discovered to be required for cell competition. We first confirmed the previous findings (Wichmann et al., 2006; McNamee and Brodsky, 2009). Irradiating third-instar larvae resulted in rapid induction of cell death in the wing imaginal disc that was largely p53-dependent (Figure 1A, C, D, E, G, H). The p53-dependent cell death, attributable to the DNA-damage response, was not much affected by the rpS12G97D mutation that interferes with cell competition (Figure 1B, F, H). While total cell death tailed off with time, p53-independent cell death increased around 18–24 hr post-irradiation, as reported previously (Wichmann et al., 2006; McNamee and Brodsky, 2009; Figure 1I–L). As expected for cell competition, 24 hr after irradiation, p53-independent cell death was reduced by 66% in the rpS12G97D p53 double mutant compared to the p53 mutant alone (Figure 1K–M). To exclude the possibility that other genetic background differences were responsible, rpS12 function was restored to the rpS12G97D p53 double mutant strain using a P element transgene encoding the wild-type rpS12 gene, and this restored p53-independent cell death (Figure 1M). Notably, a genomic transgene encoding the rpS12G97D cell competition-defective allele did not, leading to 86% less p53-independent cell death than the wild-type rpS12 transgene (Figure 1M). Thus, 66–86% of the p53-independent apoptosis was RpS12-dependent and might represent cell competition. Nuclear Xrp1 protein, which is only at low levels in control wing imaginal discs, was detected in scattered cells throughout p53 mutant wing discs 24 hr after irradiation, similar to the distribution of dying cells (Figure 1N). Strikingly, most (58%) of this Xrp1 expression was rpS12-dependent, similar to the rpS12-dependency of p53-independent cell death itself (Figure 1O–P). This is also as expected for cell competition, which is mediated through the induction of Xrp1 expression (Baillon et al., 2018; Lee et al., 2018; Ji et al., 2019). Radiation-damaged cells that have reduced Rp gene dose would be expected to differentiate short, thin bristles in adults, as reported previously in studies of DNA repair mutants (Baker et al., 1978), in studies of ionizing radiation (McNamee and Brodsky, 2009), and when aneuploidy is induced by mutation of spindle assembly checkpoint genes (Dekanty et al., 2012). We found Rp+/--like thoracic bristles at a frequency of ~1/300 following irradiation of either wild type or p53 mutant larvae (Figure 1Q, R). Their frequency increased ~three- to fourfold in rpS12G97D mutants or p53 rpS12G97D double mutants (Figure 1Q). In the absence of irradiation, only 2 Rp+/--like bristles were observed from 500 unirradiated rpS12G97D mutant flies. Since 16 macrochaetae were examined on each fly thorax, this indicated a frequency of ~1/8000 macrochaetae progenitor cells was Rp+/--like. We found none in 1000 unirradiated wild type flies (16,000 macrochaetae examined). While the actual nature of radiation-induced genetic changes in cells forming Minute-like bristles is not directly demonstrated, previous studies of DNA-repair mutants including mei-41, the Drosophila ATR homolog, demonstrated using multiply-marked chromosomes that the majority of Minute-like bristles reflect loss of heterozygosity for large, contiguous chromosome regions (Baker et al., 1978). In a small-scale experiment to compare γ-irradiation to the DNA repair defects studied previously (Baker et al., 1978), y+/- rpS12G97D larvae were irradiated (1000 Rad) and 2178 adult flies examined for phenotypically y thoracic bristles representing cells where the y+ allele had been mutated or deleted. Six times more y bristles were recovered in females than in males. Since the y locus is X-linked, this is most easily explained if y bristles generally result from deletions including essential genes linked to y that could not survive in males. In females, 37. 5% of y bristles were also phenotypically Minute, consistent with loss of chromosome regions extending at least from the y locus to the RpL36 gene 0. 3 Mb more centromere-proximal that is the nearest Rp locus (Figure 1S). Although this study was small scale, these findings support the conclusion from DNA repair mutant studies that Minute-like bristles seen following irradiation most commonly reflect loss of substantial chromosome segments including Rp genes, (Baker et al., 1978). Accordingly, many Minute-like bristles removed by cell competition genes following irradiation could represent such segmentally aneuploid cells. Taken together, these bristle results are also consistent with the notion that cell competition removes ~3/4 of the cells with genetic changes that encompass dose-sensitive Rp loci that arise after irradiation. Having confirmed that cell competition could potentially be important for removing cells following irradiation, we sought to assess the fate of sporadic cells that lose chromosome regions, using an assay where the cell genotypes would be definitively known and the dependence on competition with normal cells could be established. We used the FLP-FRT site-specific recombination system (Golic and Lindquist, 1989) to achieve this, exploiting large collections of transgenic flies that contain FRT sequence insertions at distinct chromosomal locations (Thibault et al., 2004). FLP recombination between pairs of FRT elements linked in cis excises intervening sequences to make defined deletions with a single FRT remaining at the recombination site (Figure 2A, B). Insertion elements of the Exelixis collection exist in several configurations, and FLP-mediated excision from paired FRT strains in the FRT w+ … w+ FRT configuration removes both the associated w+ genes, so that affected cells can be identified in the adult eye by loss of pigmentation (Figure 2A, B). Accordingly, we assembled a collection of genetic strains containing linked pairs of appropriate FRT w+ elements, each flanking a distinct genomic region (Supplementary file 1). It was first necessary to verify FLP recombination between FRT sequences, and investigate any cell-autonomous effects of the resulting segmental-monosomies. We used the eyFlp transgene, which confers continuous FLP expression to the eye and head primordia during larval life, so that FLP-FRT recombination is expected to approach completion (Newsome et al., 2000). Excision should result in white adult eyes, and also reveal any cell-autonomous effect of the resulting heterozygous deletion genotype on growth or differentiation of cells contributing to the adult eye. If the recombined genotype was autonomously cell-lethal, we would expect the developing animal to lack head structures and be unable to emerge from the pupa. If FLP-FRT recombination did not occur (or occurred inefficiently), we would expect adult eyes expressing the parental eye color (or with only scattered white spots that recombine late in development as cell number increases). We identified 17 paired FRT strains that were efficient FLP targets in this assay. These 17 strains were completely or substantially white-eyed in the presence of eyFlp, indicating excision between FRT sites in most or all eye cells. We also identified paired FRT strains that were poor substrates for Flp (Supplementary file 1; Figure 2C–T). These either retained the parental eye color in the presence of eyFlp or produce a salt and pepper pattern of very small clones (Supplementary file 1; Figure 2U–Z). No genotype tested was inviable in the presence of eyFlp, so there was no evidence that haploinsufficiency of any of the chromosome segments tested was incompatible with cell viability or severely impacted head development. Instead, most of the 17 genotypes that recombined differentiated heads of remarkably normal external appearance and morphology (Figure 2C–T). Although we did not measure head size, we noticed three genotypes in which eyes and heads were obviously smaller, consistent with a reduced growth rate of the recombined genotypes. The three small eye regions were 48B2-50C1,565F16-59B1, and 87B8-89E5 (Figure 2E, H, Q). Since the Drosophila genome is divided cytologically into 102 band intervals, each with lettered and numbered subdivisions, in this paper we refer to the chromosome P{XP}d09761 pBAC{WH}f00157, for example, by the cytological locations of the FRT sequences present in the P element and PiggyBac element insertions, which are at 48B2 and 50C1 respectively in this case (Supplementary file 1). This nomenclature quickly communicates the genome location under study, whether it overlaps or is distinct from that affected in another strain, and also indicates that in this case the FRT elements are likely separated by ~2% of the genome. The full description of each insertion strain is given in Supplementary file 1. The three excisions that substantially reduce eye size could delete a copy of one or more haploinsufficient genes important for growth during eye development, but there also could be a dominant effect of the novel junction generated by FLP/FRT recombination. Notably, excision between chromosome bands 87B8-93A2 led to eyes of normal size, although this excises all the sequences between 87B8-89E5 which led to reduced eyes. FLP recombination results in a different junctions in 87B8-89E5 and 87B8-93A2, however (Figure 2R). Overall, these results showed that 17 segmentally aneuploid genotypes were cell-viable, and able to grow and differentiate in the Drosophila eye, although a minority might have an effect on growth in this tissue. Recombination in these 17 strains each deleted 1. 4 Mb – 8. 5 Mb of autosomal DNA, representing 1–6% of the sequenced genome each (Supplementary file 1, Figure 3A). Together these deletions encompass 25. 3 Mb of DNA, corresponding to 21. 1% of the Drosophila euchromatin and 17. 7% of the sequenced genome. 11 of these 17 genotypes deleted one or more Rp loci (RpS11, RpS13, RpS16, RpS20, RpS24, RpS30, RpL14, RpL18, RpL28 or RpL36A), whereas six affected no Rp gene (Supplementary file 1, Figure 3A). In this paper, we use the symbol Rp to indicate a mutation affecting any of the 66 ribosomal protein genes that are dominant through haploinsufficiency, in distinction to 13 Rp encoded by Drosophila loci where heterozygous mutations have no phenotype. The 17 FRT pair strains that were efficient FLP targets were each exposed to a single burst of FLP expression using the heat-shock FLP transgene, intended to stimulate excision in a fraction of cells during early larval life (see Materials and methods). This led to mosaic eyes where segmentally aneuploid cells and diploid cells would be in competition. Clones of excised cells appeared only after heat-shock, confirming strict FLP-dependence. In contrast to eyFlp recombination, mosaic eyes containing sporadic clones of excised cells were only recovered at high frequencies for four segmental aneuploid genotypes, none of which affected Rp loci (Figure 3). This confirmed that most segmentally aneuploid genotypes were selected against in mosaic eyes where diploid cells were also present, because all had been shown to be intrinsically viable when competing diploid cells were absent (Figure 2). Importantly, no deletion that included an Rp locus showed more than minimal survival of sporadic clones induced with hsFlp, suggesting Rp loci could be the determinants of cell competition between segmental aneuploid and wild type cells (Figure 3B–D). To test this in a specific case, 5. 6 kb of genomic DNA encompassing the RpL28 locus was introduced onto the second chromosome using PhiC31-mediated transgenesis. This transgene proved completely sufficient to rescue the survival of cell clones heterozygous for Df (3L) 63A3-65A9, a deletion of 3. 2 Mb including the RpL28 locus (Figure 4A–C). Clones of Df (3L) 63A3-65A9 heterozygous cells barely survived alone, with a median contribution of 2% to the eyes of males and 0% to females (Figure 4H). In the presence of the RpL28+ transgene, however, Df (3L) 63A3-65A9/+ clones survived in 49 out of 50 eyes, with median contributions of 37% of the eye in males and 53% in females, not statistically different from clones heterozygous for Df (3L) 63C1-65A9, an overlapping deletion of 3. 0 Mb excluding the RpL28 locus (Figure 4H). Thus in this case, RpL28 gene dose alone determined whether a segmentally aneuploid genotype affecting hundreds of genes would be eliminated in competition with diploid cells. Most of the other 10 segmental aneuploid genotypes that deleted one or more Rp loci contributed to adult eyes to a very significantly lower degree that overlapping segmental aneuploidies that spared Rp loci (Figure 3). This reflected the fact that, in contrast to genotypes affecting Rp genes, 4/6 segmental aneuploidies that spared Rp loci survived in eye clones at high frequencies and large sizes (Figure 3A–C). The two exceptions were Df (3R) 87B8-89B16/+ and Df (3R) 87B8-89E5/+, for which little eye tissue was recovered (Figure 3A–C). Although neither affected any Rp gene, both deleted a locus mapping to 88E5-6 encoding the translation factor eIF2γ. Since independent studies in our laboratory already identified a role for the eIF2α protein in cell competition (Kiparaki, Khan, Chuen and Baker, in preparation), we tested the possible role of eIF2γ by restoring eIF2γ diploidy to Df87B8-89B16/+ or Df87B8-89E5/+ cells using a 11. 5 kb genomic transgene including the eIF2γ locus (Tschiersch et al., 1994). This completely rescued the growth and differentiation of these cells to the levels typical for aneuploidies not affecting Rp genes (Figure 4D–G, I). Thus, the locus encoding the translation factor eIF2γ behaved similarly to an Rp gene in triggering competitive elimination of heterozygous cells. If these studies, which tested a significant fraction of the Drosophila genome, are representative, they indicate that the normal diploid complement of Rp loci is important for sporadic segmentally aneuploid cells to evade cell competition, and that few other genes are comparably important. The one example of such another gene uncovered in our analysis encoded eIF2γ, another protein affecting translation. If the segmentally aneuploid cells were competed by virtue of their Rp+/- genotypes, the genetic pathways should be similar. Elimination of Rp+/- point-mutant cells by competition depends on apoptosis and is suppressed by a genetic deletion, Df (3L) H99, that removes three pro-apoptotic genes reaper (rpr), grim, and head-involution defective (hid) (Moreno et al., 2002; Kale et al., 2015). These genes are also required for the p53-independent cell death that follows irradiation, much of which resembles cell competition (Figure 1; McNamee and Brodsky, 2009). Using the Df (3L) 63A3-65A9, where clone loss was demonstrably due to heterozygosity for the RpL28 gene (Figure 4H), we found that recovery of Df (3L) 63A3-65A9/+ cell clones was enhanced by genetic suppression of apoptosis in the Df (3L) H99/+ background that experiences loss of heterozygosity for rpr, grim, and hid (Figure 5A, D). A similar rescue was obtained with the Df (3L) 63C1-65F5, which deletes the RpL18 locus (Figure 5A, D). The recoveries of Df (3L) 63A3-65A9/Df (3L) H99 clones and Df (3L) 63C1-65F5/Df (3L) H99 clones approached that of the overlapping genotype Df (3L) 63C1-65A5/Df (3L) H99, in which no Rp genes were affected (Figure 5A, D). Recovery was quantitatively inferior to that seen for Df (3L) 63A3-65A9/+ p{RpL28+} clones (Figure 4H), but it is to be noted that the Df (3L) H99/+ background unexpectedly reduced recovery of the control Df (3L) 63C1-65A5/+ cells (Figure 5A). Regardless of whether this reflects the recently described role for basal caspase activity in promoting imaginal disc growth in the wild type (Shinoda et al., 2019), or some other genetic interaction, it complicates assessment of whether H99 heterozygosity and RpL28+ transgenesis rescue Df (3L) 63A3-65A9/+ clones equally. We attempted to prevent apoptosis more completely using the genetic background hidWRX1/ Df (3L) H99 in which hid is homozygously affected in addition to heterozygosity for rpr and grim. Although hidWRX1/ Df (3L) H99 adult animals were recovered in the absence of other mutations, they became exceptionally rare in heat-shocked combinations with the dual FRT chromosomes: as a result, insufficient data could be obtained to address this question. In any case, it is clear from our results that pro-apoptotic genes contribute significantly to eliminating segmentally aneuploid cells (Figure 5A). Mutations in the rpS12 and Xrp1 genes are more specific for cell competition than mutations in cell death genes. The rpS12 and Xrp1 mutations prevent the elimination of Rp+/- point mutant cells from mosaics, but otherwise lead to seemingly normal flies, and do not affect other cell death processes (Kale et al., 2018; Lee et al., 2018). These genes should be required if segmental aneuploid cells are competed due to reduced Rp gene dose. Our results strongly support this conclusion in nearly all cases. Because the results are too extensive to present together in a single figure, they are presented in groups according to chromosome region (Figure 5, Figure 6, Figure 7, Figure 8). Beginning with the Df (3L) 63A3-65A9/+ genotype where clone loss was demonstrably due to heterozygosity for the RpL28 gene (Figure 4H), we found that heterozygosity for an Xrp1 mutation greatly restored contribution of Df (3L) 63A3-65A9/+ clones (RpL28+/-) to the eye (Figure 5B, D). Similar results were seen for the Df (3L) 63C1-65F5/+ genotype that is heterozygous for RpL18, but Xrp1 heterozgosity did not affect recovery of clones of the overlapping Df (3L) 63C1-65A9/+ that is Rp+/+ (Figure 5B, D). The Xrp1 mutation even improved the survival of larger segmental-aneuploidies where combinations of the RpL18, RpL28, and RpL14 genes were affected (Figure 6A–C). Because the rpS12G97D mutation that affects cell competition recessively maps to the third chromosome, it was simpler to examine in combination with segmental aneuploidies affecting chromosome 2. These aneuploid eye clones, which like those discussed above were also recovered in the presence of an Xrp1 mutation, included Df (2L) 26A1-29F8/+, heterozygous for RpL36A and RpS13, and Df (2R) 56F16-59B1/+, heterozygous for RpS16 and RpS24 (Figure 7A, B, D). Clones of the Df (2L) 26A1-28C3/+ or Df (2R) 56F16-58E2/+ cells that did not affect any Rp loci were recovered at high rates, independently of Xrp1 genotype (Figure 7A, B, D). As expected, rpS12G97D homozygosity also led to significant recovery of Df (2R) 56F16-59B1/+ clones that were heterozygous for RpS16 and RpS24, although to a quantitatively lesser degree than Xrp1 (Figure 7C, D) The contribution of Df (2R) 56F16-58E2/+ cells, where no Rp gene is affected, was unaltered by the rpS12G97D mutation (Figure 7C, D). Xrp1 also affected other segmentally aneuploid regions. Xrp1 mutations had a minor but statistically significant effect on clones of Df (2R) 48B2-50C1/+ cells, heterozygous for RpS11 (Figure 8A, D), a genotype that also had an autonomous effect on eye growth (Figure 2E). Xrp1 mutations enhanced the contributions of Df (3R) 87B8-89B16/+ and Df (3R) 87B8-89E5/+ cells that were heterozygous for the eIF2γ gene (Figure 8B–D). Crossing to Xrp1 only slightly improved survival of Df (3R) 87B8-93A2/+ clones that were heterozygous for the eIF2γ, RpS20 and RpS30 genes, and did little to enhance recovery of Df (3R) 89B13-93A2/+ genotypes that were heterozygous for RpS20 and RpS30 alone. As these deficiencies already delete the Xrp1 locus itself (within 91D3-5 region), introducing an Xrp1 mutation in trans leads to Xrp1-/- genotypes (Figure 8B–D). Heterozygous mutation of Xrp1 is already sufficient to suppress competition of Rp+/- point mutant cells (Lee et al., 2018), probably explaining why Xrp1 homozygosity had little further effect. Further evidence that segmentally aneuploid cells are eliminated by cell competition due to their Rp/+ genotypes came from studies in homotypic Rp mutant backgrounds (Figure 9). It is known from previous work that cells heterozygous at two Rp loci do not suffer more severe competition than cells heterozygous for only one Rp mutation, and therefore that cells heterozygous for two Rp loci generally cannot be eliminated by cells heterozygous at only one Rp locus (Simpson and Morata, 1981). Accordingly, clones of segmental aneuploid cells affecting the RpS11, RpS13, RpS16, RpS20, RpS24, RpS30, RpL14, RpL18, RpL28, or RpL36A genes were all recovered significantly better in an RpS3 point mutant background, ie RpS3+/- Df (Rp) /+ clones were not eliminated from RpS3+/- tissues (Figure 9A, B, D). This applied to segmental aneuploid clones heterozygous for eIF2γ as well (Figure 9C, D). On the other hand, the RpS3 point mutant background usually had no effect on the survival of clones of genotypes that did not delete other Rp loci (Figure 9C). One exception was Df (2L) 26A1-28C3/+, for which the RpS3+/- background generated significantly larger clones (Figure 9C, D). Notably, Df (2L) 26A1-28C3/+ cells had previously been recovered less than the other non-Rp segmental aneuploidies (Figure 3B, C; Figure 9C). Although this could also have reflected a lower rate of FLP-recombination between the 26A1 and 28C3 FRT sites, in the RpS3+/- background the recovery of Df2 (2L) 6A1-28C3/+ clones was similar to that of Df (2R) 48F6-50C1/+, Df (2R) 56F16-58E2/+ or Df (3L) 63C1-65A9/+, suggesting instead that Df (2L) 26A1-28C3/+ might be subject to a mild cell competition that can be rescued in the RpS3+/- background (Figure 9C). Although suppression of apoptosis, mutation of cell competition genes, or a germline-inherited RpS3 background all restored the growth and survival of cells hemizygous for Rp loci, they may not have done so equally. Suppressing apoptosis was least effective at expanding the contribution of aneuploid cells in the rescued eyes (Figure 5A). Xrp1, rpS12G97D, and the RpS3+/- background suppressed cell competition to similar extents, although the general RpS3 background often had the greatest effect, comparable to those of RpL28+ and eIF2γ+ transgenes (see statistical comparisons for the 63A3-65F5 and 56F16-59B1 regions in Figures 5 and 6 legends). Although several explanations could justify these differences, it is worth noting that the results correlate with the effects of these genetic backgrounds on translation and growth. Thus Df (3L) H99, which suppresses apoptosis with no known increase translation or cellular growth, is expected to suppress the competition of Rp+/- cells but not restore their translation. As a consequence, clones of Rp+/- Df (H99) /+ cells, although surviving, are not expected to grow as rapidly or contribute as much to the eye as clones of Rp+/+ cells. By contrast the rpS12G97D and Xrp1 mutations restore the general translation rate of Rp+/- cells, with Xrp1 mutation also restoring more normal rates of cellular and organismal growth (Lee et al., 2018; Ji et al., 2019). A background mutation in RpS3 is not expected to restore translation or growth to segmentally aneuploid cells, but by equally impairing the unrecombined cells, and systemically delaying the growth and developmental rate of the organism as a whole, it equalizes the contributions of aneuploid and control genotypes. In summary, our results strongly support the conclusion that the growth and survival of most segmentally aneuploid regions is determined by cell competition according to Rp gene copy number, and show that the RpS12/Xrp1-dependent process that eliminates Rp+/- point mutated cells also acts on cells with large losses of genetic material that include Rp genes. Experiments using the hsFLP transgene should stimulate recombination and segmental aneuploidy in all tissues, not only in the eye where excision causes loss of pigmentation. To test this, we looked for cells with deletions encompassing Rp loci in the thorax, where Rp haploinsufficiency leads to small, thin thoracic bristles (Marygold et al., 2007). This was explored using Df (2R) 56F16-59B1, which deletes the RpS16 and RpS24 loci. Minute-like bristles were not observed on the thoraces of heat-shocked flies carrying Df (2R) 56F16-58E2 heterozygous clones, which affect no Rp locus, or on the thoraces of heat-shocked 56F16-59B1 flies lacking rpS12 or Xrp1 mutations, but they represented 0. 5% of the thoracic bristles in the 56F16-59B1/+ rpS12G97D flies and 0. 25% of the thoracic bristles in the 56F16-59B1/+ Xrp1m2-73/+ background (Figure 10A, B). These findings indicate that Df (2R) 56F16-59B1/+ cells also survive to adulthood in the thorax if cell competition is suppressed, albeit at lower frequency than observed in the eye. We sought to test the hypothesis that cell competition is a mechanism that can target aneuploid cells based on their altered Rp gene dose (McNamee and Brodsky, 2009). It was already known that cells carrying point mutations at Rp loci are eliminated from developing imaginal discs by cell competition (Morata and Ripoll, 1975; Simpson, 1979; Baker, 2020). Here, we tested whether cells with more extensive genetic defects that reduce Rp gene dose also experience cell competition, and if so how significant this is for the removal of cells with damaged genomes. The idea that cell competition eliminates aneuploid cells developed from studies of cellular responses to DNA damage, where a delayed, p53-independent process follows after the acute, p53-dependent DNA damage response (Wichmann et al., 2006; Titen and Golic, 2008; McNamee and Brodsky, 2009). We found that a substantial proportion of p53-independent cell death shared genetic requirements with cell competition, consistent with cell competition being responsible (Figure 1). Accordingly, when the cell competition pathway was inhibited, more Minute-like bristles were recovered on the irradiated flies, an indication that cell competition could be removing cells that experience substantial losses of genetic material (Figure 1R). A proportion of both the p53-independent cell death and Minute-like bristles were independent of rpS12, however, suggesting that cell competition might not be the only process at work. To measure the role of cell competition on defined genotypes, where the role of surrounding wild type cells could also be assessed, we then used site-specific recombination to excise chromosome segments from isolated cells during imaginal disc development. As expected, segmental aneuploidy prevented cells contributing clones to the adult eye whenever Rp gene dose was reduced (Figure 11A). More significantly, cell competition appears to be the primary mechanism limiting the contribution of segmental aneuploidies in the tested size ranges to adult tissues, because segmental aneuploid cells easily survived and contributed large fractions of the adult tissue when they did not affect Rp loci, when diploidy for Rp loci was restored with a transgene, or when the cell competition pathway that depends on RpS12 and Xrp1 function was mutated. The segmental-aneuploid genotypes examined here were able to form entire heads of aneuploid cells when eyFlp was used to drive recombination in all the cells (Figure 2). The removal of sporadic aneuploid cells therefore depended on competition with diploid cells. In fact in the cases of Df (2R) 56F16-59B1/+, heterozygous for the RpS16 and RpS24 genes, and Df (3L) 65A5-65A9/+, heterozygous for the RpL28 gene, we bred flies that received heat-shock recombination, and recovered non-mosaic, entirely segmentally aneuploid flies in the next generation, derived from FLP-FRT recombination in the germlines of the parents. Thus, these segmentally aneuploid genotypes, which rarely survived in sporadic clones, were viable in all tissues when competing wild type cells were not present. The most effective suppression of Rp+/- segmental aneuploid clones was generally seen when the whole animal was heterozygous for a point mutation in RpS3 (Figure 9). This is further, compelling evidence that cell competition due to reduced Rp gene dose is the main mechanism eliminating segmentally aneuploid because it shows that the feature of euploid cells that enables them to eliminate aneuploid cells is their Rp+/+ genotype. In contrast to these results, segmental aneuploidy leaving Rp loci unaffected was compatible with clonal growth and differentiation for four of the five genomic regions tested (Figure 3). In the exception, we identified eIF2γ as the locus responsible for loss Df (3R) 87B8-89B16/+ clones and Df (3R) 87B8-89E5/+ clones (Figure 4I). No point mutant alleles of the eIF2γ gene are known and the locus is believed to be haplo-lethal to Drosophila (Marygold et al., 2007). It is cell competition that eliminates eIF2γ+/- aneuploid cells from the eye, however, since they could form apparently normal adult heads when no diploid competitor cells were present (Figure 2P–R). Moreover, clones of the eIF2γ+/- genotypes Df (3R) 87B8-89B16/+ and Df (3R) 87B8-89E5/+ were restored by both the Xrp1 mutant and by the RpS3+/- mutant background, as expected for cell competition (Figure 8). It is possible that the 26A1-28C3 region might also contain a non-Rp gene whose deletion leads to a cell competition, although much less severe. Out of 63 other translation factor genes examined in a systematic study of whole body, non-mosaic phenotypes, eIF2α and eIF2γ were the only haploinsufficient loci found (Marygold et al., 2007). Notably, the eIF2α gene is the only other locus known where point mutants lead to the developmental delay and thin bristle phenotype that is otherwise typical of heterozygous Rp mutants (Marygold et al., 2007), suggesting a functional relationship between Rp mutants and the eIF2 complex. Independent studies in our laboratory already indicate that eIF2α is regulated by Xrp1 and contributes directly to the cell competition mechanism (Kiparaki, Khan, Cheun and Baker, in preparation). Previous studies suggested that cells with whole chromosome aneuploidies experience a stress associated with mismatched dose of many proteins (Torres et al., 2007; Zhu et al., 2018; Terhorst et al., 2020). We cannot measure how such stresses reduced clonal growth of segmental aneuploid cells in our experiments, but the effect must be small compared to cell competition, since without cell competition, segmental aneuploid cells easily contributed half or more of the eye, whereas cell competition drastically reduces this contribution. It seems unlikely that all five independent genomic regions examined here, comprising 21. 1% of the euchromatic genome, represent exceptional cases. It is plausible, however, that additional stresses increase with more extensive loss of genetic material, eg clones heterozygous for a 6. 3 Mb deletion removing the RpL14, RpL18, and RpL28 loci were recovered less well than smaller deletions (Figure 6), as if larger monosomies experience other stresses in addition to cell competition. Since extra copies of at least two Rp genes (RpS12 and RpL36) do not trigger cell removal (Kale et al., 2018), other mechanisms would also be required to eliminate cells with triploidies, or act in tissues that lack cell competition (Ripoll, 1980). Finally, preventing apoptosis of Drosophila cells that have chromosome instability leads to invasive tumor growth that can be propagated after transplantation (Dekanty et al., 2012; Benhra et al., 2018). We did not observe invasive growth after blocking cell death of segmentally aneuploid cells, suggesting that chromosome instability may lead to different classes of aneuploidy, or to other additional effects. If cell competition is the main mechanism eliminating cells with segmental monosomies, at least up to a certain size, how important is this? The segmental aneuploidies we studied were comparable in genetic content to some whole chromosome monosomies in humans. For example, cells heterozygous for Df (2R) 56F16-58E2 were hemizygous for a 2. 2 Mb region including 1. 5% of the genome, about as large a region as can be expected to lack any Rp gene, encoding 333 protein coding genes and 55 non-coding RNAs. Human chromosome 21, which also contains 1. 5% of the genome that lacks any Rp gene, encodes 234 protein coding genes and 404 non-coding RNAs (Uechi et al., 2001). The similarity is not coincidental, because Rp number is conserved and the total gene number is also comparable, so genome segments that lack Rp loci are expected to be similar when measured by gene number or fraction of the genome. Thus, Df (2R) 56F16-58E2 is comparable in genetic terms to loss of a small human chromosome. Some of the segmental aneuploidies we studied in Drosophila were several-fold larger than Df (2R) 56F16-58E2 (Supplementary file 1; Figure 3A), Thus, our studies may best model aneuploidies affecting one or a few human chromosomes. Because ~80% of the Drosophila genome is carried on two autosomes, whole-chromosome aneuploidies in Drosophila, by contrast, better mimic complex karyotypes seen in tumors or in cells with chromosome instability, which affect many chromosomes. Little is known about what aneuploidies arise spontaneously in normal development. Ionizing radiation generates many kinds of chromosome aberration, so if more than half the p53-independent cell death following irradiation resembles cell competition (Figure 1), this suggests cell competition could be significant for removing many, although not all, the damaged cells that arise. Could cell competition be important in humans? As in Drosophila, the 80 Rp gene loci are distributed seemingly randomly around the 24 pairs of human chromosomes (Uechi et al., 2001). At least 21 human Rp genes have so far been found to be haploinsufficient and are responsible for the dominant syndrome Diamond Blackfan Anemia (DBA) (Ulirsch et al., 2018). Thus Rp genes could be sensors for aneuploidy in humans. The retention of aneuploid cells in Drosophila that inherit an Rp mutation from the germline (Figure 9) resembles the situation in human DBA patients, the majority of whom are constitutively heterozygous for a Rp gene mutation or deletion (Ulirsch et al., 2018). DBA patients experience 4. 8x higher lifetime incidence of multiple cancers, not limited to the hematopoietic system (Vlachos et al., 2018). Current hypotheses for this cancer predisposition include specific alterations to the spectrum of translation due to defective ribosome biogenesis, a loss of translational fidelity due to selection of second-site suppressor mutations, selective pressure for p53 mutations due to the chronic p53 activity in such genotypes, and oxidative stress or metabolic reprogramming in Rp+/-cells (Sulima et al., 2019). To these we can now add the possibility that DBA patients experience a diminished capacity to recognize and eliminate aneuploid cells, because their euploid cells are not Rp+/+ (Figure 11B). The nearly fivefold increase in tumor incidence suggests that if this was correct, cell competition might remove as many as 80% of pre-neoplastic cells from normal individuals due to their aneuploidy, This seems comparable to our findings that cell competition removes 58–86% of the cells with radiation-damaged genomes in Drosophila (Figure 1M, P, R). Flies were reared on standard medium at 25°C unless otherwise noted. The genetic strains used are described in the Key Resources Table. Strains carrying pairs of FRT transgenic elements in cis were obtained after meiotic recombination using appropriate genetic crosses, monitoring recombination frequency to confirm the expected transgene locations. FLP expression was induced by 37°C heat shock for 30 or 60 min at 36 ± 12 hr after egg laying. Adult flies were aged ~1 week to allow eye color to darken fully, then stored at −20°C for later measurement and photography. The fraction of each adult eye populated by unpigmented cells was estimated manually under a dissecting microscope. Samples were blinded for genotype before scoring by an independent investigator. We estimate the clonal composition of the eye by conceptually dividing each eye into segments so as to focus on the composition of the mosaic subregions. For example, an eye that is 56% white might be half white with an additional quarter of the eye that was one quarter white. Estimates are no doubt approximate although we do not think the errors are large. Importantly, the Mann Whitney procedure used to compare results statistically ranks relative clone size between genotypes rather than using the absolute values of the estimates. Many of the genetic backgrounds in which mosaics were generated carried other, distant FRT sites as part of the FRT82B Xrp1m2-73, FRT82B RpS3, rpS12G97D FRT80B, Df (3L) H99 FRT80B chromosomes. Accordingly, the control backgrounds in these experiments always included FRT82B or FRT80B, as appropriate, and as described in the figure legends. The RpL28 rescue transgene was obtained by inserting genomic sequences 3L: 3220152–3225729 (Drosophila genome Release 6) into pTL780, which uses DsRed expression as a transgenic marker (Blanco-Redondo and Langenhan, 2018). The genomic DNA was amplified from the Drosophila genomic reference strain (Adams et al., 2000). The resulting pTL780 (RpL28+) plasmid was used for integration at the VK37 landing site on chromosome 2 (Venken et al., 2009). For irradiation, food vials containing larvae were exposed to 500,1000 or 4000 rad from a γ-ray source 84 ± 12 hr after egg laying. Dissection, fixation, and immuno-labeling of wing imaginal discs with anti-active Dcp1 and anti-Xrp1 was performed as described previously (Baker et al., 2014; Lee et al., 2018). Frequencies of cell death and of Xrp1 expression were compared pairwise by t-tests (Figure 1). For multiple comparisons, one-way ANOVA was used with the Holm correction for multiple testing (Figure 1, Figure 10). Previous studies indicated that significant results could be obtained from five biological replicates, where a biological replicate is an imaginal disc preparation labeled, imaged, and quantified (McNamee and Brodsky, 2009). N for each experiment is reported in the figure legends. The extent of white tissue in mosaic eyes was compared using pairwise Mann-Whitney tests with the Benjamini-Hochberg (BH) correction for multiple testing, using FDR ≤ 0. 05. There are 109 pairwise Mann-Whitney comparisons made in the main text of this paper, their P-values and the BH corrections are summarized in Supplementary file 2. Where the extent of white tissue in mosaic eyes was compared between multiple genotypes simultaneously, the Kruskal-Wallis test was used with post-hoc follow-up tests using the method of Conover with BH correction using FDR ≤ 0. 05. No explicit power analysis was used. All flies obtained were scored in initial experiments, sometimes leading to unequal sample sizes, subsequently we considered 20 eyes of each sex generally sufficient for significant results (while the number of flies that can be obtained is rarely limiting, blinding and scoring clone sizes is time-consuming). N is given in the figures for each experiment. All the figures show experimental and control data obtained from simultaneous parallel experiments in each case, for which all the data scored were included. Some of the genotypes have been generated on multiple occasions with similar results, not all included in the figures.

Aneuploid cells emerge when cellular division goes awry and a cell ends up with the wrong number of chromosomes, the tiny genetic structures carrying the instructions that control life's processes. Aneuploidy can lead to fatal conditions during development, and to cancer in an adult organism. A safety mechanism may exist that helps the body to detect and remove these cells. Yet, exactly this happens is still poorly understood: in particular, it is unclear how cells manage to 'count' their chromosomes. One way they could do so is through the ribosomes, the molecular 'factories' that create the building blocks required for life. In a cell, every chromosome carries genes that code for the proteins (known as Rps) forming ribosomes. Aneuploidy will alter the number of Rp genes, and in turn the amount and type of Rps the cell produces, so that ribosomes and the genes for Rps could act as a'readout' of aneuploidy. Ji et al set out to test this theory in fruit flies. The first experiment used a genetic manipulation technique called site-specific recombination to remove parts of chromosomes from cells in the developing eye and wing. Cells which retained all their Rp genes survived, while those that were missing some usually died - but only when the surrounding cells were normal. In this situation, healthy cells eliminated their damaged neighbours through a process known as cell competition. A second experiment, using radiation as an alternative method of damaging chromosomes, also gave similar results. The work by Ji et al. reveals how the body can detect and eliminate aneuploid cells, potentially before they can cause harm. If the same mechanism applies in humans, boosting cell competition may, one day, helps to combat diseases like cancer.

lay_elife

By. Snejana Farberov. Images of a striking dancer from the racy Fantasy revue and her dismembered corpse were shown for the first time Thursday to a Las Vegas jury hearing opening statements in the murder trial of an ex-boyfriend accused of killing her in 2010. Jason Omar 'Blu' Griffith sat impassively with his attorneys as prosecutor Marc DiGiacomo told the jury that evidence during the next several days will prove that Griffith choked Deborah Flores Narvaez and asphyxiated her with a plastic bag over her head — and twice tried to dispose of her body in tubs of cement. Griffith defense attorney Jeff Banks told the jury the case isn't that simple. Gruesome murder: Jason Omar 'Blu' Griffith (left) is on trial in Las Vegas for allegedly murdering his girlfriend Deborah Flores Narvaez (right) and dismembering her body in 2010. Blank stare: Griffith appeared impassive in court Thursday as the prosecutor laid out the evidence for the jury. Griffith, now 35, has pleaded not guilty to first-degree murder. Banks said evidence will show he was defending himself when his ex-girlfriend died. 'They will show you pictures, and they will be gruesome and they will be shocking,' Banks said. 'But that does not change the fact... that he was scared and he was afraid that she was violent, and Jason Griffith was defending himself on Dec. 12, 2010.' Jurors were questioned for three days this week about what they remembered about news reports of Debbie Flores' disappearance in December 2010 and Griffith's arrest in January 2011. The story began as a missing person case, with posters and fliers distributed to the media. It made headlines for weeks through the Christmas and New Year's holidays while Flores' sister, Celeste Flores Narvaez, pleaded publicly for information. On Thursday, the jury heard from Flores Narvaez’s roommate and fellow Fantasy dancer Sonya Sonnenberg, who testified that on the day of the murder, the woman had gone to Griffith’s house to watch Dexter - a TV show about a serial killer - and never returned. Her dismembered, badly decomposing body was discovered weeks later in tubs of cement. Banks said the jury will hear about violence, property crimes, stalking, threats, harassment — all 'by Debbie Flores against Mr. Griffith' — as well as acts of battery and weapons. Tragic end: The up-and-coming burlesque dancer was discovered weeks after her disappearance in December 2013 hacked into pieces placed in tubs of cement. Adult entertainer: Flores Narvaez was a backup dancer in the risque Fantasy revue at Luxor hotel. A key piece of evidence is a video recording that Griffith made while confronting Flores about his car tires being slashed. In it, Flores admits to hitting Griffith, entering his house, looking on his computer, pouring egg whites on his car and slashing three tires. DiGiacomo predicted that every witness in the case would describe a volatile relationship between Flores and Griffith, who was arrested after an October domestic argument with Flores. The prosecutor spent more than an hour weaving a tale of tangled and overlapping romances — and Griffith becoming depressed and suicidal between the October fight with Flores and his 31st birthday on December 10. Griffith, a dancer in the Cirque du Soleil show Love at The Mirage, was having trouble juggling relationships at the time with Flores and with another woman, a dancer in another Cirque show at another Las Vegas Strip resort, DiGiacomo said. Police finally cracked the disappearance-turned-homicide case when they gave Griffith's housemate, Louis Colombo, immunity from prosecution. Opening statement: Prosecutor Marc DiGiacomo told the jury that evidence will prove that Griffith choked Flores Narvaez and asphyxiated her with a plastic bag over her head. Defense strategy: Griffith's lawyer Jeff Banks said his client was afraid that the dancer was violent, and he defended himself. Colombo told police he left the house after seeing Griffith put his hands around Flores' neck as they argued. When he returned, he could see Flores was dead. A friend, Kalae Casorso, told police that Griffith admitted to her that Debbie Flores was in the huge tub of cement he and Colombo tried to store at her house. DiGiacomo's description was graphic. The tub, with Flores' body and 600lbs of cement, weighed more than 700lbs as Griffith and Colombo tried to wheel it on dollies to a vacant home, the prosecutor said. He said Colombo would describe standing watch outside as Griffith chiseled the big block apart and sawed the legs off Flores' corpse to fit her remains in two smaller concrete-filled tubs that weighed less. 'The problem for Mr. Griffith is, it's painfully apparent that he never does anything alone,' DiGiacomo told the jury as he stood before a video screen showing autopsy photos of the torso and severed legs. Griffith allegedly admitted to a friend that Debbie Flores was in a huge 600lbs tub of cement. Bitter irony: Flores Narvaez’s roommate and fellow Fantasy dancer Sonya Sonnenberg testified that on the day of the murder, the woman had gone to Griffith’s house to watch Dexter - a TV show about a serial killer. Celeste Flores Narvaez, sister of Deborah Flores. Narvaez, was outraged by the defense attorney's theory that the slain dancer was the aggressor,

Jason Omar 'Blu' Griffith, 35, is on trial in the December 2010 murder of Fantasy revue show dancer Deborah Flores Narvaez. The dancer's dismembered body was discovered in several cement tubs weeks later. Griffith's roommate told police the Cirque du Soleil performer choked his girlfriend and placed a plastic bag over her head. He allegedly tried to dispose of Flores' body by submerging it in a 600lbs tub of cement. Griffith's lawyer claims the dancer was acting in self-defense because the woman was violent. Flores' roommate testified that on December 12, 2010, the dancer told her she was going to Griffith's house to watch the serial-killer show Dexter.

cnn_dailymail

1 THE THREE PRESENTS OF D'ARTAGNAN THE ELDER On the first Monday of the month of April, 1625, the market town of Meung, in which the author of ROMANCE OF THE ROSE was born, appeared to be in as perfect a state of revolution as if the Huguenots had just made a second La Rochelle of it. Many citizens, seeing the women flying toward the High Street, leaving their children crying at the open doors, hastened to don the cuirass, and supporting their somewhat uncertain courage with a musket or a partisan, directed their steps toward the hostelry of the Jolly Miller, before which was gathered, increasing every minute, a compact group, vociferous and full of curiosity. In those times panics were common, and few days passed without some city or other registering in its archives an event of this kind. There were nobles, who made war against each other; there was the king, who made war against the cardinal; there was Spain, which made war against the king. Then, in addition to these concealed or public, secret or open wars, there were robbers, mendicants, Huguenots, wolves, and scoundrels, who made war upon everybody. The citizens always took up arms readily against thieves, wolves or scoundrels, often against nobles or Huguenots, sometimes against the king, but never against the cardinal or Spain. It resulted, then, from this habit that on the said first Monday of April, 1625, the citizens, on hearing the clamor, and seeing neither the red-and-yellow standard nor the livery of the Duc de Richelieu, rushed toward the hostel of the Jolly Miller. When arrived there, the cause of the hubbub was apparent to all. A young man--we can sketch his portrait at a dash. Imagine to yourself a Don Quixote of eighteen; a Don Quixote without his corselet, without his coat of mail, without his cuisses; a Don Quixote clothed in a woolen doublet, the blue color of which had faded into a nameless shade between lees of wine and a heavenly azure; face long and brown; high cheek bones, a sign of sagacity; the maxillary muscles enormously developed, an infallible sign by which a Gascon may always be detected, even without his cap--and our young man wore a cap set off with a sort of feather; the eye open and intelligent; the nose hooked, but finely chiseled. Too big for a youth, too small for a grown man, an experienced eye might have taken him for a farmer's son upon a journey had it not been for the long sword which, dangling from a leather baldric, hit against the calves of its owner as he walked, and against the rough side of his steed when he was on horseback. For our young man had a steed which was the observed of all observers. It was a Bearn pony, from twelve to fourteen years old, yellow in his hide, without a hair in his tail, but not without windgalls on his legs, which, though going with his head lower than his knees, rendering a martingale quite unnecessary, contrived nevertheless to perform his eight leagues a day. Unfortunately, the qualities of this horse were so well concealed under his strange-colored hide and his unaccountable gait, that at a time when everybody was a connoisseur in horseflesh, the appearance of the aforesaid pony at Meung--which place he had entered about a quarter of an hour before, by the gate of Beaugency--produced an unfavorable feeling, which extended to his rider. And this feeling had been more painfully perceived by young d'Artagnan--for so was the Don Quixote of this second Rosinante named--from his not being able to conceal from himself the ridiculous appearance that such a steed gave him, good horseman as he was. He had sighed deeply, therefore, when accepting the gift of the pony from M. d'Artagnan the elder. He was not ignorant that such a beast was worth at least twenty livres; and the words which had accompanied the present were above all price. "My son," said the old Gascon gentleman, in that pure Bearn PATOIS of which Henry IV could never rid himself, "this horse was born in the house of your father about thirteen years ago, and has remained in it ever since, which ought to make you love it. Never sell it; allow it to die tranquilly and honorably of old age, and if you make a campaign with it, take as much care of it as you would of an old servant. At court, provided you have ever the honor to go there," continued M. d'Artagnan the elder, "--an honor to which, remember, your ancient nobility gives you the right--sustain worthily your name of gentleman, which has been worthily borne by your ancestors for five hundred years, both for your own sake and the sake of those who belong to you. By the latter I mean your relatives and friends. Endure nothing from anyone except Monsieur the Cardinal and the king. It is by his courage, please observe, by his courage alone, that a gentleman can make his way nowadays. Whoever hesitates for a second perhaps allows the bait to escape which during that exact second fortune held out to him. You are young. You ought to be brave for two reasons: the first is that you are a Gascon, and the second is that you are my son. Never fear quarrels, but seek adventures. I have taught you how to handle a sword; you have thews of iron, a wrist of steel. Fight on all occasions. Fight the more for duels being forbidden, since consequently there is twice as much courage in fighting. I have nothing to give you, my son, but fifteen crowns, my horse, and the counsels you have just heard. Your mother will add to them a recipe for a certain balsam, which she had from a Bohemian and which has the miraculous virtue of curing all wounds that do not reach the heart. Take advantage of all, and live happily and long. I have but one word to add, and that is to propose an example to you--not mine, for I myself have never appeared at court, and have only taken part in religious wars as a volunteer; I speak of Monsieur de Treville, who was formerly my neighbor, and who had the honor to be, as a child, the play-fellow of our king, Louis XIII, whom God preserve! Sometimes their play degenerated into battles, and in these battles the king was not always the stronger. The blows which he received increased greatly his esteem and friendship for Monsieur de Treville. Afterward, Monsieur de Treville fought with others: in his first journey to Paris, five times; from the death of the late king till the young one came of age, without reckoning wars and sieges, seven times; and from that date up to the present day, a hundred times, perhaps! So that in spite of edicts, ordinances, and decrees, there he is, captain of the Musketeers; that is to say, chief of a legion of Caesars, whom the king holds in great esteem and whom the cardinal dreads--he who dreads nothing, as it is said. Still further, Monsieur de Treville gains ten thousand crowns a year; he is therefore a great noble. He began as you begin. Go to him with this letter, and make him your model in order that you may do as he has done." Upon which M. d'Artagnan the elder girded his own sword round his son, kissed him tenderly on both cheeks, and gave him his benediction. On leaving the paternal chamber, the young man found his mother, who was waiting for him with the famous recipe of which the counsels we have just repeated would necessitate frequent employment. The adieux were on this side longer and more tender than they had been on the other--not that M. d'Artagnan did not love his son, who was his only offspring, but M. d'Artagnan was a man, and he would have considered it unworthy of a man to give way to his feelings; whereas Mme. d'Artagnan was a woman, and still more, a mother. She wept abundantly; and--let us speak it to the praise of M. d'Artagnan the younger--notwithstanding the efforts he made to remain firm, as a future Musketeer ought, nature prevailed, and he shed many tears, of which he succeeded with great difficulty in concealing the half. The same day the young man set forward on his journey, furnished with the three paternal gifts, which consisted, as we have said, of fifteen crowns, the horse, and the letter for M. de Treville--the counsels being thrown into the bargain. With such a VADE MECUM d'Artagnan was morally and physically an exact copy of the hero of Cervantes, to whom we so happily compared him when our duty of an historian placed us under the necessity of sketching his portrait. Don Quixote took windmills for giants, and sheep for armies; d'Artagnan took every smile for an insult, and every look as a provocation--whence it resulted that from Tarbes to Meung his fist was constantly doubled, or his hand on the hilt of his sword; and yet the fist did not descend upon any jaw, nor did the sword issue from its scabbard. It was not that the sight of the wretched pony did not excite numerous smiles on the countenances of passers-by; but as against the side of this pony rattled a sword of respectable length, and as over this sword gleamed an eye rather ferocious than haughty, these passers-by repressed their hilarity, or if hilarity prevailed over prudence, they endeavored to laugh only on one side, like the masks of the ancients. D'Artagnan, then, remained majestic and intact in his susceptibility, till he came to this unlucky city of Meung. But there, as he was alighting from his horse at the gate of the Jolly Miller, without anyone--host, waiter, or hostler--coming to hold his stirrup or take his horse, d'Artagnan spied, though an open window on the ground floor, a gentleman, well-made and of good carriage, although of rather a stern countenance, talking with two persons who appeared to listen to him with respect. D'Artagnan fancied quite naturally, according to his custom, that he must be the object of their conversation, and listened. This time d'Artagnan was only in part mistaken; he himself was not in question, but his horse was. The gentleman appeared to be enumerating all his qualities to his auditors; and, as I have said, the auditors seeming to have great deference for the narrator, they every moment burst into fits of laughter. Now, as a half-smile was sufficient to awaken the irascibility of the young man, the effect produced upon him by this vociferous mirth may be easily imagined. Nevertheless, d'Artagnan was desirous of examining the appearance of this impertinent personage who ridiculed him. He fixed his haughty eye upon the stranger, and perceived a man of from forty to forty-five years of age, with black and piercing eyes, pale complexion, a strongly marked nose, and a black and well-shaped mustache. He was dressed in a doublet and hose of a violet color, with aiguillettes of the same color, without any other ornaments than the customary slashes, through which the shirt appeared. This doublet and hose, though new, were creased, like traveling clothes for a long time packed in a portmanteau. D'Artagnan made all these remarks with the rapidity of a most minute observer, and doubtless from an instinctive feeling that this stranger was destined to have a great influence over his future life. Now, as at the moment in which d'Artagnan fixed his eyes upon the gentleman in the violet doublet, the gentleman made one of his most knowing and profound remarks respecting the Bearnese pony, his two auditors laughed even louder than before, and he himself, though contrary to his custom, allowed a pale smile (if I may be allowed to use such an expression) to stray over his countenance. This time there could be no doubt; d'Artagnan was really insulted. Full, then, of this conviction, he pulled his cap down over his eyes, and endeavoring to copy some of the court airs he had picked up in Gascony among young traveling nobles, he advanced with one hand on the hilt of his sword and the other resting on his hip. Unfortunately, as he advanced, his anger increased at every step; and instead of the proper and lofty speech he had prepared as a prelude to his challenge, he found nothing at the tip of his tongue but a gross personality, which he accompanied with a furious gesture. "I say, sir, you sir, who are hiding yourself behind that shutter--yes, you, sir, tell me what you are laughing at, and we will laugh together!" The gentleman raised his eyes slowly from the nag to his cavalier, as if he required some time to ascertain whether it could be to him that such strange reproaches were addressed; then, when he could not possibly entertain any doubt of the matter, his eyebrows slightly bent, and with an accent of irony and insolence impossible to be described, he replied to d'Artagnan, "I was not speaking to you, sir." "But I am speaking to you!" replied the young man, additionally exasperated with this mixture of insolence and good manners, of politeness and scorn. The stranger looked at him again with a slight smile, and retiring from the window, came out of the hostelry with a slow step, and placed himself before the horse, within two paces of d'Artagnan. His quiet manner and the ironical expression of his countenance redoubled the mirth of the persons with whom he had been talking, and who still remained at the window. D'Artagnan, seeing him approach, drew his sword a foot out of the scabbard. "This horse is decidedly, or rather has been in his youth, a buttercup," resumed the stranger, continuing the remarks he had begun, and addressing himself to his auditors at the window, without paying the least attention to the exasperation of d'Artagnan, who, however, placed himself between him and them. "It is a color very well known in botany, but till the present time very rare among horses." "There are people who laugh at the horse that would not dare to laugh at the master," cried the young emulator of the furious Treville. "I do not often laugh, sir," replied the stranger, "as you may perceive by the expression of my countenance; but nevertheless I retain the privilege of laughing when I please." "And I," cried d'Artagnan, "will allow no man to laugh when it displeases me!" "Indeed, sir," continued the stranger, more calm than ever; "well, that is perfectly right!" and turning on his heel, was about to re-enter the hostelry by the front gate, beneath which d'Artagnan on arriving had observed a saddled horse. But, d'Artagnan was not of a character to allow a man to escape him thus who had the insolence to ridicule him. He drew his sword entirely from the scabbard, and followed him, crying, "Turn, turn, Master Joker, lest I strike you behind!" "Strike me!" said the other, turning on his heels, and surveying the young man with as much astonishment as contempt. "Why, my good fellow, you must be mad!" Then, in a suppressed tone, as if speaking to himself, "This is annoying," continued he. "What a godsend this would be for his Majesty, who is seeking everywhere for brave fellows to recruit for his Musketeers!" He had scarcely finished, when d'Artagnan made such a furious lunge at him that if he had not sprung nimbly backward, it is probable he would have jested for the last time. The stranger, then perceiving that the matter went beyond raillery, drew his sword, saluted his adversary, and seriously placed himself on guard. But at the same moment, his two auditors, accompanied by the host, fell upon d'Artagnan with sticks, shovels and tongs. This caused so rapid and complete a diversion from the attack that d'Artagnan's adversary, while the latter turned round to face this shower of blows, sheathed his sword with the same precision, and instead of an actor, which he had nearly been, became a spectator of the fight--a part in which he acquitted himself with his usual impassiveness, muttering, nevertheless, "A plague upon these Gascons! Replace him on his orange horse, and let him begone!" "Not before I have killed you, poltroon!" cried d'Artagnan, making the best face possible, and never retreating one step before his three assailants, who continued to shower blows upon him. "Another gasconade!" murmured the gentleman. "By my honor, these Gascons are incorrigible! Keep up the dance, then, since he will have it so. When he is tired, he will perhaps tell us that he has had enough of it." But the stranger knew not the headstrong personage he had to do with; d'Artagnan was not the man ever to cry for quarter. The fight was therefore prolonged for some seconds; but at length d'Artagnan dropped his sword, which was broken in two pieces by the blow of a stick. Another blow full upon his forehead at the same moment brought him to the ground, covered with blood and almost fainting. It was at this moment that people came flocking to the scene of action from all sides. The host, fearful of consequences, with the help of his servants carried the wounded man into the kitchen, where some trifling attentions were bestowed upon him. As to the gentleman, he resumed his place at the window, and surveyed the crowd with a certain impatience, evidently annoyed by their remaining undispersed. "Well, how is it with this madman?" exclaimed he, turning round as the noise of the door announced the entrance of the host, who came in to inquire if he was unhurt. "Your excellency is safe and sound?" asked the host. "Oh, yes! Perfectly safe and sound, my good host; and I wish to know what has become of our young man." "He is better," said the host, "he fainted quite away." "Indeed!" said the gentleman. "But before he fainted, he collected all his strength to challenge you, and to defy you while challenging you." "Why, this fellow must be the devil in person!" cried the stranger. "Oh, no, your Excellency, he is not the devil," replied the host, with a grin of contempt; "for during his fainting we rummaged his valise and found nothing but a clean shirt and eleven crowns--which however, did not prevent his saying, as he was fainting, that if such a thing had happened in Paris, you should have cause to repent of it at a later period." "Then," said the stranger coolly, "he must be some prince in disguise." "I have told you this, good sir," resumed the host, "in order that you may be on your guard." "Did he name no one in his passion?" "Yes; he struck his pocket and said, 'We shall see what Monsieur de Treville will think of this insult offered to his protege.'" "Monsieur de Treville?" said the stranger, becoming attentive, "he put his hand upon his pocket while pronouncing the name of Monsieur de Treville? Now, my dear host, while your young man was insensible, you did not fail, I am quite sure, to ascertain what that pocket contained. What was there in it?" "A letter addressed to Monsieur de Treville, captain of the Musketeers." "Indeed!" "Exactly as I have the honor to tell your Excellency." The host, who was not endowed with great perspicacity, did not observe the expression which his words had given to the physiognomy of the stranger. The latter rose from the front of the window, upon the sill of which he had leaned with his elbow, and knitted his brow like a man disquieted. "The devil!" murmured he, between his teeth. "Can Treville have set this Gascon upon me? He is very young; but a sword thrust is a sword thrust, whatever be the age of him who gives it, and a youth is less to be suspected than an older man," and the stranger fell into a reverie which lasted some minutes. "A weak obstacle is sometimes sufficient to overthrow a great design. "Host," said he, "could you not contrive to get rid of this frantic boy for me? In conscience, I cannot kill him; and yet," added he, with a coldly menacing expression, "he annoys me. Where is he?" "In my wife's chamber, on the first flight, where they are dressing his wounds." "His things and his bag are with him? Has he taken off his doublet?" "On the contrary, everything is in the kitchen. But if he annoys you, this young fool--" "To be sure he does. He causes a disturbance in your hostelry, which respectable people cannot put up with. Go; make out my bill and notify my servant." "What, monsieur, will you leave us so soon?" "You know that very well, as I gave my order to saddle my horse. Have they not obeyed me?" "It is done; as your Excellency may have observed, your horse is in the great gateway, ready saddled for your departure." "That is well; do as I have directed you, then." "What the devil!" said the host to himself. "Can he be afraid of this boy?" But an imperious glance from the stranger stopped him short; he bowed humbly and retired. "It is not necessary for Milady* to be seen by this fellow," continued the stranger. "She will soon pass; she is already late. I had better get on horseback, and go and meet her. I should like, however, to know what this letter addressed to Treville contains." _*We are well aware that this term, milady, is only properly used when followed by a family name. But we find it thus in the manuscript, and we do not choose to take upon ourselves to alter it._ And the stranger, muttering to himself, directed his steps toward the kitchen. In the meantime, the host, who entertained no doubt that it was the presence of the young man that drove the stranger from his hostelry, re-ascended to his wife's chamber, and found d'Artagnan just recovering his senses. Giving him to understand that the police would deal with him pretty severely for having sought a quarrel with a great lord--for in the opinion of the host the stranger could be nothing less than a great lord--he insisted that notwithstanding his weakness d'Artagnan should get up and depart as quickly as possible. D'Artagnan, half stupefied, without his doublet, and with his head bound up in a linen cloth, arose then, and urged by the host, began to descend the stairs; but on arriving at the kitchen, the first thing he saw was his antagonist talking calmly at the step of a heavy carriage, drawn by two large Norman horses. His interlocutor, whose head appeared through the carriage window, was a woman of from twenty to two-and-twenty years. We have already observed with what rapidity d'Artagnan seized the expression of a countenance. He perceived then, at a glance, that this woman was young and beautiful; and her style of beauty struck him more forcibly from its being totally different from that of the southern countries in which d'Artagnan had hitherto resided. She was pale and fair, with long curls falling in profusion over her shoulders, had large, blue, languishing eyes, rosy lips, and hands of alabaster. She was talking with great animation with the stranger. "His Eminence, then, orders me--" said the lady. "To return instantly to England, and to inform him as soon as the duke leaves London." "And as to my other instructions?" asked the fair traveler. "They are contained in this box, which you will not open until you are on the other side of the Channel." "Very well; and you--what will you do?" "I--I return to Paris." "What, without chastising this insolent boy?" asked the lady. The stranger was about to reply; but at the moment he opened his mouth, d'Artagnan, who had heard all, precipitated himself over the threshold of the door. "This insolent boy chastises others," cried he; "and I hope that this time he whom he ought to chastise will not escape him as before." "Will not escape him?" replied the stranger, knitting his brow. "No; before a woman you would dare not fly, I presume?" "Remember," said Milady, seeing the stranger lay his hand on his sword, "the least delay may ruin everything." "You are right," cried the gentleman; "begone then, on your part, and I will depart as quickly on mine." And bowing to the lady, he sprang into his saddle, while her coachman applied his whip vigorously to his horses. The two interlocutors thus separated, taking opposite directions, at full gallop. "Pay him, booby!" cried the stranger to his servant, without checking the speed of his horse; and the man, after throwing two or three silver pieces at the foot of mine host, galloped after his master. "Base coward! false gentleman!" cried d'Artagnan, springing forward, in his turn, after the servant. But his wound had rendered him too weak to support such an exertion. Scarcely had he gone ten steps when his ears began to tingle, a faintness seized him, a cloud of blood passed over his eyes, and he fell in the middle of the street, crying still, "Coward! coward! coward!" "He is a coward, indeed," grumbled the host, drawing near to d'Artagnan, and endeavoring by this little flattery to make up matters with the young man, as the heron of the fable did with the snail he had despised the evening before. "Yes, a base coward," murmured d'Artagnan; "but she--she was very beautiful." "What she?" demanded the host. "Milady," faltered d'Artagnan, and fainted a second time. "Ah, it's all one," said the host; "I have lost two customers, but this one remains, of whom I am pretty certain for some days to come. There will be eleven crowns gained." It is to be remembered that eleven crowns was just the sum that remained in d'Artagnan's purse. The host had reckoned upon eleven days of confinement at a crown a day, but he had reckoned without his guest. On the following morning at five o'clock d'Artagnan arose, and descending to the kitchen without help, asked, among other ingredients the list of which has not come down to us, for some oil, some wine, and some rosemary, and with his mother's recipe in his hand composed a balsam, with which he anointed his numerous wounds, replacing his bandages himself, and positively refusing the assistance of any doctor, d'Artagnan walked about that same evening, and was almost cured by the morrow. But when the time came to pay for his rosemary, this oil, and the wine, the only expense the master had incurred, as he had preserved a strict abstinence--while on the contrary, the yellow horse, by the account of the hostler at least, had eaten three times as much as a horse of his size could reasonably be supposed to have done--d'Artagnan found nothing in his pocket but his little old velvet purse with the eleven crowns it contained; for as to the letter addressed to M. de Treville, it had disappeared. The young man commenced his search for the letter with the greatest patience, turning out his pockets of all kinds over and over again, rummaging and rerummaging in his valise, and opening and reopening his purse; but when he found that he had come to the conviction that the letter was not to be found, he flew, for the third time, into such a rage as was near costing him a fresh consumption of wine, oil, and rosemary--for upon seeing this hot-headed youth become exasperated and threaten to destroy everything in the establishment if his letter were not found, the host seized a spit, his wife a broom handle, and the servants the same sticks they had used the day before. "My letter of recommendation!" cried d'Artagnan, "my letter of recommendation! or, the holy blood, I will spit you all like ortolans!" Unfortunately, there was one circumstance which created a powerful obstacle to the accomplishment of this threat; which was, as we have related, that his sword had been in his first conflict broken in two, and which he had entirely forgotten. Hence, it resulted when d'Artagnan proceeded to draw his sword in earnest, he found himself purely and simply armed with a stump of a sword about eight or ten inches in length, which the host had carefully placed in the scabbard. As to the rest of the blade, the master had slyly put that on one side to make himself a larding pin. But this deception would probably not have stopped our fiery young man if the host had not reflected that the reclamation which his guest made was perfectly just. "But, after all," said he, lowering the point of his spit, "where is this letter?" "Yes, where is this letter?" cried d'Artagnan. "In the first place, I warn you that that letter is for Monsieur de Treville, and it must be found, or if it is not found, he will know how to find it." His threat completed the intimidation of the host. After the king and the cardinal, M. de Treville was the man whose name was perhaps most frequently repeated by the military, and even by citizens. There was, to be sure, Father Joseph, but his name was never pronounced but with a subdued voice, such was the terror inspired by his Gray Eminence, as the cardinal's familiar was called. Throwing down his spit, and ordering his wife to do the same with her broom handle, and the servants with their sticks, he set the first example of commencing an earnest search for the lost letter. "Does the letter contain anything valuable?" demanded the host, after a few minutes of useless investigation. "Zounds! I think it does indeed!" cried the Gascon, who reckoned upon this letter for making his way at court. "It contained my fortune!" "Bills upon Spain?" asked the disturbed host. "Bills upon his Majesty's private treasury," answered d'Artagnan, who, reckoning upon entering into the king's service in consequence of this recommendation, believed he could make this somewhat hazardous reply without telling of a falsehood. "The devil!" cried the host, at his wit's end. "But it's of no importance," continued d'Artagnan, with natural assurance; "it's of no importance. The money is nothing; that letter was everything. I would rather have lost a thousand pistoles than have lost it." He would not have risked more if he had said twenty thousand; but a certain juvenile modesty restrained him. A ray of light all at once broke upon the mind of the host as he was giving himself to the devil upon finding nothing. "That letter is not lost!" cried he. "What!" cried d'Artagnan. "No, it has been stolen from you." "Stolen? By whom?" "By the gentleman who was here yesterday. He came down into the kitchen, where your doublet was. He remained there some time alone. I would lay a wager he has stolen it." "Do you think so?" answered d'Artagnan, but little convinced, as he knew better than anyone else how entirely personal the value of this letter was, and saw nothing in it likely to tempt cupidity. The fact was that none of his servants, none of the travelers present, could have gained anything by being possessed of this paper. "Do you say," resumed d'Artagnan, "that you suspect that impertinent gentleman?" "I tell you I am sure of it," continued the host. "When I informed him that your lordship was the protege of Monsieur de Treville, and that you even had a letter for that illustrious gentleman, he appeared to be very much disturbed, and asked me where that letter was, and immediately came down into the kitchen, where he knew your doublet was." "Then that's my thief," replied d'Artagnan. "I will complain to Monsieur de Treville, and Monsieur de Treville will complain to the king." He then drew two crowns majestically from his purse and gave them to the host, who accompanied him, cap in hand, to the gate, and remounted his yellow horse, which bore him without any further accident to the gate of St. Antoine at Paris, where his owner sold him for three crowns, which was a very good price, considering that d'Artagnan had ridden him hard during the last stage. Thus the dealer to whom d'Artagnan sold him for the nine livres did not conceal from the young man that he only gave that enormous sum for him on the account of the originality of his color. Thus d'Artagnan entered Paris on foot, carrying his little packet under his arm, and walked about till he found an apartment to be let on terms suited to the scantiness of his means. This chamber was a sort of garret, situated in the Rue des Fossoyeurs, near the Luxembourg. As soon as the earnest money was paid, d'Artagnan took possession of his lodging, and passed the remainder of the day in sewing onto his doublet and hose some ornamental braiding which his mother had taken off an almost-new doublet of the elder M. d'Artagnan, and which she had given her son secretly. Next he went to the Quai de Feraille to have a new blade put to his sword, and then returned toward the Louvre, inquiring of the first Musketeer he met for the situation of the hotel of M. de Treville, which proved to be in the Rue du Vieux-Colombier; that is to say, in the immediate vicinity of the chamber hired by d'Artagnan--a circumstance which appeared to furnish a happy augury for the success of his journey. After this, satisfied with the way in which he had conducted himself at Meung, without remorse for the past, confident in the present, and full of hope for the future, he retired to bed and slept the sleep of the brave. This sleep, provincial as it was, brought him to nine o'clock in the morning; at which hour he rose, in order to repair to the residence of M. de Treville, the third personage in the kingdom, in the paternal estimation.

In the year 1625, in Gascony, a province of France, a young man named d'Artagnan is taking leave of his father to journey to Paris, where he will seek out the prestigious Monsieur de Treville, captain of the King's Musketeers and a childhood friend of d'Artagnan's father. D'Artagnan's father has only three gifts which he can give to his son: fifteen ecus in money, a ridiculous-looking horse about thirteen years old, and a letter of introduction to Monsieur de Treville. If d'Artagnan can convince Treville to allow him to become a musketeer, he believes that he will have his fortune made because the musketeers are a select group of swordsmen highly favored by the king. After a sentimental leave-taking from his mother, d'Artagnan begins his journey to Paris. He arrives at the market town of Meung, where he sees an unknown nobleman who he believes is laughing at him, or at least at his horse. D'Artagnan's impetuous temper causes him to insult the nobleman and pick a quarrel with him. D'Artagnan is outnumbered, however, and before long he is carried unconscious into the inn. Learning from the innkeeper that d'Artagnan has a letter to the powerful Monsieur de Treville, the nobleman steals it from d'Artagnan's doublet. When d'Artagnan recovers, he goes downstairs in time to see the nobleman talking with someone whom he addresses as "Milady." Later, d'Artagnan discovers that his letter of recommendation to Treville is missing, and after threatening the innkeeper and his servants, he learns that the mysterious nobleman ransacked his belongings and apparently stole the valuable letter of introduction. D'Artagnan departs, and when he arrives in Paris, he rents a room that he discovers is near the home of Monsieur de Treville.

booksum

The 2014–6 West African Ebola epidemic highlights the need for rigorous, rapid clinical trial methods for vaccines. A challenge for trial design is making sample size calculations based on incidence within the trial, total vaccine effect, and intracluster correlation, when these parameters are uncertain in the presence of indirect effects of vaccination. We present a stochastic, compartmental model for a ring vaccination trial. After identification of an index case, a ring of contacts is recruited and either vaccinated immediately or after 21 days. The primary outcome of the trial is total vaccine effect, counting cases only from a pre-specified window in which the immediate arm is assumed to be fully protected and the delayed arm is not protected. Simulation results are used to calculate necessary sample size and estimated vaccine effect. Under baseline assumptions about vaccine properties, monthly incidence in unvaccinated rings and trial design, a standard sample-size calculation neglecting dynamic effects estimated that 7,100 participants would be needed to achieve 80% power to detect a difference in attack rate between arms, while incorporating dynamic considerations in the model increased the estimate to 8,900. This approach replaces assumptions about parameters at the ring level with assumptions about disease dynamics and vaccine characteristics at the individual level, so within this framework we were able to describe the sensitivity of the trial power and estimated effect to various parameters. We found that both of these quantities are sensitive to properties of the vaccine, to setting-specific parameters over which investigators have little control, and to parameters that are determined by the study design. Incorporating simulation into the trial design process can improve robustness of sample size calculations. For this specific trial design, vaccine effectiveness depends on properties of the ring vaccination design and on the measurement window, as well as the epidemiologic setting. The West African Ebola epidemic highlighted the need to identify a range of trial designs to evaluate vaccine effects rapidly, efficiently and rigorously during emerging disease outbreaks. The ring-vaccination trial approach employed in the Ebola ça suffit trial in Guinea is one innovative approach [1], which produced valuable evidence that the vaccine could prevent Ebola infection [2]. Other approaches considered include individual randomization and a stepped-wedge design [3,4]. In such trials it is difficult to estimate the likely effect of an infectious disease intervention because of indirect effects, and this issue is compounded by complex trial design. Sample size calculations are based on group-level quantities such as intervention effect and are therefore potentially inaccurate. By creating a transmission dynamic model for a ring vaccination trial, we show that we can make sample size calculations based on disease characteristics and individual intervention efficacy. With this framework in place we are then able to examine the estimated vaccine effect and sample size under a range of assumptions about the properties of the vaccine, the trial, and the study population. Although the only implementation of the ring trial design has been in Guinea during the Ebola epidemic, lessons can be learned and extended to other diseases and contexts. Here, we examine the tail end of an epidemic of a disease with a latent and asymptomatic phase with effective contact tracing to illustrate a more widely-applicable set of findings. In particular, we use baseline parameters values consistent with Ebola in West Africa in 2014–6, but we vary several assumptions over broader ranges than those occurring in the Ebola ça suffit trial, with the aim of being relevant to a range of potential future situations. The simulation is based on a stochastic, susceptible-exposed-infectious-detected-removed-vaccinated (SEIDRV) model for individual disease events, and it represents progression of the disease in a small cluster (henceforth ‘ring’) with homogeneous mixing. The ring represents both contacts and contacts of contacts so the assumption of homogeneous mixing is a simplifying assumption, which we can relax by modelling ‘contacts’ and ‘contacts of contacts’ as separate compartments with the highest transmission among the contacts. New cases arise through direct contact between an infectious individual and a susceptible individual within the ring, and through external infectious pressure, denoted by F, which is constant and fixed for all members of the ring. Members of the ring undergo surveillance by the study team, meaning that infectious individuals are detected and isolated with a daily probability pH, ending their infectious period. We assume in the baseline scenario that detection rate in the trial is equivalent to routine surveillance, reflecting the fact that the trial doesn’t interrupt or enhance disease control efforts. If infectiousness ends naturally, individuals can no longer be detected. A ring is enrolled into the trial when a case is detected through routine surveillance. This first detected case is defined as the index case for the purposes of the trial, but may or may not be the true index case of the outbreak in the ring. Once a ring enters the trial all its members are randomly assigned to immediate vaccination (on day 1) or delayed vaccination (on day 22). In the baseline scenario we assume no ineligibility or non-consent, so that all susceptible and exposed individuals in the ring are vaccinated, and that there is no heterogeneity or administrative delay affecting the day of vaccination. The mechanism of the vaccine in an individual is as follows: multiplicative leaky efficacy [5] increases linearly from 0 to VE (set at baseline to be 0. 7) over a period of Dramp days following vaccination, after which there is no change in efficacy over the study period [6]. Statistical analysis of the trial is based on cumulative incidence in the rings by end of follow-up and a 95% confidence interval is calculated and reported [7]. The required sample size to test a vaccine effect with 80% power is based on a difference in cumulative incidence [8], using parameters output by a simulated trial with 15,000 rings. We chose this analysis method because of the existence of simple closed-form sample size and vaccine efficacy formulae. Because both arms receive the vaccine, cases that contribute towards the cumulative incidence in each arm are only counted during a window in which the immediate arm is presumed to be protected by the vaccine, and the delayed arm is not protected. The window length is set to 21 days, equal to the vaccination delay between the arms. Because the disease has an asymptomatic phase and the vaccine has a ramp-up period during which it is not fully efficacious, the window starts at 16 days, the sum of the average asymptomatic period length and Dramp, in an attempt to exclude cases in the immediate arm who were infected before they were fully protected by the vaccine. We did not explicitly implement clustering in the simulation, instead assuming that transmission dynamics in all rings are independent. However, clustering of cases within rings arises naturally due to dependent happenings. We measure this clustering using the intracluster correlation coefficient (ICC), calculated as per Shoukri et al [9], adjusting for the covariate of trial arm and accounting for variable ring size where appropriate. In conducting the statistical analysis we assume full knowledge of the vaccine mechanism, and that cases are only included if they are detected before their infectious period ends, and their symptoms appeared during the window. For additional details on the disease transmission model, ring initiation, and analysis of the trial see the supplementary appendix. Table 1 shows the parameters used in the model, their meanings, values under baseline assumptions, and references or justifications. In order to align this model with the presumed context of the Ebola ça suffit trial, we modelled an entirely susceptible study population at the end of an epidemic, so that Reff has fallen to below one due to behaviour change. To calibrate the model, we set Reff to reproduce a monthly detected attack rate of 2% when starting from one infected individual in a ring of 50 unvaccinated susceptible individuals, in the presence of case detection at a rate pBH. Under baseline parameters in this model, the median total vaccine effect calculated from performing 100 trials with 89 rings in each arm was 70%. This value should include direct and indirect effects, so we would expect it to exceed the direct effect of 70%. However, while direct effects begin immediately, indirect effects are only important in the second generation of preventable cases onwards. There are cases in this generation that occur in the case-counting window because Reff is small and the window duration is not much longer than a typical disease generation (17 days), so the indirect effects are small. Fig 1 shows the effect of six variables on the point estimate of vaccine effect: daily probability of detection, true individual vaccine efficacy, proportion of infections from outside the ring, baseline attack rate in the unvaccinated population, administrative delay in vaccination, and start day of case-counting window. Firstly, if there is enhanced surveillance in both arms of the trial leading to more rapid isolation of infectious cases (pH>pBH), this will modestly reduce effectiveness estimates (Fig 1A). Secondly, as individual vaccine efficacy properties increase the estimated vaccine effect increases (Figs 1B and S1). Thirdly, the percentage of infections from within the ring shows a weak negative association with the estimate of vaccine effect (Fig 1C). While the magnitude of indirect effects is modest as discussed above, they are almost negligible when most infections are from outside the ring, because preventing infections within the ring does not confer as much protection to susceptible individuals. The increase in vaccine effect with higher attack rate seen in Fig 1D is driven by the increase in indirect vaccine effects in the immediate arm. Finally, delay between ring formation and vaccination means that by the beginning of the time window the vaccine has had less time to prevent cases in the immediate arm. Thus the reduction in incidence in the immediate arm does not reflect the true effect of the vaccine and the vaccine effect estimate is reduced (Fig 1E). A major determinant of the effect estimate is the choice of time window in which to count cases, as seen in Fig 1F. Not surprisingly, starting the window too early reduces the estimated effects because it includes a period of time during which the vaccine cannot affect the incidence of cases becoming symptomatic–many cases becoming symptomatic on day 8, for example, will have been infected by the index case prior to isolation, or will have been infected by a contact on (say) day 3, before the vaccine had time to induce protection. Starting the window later than the baseline of 16 days allows the trial to capture later generations in the chain of transmission, from a vaccinated person to another vaccinated person. This increases the vaccine effect estimate as it includes indirect effects. One might expect to see that starting the window too late would reduce effect estimates because it would include a period when the delayed group was also protected by the vaccine. This does not appear to be the case, at least up to a start time of 35 days (Fig 1F) –see the supplementary material for an explanation of this phenomenon. Fig 2 shows the effect of the same six variables on the required sample size: baseline attack rate in unvaccinated population, start day of case-counting window, daily probability of detection, true individual vaccine efficacy, administrative delay in vaccination, and force of external infection. The effect of each parameter on the sample size can be understood through its effect on one or more of the three factors that determine the power of this trial: the number of events, how they are distributed between the two arms, and the level of clustering of cases within rings. Respectively these factors are represented by the attack rate in the controls, the cumulative incidence difference between the arms, and the intracluster correlation coefficient (ICC) [8]. Variables that decrease the incidence rate in the controls and cases will decrease the power because for the same sample size the trial will observe fewer events. The baseline detected attack rate among unvaccinated individuals is a simple example of such a parameter (Fig 2A). Two other parameters act on the overall incidence in the trial. Firstly, making the start of the case-counting window later decreases incidence in both arms because with Reff<1 the incidence is on average declining, so across all rings in the trial the number of cases decreases over the follow-up period (Fig 2B). Secondly, the case detection decreases detected incidence rate at both extremes (Fig 2C). When case detection is high, transmission chains are interrupted by case isolation and the true incidence decreases. When case detection is low, many cases die or recover before they can be detected and consequently the detected incidence decreases. Variables that make the two arms of the trial appear more different will increase the power of the trial as the ability to differentiate between them is increased, and Fig 1 identifies such variables. Vaccine characteristics, in particular vaccine efficacy (Fig 2D), are simple examples of such a parameter, since the immediate arm receives greater protection against disease compared to the delayed arm. Changes to two other parameters increase the incidence difference in this way, as explained above: reducing the delay between ring formation and vaccination (Fig 2E) and starting the case-counting window earlier (Fig 2B). The effect of the timing of starting to count cases thus reflects two opposing forces on the sample size: it decreases sample size by increasing the incidence difference, and it increases sample size by decreasing the overall incidence. When the window is early, the former of these effects dominates as seen by the increase in sample size for early time windows in Fig 2B. When the window is late, the latter effect dominates, as seen by the increase in sample size for late time windows in the same figure. Finally, the level of clustering within rings inflates the sample size, because more clustering means that each individual case provides less information. It is often not intuitive to predict the direction in which a parameter will cause the ICC to change, and in many cases the ICC is not sensitive to the parameter. One exception is the infection from outside the ring (Fig 2F). The most significant effect of introducing external infection and reducing within-ring transmission is to make infection probability for one individual within a ring independent from the infection prevalence within the same ring. This reduces clustering in incidence (making it more Poisson-like), thus reducing the ICC and the necessary sample size. The width of the confidence intervals is affected in the same way by the three variables described above. In particular, low incidence in either arm, high ICC and a small incidence difference between the arms all lead to a wider confidence interval. The formula for the confidence interval is different from the formula used to make the power calculation, so the trends do not completely align because the size of the effect of each of the three factors is different for the confidence interval and the sample size. For an investigation of the sensitivity of the total vaccine effect estimate and sample size to other parameters in the model, see the supplementary material. For an interactive tool to explore the sensitivity of the trial parameters, see https: //matthitchings. shinyapps. io/ShinyApps/. The ring-vaccination, cluster-randomized design has two key strengths that make it a good candidate when disease transmission exhibits spatiotemporal variation. Firstly, by including members of the study population who are contacts of cases, the trial preferentially selects those at higher risk of disease acquisition, leading to an increase in efficiency while preserving false-positive rate through randomization. Indeed, when a vaccine with 0% efficacy was tested in our simulations the false positive rate was maintained at 5%. Secondly, even those study subjects who are randomized to delayed vaccination are theoretically in close contact with the study team meaning that individuals from the source population who are at the highest risk are followed closely and benefit from the trial even in the absence of vaccination [12]. In addition, vaccination of clusters when they arise allows for gradual inclusion, meaning that this design is appropriate when logistical constraints make immediate vaccination of all participants impossible or inappropriate. In this respect it is similar to a stepped-wedge cluster trial, in which prespecified clusters within the study population are vaccinated in a random order. Although we have not made a direct comparison in this study, Bellan et al [13] showed that the stepped-wedge design is underpowered when the incidence is declining because it cannot prioritize the vaccine for those at highest risk. The ring vaccination design, on the other hand, is inherently risk-prioritized because all study participants should be at higher risk than the general population. All trials should be correctly powered in order to avoid erroneous rejection of an efficacious vaccine. For a trial design with several complexities such as the one presented here, a sophisticated approach to sample size calculation is merited. A standard approach to sample size calculation for this trial would involve specifying the attack rate among the controls, the desired effect of the vaccine on the population level, and the ICC. In the context of a serious epidemic, these parameters are unlikely to be estimated with certainty; for example, the ICC requires cluster-level data to be estimated accurately. The ICC is an important parameter in designing cluster-randomized trials, yet in the absence of data it is often assumed to be 0. 05. In our simulations the range of ICCs observed was 0. 01–0. 04, suggesting that the value of this uncertain parameter should not always be assumed to be fixed at 0. 05. Therefore, the modelling approach replaces assumptions about these cluster-level quantities with assumptions about population-level parameters and disease characteristics, which are more likely to be available through analysis of data from the outbreak. A second advantage of the modelling approach is that, based as it is on a simulating the transmission of disease within a trial, it is possible to explore the impact of parameters describing the design of the trial and the properties of the disease. The added detail gained from specifying the disease model allowed us in this study to identify some key issues with the design that are worth considering. Firstly, as seen in Fig 2C, increasing case-finding efficiency above background rate has a negative impact on power, as fast isolation of cases in both arms leads to an overall decrease in cases observed by the trial. In future trials it is worth considering if there are alternative or composite endpoints, if the disease in question permits, that can be used to allow for efficacy estimates while maintaining close follow-up. Secondly, a key design consideration in the delayed-arm ring-vaccination trial is when to count cases. An intuitively appealing approach is to place the window so that the immediate arm is receiving full protection and the delayed arm none. This should in theory minimize bias caused by misclassification of unvaccinated individuals as vaccinated and vice-versa. While this placement achieves nearly maximal power, it does not maximize the VE estimate. Indirect effects that are important later in time increase the VE estimate for later time windows, while at the same time declining incidence within each ring decreases power for later time windows. Finally, the above point draws attention to the fact that caution is required when interpreting the VE estimate produced by the trial. As seen in Fig 1, many parameters that are not characteristics of the vaccine can influence the estimated effect. Whether this is due to misclassification (for example, when the time window is too early) or due to indirect effects (for example, when the attack rate is high enough to cause long transmission chains), the context of the trial should be taken into account when interpreting the VE estimate. While in the baseline scenario the trial appears to correctly estimate the individual efficacy, this is the result of misclassification and indirect effects cancelling each other out. This claim is supported by the fact that the median VE estimate falls below the individual-level vaccine efficacy when most or all infections are from outside the ring (Fig 1C) and indirect effects are negligible. The focus of this model was to explore parameters within each ring and understand how they affect the quality of data coming from the trial. As a result, we did not consider the wider context of the population disease dynamics, and in particular how and when the rings arise. For example, we calibrated Reff to a secondary attack rate in a cluster was 2%, which is not necessarily comparable to the monthly cumulative incidence in the population. If transmission takes place mainly in clusters then population cumulative incidence could be somewhat lower than cluster secondary attack rate, increasing the efficiency of a ring-vaccination trial relative to a stepped-wedge cluster trial or individual RCT. Linking this model to a model of disease within the general population would allow us to make direct comparisons to other trial designs such as the stepped-wedge cluster trial and the individually-randomized trial investigated elsewhere [13,14], but it would require detailed information about the nature of clustering of the disease in this context, and for simplicity we focused on the within-ring dynamics only. As with every model, there are limitations to these simulation results. The strength of the modelling approach compared with a standard approach is that it better estimates the parameters on which the sample size depends. However, some of the model parameters might still be uncertain in a situation in which such a model might be useful. For example, we may have limited information about the characteristics of a disease, in particular its latent and incubation period, and its Reff. The simulation results are dependent on these assumptions, and so they cannot be used at the very outset of epidemic, or else they risk being highly inaccurate. Even at the end of the West African Ebola epidemic, there were no more than four or five reliable estimates of the latent and infectious periods of EVD, and indeed there is perhaps evidence that our understanding of the natural history of the disease remains limited [15]. In addition, we have considered only the simplest method of analysis for the trial–a comparison of attack rates between the two arms after correction for clustering of cases within rings. More sophisticated methods, including time-to-event analyses incorporating ring-level random effects, as performed in the Ebola ça suffit trial, would have somewhat different sample size requirements. However, we believe that the trends seen here would be similar for other methods, because the VE estimates returned by various methods will be similar for a rare outcome [5]. In building the model we made some simplifying assumptions, and although we tested the robustness of the results to these assumptions (see supplementary material) it is possible that a more sophisticated model would provide more accurate results, particularly if superspreading events are not rare in this study population. For a vaccine trial in an epidemic, when the level of indirect effects is hard to predict, power calculations can be sensitive to parameters about which very little is known. Simulations such as these can be important aids in understanding a range of values for these parameters before a trial is carried out, and thus ensuring that the trial has sufficient power to detect an efficacious vaccine. In this trial, a finding significantly different from the null likely indicates one or more types of vaccine efficacy at the individual level, but the magnitude of the effect and the power to detect the effect will vary across settings.

The urgency, as well as the logistical and sometimes ethical challenges of clinical trials for interventions during epidemics of emerging diseases prompts the need for novel designs and analytic strategies. The successful use of a novel cluster-randomized ring-vaccination trial to test an Ebola vaccine in Guinea raises the general question of what circumstances would favour the use of trials of similar design and how the properties of the population, the vaccine and the trial would influence the necessary sample size and the expected results. We present a generalized transmission dynamic model for a ring vaccination trial to address these questions. This work is an example of the general phenomenon that mechanistic, transmission-dynamic simulations can aid in the design and interpretation of intervention trials for infectious diseases, when the trial itself can have non-obvious effects on transmission dynamics that may not be fully captured by effect- and sample-size calculations for noncommunicable diseases.

lay_plos

Japan has been a strong supporter of a human rights investigation into North Korea. Japan has accused North Korea of kidnapping some of its citizens, and the issue remains a popular human rights and political cause. The creation of a commission of inquiry would be a victory for defectors from North Korea, including a handful of people who are said to be survivors of the isolated country’s infamous prison camps. Some of them have become among the most vocal campaigners for human rights in North Korea, holding rallies, testifying about starvation and torture in gulags and arguing that foreign governments must break their silence about the people living under one of the most repressive systems in the world. That argument has not always been popular in South Korea, where many of the defectors live. Many South Koreans believe that without an effective means of pressuring North Korea, which has defied and survived decades of international sanctions, an open challenge of its human rights record would only make it more paranoid and repressive. South Korea used to abstain from any United Nations vote on human rights resolutions on North Korea. But after a conservative party took power five years ago, Seoul began voting for and even leading such moves. It also supports the creation of the commission of inquiry. Newsletter Sign Up Continue reading the main story Please verify you're not a robot by clicking the box. Invalid email address. Please re-enter. You must select a newsletter to subscribe to. Sign Up You agree to receive occasional updates and special offers for The New York Times's products and services. Thank you for subscribing. An error has occurred. Please try again later. View all New York Times newsletters. Coupled with the new set of United Nations sanctions, the human rights report was expected to increase the pressure on the young North Korean leader, Kim Jong-un. Reports of a guarded relaxation of state control of the economy in North Korea have alternated with signs of a renewed crackdown on outside influences. Concern over its nuclear program has sharpened international attention on North Korea, but human rights activists say that the North had rebuffed criticism of its abuses and that a commission of inquiry would provide a long-overdue spotlight on them. “Increased scrutiny by international inquiry affords a measure of protection, especially when coupled with the prospect of future criminal investigations and the deterrent effect such a prospect may have on individual perpetrators,” according to Mr. Darusman’s report. Mr. Darusman, taking stock of the United Nations investigations to date, identifies nine patterns of human rights violations, including denial of access to food, arbitrary detention in prison camps that are estimated to hold up to 200,000 people, and abductions of foreigners. Many, if not all, of these patterns “may amount to crimes against humanity, committed as part of systematic and/or widespread attacks against civilian populations,” the report says. Advertisement Continue reading the main story Testimony from prisoners who have escaped the North Korean gulags appears to have played a part in the decision to take action. Ms. Pillay, in calling for an international inquiry in January, cited the “extremely harrowing” nature of the accounts she had received in a meeting with two of the escaped prisoners, describing a system that she called “the very antithesis of international human rights norms.” After eight years of inquiries, investigators had reached the point where they were forced to call for greater resources to continue their research, Mr. Darusman said in a telephone interview. Like other investigators, he has worked with a single assistant, limiting a sustained investigation. A commission of inquiry is likely to have three members, including Mr. Darusman, supported by researchers and a full-time secretariat. Even if an inquiry is commissioned, it remains unclear how effective it will be except as political symbol. North Korea does not allow outside investigators. And other countries have few means to force Pyongyang to provide access. No outsider has ever had a glimpse of a North Korean prison camp. North Korea rejected a critical General Assembly resolution in December as a “political plot” that escalated confrontation. The abuses that North Korea is accused of committing “cannot be allowed to exist in our country, where human rights and the fundamental freedom of the people are formally guaranteed by the legal system,” said Kim Song, the North Korean ambassador to the United Nations. The Obama administration bluntly warned North Korea on Monday that it will use military force if necessary to protect the United States and its allies in Asia from a North Korean nuclear strike or to prevent the impoverished North from selling nuclear weapons or expertise. Washington announced fresh sanctions against North Korea on Monday amid rising tension on the Korean Peninsula. The U.S. military also began annual joint exercises with ally South Korea, over objections from North Korea and an announcement that it would nullify the 60-year armistice that ended the Korean War. “Recently, North Korean officials have made some highly provocative statements,” White House national security adviser Thomas E. Donilon told the Asia Society in New York. “North Korea’s claims may be hyperbolic, but as to the policy of the United States, there should be no doubt.” “We will draw upon the full range of our capabilities to protect against, and to respond to, the threat posed to us and to our allies by North Korea,” he said. Donilon added that while the United States would like better relations with North Korea, it “refuses to reward bad North Korean behavior.” “The United States,” he said, “will not play the game of accepting empty promises or yielding to threats.” The United States is one of five nations that have sought unsuccessfully to negotiate a buyout of the North Korean nuclear program. The North has tested three nuclear devices and is assumed to be gradually perfecting the technology to deliver a nuclear warhead far from its own shores. On Monday, the rising tensions on the Korean Peninsula were underscored by the North’s announcement that it had “completely scrapped” the 1953 armistice agreement. The armistice has kept a shaky peace on the peninsula for 60 years, and the North’s apparent withdrawal — coupled with its severing of a communications hotline at the demilitarized border Monday — makes it more difficult for South Korea and the United States to prevent or resolve disputes with Pyongyang. North Korea has made several similar announcements in the past, most recently in 2009, and analysts cautioned this latest declaration could prove to be bluster. Experts also noted that Pyongyang — whether bound by the cease-fire or not — has occasionally ignored its terms, most notably with fatal attacks on the South in 2010. Still, anxiety about the North is particularly high now for the United States and its allies because they have little insight into the decision-making style of Kim Jong Eun, the young leader who took power of the opaque police state in December 2011 and now appears to be using the same brand of brinkmanship his father once did. Kim on Thursday visited troops on several Yellow Sea islets off North Korea’s southern coast, not far from a South Korean island that was shelled in 2010. According to a state media account of the visit, Kim “reconfirmed in detail reinforced firepower strike means and targets of the enemy.” “They’re giving all the motions to make us believe that some sort of provocation is coming,” said Ken Gause, an expert on North Korean leadership at CNA, an Alexandria-based organization. “Provocations are not necessarily imminent, but the probability is higher... and the ability to manage a provocation is much more difficult if they have withdrawn from the communication channels that existed under the armistice.” Gause added that the North’s latest threats were more specific than usual — an escalation that has experts “scratching their heads about how willing they are to push the envelope.” Many experts think that the North, by raising tensions and pulling away from existing lines of communication, is trying to pressure the United States into bilateral talks. In such talks, those experts believe, the North would seek a peace treaty, a major reduction of U.S. troops in the region and recognition as a nuclear state — a concession that Obama administration officials say they refuse to consider. Denuclearization “is still the goal of U.S. policy,” Glyn Davies, the U.S. envoy for North Korea, told the Senate Foreign Relations Committee on Thursday. The new sanctions unveiled Monday by the Obama administration target North Korea’s main foreign exchange bank and several of the country’s top economic leaders. The Foreign Trade Bank of North Korea was singled out because of its alleged support for companies linked to the country’s nuclear and ballistic missile programs, said David S. Cohen, undersecretary for terrorism and financial intelligence. The sanctions freeze any U.S. assets that may be owned by the bank, and, more significantly, they serve as a warning to other countries that trade with North Korea, Cohen said. “We urge financial institutions around the world to be particularly wary of the risks of doing business” with the trade bank, he said. Yoonjung Seo in Seoul and Joby Warrick in Washington contributed to this report. South Korean soldiers patrol along a barbed-wire fence near the demilitarized zone (DMZ) which separates the two Koreas in Paju, north of Seoul, March 11, 2013. North Korea has cut off a Red Cross hotline with South Korea as it escalates its war of words against Seoul and... A worker paints the wall with a roller next to a map bearing the demilitarized zone (DMZ) which separates the two Koreas at Imjingak pavillion in Paju, north of Seoul, March 11, 2013. North Korea has cut off a Red Cross hotline with South Korea as it escalates its war of... A South Korean soldier stands guard in front of a barbed-wire fence near the demilitarized zone (DMZ), which separates the two Koreas, in Paju, north of Seoul, March 11, 2013. North Korea has cut off a Red Cross hotline with South Korea as it escalates its war of words... South Korean soldiers patrol along a barbed-wire fence near the demilitarized zone (DMZ), which separates the two Koreas, in Paju, north of Seoul, March 11, 2013. South Korean soldiers patrol past ribbons bearing messages wishing for unification of the two Koreas, arranged in the shape of the Korean peninsula, near the demilitarized zone (DMZ), which separates the two Koreas, in Paju, north of Seoul, March 11, 2013. South Korean soldiers walk pass birds as they patrol along a barbed-wire fence near the demilitarized zone (DMZ), which separates the two Koreas, in Paju, north of Seoul, March 11, 2013. A girl runs past pinwheels arranged in the shape of the Korean peninsula at Imjingak pavillion near the demilitarized zone (DMZ) which separates the two Koreas in Paju, north of Seoul, March 11, 2013. North Korea has cut off a Red Cross hotline with South Korea as it... North Korean soldiers with weapons attend military training in an undisclosed location in this picture released by the North's official KCNA news agency in Pyongyang March 11, 2013. South Korea and U.S. forces are conducting large-scale military drills until the end of... North Korean soldiers run as they attend military training in an undisclosed location in this picture released by the North's official KCNA news agency in Pyongyang March 11, 2013. South Korea and U.S. forces are conducting large-scale military drills until the end of April,... North Korean soldiers attend military training in an undisclosed location in this picture released by the North's official KCNA news agency in Pyongyang March 11, 2013. South Korea and U.S. forces are conducting large-scale military drills until the end of April, while the... A man looks at a map bearing the demilitarized zone (DMZ), which separates the two Koreas, at Imjingak pavillion in Paju, north of Seoul, March 11, 2013. SEOUL North Korea has cut off a Red Cross hotline with South Korea as it escalates its war of words against Seoul and Washington in response to a military drill in the South and U.N. sanctions imposed for its recent nuclear test. The North had threatened to cut off the hotline on March 11 if the United States and South Korea did not abandon their joint military exercise. The Red Cross hotline is used to communicate between Seoul and Pyongyang which do not have diplomatic relations. "We called at 9 a.m. and there was no response," a government official from South Korea said. The line is tested each day. Pyongyang has also threatened to cut off a hotline with U.N. forces in South Korea, at the border "truce village" of Pammunjom. Tensions on the Korean peninsula have risen since the North conducted a third nuclear test on February 12, prompting new U.N. sanctions. South Korea and U.S. forces are conducting large-scale military drills until the end of April, while the North is also gearing up for a massive state-wide military exercise. North Korea has accused the United States of using the military drills in South Korea as a launch pad for a nuclear war and has threatened to scrap the armistice with Washington that ended hostilities in the 1950-53 Korean War. The North has threatened a nuclear strike on the United States, but such a threat has been dismissed as rhetoric by analysts, as the North does not have the military capacity to reach the United States. The North is viewed as more likely to stage some kind of attack along a disputed sea border, if it does anything at all, rather than risk a war with South Korea and the United States, which it would lose, according to most military assessments. (Reporting by Ju-min Park; Editing by Michael Perry) Image caption South Korean marines patrol Yeonpyeong Island, shelled by the North in 2010 North Korea knows how to make headlines. Over the past week, newspapers here in the South have turned Pyongyang's threats into front-page spreads: its promises of "pre-emptive nuclear strikes", "all-out war" and withdrawal from the 60-year-old Armistice Agreement that ended the Korean War. South Koreans normally approach these kinds of declarations with a weary stoicism, barely pausing over their cappuccinos as they absorb the latest threats from their neighbour. This time, though, the atmosphere here in Seoul is a little more anxious. While some dismiss it as just more of Pyongyang's bluster, others are worried by the volume and tone of the latest proclamations. That goes for older South Koreans, in particular, say some. Image caption Some see the North's Kim Jong-un (seated) as "not strong" "Young Koreans are very desensitized," one friend told me. "If it enters their heads at all, it dissipates very quickly, replaced with the latest celebrity (gossip). But the older generation do feel differently on security issues." "I didn't care about this issue until now," said another. "But I do worry this time around. The young North Korean leader is not strong, and I don't trust the new government here in the South." Even South Korea's Unification Ministry admits the mood has shifted. "Overall, there's a heightened sense of alert this time," a spokeswoman said. Not just because of the "more belligerent rhetoric" coming from Pyongyang, but because of the actions accompanying them. North Korea's launch of a long-range rocket in December, its apparently successful nuclear test in February and the fresh UN sanctions imposed on it this month have all added to the sense of tension on the peninsula. Some South Koreans are worried that the wrong lessons have been learned, and that if something small happens, it could escalate because the South Korean government doesn't want to be accused of doing nothing" John Delury, Yonsei University Joint military exercises taking place here this month between the US and South Korean forces have also antagonised the North, just as they do every year. The secretive communist state is due to begin its own large-scale military drills this week. Few people here are expecting full-blown war, but in the current climate, there is concern over accidental escalation, especially after North Korea's decision to cut the military hot-line between the two sides at Panmunjom. Even this though is not unprecedented. The telephone line at Panmunjom has been abandoned by the North before, most recently in 2010. The year before that, it also announced its withdrawal from all joint agreements involving the South. And analysts point out that an alternative military communications line, used to monitor cross-border workers at the Kaesong Industrial Complex, remains open. "I'm not so worried," another Korean friend told me. "Even though they have cut the hotline, I see their rhetoric as a plea for direct negotiations with the US." Talking tough But alongside Pyongyang's increasingly shrill denunciations, South Koreans are hearing some strong language from their own government too. Image caption Park Geun-hye was just recently sworn into office The country's new president, Park Geun-hye, who was sworn into office just two weeks ago, has vowed to respond strongly to any provocation. Her government has said it will target not only the source of any attack, but also the North's top command. That tough stance, while worrying to some, is likely to be a kind of "pre-emptive rhetorical deterrence", according to analysts such as John Delury, a professor of International Studies at Seoul's Yonsei University. The idea that strong words could act as a deterrent to North Korean actions has gained traction since the lethal shelling of South Korea's Yeonpyeong Island in 2010. As a government, Prof Delury says: "You're always fighting the last battle, and the last battle for South Korea was Yeonpyeong. The perception then was that North Korea had got away with shelling the island." By talking tough, he believes Park Geun-hye wants to avoid any initial attack by the North. But, he said, "some South Koreans are worried that the wrong lessons have been learned, and that if something small happens, it could escalate because the South Korean government doesn't want to be accused of doing nothing". For all North Korea's familiar rhetoric, its past actions against the South have seemed carefully calibrated not to provoke a full-scale conflict. The question is whether the young leader in Pyongyang will correctly judge the mood of its adversaries this time around. Adding to South Koreans’ worries, the North and its nuclear arsenal are in the hands of a young new leader, Kim Jong-un, whose brinkmanship appears to be an effort to ensure the support of his nation’s powerful military. The South also has a new president, Park Geun-hye, the daughter of a military strongman who stood firm against North Korea, who herself also faces pressure to stand fast against the North. Just two weeks after her inauguration, Ms. Park faces a crisis as the North makes vague threats interpreted by many South Koreans as the precursor to some sort of limited, conventional military provocation. Ms. Park has promised to retaliate if her nation is attacked, aware of the public anger directed at her predecessor, Lee Myung-bak, when he showed restraint after the North shelled a South Korean island in 2010, killing four people. That kind of limited skirmish is more likely than a nuclear attack, but such an episode could quickly inflame tensions and escalate out of control. Over the years, North Korea has sent armed spies across the border, dug invasion tunnels under it and infiltrated South Korean waters with submarines. But beyond the immediate fear of a military provocation, analysts say deeper anxieties are also at work in the South. One of the biggest is the creeping resurgence of old fears about the reliability of this nation’s longtime protector, the United States. Experts say the talk of South Korea’s acquiring nuclear weapons is an oblique way to voice the concerns of a small but growing number of South Koreans that the United States, either because of budget cuts or a lack of will, may one day no longer act as the South’s ultimate insurance policy. “The Americans don’t feel the North Korean nuclear weapons as a direct threat,” said Chung Mong-joon, a son of the founder of the Hyundai industrial group and the former leader of the governing party, who has been the leading proponent of South Korea’s development of a nuclear weapons program. “At a time of crisis, we are not 100 percent sure whether the Americans will cover us with its nuclear umbrella.” Video The United States, which still has 28,500 troops based in South Korea, has sought to assure its ally that it remains committed to the region as part of the Obama administration’s strategic “pivot” to Asia. But analysts say the fact that senior leaders like Mr. Chung and a handful of influential newspaper columnists now call for the need for “nuclear deterrence,” or at least hint at it, reflects widespread frustrations over the inability of the United States and other nations to end North Korea’s nuclear weapons program. Until recently the idea was too radical for most mainstream leaders and opinion makers, including both deeply pro-American conservatives and nationalistic yet antinuclear liberals. Advocacy for a nuclear-armed South Korea has been virtually taboo since the early 1970s, when the country’s military dictator, Park Chung-hee, made a serious bid to develop a nuclear weapon, fearing that the United States might pull out of Asia after its defeat in Vietnam. After catching wind of the program, Washington forced Mr. Park, the new president’s father, to stop, persuading him instead to rely on the United States, an agreement that has held ever since. Advertisement Continue reading the main story Mr. Chung and others say that if the United States does not allow South Korea to develop its own nuclear arms, it should at least restore the nuclear balance on the Korean Peninsula by reintroducing American atomic weapons, which were removed from bases in the South in 1991 in a post-cold-war effort to reduce tensions. Newsletter Sign Up Continue reading the main story Please verify you're not a robot by clicking the box. Invalid email address. Please re-enter. You must select a newsletter to subscribe to. Sign Up You will receive emails containing news content, updates and promotions from The New York Times. You may opt-out at any time. You agree to receive occasional updates and special offers for The New York Times's products and services. Thank you for subscribing. An error has occurred. Please try again later. View all New York Times newsletters. Many in the South are now convinced that the North may never give up its nuclear weapons. The South’s new level of anxiety is also apparent in the widespread speculation here about when and where the North might carry out another, non-nuclear military provocation. North Korea has stoked those fears by saying that on Monday it will drop out of the 60-year-old armistice that ended the Korean War, in a show of anger at new United Nations sanctions for its nuclear test. North Korea has threatened to terminate the armistice in the past, but the greater worry now is that it might take actions to contravene it. There have been cryptic warnings in North Korea’s state-run news media of coming “counteractions,” which have led South Korean officials to warn of an episode like the bombardment of Yeonpyeong Island in 2010. On Friday, North Korea’s state-run television showed Mr. Kim addressing the same artillery units that hit Yeonpyeong. On the same day, South Korean television stations showed President Park with heavily decorated generals, and later descending into the bunker at the Blue House, South Korea’s version of the White House, to confer with her national security advisers. The opposition parties had blocked the confirmation of her cabinet, raising concerns about her ability to respond to a crisis, but she reached a deal allowing her to fill crucial posts on Monday. Even many on the left said that the country would quickly pull together if shots were fired. “The third test was a wake-up call for the left, too,” said Lee Kang-yun, a television commentator. On the streets of Seoul, it has remained business as usual with no signs of panic, a testimony to the resilience, or perhaps resignation, of a people who have grown used to the North’s threats. Chung Eun-jin, a 26-year-old English teacher interviewed in the trendy Gangnam district, said she was not overly concerned because the North had threatened the South so often before. But Kwon Gi-yoon, 38, an engineer, said that since the North’s third test, he believed that South Korea should develop its own nuclear weapons. Opinions like Mr. Kwon’s appear to be spreading. Two opinion polls conducted after the third test, one by Gallup Korea and the other by the Asan Institute for Policy Studies, found that 64 to 66.5 percent of the respondents supported the idea that South Korea should develop its own nuclear weapons, similar to polls after the Yeonpyeong attack in 2010. “Having a nuclear North Korea is like facing a person holding a gun with just your bare hands,” said Mr. Kwon, the engineer. South Koreans should have “our own nuclear capabilities, in case the U.S. pulls out like it did in Vietnam.”

North Korea today "completely scrapped" the armistice that held a tenuous peace on the peninsula for six decades, reports the Washington Post, even as American and South Korean troops began the large-scale military drills Pyongyang had warned them to abandon. The North is playing up its unpredictability, saying in a state-run newspaper today that with the armistice gone, "no one can expect what will happen next." Further heightening tensions: The Red Cross hotline the North uses to communicate with Seoul has gone dead. "We called at 9am and there was no response," explains a South Korean official of the line, which it tries daily. The North has also threatened to cut off its hotline with UN troops-and to nuke the United States. North Korea, which is planning huge military exercises of its own, claims the US-South Korea drills are a move to launch a nuclear war. Analysts believe Pyongyang isn't capable of launching a nuclear strike on the US even if the regime was deranged enough to try. But some kind of attack along the disputed sea border with South Korea is seen as a more realistic possibility, reports Reuters. As threats fly back and forth, some South Koreans are openly discussing obtaining nuclear weapons of their own. Pyongyang's nuclear test last month, its third, "was for South Korea what the Cuban missile crisis was for the US," a professor at the Korea National Defense University tells the New York Times. "It has made the North Korean threat seem very close and very real." The hotline-closing and scrapping of peace pacts have recent precedents, but the mood in Seoul is tenser than it has been after previous outbursts from Pyongyang, the BBC finds. North Korea's tone is more belligerent, and people fear the regime is more willing to back its threats up with action. If Pyongyang was enraged by fresh UN sanctions last week, today will be more of the same as the UN examines its appalling human rights record, the New York Times reports. An investigator will present a report that is expected to lead to an inquiry into possible crimes against humanity.

multi_news

Background Under the existing, or “legacy” system, the military’s disability evaluation process begins at a military treatment facility when a physician identifies a condition that may interfere with a servicemember’s ability to perform his or her duties. On the basis of medical examinations and the servicemember’s medical records, a medical evaluation board (MEB) identifies and documents any conditions that may limit a servicemember’s ability to serve in the military. The servicemember’s case is then evaluated by a physical evaluation board (PEB) to make a determination of fitness or unfitness for duty. If the servicemember is found to be unfit due to medical conditions incurred in the line of duty, the PEB assigns the servicemember a combined percentage rating for those unfit conditions, and the servicemember is discharged. Depending on the overall disability rating and number of years of active duty or equivalent service, the servicemember found unfit with compensable conditions is entitled to either monthly disability retirement benefits or lump sum disability severance pay. In addition to receiving disability benefits from DOD, veterans with service-connected disabilities may receive compensation from VA for lost earnings capacity. VA’s disability compensation claims process starts when a veteran submits a claim listing the medical conditions that he or she believes are service-connected. In contrast to DOD’s disability evaluation system, which evaluates only medical conditions affecting servicemembers’ fitness for duty, VA evaluates all medical conditions claimed by the veteran, whether or not they were previously evaluated in DOD’s disability evaluation process. For each claimed condition, VA must determine if there is credible evidence to support the veteran’s contention of a service connection. Such evidence may include the veteran’s military service records and treatment records from VA medical facilities and private medical service providers. Also, if necessary for reaching a decision on a claim, VA arranges for the veteran to receive a medical examination. Medical examiners are clinicians (including physicians, nurse practitioners, or physician assistants) certified to perform the exams under VA’s Compensation and Pension program. Once a claim has all of the necessary evidence, a VA rating specialist determines whether the claimant is eligible for benefits. If so, the rating specialist assigns a percentage rating. If VA finds that a veteran has one or more service- connected disabilities with a combined rating of at least 10 percent, the agency will pay monthly compensation. In November 2007, DOD and VA began piloting the IDES. The IDES merges DOD and VA processes, so that servicemembers begin their VA disability claim while they undergo their DOD disability evaluation, rather than sequentially, making it possible for them to receive VA disability benefits shortly after leaving military service. Specifically, the IDES: Merges DOD and VA’s separate exam processes into a single exam process conducted to VA standards. This single exam (which may involve more than one medical examination, for example, by different specialists), in conjunction with the servicemembers’ medical records, is used by military service PEBs to make a determination of servicemembers’ fitness for continued military service, and by VA as evidence of service-connected disabilities. The exam may be performed by medical staff working for VA, DOD, or a private provider contracted with either agency. Consolidates DOD and VA’s separate rating phases into one VA rating phase. If the PEB has determined that a servicemember is unfit for duty, VA rating specialists prepare two ratings—one for the conditions that DOD determined made a servicemember unfit for duty, which DOD uses to provide military disability benefits, and the other for all service-connected disabilities, which VA uses to determine VA disability benefits. Provides VA case managers to perform outreach and nonclinical case management and explain VA results and processes to servicemembers. Pilot Evaluation Results Were Promising, but Degree of Improvement was Unknown, and Timeliness Has Since Worsened In August 2010, DOD and VA officials issued an interim report to Congress summarizing the results of their evaluation of the IDES pilot as of early 2010 and indicating overall positive results. In that report, the agencies concluded that, as of February 2010, servicemembers who went through the IDES pilot were more satisfied than those who went through the legacy system, and that the IDES process met the agencies’ goals of delivering VA benefits to active duty servicemembers within 295 days and to reserve component servicemembers within 305 days. Furthermore, they concluded that the IDES pilot has achieved a faster processing time than the legacy system, which they estimated to be 540 days. Although DOD and VA’s evaluation results indicated promise for the IDES, the extent to which they represented an improvement over the legacy system could not be known because of limitations in the legacy data. DOD and VA’s estimate of 540 days for the legacy system was based on a small, nonrepresentative sample of cases. Officials planned to use a broader sample of legacy cases to compare against pilot cases with respect to processing times and appeal rates; however inconsistencies in how military services tracked information and missing VA information (i.e., on the date VA benefits were delivered) for legacy cases, precluded such comparisons. While our review of DOD and VA’s data and reports generally confirmed DOD and VA’s findings as of early 2010, we found that not all of the service branches were achieving the same results, case processing times increased between February and August 2010, and other agency goals are not being met. Since our December report, processing times have worsened further and the agencies have adjusted some goals downward. Servicemember satisfaction: Our reviews of the survey data as of early 2010 indicated that, on average, servicemembers in the IDES pilot had higher satisfaction levels than those who went through the legacy process. However, Air Force members—who represented a small proportion (7 percent) of pilot cases—were less satisfied. Currently, DOD and VA have an 80-percent IDES satisfaction goal, which has not been met. For example, 67 percent of servicemembers surveyed in March 2011 were satisfied with the IDES. Satisfaction by service ranged from 54 percent for the Marine Corps to 72 percent for the Army. Average case processing times: Although the agencies were generally meeting their 295-day and 305-day timeliness goals through February 2010, the average case processing time for active duty servicemembers increased from 274 to 296 days between February and August 2010. Among the military service branches, only the Army was meeting the agencies’ timeliness goals as of August, while average processing times for each of the other services exceeded 330 days. Since August 2010, timeliness has worsened significantly. For example, active component cases completed in March 2011 took an average of 394 days—99 days over the 295-day target. By service, averages ranged from 367 days for the Army to 455 days for the Marine Corps. Meanwhile, Reserve cases took an average of 383 days, 78 days more than the 305-day target, while Guard cases took an average of 354 days, 49 days more than the target. Goals to process 80 percent of cases in targeted time frames: DOD and VA had indicated in their planning documents that they had goals to deliver VA benefits to 80 percent of servicemembers within the 295-day (active component) and 305-day (reserve component) targets. For both active and reserve component cases at the time of our review, about 60 percent were meeting the targeted time frames. DOD and VA have since lowered their goals for cases completed on time, from 80 percent to 50 percent. Based on monthly data for 6 months through March 2011, the new, lower goal was not met during any month for active component cases. For completed Reserve cases, the lower goal was met during one of the 6 months and for Guard cases, it was met in 2 months. The strongest performance was in October 2010 when 63 percent of Reserve cases were processed within the 305-day target. Pilot Sites Experienced Several Challenges Based on our prior work, we found that--as DOD and VA tested the IDES at different facilities and added cases to the pilot--they encountered several challenges that led to delays in certain phases of the process. Staffing: Most significantly, most of the sites we visited reported experiencing staffing shortages and related delays to some extent, in part due to workloads exceeding the agencies’ initial estimates. The IDES involves several different types of staff across several different DOD and VA offices, some of which have specific caseload ratios set by the agencies, and we learned about insufficient staff in many key positions. With regard to VA positions, officials cited shortages in examiners for the single exam, rating staff, and case managers. With regard to DOD positions, officials cited shortages of physicians who serve on the MEBs, PEB adjudicators, and DOD case managers. In addition to shortages cited at pilot sites, DOD data indicated that 19 of the 27 pilot sites did not meet DOD’s caseload target of 30 cases per manager. Local DOD and VA officials attributed staffing shortages to higher than anticipated caseloads and difficulty finding qualified staff, particularly physicians, in rural areas. These staffing shortages contributed to delays in the IDES process. Two of the sites we visited—Fort Carson and Fort Stewart—were particularly challenged to provide staff in response to surges in caseload that occurred when Army units were preparing to deploy to combat zones. Through the Army’s predeployment medical assessment process, large numbers of servicemembers were determined to be unable to deploy due to a medical condition and were referred to the IDES within a short period of time, overwhelming the staff. These two sites were unable to quickly increase staffing levels, particularly of examiners. As a result, at Fort Carson, it took 140 days on average to complete the single exam for active duty servicemembers, as of August 2010—much longer than the agencies’ goal to complete the exams in 45 days. More recently, Fort Carson was still struggling to meet goals, as of March 2011. For example, about half of Fort Carson’s active component cases (558 of 1033 cases) were in the MEB phase, and the average number of days spent in the MEB phase by active component cases completed in March 2011 was 197 days, compared to a goal of 35 days. Exam summaries: Issues related to the completeness and clarity of single exam summaries were an additional cause of delays in the VA rating phase of the IDES process. Officials from VA rating offices said that some exam summaries did not contain information necessary to determine a rating. As a result, VA rating office staff must ask the examiner to clarify these summaries and, in some cases, redo the exam. VA officials attributed the problems with exam summaries to several factors, including the complexity of IDES pilot cases, the volume of exams, and examiners not receiving records of servicemembers’ medical history in time. The extent to which insufficient exam summaries caused delays in the IDES process is unknown because DOD and VA’s case tracking system for the IDES does not track whether an exam summary has to be returned to the examiner or whether it has been resolved. Medical diagnoses: While the single exam in the IDES eliminates duplicative exams performed by DOD and VA in the legacy system, it raises the potential for there to be disagreements about diagnoses of servicemembers’ conditions. For example, officials at Army pilot sites informed us about cases in which a DOD physician had treated members for mental disorders, such as major depression. However, when the members went to see the VA examiners for their single exam, the examiners diagnosed them with posttraumatic stress disorder (PTSD). Officials told us that attempting to resolve such differences added time to the process and sometimes led to disagreements between DOD’s PEBs and VA’s rating offices about what the rating should be for purposes of determining DOD disability benefits. Although the Army developed guidance to help resolve diagnostic differences, other services have not. Moreover, PEB officials we spoke with noted that there is no guidance on how disagreements about servicemembers’ ratings between DOD and VA should be resolved beyond the PEBs informally requesting that the VA rating office reconsider the case. While DOD and VA officials cited several potential causes for diagnostic disagreements, the number of cases with disagreements about diagnoses and the extent to which they have increased processing time are unknown because the agencies’ case tracking system does not track when a case has had such disagreements. Logistical challenges integrating VA staff at military treatment facilities: DOD and VA officials at some pilot sites we visited said that they experienced logistical challenges integrating VA staff at the military facilities. At a few sites, it took time for VA staff to receive common access cards needed to access the military facilities and to use the facilities’ computer systems, and for VA physicians to be credentialed. DOD and VA staff also noted several difficulties using the agencies’ multiple information technology (IT) systems to process cases, including redundant data entry and a lack of integration between systems. Housing and other challenges posed by extended time in the military disability evaluation process: Although many DOD and VA officials we interviewed at central offices and pilot sites felt that the IDES process expedited the delivery of VA benefits to servicemembers, several also indicated that it may increase the amount of time servicemembers are in the military’s disability evaluation process. Therefore, some DOD officials noted that servicemembers must be cared for, managed, and housed for a longer period. The military services may move some servicemembers to temporary medical units or to special medical units such as Warrior Transition Units in the Army or Wounded Warrior Regiments in the Marine Corps, but at a few pilot sites we visited, these units were either full or members in the IDES did not meet their admission criteria. In addition, officials at two sites said that members who are not gainfully employed by their units and left idle are more likely to be discharged due to misconduct and forfeit their disability benefits. However, DOD officials also noted that servicemembers benefit from continuing to receive their salaries and benefits while their case undergoes scrutiny by two agencies, though some also acknowledged that these additional salaries and benefits create costs for DOD. Deployment Plans Address Many, but not All, Challenges DOD and VA are deploying the IDES to military facilities worldwide on an ambitious timetable—expecting deployment to be completed at a total of about 140 sites by the end of fiscal year 2011. As of March 2011, the IDES was operating at 73 sites, covering about 66 percent of all military disability evaluation cases. In preparing for IDES expansion militarywide, DOD and VA had many efforts under way to address challenges experienced at the 27 pilot sites. For example, the agencies completed a significant revision of their site assessment matrix—a checklist used by local DOD and VA officials to ascertain their readiness to begin the pilot—to address areas where prior IDES sites had experienced challenges. In addition, local senior-level DOD and VA officials will be expected to sign the site assessment matrix to certify that a site is ready for IDES implementation. This differs from the pilot phase where, according to DOD and VA officials, some sites implemented the IDES without having been fully prepared. Through the new site assessment matrix and other initiatives, DOD and VA planned to address several of the challenges identified in the pilot phase. Ensuring sufficient staff: With regard to VA staff, VA planned to increase the number of examiners by awarding a new contract through which sites can acquire additional examiners. To increase rating staff, VA filled vacant rating specialist positions and anticipates hiring a small number of additional staff. With regard to DOD staff, Air Force and Navy officials told us they added adjudicators for their PEBs or planned to do so. Both DOD and VA indicated they plan to increase their numbers of case managers. Meanwhile, sites are being asked in the assessment matrix to provide longer and more detailed histories of their caseloads, as opposed to the 1-year history that DOD and VA had based their staffing decisions on during the pilot phase. The matrix also asks sites to anticipate any surges in caseloads and to provide a written contingency plan for dealing with them. Ensuring the sufficiency of single exams: VA has been revising its exam templates to better ensure that examiners include the information needed for a VA disability rating decision and to enable them to complete their exam reports in less time. VA is also examining whether it can add capabilities to the IDES case tracking system that would enable staff to identify where problems with exams have occurred and track the progress of their resolution. Ensuring adequate logistics at IDES sites: The site assessment matrix asks sites whether they have the logistical arrangements needed to implement the IDES. In terms of information technology, DOD and VA were developing a general memorandum of agreement intended to enable DOD and VA staff access to each other’s IT systems. DOD officials also said that they are developing two new IT solutions—one intended to help military treatment facilities better manage their cases, another intended to reduce multiple data entry. However, in some areas, DOD and VA’s efforts to prepare for IDES expansion did not fully address some challenges or are not yet complete. For these areas, we recommended additional action that the agencies could take, with which the agencies generally concurred. Ensuring sufficient DOD MEB physician staffing: DOD does not yet have strategies or plans to address potential shortages of physicians to serve on MEBs. For example, the site assessment matrix does not include a question about the sufficiency of military providers to handle expected numbers of MEB cases at the site, or ask sites to identify strategies for ensuring sufficient MEB physicians if there is a caseload surge or staff turnover. We recommended that, prior to implementing IDES at MTFs, DOD direct military services to conduct thorough assessments of the adequacy of military physician staffing for completing MEB determinations and develop contingency plans to address potential shortfalls, e.g. due to staff turnover or caseload surges. Ensuring sufficient housing and organizational oversight for IDES participants: Although the site assessment matrix asks sites whether they will have sufficient temporary housing available for servicemembers going through the IDES, the matrix requires only a yes or no response and does not ensure that sites will have conducted a thorough review of their housing capacity. In addition, the site assessment matrix does not address plans for ensuring that IDES participants are gainfully employed or sufficiently supported by their organizational units. We recommended that prior to implementing the IDES at MTFs, DOD ensure thorough assessments are conducted on the availability of housing for servicemembers and on the capacity of organizational units to absorb servicemembers undergoing the disability evaluation; alternative housing options are identified when sites lack adequate capacity; and plans are in place for ensuring that servicemembers are appropriately and constructively engaged. Addressing differences in diagnoses: According to agency officials, DOD is currently developing guidance on how staff should address differences in diagnoses. However, since the new guidance and procedures are still being developed, we cannot determine whether they will aid in resolving discrepancies or disagreements. Significantly, DOD and VA do not have a mechanism for tracking when and where disagreements about diagnoses and ratings occur and, consequently, may not be able to determine whether the guidance sufficiently addresses the discrepancies. Therefore, we recommended that DOD and VA conduct a study to assess the prevalence and causes of such disagreements and establish a mechanism to continuously monitor diagnostic disagreements. VA has since indicated it plans to conduct such a study and make a determination by July 2011 regarding what, if any, mechanisms are needed. Further, despite regular reporting of data on caseloads, processing times, and servicemember satisfaction, and preparation of an annual report on challenges in the IDES, we determined that DOD and VA did not have a systemwide monitoring mechanism to help ensure that steps they took to address challenges are sufficient and to identify problems in a more timely basis. For example, they did not collect data centrally on staffing levels at each site relative to caseload. As a result, DOD and VA may be delayed in taking corrective action since it takes time to assess what types of staff are needed at a site and to hire or reassign staff. DOD and VA also lacked mechanisms or forums for systematically sharing information on challenges, as well as best practices between and among sites. For example, DOD and VA have not established a process for local sites to systematically report challenges to DOD and VA management and for lessons learned to be systematically shared systemwide. During the pilot phase, VA surveyed pilot sites on a monthly basis about challenges they faced in completing single exams. Such a practice has the potential to provide useful feedback if extended to other IDES challenges. To identify challenges as they arise in all DOD and VA facilities and offices involved in the IDES and thereby enable early remedial action, we recommended that DOD and VA develop a systemwide monitoring mechanism. This system could include continuous collection and analysis of data on DOD and VA staffing levels, sufficiency of exam summaries, and diagnostic disagreements; monitoring of available data on caseloads and case processing time by individual VA rating office and PEB; and a formal mechanism for agency officials at local DOD and VA facilities to communicate challenges and best practices to DOD and VA headquarters. VA noted several steps it plans to take to improve its monitoring of IDES workloads, site performance and challenges—some targeted to be implemented by July 2011—which we have not reviewed. Concluding Observations By merging two duplicative disability evaluation systems, the IDES has shown promise for expediting the delivery of VA benefits to servicemembers leaving the military due to a disability. However, we identified significant challenges at pilot sites that require careful management attention and oversight. We noted a number of steps that DOD and VA were undertaking or planned to undertake that may mitigate these challenges. However, the agencies’ deployment schedule is ambitious in light of substantial management challenges and additional evidence of deteriorating case processing times. As such, it is unclear whether these steps will be sufficiently timely or effective to support militarywide deployment. Deployment time frame notwithstanding, we continue to believe that the success or failure of the IDES will depend on DOD and VA’s ability to quickly and effectively address resource needs and resolve challenges as they arise, not only at the initiation of the transition to IDES, but also on an ongoing, long-term basis. We continue to believe that DOD and VA cannot achieve this without a robust mechanism for routinely monitoring staffing and other risk factors. Chairman Chaffetz and Ranking Member Tierney, this concludes my prepared statement. I would be pleased to respond to any questions that you or other Members of the Subcommittee may have at this time. GAO Contact and Staff Acknowledgment For further information about this testimony, please contact Daniel Bertoni at (202) 512-7215 or bertonid@gao.gov. Contact points for our Offices of Congressional Relations and Public Affairs may be found on the last page of this testimony. In addition to the individual named above, key contributors to this testimony include Michele Grgich, Greg Whitney, and Daniel Concepcion. Key advisors included Bonnie Anderson, Mark Bird, Sheila McCoy, Patricia Owens, Roger Thomas, Walter Vance, and Randall Williamson. Related GAO Products Military and Veterans Disability System: Pilot Has Achieved Some Goals, but Further Planning and Monitoring Needed. GAO-11-69. Washington, D.C.: December 6, 2010. Military and Veterans Disability System: Preliminary Observations on Evaluation and Planned Expansion of DOD/VA Pilot. GAO-11-191T. Washington, D.C.: November 18, 2010. Veterans’ Disability Benefits: Further Evaluation of Ongoing Initiatives Could Help Identify Effective Approaches for Improving Claims Processing. GAO-10-213. Washington, D.C.: January 29, 2010. Recovering Servicemembers: DOD and VA Have Jointly Developed the Majority of Required Policies but Challenges Remain. GAO-09-728. Washington, D.C.: July 8, 2009. Recovering Servicemembers: DOD and VA Have Made Progress to Jointly Develop Required Policies but Additional Challenges Remain. GAO-09-540T. Washington, D.C.: April 29, 2009. Military Disability System: Increased Supports for Servicemembers and Better Pilot Planning Could Improve the Disability Evaluation Process. GAO-08-1137. Washington, D.C.: September 24, 2008. DOD and VA: Preliminary Observations on Efforts to Improve Care Management and Disability Evaluations for Servicemembers. GAO-08-514T. Washington, D.C.: February 27, 2008. DOD and VA: Preliminary Observations on Efforts to Improve Health Care and Disability Evaluations for Returning Servicemembers. GAO-07-1256T Washington, D.C.: September 26, 2007. Military Disability System: Improved Oversight Needed to Ensure Consistent and Timely Outcomes for Reserve and Active Duty Service Members. GAO-06-362. Washington, D.C.: March 31, 2006. This is a work of the U.S. government and is not subject to copyright protection in the United States. The published product may be reproduced and distributed in its entirety without further permission from GAO. However, because this work may contain copyrighted images or other material, permission from the copyright holder may be necessary if you wish to reproduce this material separately.

This testimony discusses the efforts by the Departments of Defense (DOD) and Veterans Affairs (VA) to integrate their disability evaluation systems. Wounded warriors unable to continue their military service must navigate DOD's and VA's disability evaluation systems to be assessed for eligibility for disability compensation from the two agencies. GAO and others have found problems with these systems, including long delays, duplication in DOD and VA processes, confusion among servicemembers, and distrust of systems regarded as adversarial by servicemembers and veterans. To address these problems, DOD and VA have designed an integrated disability evaluation system (IDES), with the goal of expediting the delivery of VA benefits to servicemembers. After pilot testing the IDES at an increasing number of military treatment facilities (MTF)--from 3 to 27 sites--DOD and VA are in the process of deploying it worldwide. As of March 2011, the IDES has been deployed at 73 MTFs--representing about 66 percent of all military disability evaluation cases--and worldwide deployment is scheduled for completion in September 2011. This testimony summarizes and updates our December 2010 report on the IDES and addresses the following points: (1) the results of DOD and VA's evaluation of their pilot of the IDES, including updated data as of March 2011 from IDES monthly reports, where possible; (2) challenges in implementing the piloted system to date; and (3) DOD and VA's plans to expand the piloted system and whether those plans adequately address potential challenges. In summary, DOD and VA concluded that, based on their evaluation of the pilot as of February 2010, the pilot had (1) improved servicemember satisfaction relative to the existing "legacy" system and (2) met their established goal of delivering VA benefits to active duty and reserve component servicemembers within 295 and 305 days, respectively, on average. However, 1 year after this evaluation, average case processing times have increased significantly, such that active component servicemembers' cases completed in March 2011 took an average of 394 days to complete--99 days more than the 295-day goal. In our prior work, we identified several implementation challenges that had already contributed to delays in the process. The most significant challenge was insufficient staffing by DOD and VA. Staffing shortages and process delays were particularly severe at two pilot sites we visited where the agencies did not anticipate caseload surges. The single exam posed other challenges that contributed to delays, such as disagreements between DOD and VA medical staff about diagnoses for servicemembers' medical conditions that often required further attention, adding time to the process. Pilot sites also experienced logistical challenges, such as incorporating VA staff at military facilities and housing and managing personnel going through the process. DOD and VA were taking or planning to take steps to address a number of these challenges. For example, to address staffing shortages, VA is developing a contract for additional medical examiners, and DOD and VA are requiring local staff to develop written contingency plans for handling caseload surges. Given increased processing times, the efficacy of these efforts at this time is unclear. We recommended additional steps the agencies could take to address known challenges--such as establishing a comprehensive monitoring plan for identifying problems as they occur in order to take remedial actions as early as possible--with which DOD and VA generally concurred.

gov_report

Rotavirus (RV) is the major cause of severe gastroenteritis in young children. A virus-encoded enterotoxin, NSP4 is proposed to play a major role in causing RV diarrhoea but how RV can induce emesis, a hallmark of the illness, remains unresolved. In this study we have addressed the hypothesis that RV-induced secretion of serotonin (5-hydroxytryptamine, 5-HT) by enterochromaffin (EC) cells plays a key role in the emetic reflex during RV infection resulting in activation of vagal afferent nerves connected to nucleus of the solitary tract (NTS) and area postrema in the brain stem, structures associated with nausea and vomiting. Our experiments revealed that RV can infect and replicate in human EC tumor cells ex vivo and in vitro and are localized to both EC cells and infected enterocytes in the close vicinity of EC cells in the jejunum of infected mice. Purified NSP4, but not purified virus particles, evoked release of 5-HT within 60 minutes and increased the intracellular Ca2+ concentration in a human midgut carcinoid EC cell line (GOT1) and ex vivo in human primary carcinoid EC cells concomitant with the release of 5-HT. Furthermore, NSP4 stimulated a modest production of inositol 1,4, 5-triphosphate (IP3), but not of cAMP. RV infection in mice induced Fos expression in the NTS, as seen in animals which vomit after administration of chemotherapeutic drugs. The demonstration that RV can stimulate EC cells leads us to propose that RV disease includes participation of 5-HT, EC cells, the enteric nervous system and activation of vagal afferent nerves to brain structures associated with nausea and vomiting. This hypothesis is supported by treating vomiting in children with acute gastroenteritis with 5-HT3 receptor antagonists. Rotavirus (RV) is the major cause of infantile gastroenteritis worldwide and the infection is associated with approximately 600,000 deaths every year, predominantly in developing countries [1]. Most of the deaths result from excessive loss of fluids and electrolytes through vomiting and diarrhoea. Despite its significant clinical importance and the research conducted over several decades, knowledge of the pathophysiological mechanisms that underpin this life-threatening disease remains limited. Several mechanisms have been proposed to account for the watery diarrhoea associated with RV infection. These include osmotic diarrhoea following a virus-induced loss of epithelial absorptive function, the effect of NSP4, a virus-encoded enterotoxin, and an active role of the enteric nervous system (ENS) and serotonin (5-hydroxytryptamine, 5-HT) [2]–[5]. However, the pathophysiological basis of virus-induced emesis, a hallmark of illnesses caused by RV and norovirus, is poorly understood. The human ENS contains about 100 million neurones which are sensory-, inter- and motor neurons [6]. The luminal enterochromaffin (EC) cells “taste” and “sense” the luminal contents and can release mediators such as 5-HT to activate ENS as well as extrinsic vagal afferents to the brain. 5-HT is located in the secretory granules of the EC cells, which are most abundant in the duodenum and comprise the single largest enteroendocrine cell population. They are strategically positioned in the intestinal mucosa to release mediators of endocrine signalling from the basolateral surface to activate afferent neuron endings within the lamina propria [7], [8]. Following stimulation by several agents (e. g. hyperosmolarity, carbohydrates, mechanical distortion of the mucosa, cytostatic drugs) including the cholera toxin [8], [9], EC cells mobilize intracellular Ca2+ and release 5-HT [10]. 5-HT is involved in the regulation of gut motility, intestinal secretion, blood flow and several gastrointestinal disorders [11]–[15] including RV diarrhoea [3], chemotherapy-induced nausea and vomiting [16], [17] and Staphylococcal enterotoxin-induced vomiting [18]. We have previously shown that RV infection results in stimulation of the ENS and that RV diarrhoea in mice can be attenuated with 5-HT3 receptor antagonists, such as granisetron [3]. This drug is frequently used to reduce vomiting in humans [19], including children with acute gastroenteritis [20]–[23], suggesting that EC cells could be an important mediator of RV-induced diarrhoea and vomiting, symptoms regarded as naturally inherited host responses [24]. The proposed anti-emetic mechanism of 5-HT3 receptor blockade involves an action on receptors located on vagal afferents communicating with the medullary vomiting centre [25], [26] which is supported by observations that the stimulation of vagal 5-HT3 receptors in ferrets, shrews and dogs triggers an emetic reflex, evoking reverse peristalsis in response to chemotherapeutic agents and radiation treatment [19], [27]–[29]. The present study was designed to test the novel hypothesis that RV can stimulate human EC cells in the gut, causing release of 5-HT, which activates vagal afferents and the brain stem vomiting centre, a reaction cascade associated with vomiting. To determine whether RV can infect EC cells, primary EC tumor cells (t. c.) were harvested from patients with liver metastasis undergoing surgery for the midgut carcinoid syndrome. In addition, a human midgut carcinoid t. c. line (GOT1), was examined for susceptibility to RV infection. The EC cell phenotype was confirmed by staining for 5-HT and the neuroendocrine granule marker chromogranin A. Immunofluorescence microscopy revealed that 100% of the GOT1 cells and 95% of the primary EC t. c. were positive for chromogranin A, and 68% of GOT1 cells and 40% of the primary EC t. c. were positive for 5-HT (Figure. S1); this confirmed that the vast majority of the cells were EC specific. To determine whether RV replicates in EC cells, we infected confluent GOT1 cells (200,000 cells/well) with trypsin-activated Rhesus rotavirus (RRV) (MOI 0,1), collected cells and media at 3,24 and 48 h post infection (p. i.) and determined the progeny viral titre. The EC t. c. did indeed support RRV replication (Figure. S2) and the viral titres increased >100 fold from ≤1×101 plaque forming units (pfu) /ml in samples collected 3 h p. i. to 2×103 pfu/ml at 48 h p. i. RV infection has previously been shown to increases the intracellular Ca2+ concentration in human intestinal cells [30]–[34]. To investigate whether RRV has a similar effect on GOT1 and primary t. c., the cells were loaded with the ratiometric fluorescent Ca2+ indicator Fura-2 prior to infection with trypsin-activated RRV at an MOI of 10 and 3 respectively (Protocol. S1). The Ca2+-images were captured at 1,5 and 23 h p. i. These experiments showed that RV infection caused an elevation in intracellular Ca2+ in EC t. c. within 60 min. (Protocol. S2, Figure. S3). GOT1 cells had a maximum release of Ca2+ at 60 min p. i. and primary cells had a maximum release at 5 h p. i. This observation is important since the release of 5-HT is known to be a Ca2+-dependent process in EC cells [35], including human enterochromaffin-like cells [36]. Using the cholera toxin (2 nM, 20 nM, 200 nM) as an agonist we observed a dose-dependent release of 5-HT from primary EC t. c. within 24 h (Figure. S4). RV was shown to stimulate the release of 5-HT in a dose and time-dependent manner with an increase of 5-HT release after 6 h p. i. in primary EC t. c. (Figure. 1A). Furthermore, when primary EC t. c. and GOT1 cells were incubated with supernatant from RRV-infected MA104 cells, a release of 5-HT was observed within 60 min (Figures. 1B, 1C). Such an early 5-HT response indicated that viral replication was unlikely to be required for 5-HT release. More likely, a release of viral protein (s) from infected cells could explain this effect. Purified double- and triple-shelled RV [37] (1 and 2 µg/ml, respectively) and virus particle-free supernatant (130000× g; 4 h, SW40) were individually added to GOT1 (500,000 cells/well) and primary EC t. c. (200,000 cells/well) for 60 min. While the ultracentrifuged supernatant stimulated 5-HT release within 60 min (Figures. 1B, 1C), no such effect was observed using the purified particles (data not shown). Therefore, it was concluded that double- and triple-shelled RV were not responsible for this early effect on 5-HT secretion. The enterotoxic glycoprotein NSP4 is secreted from human intestinal cells following RV infection [38], [39] and has been reported to mobilize intracellular Ca2+ from human HT-29 cells [40]. Therefore, we examined whether NSP4 secreted from polarized human intestinal Caco-2 cells infected with rotavirus [39] or recombinant NSP4 [41] could stimulate the release of 5-HT from GOT1 and primary EC t. c. Indeed NSP4 at concentrations ranging from 0. 25–2. 5 µM resulted in the secretion of 5-HT from primary EC t. c (Figure. 2). In a subsequent experiment, the addition of 2 µM NSP4 to GOT1 cells for as short time as 60 min revealed an increase of 204% of 5-HT in the media from cells stimulated with recombinant NSP4 (data not shown), and an increase of 84% of 5-HT from the secretory NSP4 (data not shown). Thus, both recombinant NSP4 and NSP4 secreted from rotavirus-infected Caco-2 cells were capable of stimulating the release of 5-HT from human EC t. c. Both GOT1 and primary EC t. c. were loaded with the ratiometric fluorescent Ca2+ indicator Fura-2 and stimulated with NSP4. Following the establishment of a stable Fura-2 fluorescence baseline, recombinant NSP4 (2 µM) was added to cells and the Ca2+ signal measured continuously for 40–50 min. The intracellular Ca2+ concentration increased 1. 5–2-fold in EC t. c. (GOT1 n = 8 cells, primary EC t. c. n = 10 cells) within 30 min of NSP4 stimulation (Figure. 3), which was consistent with observations in Caco-2 cells [34]. Ionomycin (1 µM) was added at the end of the experiment as a control to compare the magnitude of the increase, but it had little further effect, suggesting that NSP4 is a strong mobilizer of intracellular free Ca2+ (Figure. 3). Exogenous NSP4 can stimulate IP3 production in human intestinal HT-29 cells [40], and a human pancreatic carcinoid cell line (BON) was shown to produce IP3 after mechanical stimulation and activation of purinergic receptors [42]. We therefore determined weather NSP4 caused IP3 production in EC cells. The production of IP3 was indeed stimulated in GOT1 cells (400,000 cells/well) when incubated for 60 min with recombinant or secretory NSP4 (2 µM). Caracole (100 µM), a muscarinic receptor agonist known to activate the PLC pathway, was also used as an agonist. Both forms of NSP4 activated the PLC pathway, albeit to a less degree than carbachol (Figure. 4A). Since activation of adenylyl cyclase (AC) and cyclic adenosine monophosphate (cAMP) signalling has been previously associated with Ca2+ increase and 5-HT release in GOT1 [43], KRJ-I [44] and BON cells [36], we next determined whether NSP4 could stimulate this pathway. GOT1 cells (650,000 cells/well) were thus stimulated with 2 µM NSP4 and the AC agonist isoproterenol (10 µm) for 5 and 30 min, followed by collection of the supernatant and quantification of cAMP as an indicator of AC activation. While isoproterenol stimulated cAMP in GOT1 cells, no effect of NSP4 was observed at 5 min (Figure. 4B) or 30 min (data not shown), suggesting that the cAMP pathway in GOT1 cells was not activated by NSP4. The distribution and occurrence of neuroendocrine cells in the small intestine of the mouse duodenum, jejunum and ileum was assessed by immunohistochemistry for the neuroendocrine secretory granular marker chromogranin A. The duodenum, jejunum and ileum all contained neuroendocrine cells (Figures. 5A–D). They were observed in the crypts as well as among mature villous enterocytes, most abundantly in the duodenum. To investigate whether RV could infect enterocytes in the close vicinity of EC cells or infect chromogranin/5-HT-containing EC cells, infant mice were infected with murine rotavirus (strain EDIM) as described [5] and processed for histopathology and immunohistochemistry at different time points p. i. Figure 6A illustrates the typical vacuolization of infected mature enterocytes 48 h p. i. The vacuoles were occasionally found in the close vicinity of chromogranin-containing cells (Figure. 6B). No infected crypt cells were seen. Moreover, confocal microscopy revealed a co-localization between RV proteins and 5-HT-containing EC cells in jejunum of infected mice (Figures. 6C, 6D). Evidently, RV can infect enterocytes in the close vicinity of EC cells in the small intestine, and occasionally EC cells as well. To further support our previous observations [3], that 5-HT participates in RV-induced illness, 50 µL 5-HT at 5 mg/kg (Serotonin creatinine sulfate monohydrate, Sigma Aldrich, Code: 85030) were administered intraperitoneally to five to seven days old Balb/C pups. Our objective was to investigate whether diarrhoea would occur. Following 30 min post administration, 5 of the 7 pups responded with diarrhoea, after 45 min 6 out of 7 and after 60 min all 7 responded with diarrhoea indistinguishable from RV–induced diarrhoea [3]. To determine whether RV infection leads to activation of brain structures involved in sickness symptoms such as nausea and vomiting, immunohistochemical detection of Fos, a commonly used marker of neuronal activity was carried out on brain sections of RV-infected and uninfected mice [45]. Fos is an immediate early gene that is expressed upon repeated depolarization of neurons. In brains harvested 48 h. p. i., there was a robust activation of nucleus of the solitary tract, the main target structure for incoming fibers from the vagal nerve. This was seen in 3/5 of the infected animals. Thus, RV infection activated brain areas considered to be the vomiting centre. No activity was seen in uninfected animals (0/6) (Figure. 7). Our observation shows for the first time that RV activates brain structures associated with vomiting, presumably through activation of vagal afferents. No clear infection-induced Fos expression was seen in other parts of the brainstem. Despite the clinical importance of vomiting induced by RV infection and its contribution to severe dehydration, no mechanism has yet been proposed for emesis. Progress in elucidating the RV-induced emetic mechanism has been hindered by lack of appropriate small animal models, because most commonly used animal models that are susceptible to RV, i. e. mice and rats, do not exhibit an emetic response [24]. Progress has been further restricted by the limited availability of human primary and established EC cell lines. Primary human carcinoid EC cells as well as the carcinoid EC cell line (GOT1), enable investigatigation of the role of EC cells in RV pathogenesis. The EC cells have been identified from the mid- to the top of villi in the duodenum, jejunum and ileum, which are the segments associated with RV replication and histopathological lesions [46]. A cross-talk between EC cells and infected enterocytes is supported by immunohistochemistry and confocal microscopy experiments. The neuroendocrine cells were found in the close vicinity of cells with vacuoles, a characteristic feature of RV-infected enterocytes [46]–[48]. Moreover, RV sometimes co-localized with EC cells, suggesting a paracrine signalling of NSP4 and a cross-talk between EC cells and NSP4 in vivo. Consistent with the proposal of EC cells being key cells in RV pathophysiology, we demonstrated that: (i) RV cause release of 5-HT from primary EC t. c., (ii) crude and virus particle-free supernatants from RV-infected MA104 cells stimulated 5-HT release within 60 min in primary and GOT1 EC t. c., (iii) recombinant and secretory NSP4 caused release of 5-HT from primary and GOT1 EC t. c. The latter finding is particularly interesting, and is to the best of our knowledge, the first observation of a virus/viral protein-mediated effect on human EC t. c., stimulating 5-HT release. This release exhibited similar time kinetics as the Ca2+ mobilization, which is consistent with previous findings in EC cells and BON cells [35], [36]. RV also stimulated 5-HT release from EC t. c. in a time and dose-dependent manner beginning about 6 h p. i. It is reasonable to believe that the 5-HT release and Ca2+ mobilization were associated with expression of NSP4. Exogenous NSP4 can also cause the mobilization of intracellular Ca2+ [30], [31] via PLC-mediated IP3 production in human intestinal cells [40], [49]. Moreover, NSP4 is secreted from human intestinal cells [38] in a polarized fashion [39]. Therefore, we explored the novel hypothesis that RV, and particularly NSP4, stimulate EC t. c. via mobilization of intracellular Ca2+ and release of 5-HT. Since the elevation of Ca2+ after addition of NSP4 occurred after approx 20 min, similar to RV infections in Caco-2 cells [34], we speculate that the effect was not mediated by the opening of ion channels in the plasma membrane. It is more likely that it originated from internal ER stores as previously shown [31], although we cannot exclude that other possible mechanisms were involved. The kinetics of the Ca2+ response were similar to those of non-infected human intestinal Caco-2 cells inoculated with supernatant from RRV-infected Caco-2 cells at 18 h p. i. [34]. As the presence of proteins in the supernatant at this time point could not be explained by cell lysis, it was suggested that the Ca2+ mobilization is stimulated by viral proteins or peptides secreted from RV-infected cells [34]. Indeed, release of soluble NSP4 from RV-infected human intestinal cells has been demonstrated [38], [39], and we can now report that both recombinant NSP4 and NSP4 secreted from virus infected intestinal cells mobilize Ca2+ in EC t. c. and stimulate 5-HT release. The EC cells express an ensemble of ligand-gated ion channels, chemo- and mechanosensitive-ion channels and G-protein-coupled receptors on their surface [50], [51]. G-protein-coupled AC and PLC are key enzymes involved in 5-HT release in EC cells [36], [44], [50], [52], [53]. Kolby and co-workers previously showed that GOT1 cells weakly responded to carbachol, a muscarinic receptor agonist and activator of PLC, suggesting that these cells lacked functional muscarinic receptors or had an impaired PLC-dependent formation of IP3 [43]. Consistent with this observation, we found only a modest response to carbachol and NSP4, indicating that NSP4 increases Ca2+ in an PLC-independent way [54]. In order to explain the mechanism, by which NSP4 alter the ER, it has been proposed that NSP4 stimulates Ca2+ signal transduction mechanisms by binding to specific surface membrane receptors, activates PLC [31], [40] and thus creating IP3. Suggested membrane receptors for NSP4, increasing intracellular Ca2+ are muscarinic receptors [31], [40] but also α1β1 and α2β1 integrins [55]. The mechanism by which NSP4 may alter ER is unknown. It has been hypothesized that NSP4 acts as a viroporin [54], forming a cation channel in the ER membrane or having a direct action on IP3 receptors in the ER membrane, with action or no action on the membrane itself [34]. Brunet and co-workers [34] reported that at a late stage of infection in Caco-2 cells, Ca2+ is partially increased by a PLC-dependent Ca2+ release from the ER through the opening of IP3-sensitive channels. However, they did not exclude an efflux of Ca2+ due to a direct alteration of the ER membrane. GOT1 cells are believed to respond with an increase of intracellular Ca2+ concentration upon stimulation with the AC activator isoprotenerol [43]. We confirmed that isoprotenerol did indeed stimulate the formation of cAMP, but no such effect was observed with NSP4, suggesting that NSP4 does not induce the release of 5-HT through the AC pathway in these cells. Activation of the vagal afferent fibres by toxins in the gut appears to operate via detection of toxins by EC cells, which release 5-HT to activate 5-HT3 receptors on vagal afferent fibres [56]. Induction of Fos has previously been observed in vomiting animals [57], [58] at the nucleus of the solitary tract (NTS) of CNS [58]. Our finding that RV-infected mice responded with a strong neural activation of the primary target site of vagal afferents, i. e. the NTS, is in line with the hypothesis that RV-induced activation of vagal afferents triggers vomiting. The fact that not all infected mice showed such activation (3/5) may be due to the time-kinetic variation of Fos activation between different animals. The peak period of Fos expression has shown to be 60–120 min after stimulation [59], and prolonged for up to 6 hours. Another study showed a long-term increase in Fos expression in the area postrema, the NTS and the nucleus amygdalae in conjunction with vomiting after cisplatin treatment in a animal species with an emetic response (the house musk shrew, Suncus murinus) [60]. They also showed that Fos activation in these brain stem areas could be suppressed by palonosetron, a 5-HT3 receptor antagonist, which is used as an anti-emetic drug. The time kinetics of RV-induced Fos expression needs to be further investigated, since the onset of clinical symptom (diarrhoea) in mice started around 24 h p. i and persisted, at least up to 96 h. A limitation of using mice in these studies is the absence of a functional emetic reflex, but there are reports of “retching” but not vomiting, which may suggest that they have a degenerate reflex rather than none at all [56], [61]. Further support that 5-HT is associated with RV disease was provided by the observation that 5-HT induced diarrhoea in 7/7 animals within 60 min which is consistent with our previous observation that 5-HT3 receptor antagonists attenuate RV-induced diarrhoea [3]. The participation of EC cells in diarrhoea, as revealed by studies of 5-HT release and/or use of pharmacological blocking agents, has been demonstrated in such diverse fluid secretory states as those caused by cholera toxin [62], [63], the enterotoxins produced by E coli [64], bile salts [65] and Salmonella typhimurium [66]. Furthermore, an ENS involvement has been associated with cholera toxin [67], E coli heat stable toxin [68], certain bile salts [69], and gut inflammation [70]. These observations indicate that the interaction between EC cells and ENS is a pathophysiological mechanism common to many intestinal secretory states. A Staphylococcal enterotoxin was recently reported to induce emesis by releasing 5-HT into the intestine, an effect inhibited by a 5-HT3 receptor antagonist [18]. Similarly, our data show that the NSP4 enterotoxin stimulated release of 5-HT and, sometimes, RV was localized to EC cells in the small intestine. Support for our hypothesis that RV-induced vomiting includes stimulation of EC cells, 5-HT and activation of vagal afferents is derived from several clinical studies with 5-HT3 receptor antagonists. For example, ondansetron, a 5-HT3 receptor antagonists, has successfully been used to attenuate vomiting in paediatric gastroenteritis [20], [21], [23], [71], [72]. Furthermore, it has also been reported that ondansetron-treatment of vomiting in American children has become quite common [21]. While it is established that vomiting during acute gastroenteritis in young children can be treated with 5-HT3 receptor antagonists, it remains to be shown in clinical studies that children with RV-induced vomiting can be successfully treated. Moreover, the emetic responses to the Staphylococcus toxin in the musk shrew animal model [18], suggests that this animal model might be explored to study RV–induced vomiting. Our present and previous studies of the pathophysiology of RV infections [2], [3] suggest a common triggering mechanism for the fluid loss and the emesis as schematically illustrated on Figure 8. The results of the present study strongly suggest that RV per se and/or NSP4 released from adjacent virus-infected enterocytes increase intracellular Ca2+concentration in the EC cells, which, in turn, stimulates the release of 5-HT from EC cells. We propose that the released 5-HT activates both intrinsic and extrinsic afferent nerve fibres located in close vicinity to the EC cells. Hence, EC cells function as sensory transducers of different luminal stimuli. Such a mechanism has been demonstrated in several experimental models [8], [9], [42]. As pointed out above intrinsic afferent nerves stimulated by the released 5-HT are part of intramural nervous reflex (es), which in the case of RV infection increase fluid secretion from intestinal crypts via the release of VIP (vasoactive intestinal peptide) at the crypt epithelium [3], [4]. The released 5-HT also activates vagal afferents that project to the medullary vomiting centre of the central nervous system, triggering the emetic reflex [25], [26], [42], [50]. It is apparent that EC cells and nerves play an important role for RV-induced diarrhoea and vomiting and the present findings may be of more general importance for our understanding of pathophysiological mechanisms of many different types of infection-induced diarrhoea and vomiting. Our observations may be of clinical importance, since the possibility to reduce vomiting by 5-HT3 receptor antagonists in acute viral gastroenteritis will both favour oral rehydration therapy by preventing vomiting and attenuate fluid loss, thus reducing hospitalisation of children [21]. Primary tumor EC cells [43] and a human midgut carcinoid tumor cell line (GOT1), previously characterized for specific EC cell markers [43], were cultured in RPMI 1640 medium supplemented with 10% FCS, 0. 73 mg/ml L-glutamine, 5 µg/ml apo-transferrin, 5 µg/ml insulin, and PEST (100 U penicillin, 100 µg/ml Streptomycin). Primary EC cells were obtained from patients with liver metastasis and midgut carcinoid syndrome [43]. RRV was cultivated, quantified and purified as described [37], [73]. The GOT1 cells (200,000/well) were infected with trypsin-activated RRV at an MOI of 0. 1 as previously described [73]. Briefly, after infection the cells were washed twice and then incubated with Minimal Essential Medium (MEM) containing trypsin (T8353, bovine pancreas, type III, Sigma Aldrich), 1 µg/ml medium, for 3,24 and 48 h. At each time point cells and supernatants were collected and frozen at −80°C. Cell suspensions were freeze-thawed twice, centrifuged to remove cell debris, followed by determination of the progeny virus, as previously described [74]. The GOT1 cells (450,000 cells/plate) and primary EC t. c. (200,000 cells/plate) were seeded onto coverslip-bottomed plastic Petri dishes used for fluorescent microscopy (MatTek Corporation). The cells were washed twice with MEM without FCS and then loaded with the fluorescent Ca2+ indicator Fura-2-AM (Molecular Probes), 10 µM in the presence of Pluronic-F 127 (Sigma Aldrich), 20% w/v in DMSO, 10 µl/plate in a total volume of 1 ml MEM without FCS for 45 min at 37°C. The cells were washed twice with MEM without FCS and incubated in fresh medium. Ratiometric imaging of Fura-2-loaded cells was performed using a Photon Technology International (Monmouth Junction, NJ) system and a Zeiss Axiovert 100 M (Jena, Germany) microscope equipped with a ×100 glycerol-immersion Fluar objective (1. 3 numerical aperture) and a PTI IC-200 camera for fluorescence imaging. Before adding NSP4 to the cell cultures, initial 10 min fluorescence (F340/F380) was captured to obtain a Ca2+-baseline. Bright-field images were taken simultaneously using a PTI IC-100 camera by passing the transmission light through a 700 nm band-pass filter in front of the halogen lamp to avoid stray light in the fluorescence channel. NSP4 were added to a final concentration of 125 nM and 2 µM, respectively, and continuously measured in real time for further 30 min. For measuring of intracellular Ca2+ using RRV see supporting information (Protocol. S1 and S2). The GOT1 and primary EC-cells were stained for 5-HT and chromogranin A. Cells were fixed with 4% paraformaldehyde/PBS on microscope slides (Histolab, Göteborg, Sweden) overnight at 4°C in a humidity chamber and then processed for immunofluorescence (Protocol. S3). Histidine-tagged NSP4 from a simian rotavirus strain (SA11) was produced using the baculovirus expression vector system and Spodoptera frugiperda (Sf9) insect cells. The NSP4 was purified by column chromatography, as previously described [41]. Secreted NSP4 was purified from the media of polarized epithelial Caco-2 cells infected with bovine rotavirus (UK strain) [39]. Briefly, the medium was ultracentrifuged to remove virions and NSP4 purified by sequential concanavalin A affinity chromatography and monoS cation exchange chromatography. The protein was judged as >99% pure by SDS PAGE and silver staining. The cholera toxin was purchased from List Biological Laboratories, Campbell, California; Code 101A. The 5-HT in cell culture medium from primary EC t. c. and GOT1 cells was quantified using a commercial serotonin ELISA kit (IBL International, Hamburg, Germany; Code: RE59121) according to the manufacturer' s instructions, or by HPLC [43]. GOT1 cells (650,000/well) were pre-incubated for 30 min in MEM without FCS with (100 µM) or without the AC inhibitor 2′, 5′-Dideoxyadenosine, (Sigma Aldrich; Code: D7408), at 37°C with 5% CO2. Recombinant or secretory NSP4 (2 µM), were added for 5 and 30 min. Cells were lysed and analysed for intracellular cAMP with a commercial cAMP EIA kit as described by the manufacture (R&D systems, United Kingdom; Cat. No KGE002B). MEM without FCS was used as a negative control and Isoprotenerol (10 µM; Sigma Aldrich; Code: I6504) as a positive control. IP3 production was measured indirectly through the accumulation and analysis of the metabolite IP-one. GOT1 cells (650,000/well) were stimulated with recombinant NSP4 or secretory NSP4,2 µM, for 60 min at 37°C with 5% CO2 in a buffer containing LiCl (50 mM) to prevent degradation of IP-one. Carbachol (Sigma Aldrich, USA), 100 µM, were used as positive control. Cells were lysed and analysed using a commercial IP-one ELISA kit (Cisbio Bioassay, France; Code: 72IP1PEA), according to the manufacturer' s instructions. RV naïve, five to seven days old BALB/c mice (B&K Laboratories, Sollentuna, Sweden) were orally infected with 10 µL/animal (100DD50 diarrhoea doses) of murine rotavirus strain EDIM [3], [46]. For mice receiving 5-HT (Sigma Aldrich), 50 uL at 5 mg/kg were administered intraperionally and observed for signs of diarrhoea at 30,45 and 60 minutes after administration. The small intestines were removed and processed as previously described [3], [46]. For immunohistochemistry of mice intestinal tissue, paraffin-embedded specimens were cut into thin sections as previously described [46]. Intestinal sections were hydrated and processed for immunohistochemistry (Protocol. S4). For immunoflourescence staining, the paraffin-embedded intestinal specimens were cut into thin sections, hydrated and processed for immunohistochemistry (Protocol. S5). Brains were cut at a freezing microtome at 30 micrometer and further processed for free floating immunohistochemistry. Sections were incubated with a primary antibody directed against Fos (Santa Cruz Biotechnology; sc-52; 1∶1000) and the antibody was visualized using avidin-biotin complex amplification and DAB as chromogen according to previously published protocols [75]. The results are expressed as mean ± standard errors of the mean (SEM), unless indicated. Statistical analysis of all data was performed using the repeated measures analysis. The Mann Whitney test was used unless stated. P-value of ≤0. 05 was considered significant. All animal experiments in this study were carried out in strict accordance with the recommendations in the guide for the care and use of laboratory animals conformed to Swedish animal protection laws and applicable guidelines (djurskyddslagen 1988: 534; djurskyddsförordningen 1988: 539; djurskyddsmyndigheten DFS 2004: 4). Animal experiments were approved by the local Ethical Committee (Stockholm Norra Djurförsöksetiska nämnd, Stockholm, Sweden; Approval No: N289/09). All efforts were made to minimize suffering and surgical operations were performed afterwards the animals were euthanized by overdose of Isoflurane.

Rotavirus (RV) can cause severe dehydration and is a leading cause of childhood deaths worldwide. While most deaths occur due to excessive loss of fluids and electrolytes through vomiting and diarrhoea, the pathophysiological mechanisms that underlie this life-threatening disease remain to be clarified. Our previous studies revealed that drugs that inhibit the function of the enteric nervous system can reduce symptoms of RV disease in mice. In this study we have addressed the hypothesis that RV infection triggers the release of serotonin (5-hydroxytryptamine, 5-HT) from enterochromaffin (EC) cells in the intestine leading to activation of vagal afferent nerves connected to brain stem structures associated with vomiting. RV activated Fos expression in the nucleus of the solitary tract of CNS, the main target for incoming fibers from the vagal nerve. Both secreted and recombinant forms of the viral enterotoxin (NSP4), increased intracellular Ca2+ concentration and released 5-HT from EC cells. 5-HT induced diarrhoea in mice within 60 min, thereby supporting the role of 5-HT in RV disease. Our study provides novel insight into the complex interaction between RV, EC cells, 5-HT and nerves.

lay_plos

Summarize the discussion about Marketing's presentation on user requirement. Project Manager: {vocalsound} Industrial Designer: {vocalsound} {vocalsound} Project Manager: {vocalsound} Industrial Designer: Okay. Project Manager: Okay everyone's ready. User Interface: Hello. Project Manager: So we are here for uh for uh functional design. User Interface: {gap} Project Manager: Okay? So we are here for the functional design meeting mm {vocalsound} so first I will show the agenda so we will uh I will take notes during this meeting so I will try to summarise it and put that summary in the shared folder if you want to look at it afterwards User Interface: {vocalsound} Project Manager: so then uh each of you will uh lead a presentation on the task that has been required last time so user requirement specification, technical function design and working design. Then I will uh present you some new project requirements I received from uh the management board. Then we will take uh the decision on on the remote control uh needed functions and then I will assign you the task for the next part of the meeting. Of the {disfmarker} of the process. So uh who want to start the the presentation of what they did? Industrial Designer: F do you want to start? User Interface: Make a start yeah. Project Manager: You can start. User Interface: So. {vocalsound} Project Manager: {vocalsound} User Interface: Cable, camera. Project Manager: You have uh PowerPoint? User Interface: Should be in my {disfmarker} in their folder no? Project Manager: Ah yeah maybe there. Okay. User Interface: Up. Project Manager: Who are you? {vocalsound} User Interface: Um at three I think. Project Manager: {vocalsound} User Interface: No? {vocalsound} Mm. Project Manager: Ouch. And {disfmarker} User Interface: {vocalsound} Industrial Designer: Okay. {vocalsound} Project Manager: We have a technical problem uh. User Interface: Do we think w s in the {disfmarker} in the wrong folder maybe? {vocalsound} It is possible. Project Manager: You put it on {disfmarker} Industrial Designer: No. User Interface: It was somewhere in something like this. I don't remember the name actually must be something like messenger AMI or something. Industrial Designer: What do you have in short cut? User Interface: Go up. Industrial Designer: Participant two. User Interface: Yeah go up. Project Manager: Yeah. User Interface: Again. No. Go back. Project Manager: You have no {disfmarker} User Interface: Uh maybe messenger AMI. Messenger. Project Manager: Over. Okay. User Interface: No. There is nothing. Project Manager: There's no {disfmarker} We have a technical problem. User Interface: Let's go and check. Industrial Designer: Yeah. User Interface: I'll go and check. Industrial Designer: Otherwise, could you just describe by hand? User Interface: Okay. Industrial Designer: With the the whiteboard? Project Manager: If you remember yeah User Interface: Yeah. Yeah. Project Manager: but that's {disfmarker} User Interface: So uh. Basically {vocalsound} what we want here is a remote control right. Project Manager: Yeah. User Interface: {vocalsound} So um the question well first of all what to control. So {vocalsound} most people want to have a a remote for their hi-fi and T_V_ and stuff like that. Industrial Designer: Mm-hmm. User Interface: And {disfmarker} but other people want th also remotes for {vocalsound} controlling uh and toys like robotic pets and little robots and stuff Industrial Designer: Mm-hmm. User Interface: and other people also want to have remotes for controlling um whole house. Project Manager: Yeah. Industrial Designer: Mm-hmm. User Interface: Yeah, so there's a project I think called X_ house or something like that that does that, uh you can integrate your remote with uh computers stuff. So {vocalsound} Industrial Designer: Mm-hmm. User Interface: there is one {disfmarker} that is one thing. The other is the the finder feature yeah by whistling or whatever. Uh if you have the finder feature then you can also have Industrial Designer: Okay. User Interface: uh at the same time as {disfmarker} and general voice commands if you want yeah. {vocalsound} So I think it should be a package in that case. Industrial Designer: Mm-hmm. User Interface: Uh so the user interface will consist of two parts. {vocalsound} One is the voice command part and on one is the actual buttons part. {vocalsound} Uh and th the buttons part would be uh a set of buttons for choosing devices, a set of buttons for special navigation in space, Industrial Designer: Mm-hmm. Mm-hmm. User Interface: a set of buttons for {vocalsound} linear access of medium and a set of buttons for random access. Project Manager: Okay. User Interface: Yeah? Industrial Designer: What do you mean by linear access then? User Interface: Like a video tape goes forward, backwards, uh fast and stuff yeah. Industrial Designer: Ah. Project Manager: Okay so special navigation, linear access, random access User Interface: Um. Project Manager: and there's a fourth one no? User Interface: Mm? Project Manager: So the better now for special navigation? User Interface: Yeah. For special navigation for example you might have a T_V_ in the menu and you going to change yeah? Project Manager: Okay. Then linear access User Interface: Uh. Project Manager: then random access. User Interface: Mm. Yeah and also parameter changing. Project Manager: Ah yeah parameter okay. User Interface: {vocalsound} So if there are common parameters maybe we should put special buttons for that um Project Manager: Okay. User Interface: or maybe we could have everything uh generic but uh there are a lot of uh remotes on the market right now and {vocalsound} basically this is most of the {disfmarker} almost everybody has this stuff. Project Manager: Okay. Okay and and voice command did you uh {disfmarker} User Interface: Voice command w we could specify anything. We could assign any button {disfmarker} a command to any button, if we have enough processing power, Project Manager: Okay. Okay. User Interface: I guess so. {vocalsound} Yeah. Project Manager: Okay. User Interface: Yeah. Project Manager: So that's uh that close your investigations? User Interface: Yeah. Project Manager: Okay. User Interface: Uh yeah I think so. Project Manager: Okay. User Interface: Not so far. Project Manager: Maybe we can have a look at the user requirements with {disfmarker} Marketing: Yeah. {vocalsound} Um I dunno if you can open the {disfmarker} Project Manager: I dunno if I can open it. Maybe you can s Marketing: uh m is not here. Project Manager: It's {disfmarker} Marketing: Uh in {disfmarker} yeah okay. Project Manager: Messenger no? Marketing: No. In document {gap}. Mm computer yeah. Project Manager: In which folder? User Interface: Where did you put it? Marketing: Here. Here. Project Manager: {vocalsound} Short-cut to AMI shared folder? User Interface: {gap} mm. Marketing: But it's not {disfmarker} Um. Project Manager: Maybe you can send it to me by email. Just to participant one. At AMI. Marketing: Mm-hmm. Yeah, I can do that. Project Manager: I will try to show it to everyone, that would be more comfortable. Marketing: Okay. Um. Project Manager: You send it? Marketing: {vocalsound} It's participant one? Project Manager: Yeah. Uh this is this email. User Interface: I'm designing the user interface. {vocalsound} Marketing: Okay. You can uh. Project Manager: Okay. So maybe I can switch slides when you {disfmarker} whenever you ask, Marketing: Yeah. Project Manager: that will be more convenient. So okay, functional requirements. Marketing: Okay so you can {disfmarker} you can go. Okay so {vocalsound} in our usability lab we observed the remote control use among one hundred subjects Project Manager: Mm. Marketing: and the subjects also filled a questionnaire Project Manager: Yeah. Marketing: okay? And here I have the results so you can see that um seventy five per cent of users find most remote controls ugly so we have to find something to make them more {vocalsound} more nice, more kind. Eighty per cents of users would spend more money when the remote control would look fancy. {vocalsound} Eighty hundred per cent of users would spend more money when the remote control would look {disfmarker} Project Manager: {vocalsound} User Interface: {vocalsound} Marketing: oh {disfmarker} to {disfmarker} it's not good. {vocalsound} So okay. Project Manager: We can just keep doing that? Marketing: So it's not in theory {disfmarker} but I I can I can say yeah. {vocalsound} Project Manager: Yeah. Marketing: Fifty f uh seventy five per cent of users say they s zap a lot. So mm {vocalsound} we have to have a remote control uh very um {vocalsound} out for that. {vocalsound} Uh the buttons have {disfmarker} are to be um uh uh like you say resist resisting to to shocks. Project Manager: Okay. Marketing: {vocalsound} Um and fifty per cents of users say they only use uh {vocalsound} ten per cents of but of the buttons in the {disfmarker} in the remote control. Industrial Designer: {vocalsound} Project Manager: Okay. Marketing: So all the buttons we we have to put are {disfmarker} have to to have um a use a real use Project Manager: Okay. Marketing: and not only or {disfmarker} Project Manager: Okay, so fewer buttons maybe would be good? Marketing: Yeah. F not many buttons, and uh and uh uh u useable buttons {vocalsound}. Project Manager: Okay. User Interface: But what kind of remote controls did you look at? Marketing: Sorry? User Interface: What kind of task was it? It was a T_V_? Marketing: Yeah. Uh {vocalsound} most for most is T_V_. Project Manager: Yeah. Yeah but in fact we {disfmarker} it it seems that we are going to make a T_V_ remote control according to new requirements I received from the management User Interface: Huh. Project Manager: bo I will present them in the following. User Interface: Uh-huh. Ah! Good. Marketing: {vocalsound}'Kay you can go so. Project Manager: Okay. Marketing: So there are other frustrations expressed by users, so they said uh they lost uh often the remote control in in the room so they want to have a way to {vocalsound} to Project Manager: Yeah. To find it. Marketing: to find it. Project Manager: Yeah. Marketing: Um and um lot of the time they {disfmarker} it takes too much time to learn how to use a new remote control. Project Manager: Yeah. Marketing: So they want something s really very simple and uh easy to use. Industrial Designer: Mm-hmm. Project Manager: Okay. Marketing: And uh remote controls are bad for Project Manager: What is her other side? Marketing: R_S_I_ {vocalsound} um {disfmarker} User Interface: Other side yeah, yo wa your wrist Marketing: I dunno. Project Manager: {vocalsound} User Interface: It i can become painful you can have tendonditis. Project Manager: Oh yeah? User Interface: Yeah. Project Manager: I did not knew that. User Interface: If you also {gap} up on a computer in a strange position. Project Manager: Okay so you {disfmarker} we have to make it uh more ergonomic yeah. User Interface: Ergonomic. But uh {vocalsound}. Marketing: Yeah. User Interface: Have to say ha ha. {vocalsound} Project Manager: {vocalsound} It's your job {vocalsound} Marketing: {vocalsound} Project Manager: Oh. {vocalsound} Marketing: {vocalsound} Uh. Project Manager: Uh sorry {vocalsound} got a message from Microsoft. {vocalsound} Industrial Designer: {vocalsound} Marketing: {vocalsound} Okay um before that I I have some some {vocalsound} some thing {vocalsound} uh to say before um {disfmarker} Project Manager: Yeah. Yeah yeah. Marketing: {vocalsound} We know that uh the user use uh a lot their um remote control um to to change channel. Project Manager: Yeah. Marketing: {vocalsound} Um and um to to change uh volume selection of the {disfmarker} Project Manager: Okay. Marketing: and uh and not uh a lot for setting {disfmarker} for setting the the channels and uh thing things like that. Project Manager: Yeah. Industrial Designer: Mm-hmm. Project Manager: Okay. User Interface: Mm-hmm. Marketing: So it's better to put uh uh uh something very easy to set and uh and {disfmarker} Project Manager: Yeah. This function should be very uh accessible. Marketing: Very accessible yes. Project Manager: Yeah, okay. This is the main function okay. Marketing: That's right. {vocalsound} So then we asked some questions to them Project Manager: Yeah. Marketing: and um we asked this question if they prefer an L_C_D_ screen or on their remultific function remotes control Project Manager: Yeah. Marketing: and if they mm pay more for speech recognition in remote control Project Manager: Yeah. Marketing: and you can go {vocalsound} we have here the results of User Interface: The first question. Marketing: of the questions. So you know that um Project Manager: {vocalsound} Marketing: {vocalsound} for the younger it's very important Project Manager: To have L_C_D_ and voice. Marketing: to have the s yes and speech recognition. And uh and the others is not so important but uh we know that uh uh people between fifteen and twenty five are people who watch a lot T_V_ and uh who who wh can use a lot this uh. Project Manager: Okay. Marketing: So maybe we we can have a speech recognition in. Project Manager: Yeah maybe this this is important. Marketing: Yeah. Project Manager: Moreover th maybe those uh like those teenager customer could advice their parents to buy this equipment and so we can {disfmarker} User Interface: Mm. Project Manager: we have to take care of that point of view I think or so. Marketing: Mm. Project Manager: Yeah. Marketing: Okay and if there is th the conclusion now. So as we say before, I think uh um a remote control lightening in the dark it's it's a good thing. Project Manager: Okay. Marketing: Uh not to many mud buttons like we we said before, Project Manager: Yeah. Marketing: e easy to use uh a way to find it easily in the room and uh uh resistant to to shock and to to {disfmarker} Industrial Designer: Mm-hmm. Project Manager: Okay. Marketing: {vocalsound} An I s no, yeah. Project Manager: {vocalsound} Okay these are the user requi Marketing: {vocalsound} I dunno if you see something else important or {disfmarker} Industrial Designer: {vocalsound} Project Manager: {vocalsound} Industrial Designer: I'm just thinking of some thing. Project Manager: Yeah. Marketing: Mm. Industrial Designer: Um {disfmarker} We want to have a {disfmarker} no, I don't know if this is a good idea. We want to have a a general remote control for everything. Project Manager: No no no. We {disfmarker} w it seems that we no want to have a T_V_ remote control. From the management board I receive an email. User Interface: Mm. Industrial Designer: Okay. Yeah. Project Manager: Cos it would be costly uh and and also it it would take more time to develop to have a a general generic remote control. Industrial Designer: Yeah. Yeah yeah. Yeah. User Interface: Mm {disfmarker} I {disfmarker} it's not true I think. The the second claim that you put. Industrial Designer: No no. {vocalsound} Project Manager: That it would be too long to develop. User Interface: Yeah. Marketing: {vocalsound} User Interface: I think that should be the same. Project Manager: Oh yeah. Because I received that email from management board and they seems to tell that that if we want to be on the market as early as possible we should uh focus on T_V_ more where it seems that the market is more important. So maybe it's a good decision. I dunno. What's your opinion? User Interface: I have uh I've no idea I mean I should know a bit more about how fast we can uh design it. Marketing: {vocalsound} User Interface: I don't think {disfmarker} Project Manager: {vocalsound} Industrial Designer: {vocalsound} Marketing: {vocalsound} User Interface: Uh yeah. Industrial Designer: Finish tonight. Project Manager: {vocalsound} User Interface: Yeah. Okay. Yeah. Industrial Designer: {vocalsound} But basically yeah maybe I can continue with my presentation, it would be al you {disfmarker} you {disfmarker} Project Manager: Okay. User Interface: Yeah. Marketing: Yeah. Industrial Designer: but I think we have some technical problem or so. So I'm just going to describe briefly what we do in the remote control. Project Manager: Maybe you can go to the whiteboard if you have some drawings to do Industrial Designer: If fact {disfmarker} Project Manager: I don't know. Industrial Designer: Yeah but {disfmarker} Marketing: Mm. Industrial Designer: Do I have {disfmarker} oh yeah. Project Manager: {vocalsound} Industrial Designer: Now I have enough cables. User Interface: Like a {disfmarker} you feel a bit like a dog with this stuff. Project Manager: {vocalsound} Industrial Designer: {vocalsound} Okay so I'm just going to describe {disfmarker} in fact for for a remote control this is quite easy. We just have {disfmarker} Project Manager: {vocalsound} User Interface: {vocalsound} Industrial Designer: sorry, I'm going {vocalsound} Project Manager: Okay. User Interface: {vocalsound} Project Manager: Are you okay? {vocalsound} Industrial Designer: Like that. I'm just going to describe. Basically we have a a battery a power supply here. After that we just have um user interface. Let's say that um something like that, which could be um a L_C_D_ let's say or um an array of push button, something like that. Push button or a L_C_D_. After that we we feed that into um uh an electronic chip. So I say U_C_ and I feed that to uh L_E_D_ which is uh infrared {disfmarker} um which is a an infrared um component. And so what we {disfmarker} for for myself this {disfmarker} for for us this is quite easy. Project Manager: U_C_ is the central unit? Industrial Designer: Yeah. Project Manager: Okay yeah. Industrial Designer: Y it's a {disfmarker} it's {disfmarker} this just a chip which does all the um numerical Project Manager: Computation. Industrial Designer: numerical computation according to your display. Project Manager: Okay. Industrial Designer: And so for us uh this is quite easy. We just need to take {disfmarker} to define what we want to do when the user interface um wants something and after that we just do the coding to s and send that to to to the {disfmarker} not the {disfmarker} to the television. Project Manager: Okay. Industrial Designer: So for us this is quite easy. Project Manager: Okay so this is quite easy. There is not that much constraints. Industrial Designer: Yeah. Um we just have to define the processing power that we need uh especially if we want to do some uh speech recognition, in that case that mean that we are going to use more for simple {disfmarker} User Interface: {vocalsound} This will {disfmarker} think this will take more time to develop also. Industrial Designer: Yeah of course of course. User Interface: Yeah. {vocalsound} Industrial Designer: And um but for a standard one this is really easy. It's a question of one month and so on s User Interface: Soon. Project Manager: To have a {disfmarker} you s you speak about with voi voice control? Industrial Designer: No no no no, Project Manager: Standard button one. Industrial Designer: I say {disfmarker} yeah {disfmarker} standard uh standard remote control takes maybe uh one month to to do that. User Interface: Yeah. So the only time problem is the sp voice recognition. Marketing: {vocalsound} Industrial Designer: Yeah. User Interface: Yeah. Industrial Designer: Definitely. User Interface: Yeah. Project Manager: So do you have any idea of how long it would take to have voice recognition now? User Interface: {vocalsound} Ten years. Industrial Designer: I would say {vocalsound}. Project Manager: {vocalsound} {vocalsound} User Interface: {vocalsound} Industrial Designer: I would say uh about eight months to have the first results. Project Manager: {vocalsound} Okay so i it's a bit long yeah. Industrial Designer: Yeah. I can {disfmarker} Um. Project Manager: One month for the standard one with button. Industrial Designer: Yeah. Project Manager: Even if we have a L_C_D_ display? Industrial Designer: Yeah even. I mean that this is really standard devices now. Um eight. For uh speech recognition. Project Manager: Okay yeah. Okay so we can take this into account. So who think it would be good to go for uh like speech recognition? User Interface: But we don't have time to market. Project Manager: {vocalsound} Yeah it will. Industrial Designer: And also {disfmarker} how much uh I think User Interface: I think we should contact management. Industrial Designer: during the kickoff meeting you say that we we shouldn't {disfmarker} we shouldn't go up to twelve point five Euro per unit Marketing: {vocalsound} Project Manager: Euros. Yeah, yeah. Industrial Designer: so how many units should we sell to have a {disfmarker} User Interface: Well. Uh {disfmarker} Project Manager: Well each unit is is sell uh twenty five Euros. Industrial Designer: Yeah but how many {disfmarker} yeah. User Interface: How muc how much do you get {disfmarker} how much do you {disfmarker} if you buy one million units h no, one hundred thousand units. Marketing: {vocalsound} User Interface: Eh chips. We're gonna need chips right. Industrial Designer: Yeah. User Interface: Yeah. How much will it cost for one hundred thousand? Industrial Designer: {vocalsound} Usually this is less tha at two dollars per chip. User Interface: Okay. Project Manager: Because we are {disfmarker} User Interface: So you have any idea for a powerful one that has uh good enough for do speech recognition. Industrial Designer: Yeah, User Interface: Yeah? Okay. Industrial Designer: we can {disfmarker} User Interface: So. Project Manager: It seems that that we want to sell like four million units from the first meeting. User Interface: No it doesn't. Industrial Designer: Mm-hmm. User Interface: Okay. Four million. {gap} Project Manager: Okay. Maybe we can uh we can look at the new requirement I receive from the management board and discus discuss all function we want to have. Marketing: Mm. Industrial Designer: Um I just had a question uh do you want to continue with your presenta? Marketing: {vocalsound} Project Manager: Yeah I I will continue. Industrial Designer: Is it {disfmarker} Project Manager: Well ask your question if you want. Industrial Designer: {vocalsound} Um you say that I don't remember by heart but thirty per cent Marketing: Mm? Industrial Designer: of the tested people say that's it's quite difficult to to to use the remote control. Marketing: Yeah. Industrial Designer: Do they say that this is difficult but for the same reason or do they have other reason? To to {disfmarker} Marketing: Oh. Industrial Designer: maybe to keep in mind maybe to access to that menu you should do something like that. Marketing: Would j Uh {vocalsound} yeah w I I think they they say that it's uh difficult to learn how to use it but i when you know how to use it, it's it's okay. Industrial Designer: Mm-hmm. Mm-hmm. Project Manager: Okay. It's not intuitive first. Industrial Designer: And {disfmarker} Marketing: But {disfmarker} Yeah. Industrial Designer: But yeah maybe and what about if we design a remote control which can be configure as you want? You say that I want, I have six button {disfmarker} User Interface: Mh-hmm. A a lot of people are uh {disfmarker} if you have the L_C_D_ screen if you can do it completely the way you want because the buttons also look the way you want them Industrial Designer: Yeah. Project Manager: Yeah. But, but also it seems that {disfmarker} Industrial Designer: Yeah. User Interface: but it will be hard to configure I mean imagine i uh {disfmarker} Project Manager: Yeah. Industrial Designer: Yeah. User Interface: so it's really something for the expert user. So {disfmarker} Industrial Designer: Okay. User Interface: I mean there are markets and markets. I think the young people are th uh are uh {disfmarker} Marketing: {vocalsound} User Interface: Christine here said uh you have a {disfmarker} Industrial Designer: Yeah. Project Manager: Yeah. User Interface: {vocalsound} uh it is yeah. {vocalsound} So for our young people uh it will be cool, they can be able to use it. Th maybe their parents will not but they will configure it. I guess. I don't know if there is study about that. Project Manager: Yeah. Maybe it would be more complex to configure it to be simple {vocalsound} than creating a simple product. Industrial Designer: Mm-hmm. Marketing: Mm. Industrial Designer: Yeah. Yeah. User Interface: Mm. Industrial Designer: Yeah. Marketing: Mm. User Interface: And there are {disfmarker} another thing is that if we make something that's simple and easy to use that's bas to use immediately that means that it will be exactly the same as everything else. Industrial Designer: Yeah. User Interface: All right? Industrial Designer: Yeah. Yeah. User Interface: Otherwise, if it's different then of course everybody has {disfmarker} somebody has to learn to use it first. Project Manager: Yeah. Industrial Designer: Yeah. Project Manager: But also we we see that that most people find it {disfmarker} find remote controls too complex because they have too many buttons and they mainly use only channels and volume buttons. User Interface: Mm-hmm. Marketing: Mm. Project Manager: So we may just uh make a very easy to use remote control with mainly those buttons User Interface: Yeah. Project Manager: and maybe also um some lightning stuff too because most people find also hard to to find the remote control. Losed lose it etcetera. User Interface: Mm-hmm. Project Manager: These {disfmarker} these are {disfmarker} these two points are the main frustrations so maybe if we design something very simple and easy to uh find when lost it will uh add uh a serious competitive advantage without making something too complex and too long to develop. Marketing: Mm. User Interface: Mm-hmm. Project Manager: So but le let us see first the new requirement. So we don't have to {disfmarker} so this this uh is uh is um in the this is in the same direction as we were speaking so we don't have to make a very complex remote controls to access teletext and stuff like that {disfmarker} User Interface: But teletext is just one button. Project Manager: Yeah but then you have to {disfmarker} you have to define the buttons to surf amongst pages and stuff. User Interface: You you just write the {disfmarker} write the numbers. Project Manager: Yeah. So well {disfmarker} User Interface: So will you add with the channel keys, right? Project Manager: Yeah. So anyway we don't have to include this feature because it's it's not used any more by users, User Interface: So. {vocalsound} Project Manager: they prefer to s User Interface: I am. I'm sure that uh it don't like but uh I don't see just one button. Project Manager: Yeah. I dunno. User Interface: So. Project Manager: If i one button is still one more button. If {disfmarker} if {disfmarker} if we want to make it very simple we have to reduce number of buttons compared to th to our competitors. User Interface: Mm-hmm. Project Manager: Well anyway I have this point. We can discuss. Also um {disfmarker} so as as I told before uh it would be better if it's only for the T_V_ um because we want to be quick on on the market. And then also we have to make very uh uh clear that this uh this remote control is is part of of our products and show our corporate uh logo and and colours on the {disfmarker} on the design as well so that uh they identify it as one of our product. So this is the the key point. Marketing: {vocalsound} Project Manager: So before uh finishing we can uh define uh what would be the characteristic of the {disfmarker} o th of the control {disfmarker} of the remote control and which button do we need which function do we want etcetera. User Interface: Capital. Marketing: Mm. Project Manager: {vocalsound} So do you um so so from from the the Marketing Expert I think a key aspect is the easy to use aspect, it should be very simple and most button are never used Marketing: Mm. Project Manager: only ten per cent of the button are are used often so I think we have to do something very simple and I think we all agree on that point, no? Industrial Designer: Mm-hmm. User Interface: Well if it is going to be just a T_V_ remote control it is going to be very simple. Marketing: Mm. Project Manager: Yeah okay. So yeah the key point here is simple. Maybe. {vocalsound} So few buttons, channel, volume control and what el what function do you see in addition to that? User Interface: Well if it's going to be as simple as possible then just have the remote control, there is no other function that I can see Project Manager: Yeah. User Interface: really. Project Manager: Maybe switch T_V_ on and off {vocalsound} User Interface: {vocalsound} Uh Industrial Designer: {vocalsound} Project Manager: {vocalsound} User Interface: no you want to keep television on so that the advertising can {disfmarker} revenue can come back to us. Project Manager: {vocalsound} Yeah. Yeah. User Interface: Or something. Marketing: {vocalsound} Project Manager: Volume, maybe a mute button, and then on off button. Industrial Designer: Mm. Project Manager: And that's all? Industrial Designer: Uh I know that som you say that many people are doing plenty of {disfmarker} a lot of zapping. Project Manager: Mm. Marketing: Mm-hmm. User Interface: {vocalsound} Industrial Designer: I know tha I discovered that when I did a quick look, uh they do now som they do something which is quite nice now, User Interface: It's a memory, yeah. Industrial Designer: you have a button, you you press it, and this is uh the previous channel which has come back. Project Manager: Okay. Yeah this is cool. Maybe we can include that also. Previous previous channel button. So we have like channel button, the previous channel button, the volume button, plus a mute button, and uh just the the traditional on off button. Industrial Designer: Mm-hmm. Mm-hmm. User Interface: And uh and of course the channel changing buttons. Industrial Designer: Mm-hmm. {vocalsound} Project Manager: Yeah. I I talk about that, yeah? User Interface: How should they how should we implement that? Because uh could be numerical only or could be also incremental. Project Manager: Yeah. Incremental definitely because zapping you you switch them. Industrial Designer: {vocalsound} User Interface: Mm-hmm. Industrial Designer: Let's say that we can do something like that. This is uh incremental, but once you press it for a long time, you go five by five. Project Manager: We go faster? Industrial Designer: To go fa to go faster. Project Manager: Mm. User Interface: Mm. It's an interesting idea, that if you press it for a long time it does something else, in general. So if you you have your ten buttons for the {disfmarker} for the numeric the numerical buttons and you have {disfmarker} instead of having just one memory Industrial Designer: Yeah. Marketing: {vocalsound} User Interface: you have if you press them for a long time {disfmarker} No. Doesn't work {vocalsound} does it. Project Manager: Maybe we should have also a digit button {disfmarker} User Interface: Maybe we should have a complete keyboard and just type console commands. Project Manager: {vocalsound} Industrial Designer: {vocalsound} Project Manager: Yeah. User Interface: {vocalsound} Change channel to eight. Project Manager: {vocalsound} Maybe we have also to have digits or only incremental. User Interface: No. {vocalsound} Project Manager: I dunno bec because if you have the {disfmarker} User Interface: Well if it's useful like if if you want to change between three channels for example then you h you cannot {disfmarker} you cannot cannot work with just memory being incremental. Project Manager: Uh. Yeah. Because you have your previous channel button if you have incremental only it's not uh it isn't worth it because the previous channel is eith either minus one or plus one. Industrial Designer: Yeah. User Interface: Yeah. Project Manager: {vocalsound} So I think we need also digits. User Interface: Yeah. {vocalsound} Industrial Designer: Okay. Project Manager: Maybe we we can make very obvious the channel and volume button button and smaller button down there with the the digits. User Interface: Yeah when you zap usually you will have to press the same button all the time {vocalsound} Project Manager: Yeah. User Interface: and {disfmarker} Industrial Designer: {vocalsound} Or we can do something like that. User Interface: Yeah. Industrial Designer: We can design the remote control to have access. You know some remote control have uh protection Marketing: {vocalsound} User Interface: Mm-hmm. Industrial Designer: and so you you y Project Manager: Yeah. User Interface: Hey I just thought this thing {disfmarker} Industrial Designer: You {disfmarker} User Interface: there is a {disfmarker} I mean you know there is are some {gap} with a wheel like this. Instead of having the up down buttons for uh you can have the wheel. Project Manager: Yeah. Industrial Designer: Yeah, a kind of joystick. User Interface: {vocalsound} Yeah. Project Manager: Yeah. Maybe we can have a wheel for incremental. User Interface: {vocalsound} Project Manager: Okay. User Interface: Yeah. Project Manager: So have a wheel for incremental, have the digits uh on the lower side that can be closed so as you say protected, Industrial Designer: Mm-hmm. User Interface: Mm-hmm. Project Manager: {vocalsound} and uh yeah I think this is the basi User Interface: On the lower side I think it {disfmarker} you have to turn it. Project Manager: And do we {disfmarker} do we have a {disfmarker} User Interface: No? If we do that. Industrial Designer: Or a {disfmarker} or a ball, yeah, not a a wheel but a ball, and you say uh to {disfmarker} Project Manager: No, a wheel is better. User Interface: Yeah. Project Manager: I would say the wheel is better. User Interface: Because of that {vocalsound} Project Manager: What is the expert of uh {disfmarker} Industrial Designer: Yeah. User Interface: The channels change one by one. Industrial Designer: Yeah. User Interface: So you have to the user has to like to should feel the the the discrete sense a bit. Industrial Designer: Yeah, the {disfmarker} Project Manager: Okay. Industrial Designer: yeah. Project Manager: Yeah. That's a good idea. User Interface: Yeah. Project Manager: Well also we have to decide uh so it should be lightening in the dark I think because most people lost their remote control. Industrial Designer:'S quite {disfmarker} it's quite easy we do that w with back light on the {disfmarker} on the wheel. Project Manager: Okay. Marketing: {vocalsound} User Interface: Yeah. Even if i L_E_D_ uh or a if if it's the L_C_D_ feature uh Industrial Designer: A blue {disfmarker} a blue L_E_D_ and we sell that um. Project Manager: Yeah, User Interface: whatever, yeah. Project Manager: and do we put an L_C_D_ display? Marketing: {vocalsound} User Interface: Yeah. Project Manager: Because it was important for young customers if you remember. User Interface: {vocalsound} I think it's only put on if cou have multi function. If you do not multi function then there is no p point in having L_C_D_. Industrial Designer: Yeah. User Interface: Just increase the cost. Project Manager: Okay. User Interface: {vocalsound} The user does not have an advantage really. Project Manager: So no L_C_D_? Industrial Designer: Mm-hmm. Marketing: Hmm. Project Manager: And so no speech as well because it w it would delay too much the development process User Interface: Well if it's going to delay yeah Industrial Designer: Okay. User Interface: but uh {vocalsound} it will be cool. It would. Because a user could say C_N_N_ for example and it would go C_N_N_. Mm. Marketing: Mm-hmm. Project Manager: Yeah. That would be cool. But eight months is really long User Interface: Yeah. Project Manager: and {disfmarker} Maybe we can just uh {disfmarker} Industrial Designer: {vocalsound} Ten years {vocalsound} Project Manager: {vocalsound} User Interface: {vocalsound} Project Manager: Okay. So um I I will uh {disfmarker} I will {disfmarker} so I we will move to next meeting so in {disfmarker} after the lunch break uh here are the individual uh action uh you are required to do Industrial Designer: Mm-hmm. Project Manager: but you will be uh recalled to the actions by uh email I think but you can take notes if you {disfmarker} if you want but well the instruction will be sent. So thank you for uh your suggestion Industrial Designer: Okay. Project Manager: and I I will make a summary of that meeting that I will put in the shared folder you can't see {vocalsound} Industrial Designer: Okay. Okay. Marketing: {vocalsound} Project Manager: and uh and then you will um you will be able to to see what has been uh has been said on on this meeting and and what has been decided. Uh maybe for next meeting uh send me your presentation before by email so that we can see them uh altogether. That would be easier. User Interface: Yeah. Marketing: Mm. User Interface: What is the folder that you put yours in? And did it it did work? Marketing: {vocalsound} No. Project Manager: No it did not work. Marketing: No no. Project Manager: She send it to me by email. User Interface: Ah yeah. Mm. Marketing: I dunno, I dunno it. User Interface: Okay. Project Manager: So maybe this is better, to send it by email. Okay? User Interface: Okay what is your email? Project Manager: So yeah I I'm {disfmarker} it's in the first uh email so I'm participant one at AMI User Interface: At participant one. Okay. Project Manager: uh where is that, it's here. Participant one at AMI. {vocalsound} Okay. So see you after lunch break. User Interface: Well during lunch break actually. {vocalsound} Marketing:'Kay thank you. User Interface: So next time we should have a fight. Project Manager: {vocalsound} User Interface: How about uh {gap} management or something. Who happens to be your friend. Project Manager: {vocalsound} User Interface: {gap}

Marketing mainly focused on users'frustrations and expectations for remote controls. Apart from the requirements for a fancier look, shock-resistance, and limited but usable buttons, other expectations were also raised by users, such as the need to be found, to easily use, to light in the dark, and the need of ergonomic design. Moreover, LCD and speech recognition also seemed important, especially for people between 15 and 25 who watched TV a lot. Last but not least, the previous plan for a generic remote control for everything failed due to the imbalance between the long developing time and the timely need to be on the market.

qmsum

O'Donnell: Did you see that spaceship in the hangar? Yeah, we found it on the lake bed. O'Donnell: In here! Clara: Wait, are they saying something? The Doctor: The dark, the sword, the forsaken, the temple. These words actually rewrite the synaptic connections in your brain. They're coordinates. Someone is deliberately getting people killed and turning them into transmitters. I can save you. Clara: A new ghost, oh, no, no, no, no... [SCENE_BREAK] [ TARDIS ] [SCENE_BREAK] The Doctor: So there's this man. He has a time machine. Up and down history he goes, zip zip zip zip zip, getting into scrapes. Another thing he has is a passion for the works of Ludwig van Beethoven. And one day he thinks, "What's the point of having a time machine if you don't get to meet your heroes?" So off he goes to 18th-century Germany. But he can't find Beethoven anywhere. No-one's heard of him, not even his family have any idea who the time traveller is talking about. Beethoven literally doesn't exist. This didn't happen, by the way. I've met Beethoven. Nice chap. Very intense. Loved an arm-wrestle. No, this is called "The Bootstrap Paradox". Google it. The time traveller panics, he can't bear the thought of a world without the music of Beethoven. Luckily he'd brought all his Beethoven sheet music for Ludwig to sign. So he copies out all the concertos and the symphonies... and he gets them published. He becomes Beethoven. And history continues with barely a feather ruffled. But my question is this. "Who put those notes and phrases together?" Who really composed Beethoven's 5th? (Plays the opening of Beethoven's 5th Symphony) [SCENE_BREAK] [ Train Station ] [SCENE_BREAK] The Doctor: Where's Bennett? We need to get going. O'Donnell: Oh, he's still throwing up. "One small step for man, one giant... Bleaurgh." The Doctor: Time travel does that sometimes. O'Donnell: Somehow I doubt that Rose or Martha or Amy lost their breakfast on their first trip. The Doctor: You seem to know an awful lot about me. O'Donnell: I used to be in military intelligence. I was demoted for dangling a colleague out of a window. The Doctor: In anger? O'Donnell: Is there another way to dangle someone out the window? What year are we in? The Doctor: 1980. O'Donnell: So, pre-Harold Saxon. Pre-the Minister of War. Pre-the moon exploding and a big bat coming out. The Doctor: The Minister of War? O'Donnell: Yeah. The Doctor: Never mind. I expect I'll find out soon enough. Bennett: Sorry about that. Had a prawn sandwich. Might have been off. The Doctor: Don't worry. Shall we go? O'Donnell: Just one sec, I've just got something in my boot... O'Donnell: It's bigger on the inside, it's bigger on the inside, it's bigger on the inside. How can it be bigger on the inside, Bennett? OK, let's roll. [SCENE_BREAK] [ Town ] [SCENE_BREAK] Bennett: Why have we gone to Russia? The Doctor: Er, we haven't. We're still in Scotland. This is the town before it flooded. The TARDIS has brought us to when the spaceship first touched down. But here and now, it's the height of the Cold War. The military were being trained for offensives on Soviet soil. [SCENE_BREAK] [ Spaceship ] [SCENE_BREAK] O'Donnell: Oh, is that the pilot? God, look at size of it. The Doctor: No, that's the body. O'Donnell: What do you mean, "the body"? The Doctor: This isn't just any spaceship. It's a hearse. Bennett: The suspended animation chamber is still here, and the power cells for the engine. O'Donnell: And there are no markings on the wall. The Doctor: Yet. [SCENE_BREAK] [ EXT. Church ] [SCENE_BREAK] Prentis: Greetings! O'Donnell: It's him. That's the ghost from the Drum. Prentis: Remarkable. Oh, and humans, too... Albar Prentis, Funeral Director. Bennett: You're from Tivoli, aren't you? Prentis: The most invaded planet in the galaxy! Our capital city has a sign saying, "If you occupied us, you'd be home by now." The Doctor: Yes, I've had dealings with your lot before. I can't say I'm a fan. Prentis: No, we do tend to antagonise. The Doctor: What are you doing here? Prentis: Ah, yes. Of course. This... is the Fisher King. He and his armies invaded Tivoli and enslaved us for ten glorious years! Until we were liberated by the Arcateenians. But, thank the Gods, soon we'd irritated them so much, they enslaved us, too! Bennett: My first proper alien, and he's an idiot. Prentis: And now, in accordance with Arcateenian custom, I've come to bury him on a barren, savage outpost. O'Donnell: You mean the town? The Doctor: He means the planet. Prentis: Although, at the risk of starting a bidding war, you could enslave me. In the ship I have directions to my planet and a selection of items that you can oppress me with. The Doctor: Listen. We've come from the future. You're about to send some sort of signal. How do you do it? Is it a special pen? Prentis: What are you talking about? The Doctor: The technology you use, the thing that wrenches the soul out of the body and makes it repeat your coordinates for eternity. Give it to me now, I'm going to take the batteries out. Prentis: We don't have anything like that. Even this belongs to the glorious Arcateenians. The Doctor: So who sends the message? (He sniffs the Doctor) The Doctor: Back to the TARDIS. I need to talk to Clara. [SCENE_BREAK] [ Mess hall ] [SCENE_BREAK] Lunn: You've been here before, in situations like this before. Clara: Yeah, not exactly like... But, yeah, once or twice. Lunn: So you've had to deal with people who are scared. What do you say to them? I'm asking what I should say to you. Clara: That it will be all right. That the Doctor will save us. Lunn: And when you say it, do you believe it? Clara: Yeah. Yeah, I do. Lunn: And now? Lunn: Cass thinks the Doctor is saying something different to the others. He's saying... "Moran... Pritchard... Apprentice"? No, "Prentis... O'Donnell... Clara... Doctor... Bennett... Cass..." It's a list of all our names and when he finishes, he just goes back to the beginning again, over and over. That's it. Clara: Who's Prentis? (Phone rings) Clara: It's the Doctor. Lunn: He's alive? Clara: For the moment. Clara: Doctor? Doctor, are you all right? The Doctor (O.C.): Yeah, fine. So listen, the spaceship, it's a hearse. [SCENE_BREAK] [ TARDIS / Mess hall ] [SCENE_BREAK] The Doctor: Clara, what's wrong? Clara: Another ghost has appeared. The Doctor: What? Who? Has someone died? Clara: Doctor... it's you. Are you OK? The Doctor: Yeah. Well... currently. Clara: What does it mean? The Doctor: It means I die. Clara: No, not necessarily. We can change the sequence of events so... The Doctor: This isn't a potential future... this is the future now. It's already happened. The proof is right there in front of you. I have to die. Clara: No. You can change things. The Doctor: I can't. Even the tiniest change, the ramifications could be catastroph... could spread carnage and chaos across the universe like ripples on a pond. Ah, well, I've had a good innings. This regeneration is a bit of a clerical error anyway. I've got to go sometime. Clara: Not with me! Die with whoever comes after me. You do not leave me. The Doctor: Clara, I need to talk to you, just on your own. The Doctor: Listen to me. We all have to face death eventually, be it ours... or someone else's. Clara: I'm not ready yet. I don't want to think about that, not yet. The Doctor: I can't change what's already happened. There are rules. Clara: So break them. And anyway, you owe me. You've made yourself essential to me. You've given me something else to to be. And you can't do that and then die. It's not fair. The Doctor: Clara. Clara: No. Doctor, I don't care about your rules or your bloody survivor's guilt. If you love me in any way, you'll come back. Doctor, are you...? The Doctor: I can't save Moran or Pritchard. Clara: No... but like you said, if you can find out why this is happening, maybe you can stop them killing anybody else, you can save us. And you can stop it happening to you. The Doctor: I'll do what I can, but the future has already happened. We've just met the Undertaker and he's still alive. (He gasps) (Heavy footsteps) (Loud roar) The Doctor: So. Ghost me. You've got a better view than me. How do I look? Any signs of trauma, any scars? Any clues as to how I die? Clara: No, nothing. You're the same as all the other ghosts with the weird black eyes and... No. No, wait. Your coat. It's torn. The right shoulder. The Doctor: I assume I'm just saying... the same thing as the others. Clara: No. You're saying a list of names. Our names, mainly. "Moran, Pritchard, Prentis, O'Donnell, Clara, Doctor, Bennett, Cass." Who's Prentis? The Doctor: The mole-faced chap. The Doctor: What's the matter, Clara, what's happening? Clara: You've moved inside. You're inside here now. The Doctor: What am I doing? (He mouths) Clara: Er, nothing. You're, you're just standing there. The Doctor: I'm not trying to kill you? Why am I not trying to kill you? Clara: No, wait, you're moving, going toward the control panel. Clara: Oh, no. He's opened the Faraday Cage. He's let the other ghosts out. The Doctor: I need to talk to me now. Clara: Didn't you hear me? You opened the Faraday Cage. The other ghosts are outside. Shouldn't we be hiding? The Doctor: In a minute. I need to talk to the ghost me. Clara: OK, Doctor, you're on. The Doctor: Doctor. Such an honour. I've always been a huge admirer. This is really a delight. Finally someone worth talking to. So firstly, why are you here? The Doctor: Clara? Clara, what's happening? Clara: Erm... Er... you've just stopped. Oh, no, wait, you've started again. Cass via Lunn: His message has changed. He's saying something different. He's saying... Clara: What? The Doctor: What? Lunn: What? Cass via Lunn: He's saying... "The chamber will open tonight." The Doctor: Clara, now the ghosts are out, go to the Faraday Cage. They won't be able to get you in there. Oh, there's a problem. Clara: Problem? What problem? Oh, really? Because everything else is going so smoothly... The Doctor: The phone signal won't be able to get through. What you'll have to do... Clara, put the phone outside, and you can watch it through the little round porthole, and when you see it ringing, if it's safe to do so, go out and answer it. Clara: OK, how long are you going to...? The Doctor: Clara, listen to me. Don't let that phone out of your sight. I need to be able to reach you, I need to know everything my ghost does. Do you understand? I'll come back for you. I swear. [SCENE_BREAK] [ TARDIS ] [SCENE_BREAK] The Doctor: Come on. Oh, wait a minute. Not you, O'Donnell. O'Donnell: Why not? The Doctor: Someone needs to stay here and mind the shop. What if Clara calls? O'Donnell: The last bloke that said something like that to me got dangled out of a window. Bennett: Maybe the Doctor's right. Maybe it's best if you stay here. O'Donnell: Never going to happen. Seriously, have you two met me? [SCENE_BREAK] [ Spaceship ] [SCENE_BREAK] The Doctor: Prentis. Prentis! O'Donnell: Guess that dead body wasn't so dead after all. Bennett: And now we've got the writing. The Doctor: The Fisher King did it himself. The future is still coming. [SCENE_BREAK] [ EXT. Church ] [SCENE_BREAK] The Doctor: He's taken the suspended animation chamber to the church. (Huge roar) O'Donnell: What was that? The Doctor: We need to get back to the TARDIS. Now! (Louder roars and screams) [SCENE_BREAK] [ Building ] [SCENE_BREAK] The Doctor: It's cut us off. O'Donnell: Let's split up. Go on, Bennett. (Heavy breathing and footsteps) (Loud roar) The Doctor: No, Bennett! Wait! (O'Donnell moans) Bennett: Why did you come? You shouldn't have come. I mean, you never listen to anyone. It drives me mad. O'Donnell: To keep an eye on you, idiot. So, don't die. Bennett: Who's next on the list? Bennett: That list your ghost was saying, that's the order in which people are going to die, isn't it? I mean, I've only just figured that out. But you knew that all along, didn't you? Moran, Pritchard, Prentis, O'Donnell. The Doctor: I thought perhaps, because her ghost wasn't there in the future, like Prentis's was, I thought maybe... maybe it wouldn't happen. Maybe she stood a chance. Bennett: Yeah, but you didn't try very hard to stop her, though, did you? It was almost like you wanted to test your theory. So who's next? The Doctor: Clara. Bennett: Yeah. Yeah. Except now you're going to do something about it, aren't you? Yeah, because it's getting closer to you. You're going to change history to save yourself but not to save O'Donnell. You wouldn't save her. The Doctor: This isn't about saving me. I'm a dead man walking. I'm changing history to save Clara. [SCENE_BREAK] [ Faraday Cage ] [SCENE_BREAK] Clara: O'Donnell's dead. Clara: Oh, no. No, no, no, no, don't you dare... Don't you dare... [SCENE_BREAK] [ TARDIS ] [SCENE_BREAK] The Doctor: Big day for you. Time travel - twice! Bennett: Whoa, really? When are we going to? The Doctor: Off the map! Out of the rule book. What if I don't die? What if I refuse? I'm going to go back to the base and I'm going to save Clara, because that's what I do. And I don't see anyone here who's going to stop me. [SCENE_BREAK] [ Town ] [SCENE_BREAK] The Doctor: Why are we still here? The Doctor: No, no, no, no, no, no. Bennett: What? The Doctor: We've moved half an hour backwards. I'm locked in my own time-stream. The TARDIS won't let me leave. Bennett: What do we do? The Doctor: Now we have to keep out of sight, until time catches up. Later Doctor: Where's Bennett? We need to get going. Oh, he's still throwing up. Bennett: Prentis. He's alive. The Doctor: No, he's just not dead yet. And we don't tell him. Bennett: Yeah, but he's right there. I mean, we can just... The Doctor: No. However that sentence ends, no, we can't. Save him, and you'll want to save O'Donnell. You can't cheat time. I've just tried. You can't just go back and cut off tragedy at the root. Because you find yourself talking to someone you just saw dead on a slab. Because then you really do see ghosts. We don't tell him. Understand? Not a word. We don't have that right. [SCENE_BREAK] [ EXT. Church ] [SCENE_BREAK] Later Prentis: Albar Prentis, Funeral Director. Later Bennett: You're from Tivoli, aren't you? Later Prentis: The most invaded planet in the galaxy! Our capital city has a sign saying, "If you occupied us, you'd be home by now." Later Doctor: Yes, I've had dealings with your lot before. I can't say I'm a fan. Later Prentis: No, we do tend to antagonise! (Gunshot) Later Doctor: What are you doing here? Later Prentis: Ah, yes. Of course. [SCENE_BREAK] [ Faraday Cage ] [SCENE_BREAK] Clara: The dark. The sword. The forsaken. The temple. When we found out what the ghosts were saying, we weren't surprised because the words, they were already inside us. (He mouths) Clara: But you, you were, weren't you? You didn't know what the words were going to be. Lunn: No, I didn't. How did you know? Clara: Who was the one person who didn't see the writing in the spaceship? Lunn: Me. Cass wouldn't let me go inside. Clara: That's why the ghosts didn't hurt you when they had the chance. The message isn't inside you. Lunn: Yes, I suppose that makes sense. Clara: So you can get the phone back. Lunn: What? Lunn: She's saying I should go and get the phone back. Cass: No. Clara: Listen, listen. I need... We, we need to be able to contact the Doctor and you are the only one who can do this. Lunn: OK. No, she's right. Neither of you can get it back. Clara: What? What is it? What did she say? Lunn: It doesn't matter. Clara: Please. Lunn: She said to ask you whether travelling with the Doctor changed you, or were you always happy to put other people's lives at risk. Clara: He taught me to do what has to be done. You should get going. [SCENE_BREAK] [ EXT. Church ] [SCENE_BREAK] The Doctor: Oh. I need more time. It's too soon. I haven't saved her yet. Tick tock, tick tock, tick tock, tick tock, tick tock. I've got no choice now, I have to face the Fisher King. You, back to the TARDIS. [SCENE_BREAK] [ Outside the Faraday Cage ] [SCENE_BREAK] Clara: Look, he'll be fine, I promise. Clara: OK. Didn't need anyone to translate that. [SCENE_BREAK] [ EXT. Church ] [SCENE_BREAK] The Doctor: Now I'm ready. (Wind howls) [SCENE_BREAK] [ Basement ] [SCENE_BREAK] The Doctor: I've come from the future. I've seen the chaos you cause. The bloodshed. Fisher King (O.C.): Tell me what you have seen. The Doctor: Ghosts. Fisher King (O.C.): Ghosts? The Doctor: Souls wrenched from the dead. Repeating directions to here, to this spot, over and over. Fisher King (O.C.): How many ghosts do I create? How many? The Doctor: Four that I know of. Maybe five by now. Probably more since I left. Fisher King (O.C.): My ghosts will make more ghosts. Enough to bring an armada. Enough to wake me from my sleep. (Machine dings) (Machine beeps) (Indistinct hissing) (Rhythmic thumping) The Doctor: What will happen when your people arrive? Fisher King (O.C.): We will drain the oceans and put the humans in chains. The Doctor: This world is protected by me. Fisher King (O.C.): Yes. One man, lost in time. [SCENE_BREAK] [ Mess hall ] [SCENE_BREAK] Computer: Door emergency security lock. [SCENE_BREAK] [ Basement ] [SCENE_BREAK] Fisher King: The seed of their destruction is already sown. They will die. The message will be sent. My people will come, and you will do nothing to stop it, Time Lord. [SCENE_BREAK] [ Faraday Cage ] [SCENE_BREAK] Clara: Hey, no, no, no, no. Cass! Wait, what are you doing? Look, Lunn, he is going to be fine, I promise. We have to stay here. I know that look. I do that look. OK, fine. But we stick together. [SCENE_BREAK] [ Basement ] [SCENE_BREAK] Fisher King: Time Lords. Cowardly, vain curators who suddenly remembered they had teeth and became the most warlike race in the galaxy. But you, you! You are curious. You have seen the words, too. I can hear them tick inside you. But you are still locked in your history. Still slavishly protecting Time. Willing to die rather than change a word of the future. [SCENE_BREAK] [ Corridors ] [SCENE_BREAK] Clara: Lunn. Lunn. Lunn. Oh, Cass. Cass. Cass. Idiot. I'm an idiot. (Metallic scraping) [SCENE_BREAK] [ Basement ] [SCENE_BREAK] Fisher King: You will be a strong beacon. How many ghosts can I make of you? The Doctor: You know, you've got a lot in common with the Tivoleans; you'll both do anything to survive. They'll surrender to anyone. You will hijack other people's souls and turn them into electromagnetic projections. That will to endure. That refusal to ever cease. It's extraordinary. And it makes a fella think. Because you know what? If all I have to do to survive is tweak the future a bit, what's stopping me? Oh, yeah, yeah, yeah, yeah, the ripple effect. Maybe it will mean that the universe will be ruled by cats or something, in the future. But the way I see it, even a ghastly future is better than no future at all. You robbed those people of their deaths. Made them nothing more than a message in a bottle. You violated something more important than Time. You bent the rules of life and death. So I am putting things straight. Here, now, this is where your story ends. (Fisher King growls) [SCENE_BREAK] [ Corridor ] [SCENE_BREAK] (Machine beeps) (Metallic scraping) (Silence) (Metallic scraping) (Silence) (Metallic scraping) (Silence) (Metallic scraping) Clara: Oh, there you are. [SCENE_BREAK] [ Basement ] [SCENE_BREAK] Fisher King: There is nothing you can do. The Doctor: I've already done it. The words have gone. I got rid of them. The future I saw, none of that will happen now. The message will never contaminate my friends, no-one will die, no-one is coming to save you. That's the thing about knowing you're going to die. You've got nothing left to lose. (Fisher King growls) [SCENE_BREAK] [ Mess hall ] [SCENE_BREAK] Lunn: No, no, you've got to get out of here. The ghosts locked me in. It's a trap. Clara: Come on. Faraday Cage now! [SCENE_BREAK] [ Spaceship ] [SCENE_BREAK] Fisher King: The Time Lord... lied. (Device beeps) (Beeping intensifies) (Fisher King growls) (Rumbling) (Wind howls) [SCENE_BREAK] [ TARDIS ] [SCENE_BREAK] The Doctor (hologram): This is security protocol 7-1-2. The echelon circuit has been activated. Please stow any hand luggage and prepare for departure. [SCENE_BREAK] [ Main hangar ] [SCENE_BREAK] Clara: Back, get back. (Machine beeps) (Rumbling) (Fisher King growls) (Fisher King roars) The Doctor: Don't kiss me. Morning breath. Clara: Doctor? The Doctor: Follow me. [SCENE_BREAK] [ Bridge ] [SCENE_BREAK] (Fisher King roars) Clara: What's that noise? The Doctor: It's the call of the Fisher King. The call of their master. (Fisher King roars) Clara: Where are they going? So what was it? Your ghost. The Doctor: A hologram. Like the one we made of you to lure the ghosts into the Faraday Cage. With a soupcon of artificial intelligence, and a few prerecorded phrases thrown in. Uh... All beamed from the sonic glasses. As soon as you brought me and the chamber on board, it connected with the base's wi-fi and Bob's your uncle, you've got a ghost Doctor. Clara: Why did they only come out at night? The Doctor: They're electromagnetic projections that were out of phase with the base's day mode. Right. That's it. I've erased the memory of the writing. Though you might find you've lost a couple of other memories too. You know, like people you went to school with, or previous addresses or how to drink liquids. That's you two done. Where's Bennett? [SCENE_BREAK] [ Outside the Faraday Cage ] [SCENE_BREAK] Bennett: What will happen to them? The Doctor: UNIT will cut out the Faraday Cage with them inside and take it away. Then the space-hearse will be destroyed, so the writing doesn't infect anyone else. Bennett: What do I do now? The Doctor: I don't understand. Clara: I do. You keep going. You have to. Take it from me, there is a whole world out there. A galaxy, a life. What would O'Donnell have wanted? The Doctor: I need to erase that message from your mind, but it's fine, I'll do it later. Bennett: Lunn. Will you translate something to Cass for me? Lunn: Of course. Bennett: Tell her that you're in love with her and that you always have been. Lunn: What? Bennett: Tell her there is no point wasting time because things happen and then it's too late. Tell her I wish someone had given me that advice. Lunn: Oh, God. I was just passing on what he said. Please, don't feel... [SCENE_BREAK] [ TARDIS ] [SCENE_BREAK] Clara: What will UNIT do with the ghosts? The Doctor: Drag the cage into space, away from the Earth's magnetic field. With nothing to sustain them, the ghosts will eventually fade away. Clara: Here's what I don't understand. You did change the future. You stopped the Fisher King from returning. The Doctor: The Fisher King had been dead for 150 years before we even got here. But once I went back I became part of events. But here's the thing. The messages my ghost gave, they weren't for you, they were for me. That list. Everyone after you was random, but you being the next name, that's what made me confront the Fisher King. Clara: And saying the chamber will open? The Doctor: That was me telling me to get inside and when to set it for. Clara: Smart. The Doctor: Except that's not why I said them. Clara: How do you mean? The Doctor: I programmed my ghost to say them because that's what my ghost had said. And the only reason I created my ghost-hologram in the first place was because I saw it here. I was reverse engineering the narrative. Clara: OK, that's still pretty smart. The Doctor: You do not understand. When did I first have those ideas, Clara? Clara: Well, it must have been... Wow. The Doctor: Exactly. Who composed Beethoven's 5th? [SCENE_BREAK] Doctor, help me! Yes, you're the very next thing on the list. We are Vikings! (Cheering) You fight or you die. They're called the Mire. You will beg for mercy. The deadliest warrior race in the galaxy? One of them, yes, why? This village declared war on them. There's going to be a war and here's some news. We'll win the hell out of it. Why are you here? I'm the Doctor, and I save people.

The Doctor, Bennett, and O'Donnell land in 1980. They discover that the Tivolian, Prentis, is still alive, the writing has not yet been scratched into the wall, and Prentis landed on Earth to bury his former enslaver the Fisher King. The Doctor contacts Clara in 2119, who tells him the Doctor's ghost is mouthing their names. The ghost releases the others from the Faraday cage. In 1980, the Fisher King awakes, killing Prentis and writing the glyphs on the wall. O'Donnell is killed, confirming the suspicion that the Doctor's ghost is saying the order in which they will die. Attempting to save Clara, the Doctor confronts the Fisher King. He tells the Doctor the ghosts created by the glyphs will send a signal that will draw an armada. The Fisher King is drawn away from the chamber, and the Doctor destroys the dam wall, flooding the town. The stasis chamber opens in 2119 to reveal the Doctor inside; his "ghost" is a hologram, programmed by the Doctor. The hologram lures the ghosts back inside the Faraday cage. The plot is a bootstrap paradox -the Doctor programmed the ghost to say the names because that is what the ghost's message said.

summ_screen_fd

[Darren's (a/k/a Shanda Leer's) apartment. His arm and foot are in casts. His face is battered, bruised and swollen. His eye looks really gross. Justin has brought him some food from the diner.] Darren: Better not be a parakeet in there, Jane! Justin: Huh? Darren: Obviously you're not up on your gay film history. Whatever Happened to Baby Jane? Starting the two greatest bitch goddesses in the history of the human race, Miss Joan Crawford and Miss Bette Davis. 'Miss Crawford and I neeever got along.' Justin: I neeever saw it! Darren: I suppose there's no point in even mentioning Esther Blodgett. Justin: I know that one. It's A Star is Born, right? Darren: Oh, thank God! He really is gay! Justin: Eat. Darren: No, thanks. Justin: It's tuna salad, not parakeet. Darren: I'm not hungry. Justin: You're gonna need your strength. Darren: For what? The doctor says I might have a permanent limp. That should brighten up my act! Justin: What are you listening to the doctors for? After they stuffed my brain back in my head, they told me I'd never draw again. Darren: Well, do you? Justin: Yes. For about 15 minutes, then my hand starts to shake. So I use a computer. But the point is - Darren: You went on! Justin: Right. So will you. Darren: You're so reasonable. I keep hoping when they catch my bashers, they'll sentence them to life in prison and they'll get f*cked nightly by prisoners with AIDS. Justin: That's gruesome! Darren: It doesn't cost anything to dream! When it happened to you, weren't you angry? Justin: I tried not to think about it. Darren: I can't stop thinking about it. I see their faces, their boots smashing into me. I even hugged this one guy's foot to my stomach, hoping it would stop him from kicking me. It didn't. Justin: If you're still holding onto that boot, then you're still on the ground, taking it. Forget about it. Darren: Listen, if you'd been reduced to jerking off with your left hand, you'd want him dead, too! [Darren laughs. Justin smiles and shakes his head.] [At the diner, Michael is whining about pain from an impacted wisdom tooth.] Debbie: What's the matter baby? Tummy-ache? Ben: What is it, Michael? Migraine? Michael: Impacted wisdom tooth. Debbie: But you've have them all extracted. Michael: Emmett doesn't know that. What possessed me? Why did I ever agree to go with him? Debbie: Because you're a goodhearted human being who would do anything for his friend. Michael: Because I'm a lame-brained doormat who doesn't know how to say now! Ben: It'll only be a couple of days. They're supposed to be a very empowering and enlightening experience. Michael: Well, if they're so empowering and enlightening, why don't you go? Ben: I have classes. There's Hunter - anyway, Emmett asked you, not me. Michael: I'm not going. No way. No way. [Enter Justin, back from his visit with Darren.] Justin: (to Debbie) Darren asked me to thank you for all the food you sent him. Debbie: I'll get what I can do. Ben: How's he doing? Justin: I'm afraid it's gonna be awhile before Shanda Leer appears in public. Debbie: Well, at least he has one ray of sunshine in his life! Ben: Tell him we'll miss him at the Center tonight, but we're gonna make sure this never happens again. [Justin leaves, Emmett comes in. At the sight of Emmett, Michael starts to launch into his sick act. Emmett sits down at the booth with Michael and Ben.] Debbie: Are you ready to find your inner fairy? Emmett: I do. I just feel like Thinkerburt and Captain Hook. Michael: Em, I - Emmett: Before you say anything, as we all know, I've been going through some really tough times lately. I guess you could say my headlights are on low beam. But if this faerie frolic will turn the brights back on, well - what the f*ck. I'll give it a try. Debbie: More power to ya, sweetheart. Emmett: Whatever happens, the important thing is, I will always always be grateful that your son - your boyfriend - my best pal could be there with me, offering his love and support. (getting all choked up). [Deb and Ben give Michael pointed looks. Mikey's cornered and he knows it.] Michael: Well, we'd better get a move on. Otherwise these faeries won't get their wings. Emmett: Bye, honey. Debbie: Bye. Bye, honey. [she waves with her wings.] [Meanwhile, back at the rehab center, Ted's in group therapy. Once again, Blake is leading the group.] Ted: So it was actually a good thing I left here that night. See, my condo needed dusting and I don't really trust my cleaning lady and uh... It gave me time to think about where I should be and what I should be doing. So I came back. Although it hasn't been easy and every day's been a challenge - Blake: But you did it. Ted: And now it's time for me to leave with clear eyes and my head held high. I guess that's what happens when you have a great counselor. And your health insurance runs out. [That gets a laugh. Then one of the other patients mutters something.] Blake: What'd you say, Jonathan? Jonathan: You'll be back. Ted: Sure, for group, as an out-patient. Jonathan: I mean back using. And then back here. Woman in group: Jesus, Jonathan, can't you be positive? Jonathan: It doesn't mean I have to laugh at Billy Crystal here doing his stand-up. It's not gonna be any different for you than it is for the rest of us. Ted: You're wrong. Jonathan: You'll find out soon enough. It's not the same world you left when you came here. Ted: I'm gonna be alright. I'll make sure of it! Jonathan: Well, just in case - we'll leave the light on for ya. [Cut to naked Brian and Justin in bed. Brian is lying on the bed. Brian is on the phone. As he's talking, Justin comes over and shakes his wet hair on Brian. Brian grabs him and pulls him down on the bed.] Brian: It's an interesting offer. Headhunter: "It's a first-rate company, Mr. Kinney. You'd fit right in." Brian: Interesting in that they think I'd actually agree to it. Two-thirds of what I was previously making, a lesser title, and no profit-sharing until the fifth year. Headhunter: "Times are tough, Mr. Kinney. It's a job." Brian: You mean indentured servitude . Headhunter: "So you're passing?" Brian: You can tell them I've just taken a new position. Bye-bye. (Hangs up). Justin: I can't believe you just did that. Brian: Flipped you on your back? Justin: Flipped off that headhunter. Brian: I can do better. Justin: Doing what? Brian: Working for myself instead of other people. Justin: But all your clients turned you down. Brian: f*ck 'em. I'll get others. Justin: What if you can't? You'll be destitute. You'll have nothing. Brian: (laughs) Since when did you turn into a Jewish mother? Or Michael? Justin: You're just taking an awfully big risk, that's all. Brian: Well, what's one more? Besides, if I don't do this now, I never will. [Michael and Emmett at the faerie gathering. They're in the woods. There are lots of naked men wandering around in weird costumes.] Emmett: Something tells me we're not in Pittsburgh anymore. Michael: Something tells me we're not on the planet anymore. [They're greeted by a faerie who introduces himself as Periwinkle] Periwinkle: You two must be virgins! Emmett: I haven't been a virgin since I was 15. Periwinkle: Well, maybe out there but in here, your life as a faerie has just begun! I'm Periwinkle - the Queen Registrar and Official Greet-ar. Michael: Periwinkle? Periwinkle: It's my faerie name. Every when you has one... Emmett: It's strong about real names. Periwinkle: Real names are fine for the real world but this is a special place. This is a magical place were you leave your real life behind. At least for a little while. So, take your time, get some cloth. So get settle in to your cabin, dinners coming up. Oh, there is a middle massage in the year. You don't miss that. Michael: See? Emmett: If we back up slowly, no one will know we were there. Michael: Hey, you dragged me here. We're staying. What's the worse that could happen? Emmett: That? [He's looking at two naked men walking together holding hands in the woods with paintings of lizards, etc on their bodies.] [Slings & Eros, a shop specializing in adult toys.] Brian: We'll start with a new logo, launch an aggressive ad campaign with the locals and set up some high-profile sponsorships. Trainer: That sounds bigger than we can handle. Brian: It's simple branding. Trainer: Our customers aren't into branding. Tattoos, piercings maybe. Brian: I'm talking about a new image. Trainer: I adore makeovers, but our ad budget's only $200 a month. Brian: (Grimacing) Great! Then we'll run a lean campaign. No fat! Trainer: Just like our customers. Perfect! [Brian looks like he's getting a headache.] [Ted is leaving the rehab. Blake, of course is seeing him off.] Blake: Just between us, around here we refer to Jonathan as D&G. Ted: Dolce & Gabbana? Blake: No, doom and gloom. Ted: He doesn't bother me. Blake: That's good. To be honest, he's not entirely wrong. A lot of patients do wind up coming back here. Ted: Not me. Blake: That's good. Just remember what we talked about. Ted: Give myself time to adjust. Blake: Not just you, but people in you life are gonna need some time, too. [They get to the front door.] Blake: Here we are. Ted: I never thought I'd be so scared to see a door. Blake: Don't think of it as a door. Think of it as a threshold to your life. Ted: Right. Well, I want to thank you for everything. Blake: You don't have to thank me. You did it all. Ted: With your help. Listen, I was wondering if I could well, give you a call sometime. Strictly on a professional basis. If I suddenly find myself standing on a ledge with a rope around my neck or something. Blake: Or maybe just to say hello. [Ted nods. Blake writes down his digits. Ted gives Blake a hug.] Blake: Good luck. [Ted walks over the threshold of the rest of his life.] [Cut to Cynthia, Brian and Justin at the loft. Cynthia takes a poster out of a portfolio and holds it up.] Cynthia: Recognize this? Brian: Yes. Remsen Pharmaceutical account. My last campaign. Justin: Before the old warrior was unceremoniously escorted from the building, without so much as a ballpoint pen. [Brian mouths the words along with him] Justin: So what does this magic pill do? Brian: Well, it's guaranteed to turn you into a hottie. You can climb the Matterhorn. It also reduces your viral load. What are you showing it to me for? Cynthia: Vance is taking your idea and pitching it to Remsen tomorrow. Brian: Well, he should. He owns it. Justin: But you thought it up. Brian: Basic rule of advertising and eternal damnation: once you sell your soul to the Devil, he holds the copyright. Justin: So come up with another idea. Cynthia: Then you can steal the account from Vance and your former assistant along with it! Brian: Well that's an ingenious plan, Cynthia. But what pharmaceutical company is going to trust their wonder weapon to a one - man band working out of his house? Justin: It's not the size that matters! Brian: Have I taught you nothing? Justin: It's the vision! If you can win this account, then you'll be back on top. Brian: For once, I'll stick to the bottom. [He picks up the Slings & Eros sign as he speaks.] [Back to wacky wonkers in the woods - or Camp Faerie Peri is showing Em and Michael the layout of the camp.] Periwinkle: You have to draw up your civilian dress to become true faeries. So the magic doesn't work. [He's taking yellow ball gown an say to Michael] You looking enchanting in this. Michael: Strapless makes me look fat. Periwinkle: Well then use your own talents to come up with something fabulous. Every faerie has a god given gift to take odds ugly and makin' in something beautiful. You'll soon discovered when you can do. So, I really must fly. I wait for you at candleling. If you care to join me, it down by the River. Emmett: Where is the book? Periwinkle: Make a way to the yard. Michael: When I agree to come here with you, I didn't agree to look like my mother. Emmett: Well, maybe Periwinkle's right and we put something on and feel the magic. [Emmett wearing a green turban and animal print dress. Michael in a sleeveless denim jacket red plaid mini skirt! ] Emmett: We look like Virginia Maye in "Sweet Woman". You know just let's forfeit their registration fee, and go home? Michael: Now you tell me? You look completly and ugly... men#1: Hot. [Two men come out of nowhere.] Men#2: Totally hot. Michael: It's just something I picked off the rack. Piston: I'm Piston. Wolfen: And I Wolfen... Michael: I'm... Dumpling. Piston: Hello Dumpling. Wolfen: See ya later, Dumpling. Emmett: Dumpling? Were you get that? Michael: It's suddenly came to me. Men#3: Yee, yah, wanna play? [They play naked volleyball!] Michael: This must be the naked bubbly ball game. Emmett: You're intuition are remarkable. [Michael immediately runs to check it out, while Em stays behind by his own choice.] [Cut to the GLC. Ben is leading a community meeting about Darren's bashing.] Ben: Thank you for you all to coming up here. I do now the police are still working to find the mens who attacted Darren. Melanie: In the meantime we met our contact officer who's look out for any one matching descriptions. Ben: Keeping that in mind we're like to think how can Liberty Avenue be safer and enviroment for all of us. Debbie: I never though I say this but the first thing we need is more cops on the street. Mel: We're already talked to the new police chief. He said he do his best. Man#1: Yeah, sure. We're already heared that one before. Lindsay: Let's send a petition to the major to demending something we're done. Man#2: We're need more lighting, especially on the side streets. Ben: You're idea, we will take note of that. Woman: We should wear little whistles around our necks that way if someone attacks us, all we have to do is blow! [There's a whistle from the back of the room. It's Cody. He's angry.] Cody: Like that? Mel: Would you like to say something? Cody: Who do you think will come running? Them? They cant even defend themselves. The cops? If a bunch of homo haters wanna beat the sh1t out of you, you're not gonna stop them by blowing a little whistle. Ben: Then what do you suggest? Cody: We protect ourselves. Patrol the streets. Lindsay: You mean a vigilante group? Cody: Yeah! The Pink Posse! Mel: Isn't that taking the law into your own hands? Cody: Which law is that? The one that says you can't get married? That if they find out you're gay, they can fire your ass? That you can't adopt kids? Hell, they can even arrest you for f*cking! Ben: That was changed. Cody: Riiight. You can now copulate in Texas. Whoa, yippee-yi-yo-ki-yay! [laughter] Cody: You think anyone would dare call a black man a nigger? And try calling an Israeli a kike! Man, they'd blow your ass right off the face of the earth. But they have no prob calling us fags. Why? Because they can. Because they know we're all sissies, that we're too chickenshit to do anything. So go on. Sign your petitions and write your letters. And blow your little whistles. But nothing's going to change until you fight back. Until you learn to say Don't. f*ck. With. Me! [He strides out. Justin looks impressed.] [At the diner, Deb rushes over to show Mel and Lindz the headlines.] Debbie: Did you see this? They actually caught the f*cking bashers! Mel: Mmmh, it's fantastic. Lindsay: I think it's wonderful and what's Justin worked. Justin: It's all thanks to Darren's description. Lindsay: I bet, he must feel relieved. Justin: More like bloodthirsty. Debbie: Well, we all know there's nothing more terrifying than a bloodthirsty drag queen! [Suddenly, a voice from the not-so-distant past. It's Ted, fresh out of rehab.] Ted: Hey Deb. How about a cup of coffee? Debbie: Sure, honey. Have a seat... [she noticed it's Ted] Teddy, hi! How you doin'? Ted: I'm doin' great. Debbie: You do look good. Lindsay: How did it go? Ted: I just get to rehab. It was probably the most significant experience of my life. The first day I was there, I was totally resistant. Furious, ashamed, certain I didn't belong there. The second day, I started accepting help. I went to group and individual sessions at the end , sharing the pain. The third day... Debbie: Honey, I'd love to stay for the whole seven days, but I've got a grilled cheese that's about to turn to cement. Welcome home! [Lindz and Mel, who have been fidgeting and looking at their watches, make their excuses.] Lindsay: I've got to get back to the gallery. Mel: I'd love to hear more, but Gus is waiting at daycare. Bye! Ted: OK. Bye. [He turns to Justin, who is still there.] Ted: Don't you have somewhere to be? Justin: Thanks for reminding me. Welcome back! [And Justin leaves. But wait...Brian is sitting at the counter. ] Brian: Well, I for one think you're to be commended. Ted: (warily) Well, thank you, Brian. Brian: What you've accomplished is an amazing achievement. Ted: I wouldn't go so far as to - Brian: To sink so low, to hit bottom with such a resounding thud! Ted: You should be a guest motivational speaker in rehab. Brian: Of course, the good news is, once you've hit the bottom, you can't go any lower. So that means there's only way to go. And that way is...? Rhymes with...? (he raises his coffee cup) Ted: Up? [Brian pats him on the shoulder and leaves.] [At the gym, Brian is trying to drum up more business.] Brian: I have an corrospondation campaign with Torso. A new membership here and a 10% there and you have a box full of queers. Trainer: Sounds like an interesting idea. I'll get back to you. [Ben and Hunter come in.] Brian: Batman and the new Robin! Ben: Better not tell the old Robin that. I just got Hunter a membership. Hunter: (to Brian) Ya wanna hook up in the steamroom? Brian: (laughs) Ah - yeah. (To Ben) He should fit in here just fine. Ben: We did not come here so you could hit on the guys and that includes Brian. We came here so you could stay healthy. Hunter: Too late for that! Ben: No, it's never too late to get in shape. Brian: Yeah, you want to develop that six-pack before you drink it! I can use that for the ad! Hunter: Why work out when I can take some meds and - kapow! [He's looking at an poster advertising an HIV med, featuring a buff guy.] Ben: Because it's better to build your immune system naturally. Hunter: That guy looks pretty healthy to me. Ben: That's not what HIV looks like. And the meds don't fix anything. They buy you time if they work. And then there are dozens of side effects. Skin rashes, liver failure, cardiac arrest. Brian: It's a little early to be freaking the kid out, don't you think? Ben: I just don't want him believing some misleading ad that makes it seem like all you have to do is pop some pill and you're as good as new. Anybody who's ever taken one knows what a crock of sh1t that is. [Ben and Hunter go off to work out, leaving Brian looking thoughtfully at the poster.] [Back to the wild wonders of willies in the woods. Em's sitting at the water's edge, watching as two guys have at it on the opposite side of the bank. And Em's not even happy about the sightseeing opportunity. Enter Periwinkle again.] Periwinkle: Mushrooms? Emmett: Sure. Why not? [Em eats one. And what fallows is view after view of a hallucinating Honeycutt, wandering in distraction through the flora and fauna of Mother Nature's backyard. Finally he runs out and sees an older man who's busy putting rocks into a circle - for a group bon fire to be held that night.] Ariel: Looking for the naked twister competition? Emmett: All other I looking for. Ariel: And how about giving me a hand here? Emmett: What are you doin'? Ariel: Important work. Emmett: Moving rocks? Ariel: Depends on why you're moving on? Hey, help my place them in a circle. You a new faerie, aren't you? Emmett: Aren't here, suppose I am. Ariel: What's your name? Emmett: Emmett. Ariel: I don't mean your real name. Back there I'm Harry, but here I'm Ariel. So, what's your faerie name? Emmett: I don't though about one. Ariel: What stoppin' ya? Emmett: I don't know. Ariel: I do. You don't believe in fairies. You think this is all nonsense, don't ya? Emmett: No, I don't! Really. [Pause] Yeah, I do. Ariel: Well, maybe you stop thinking that way and the faerie spirit comes to you. But somehow I doubt it. You see, I can always tell you, you are not one of us - a proud faerie. Emmett: I have want to be a very proud faerie. Ask anyone who knows me! My flame burns bright. Ariel: Right now it wouldn't toast a marshmallow... Emmett: It maybe a little low. Ariel: I guess you be hurt by someone you loved. Now all you can feel is anger and pain. Emmett: How do you know? Ariel: The way you though like that aren't fit. Which isn't by the way very flattering. Emmett: Thanks. Ariel: Hey, give me that. Emmett: Very strong for a... Ariel: ...old faerie? It's called faerie power. It's giving to you to survive. Sometimes we forget we have it. That's why the catering was start it. To help us remember, to celebrate. Emmett: What a aim to renew my spirit with a little working. Ariel: Cause you start looking for isn't here. Emmett: Then where is it? Ariel: There. The circle is complete. [At home, Ted logs onto his laptop to check his email, which is all from Dr. Crystal, of course. He goes to the kitchen, opens a drawer and gets out his pipe. He's saved by the bell. Enter Lindsay and Mel with his mail, Droopidus Orchidus and some groceries.] Lindsay: Hi, can we come in? Ted: Uh. Yeah, yeah, sure. Mel: We wouldn't to run away. Lindsay: We were just in a hurry. Ted: It's okay. I understand. Mel: So, you're alright? Ted: Yeah, fine, fine. Don't remind me. Lindsay: We brought Droopy back. You rename him because he's not droppy anymore. Look, he got a new buttom. Let's pretend the lesbian care. Mel: And you're mail. Lindsay: We brought you a few things you may need. Mel: Milk, coffee. [As they're putting away the groceries, they see the pipe.] Ted: I was just getting rid of my triggers. Anything that reminds you of what you don't want to be reminded of. In my case, well, let's just say I should move to a desert island and start from scratch. I don't want you to think I was - because I wasn't - Mel&Lindsay: no, no, of course not! Nobody said you were! [They don't believe him. Ted doesn't even believe himself.] [At Vanguard, Gardner is unveiling his (Brian's) Endovir campaign to the client, Remsen. The theme is "Back on top" and it's a lot like that ad in the gym.] Gardner: What is the end of view? Is it a drug? A pill? Or the latest miracle of medical technology? Of course, it's all of these things and more. It is the promise of health, of a future bright with hope, of dreams fulfilled. Of being (Cynthia unveils poster) "Back on Top". So we've designed a campaign to accentuate the positive aspects of being positive. A campaign based on one simple word and that word is - [Brian walks in. He always did know how to make an entrance.] Brian: - Bullshit? Gardner: I was going to say optimism. How the hell you know about the meeting? Brian: I'm the one who set it up. Before I left. Remsen: I like what you've done. Downplayed the disease , emphasized quality of life. Brian: I should be so lucky as to have HIV. Then I, too, could go play volleyball with my shirtless, hunky buds. [Vance whispers in Brian's ear] Gardner: If you don't get the hell out or I call the security. Brian: Regretfully, Mr. Remsen, I must be going. But before I leave, I'd like - with your permission - to show you a new campaign that I've designed based on one simple word and that word is honesty. Ladies and gentlemen, the real face of HIV. Gardner: (reading) "Some days I feel like hell. But at least I'm still alive." - "I don't want to climb a mountain. I just want to live another day." - "Sure, there are side effects. But nothing I can't handle." Remsen: You've got to be kidding! Brian: It's strong medicine. I'll admit that . Remsen: We spent six years and $50 million developing this drug! I personally worked my ass off getting it through the FDA. You think I'm gonna throw it away on some ad campaign that tells people they're gonna feel like hell? Brian: They already know that! And they're willing to accept it, provided your drug can buy them another year, another month. That's all they're hoping for, that's all they want. And that's all you can honestly offer. Gardner: Our job is to make your drug as attractive as possible to the consumer. And that's exactly what we did. Brian: You mean what I did. This was my idea and let me be the first to say - it stinks. Having HIV may not be a ride in the park, but with Endovir it's not a death sentence. So why don't you say that? [Remsen looks thoughtful.] [At Darren's apartment, Justin reads him the news article about the arrest of his attackers.] Justin: Isn't that great? They finally caught the M*therf*ckers! Now all you have to do is finger them - metaphorically speaking of course - so they can rot behind bars for 20 years. Or, better yet, get gang raped nightly by prisoners with AIDS. Darren: I was thinking maybe Chanda should go blonde for her comeback. Justin: Didn't you hear me? Darren: Of course I heard you. Gang raped nightly by prisoners with AIDS. Where did you come up with such gruesome thoughts? Justin: So when's the lineup? Darren: If you're referring to what goes on in the backroom of a certain club, I don't engage in such activity! Justin: I'm referring to the police lineup. When are you going to identify them? Darren: I'm not. I don't really have a very clear memory of what they looked like. Justin: But you're the one who described them. Darren: I guess I've forgotten. Justin: Well, maybe seeing them again will jog your memory. Darren: Look, I already told the police, 'Sorry, wish I could help but I really can't. Thanks for asking.' Justin: (angry) If you don't point them out, they're just gonna walk. Darren: I really don't want to discuss this any further. Now what's for lunch. Justin: Chicken! [Darren gives him a look. Justin relents a little.] Justin: Darren, why did you change your mind? Darren: I guess I had time to think. And I decided cowardice is the better part of valor. Justin: Nothing is going to happen! Darren: (angry) How do you know that? Say I identify them. Say they go to trial. Say they get off. Say they come looking for me! Justin: Say they're back on the streets tomorrow because you didn't do anything! Say they attack someone else! Darren: Look, you're the one who told me to put all this behind me, to get on with my life! Yes! I do think blond! Justin: Maybe I was wrong. Maybe it's time we stood up for ourselves. Fought back! Darren: And when your attacker bashed you, and then they got off practically scot-free, what exactly did you do? [Justin has no answer for that.] [SCENE_BREAK] [Cut to Ted and Blake at the diner.] Ted: I know I said I'd only call you if I was on a ledge with a rope around my neck, but I figured a curb and a scarf would suffice. Blake: It's no problem, I told you you could call me anytime. More coffee? Ted: No thanks. If I was any more wired, I'd light up Times Square. Blake: Be patient. You've only been back for one day. Ted: And I'm already a wreck. Look, I have sweety palms. I make my friends nervous. They don't know what to say. They see me, they wanna flee. They don't trust me. The think I'm still using. Blake: Same thing happened with me. Eventually, they came around and even forgave me. Except one. Ted: Your mother? Your father? Blake: (shakes his head) The first guy who ever believed in me. I never really made amends to him. Ted: Did you try? Blake: We'd lost touch. But recently, we reconnected. Maybe now I'll get my chance. [Cut to angry Justin drawing at the loft, crumpling up pictures in frustration. His hand is bothering him.] Brian: Since when did our heroes become the merry butchers of Gayopolis? Justin: Someone has to do it, since fags are too cowardly to stand up for themselves! Brian: Somebody's pissed off. Justin: Well, you would be too if you'd got your head bashed in. Brian: I know, I was there. I thought you'd put that behind you and moved on. Justin: I don't wanna talk about it. [Brian strokes his hair. Justin takes his hand and puts it away.] Justin: Darren refused to identify his attackers. They're gonna get off. When I told him to be brave, stand up for himself, he said to me, what did you do? I was a coward. I should have done something and I didn't. Brian: Well, you wanna get even? I'll tell you how to get even Become the biggest f*cking success you can possibly be. Justin: (disgusted) I already know. Brian: Well if you know, take that anger and put it into your work. Use it! Have more money, more power, more s*x than any poor hetero schmuck because trust me, nothing pisses off a straight guy more than a successful fag. Justin: You know Guernica? People say it's the most powerful anti-war statement ever made. I say bullshit. It hangs in a f*cking museum, collecting dust. And this is all bullshit. It doesn't do a motherfucking thing. [He crumples up the drawing he was working on and stomps off.] [Back to Blake and Ted. They're at Ted's apartment. Blake is helping Ted dispose of his "triggers." p0rn. f*ck clothes. A picture of some opera guy.] Ted: f*ck clothes! Ah, they stink! And these. Or my p0rn books and my p0rn tapes. Blake: Wait! How's Giuseppe Verdi a trigger? Ted: He was here watching the whole time. Blake: He was also here the first time you played La Traviata for me. I say we give him a reprieve. That it? Ted: Almost. [He gets the laptop.] Blake: You sure you wanna trash that? Ted: Never gave me anything but trouble. Hookups, p0rn sites, not to mention endless e-mails to increase my dick size. Blake: I don't recall that being one of your shortcomings. Ted: Even if I got rid of every trigger, every reminder, I'd still remember what I did. Blake: You gonna be alright? Ted: To tell you the truth, after rehab where they never left you alone, I'm feeling a little freaky being here by myself. Blake: I could stay with you, if you'd like. Ted: No, I couldn't ask you - Blake: If it'd make you feel better. Ted: Well, uh, where do we, uh - Blake: I'd crash on the sofa. Ted: Of course. The sofa - uh, I'll get you some blankets. [He goes into another room to gets some blankets.] Ted: Sleep tight. Blake: Yeah, you too. And if you need anything - [Ted nods. Goes into bedroom, closes the door.] [Back in the woods. It's a group circle bon fire. Everyone's passing a stick around and repeating, "I feel the power." Em rushes into the group] Emmett: Hold it! I take that, Dumpling. I feel the power, to. My name is "On A Clear Day You Can See Forever." It just overcame to me. You know what I mean. But you can call me Clear Day. Man#1: This is a heart circle, Clear Day. We share our feelings here. Is there anything you like to share? Emmett: Well, the truth is I donna want really come to here. But then somehow something magical happened. I realized that if it's true that faeries have the ability to create beauty out of ugliness and joy out of pain then that power come from one place. Which I know it's come from all along. Man#1: Thank you. Rise faeries. We thank the powers of the Earth and the Heavens for giving us our special gifts. We celebrate our faeries forfathers and the generations to follow. And our faerie folder Harry. Emmett: [whispers to Periwinkle] Where is he, anyway? Periwinkle: Harry passed away two years ago. [Cut to a focus group watching Brian's old ad and Brian's new ad. Apparently, First, the "old" ad.] moderator: So, how do you think about these ads? When you see this in the newspaper and magazine or on a billboard. Would you make those to ask your doctor to an interview? Man#1: Sure, if he gave me his phone number. woman#1: Pretty much the same old, same old. Man#2: Hunky white dudes having fun. Man#3: I get the message of hope. Man#1: And being on top. moderator: What about these? [She brings out Brian's new ads.] Man#1: You gotta be kidding! Woman#1: I don't believe it. Man#3: Who would run ads like these? Woman#1: It's the truth. Some days I do feel like hell. Man#1: But actually see that? Man#3: But you think you're ever gonna see that? Man#2: That is one ad you'll never see. Man#3: They wouldn't have the balls. [Brian and Remsen are watching the whole thing through a one-way mirror. Obviously Remsen was sufficiently impressed by Brian's presentation to take it one step further.] [Cut to Babylon and an overly long system of Emmett dancing. Apparently, his flame was ignited around the campfire at the faerie gathering and is once again burning brightly. At the bar, Brian, Michael and Ben look at Michael's pictures from the faerie gathering.] Michael: This is me and Wolfen and Periwinkle. And there is Emmett, I mean "Clear Day". Ben: Don't forget the cutie in the kilt. Brian: I think I got these on your Christmas Card. Ben: Didn't I say to you it isn't a life-time experience, Dumpling? Brian: Dumpling? Michael: That's my faerie name. And if you ever say about this... Emmett: Cosmo, please. Brian: Why did you do that? Emmett: Just try to spreat the magie. Ben: Maybe next year we all should go. Michael: Yeah! Emmett: Absolutely! Ben: Brian, how about you? Brian: I'd rather have my tongue super-glued to a lesbian's twat. Besides, I'm gonna be way too busy with my new business. And my new account! Michael: The Circus of p0rn account isn't exactly going to buy you a summer home in P-town. Brian: But the Remsen Pharmaceuticals account will! Here's to Kinnetic! Drinks are on me, boys! All: To Kinnetic! [Meanwhile, at Woody's, Justin is drinking alone, looking morose. He walks over to Cody, who's is at a table with his crew, doing what he does best: being angry.] Cody: Well, if it isn't Meg Ryan! Justin: I heard what you said at the Center and I agree. Cody: (unimpressed) That's nice. (Goes on talking to his gang) You need to understand, it's not about being a victim - Justin: It's about not allowing yourself to be victimized. [Now Cody realizes that Justin gets it. Justin is allowed to sit down with the cool kids.] Cody: (extends his hand) Cody Vail. Justin: (shakes it) Justin Taylor. Cody: Ready to kick some straight ass? [Black Screen.]

Brian decides to start his own ad agency. Ted struggles to re-enter life after rehab. Emmett and Michael discover their inner Faerie. Justin encourages a friend to fight back against his bashers.

summ_screen_fd

Cancer poses danger because of its unregulated growth, development of resistance, and metastatic spread to vital organs. We currently lack quantitative theory for how preventive measures and post-diagnostic interventions are predicted to affect risks of a life threatening cancer. Here we evaluate how continuous measures, such as life style changes and traditional treatments, affect both neoplastic growth and the frequency of resistant clones. We then compare and contrast preventive and post-diagnostic interventions assuming that only a single lesion progresses to invasive carcinoma during the life of an individual, and resection either leaves residual cells or metastases are undetected. Whereas prevention generally results in more positive therapeutic outcomes than post-diagnostic interventions, this advantage is substantially lowered should prevention initially fail to arrest tumour growth. We discuss these results and other important mitigating factors that should be taken into consideration in a comparative understanding of preventive and post-diagnostic interventions. Mathematical models play an important role in describing and analysing the complex process of carcinogenesis. Natural selection for increases in tumour cell population growth can be represented as the net effect of increased cell division rates and/or decreased apoptosis (e. g., Wodarz and Komarova, 2007). Relatively rare driver mutations confer such a net growth advantage, whereas numerically dominant passenger mutations with initially neutral or mildly deleterious effects (Marusyk et al., 2012; Bozic et al., 2013; McFarland et al., 2013) can increase in frequency due to genetic hitchhiking or subsequent positive selection. Amongst the many passengers in a growing tumour, some can contribute to chemoresistance, and sufficiently large tumours could contain different clones that, taken as a group, can resist some, if not most, chemotherapies (see Michor et al., 2005 for resistance to imatinib). Chemotherapeutic remission followed by relapse suggests that these resistant cells are often present at low frequencies prior to therapy, either due to genetic drift or costs associated with resistance. Resistant phenotypes subsequently increase in frequency during radiotherapy or chemotherapy, and through competitive release they may incorporate one or more additional drivers, resulting in accelerated growth compared to the original tumour (for related discussion on pathogens, see Huijben et al., 2013). Previous mathematical studies have considered alternatives to attempting to minimize or eradicate clinically diagnosed cancers with maximum tolerated doses (MTDs) of chemotherapeutic drugs. This body of work indicates that MTD is particularly prone to select for chemoresistance (e. g., Foo and Michor, 2009; Foo and Michor, 2010; Lorz et al., 2013), and what little empirical work exists supports this basic prediction (Turke et al., 2010), but see (Kouyos et al., 2014) for other disease systems. Numerous alternatives to the goal of cancer minimization/eradication have been proposed and investigated (e. g., Maley et al., 2004; Komarova and Wodarz, 2005; Foo and Michor, 2009; Gatenby et al., 2009a, 2009b; Bozic et al., 2013; Jansen et al., 2015). For example, Komarova and Wodarz (2005) considered how the use of one or multiple drugs could prevent the emergence or curb the growth of chemoresistance. They showed that the evolutionary rate and associated emergence of a diversity of chemoresistant lineages is a major determinant in the success or failure of multiple drugs vs a single one. Lorz and co-workers (Lorz et al., 2013) recently modelled the employment of cytotoxic and cytostatic therapies alone or in combination and showed how combination strategies could be designed to be superior in terms of tumour eradication or managing resistance than either agent used alone. Foo and Michor (2009) evaluated how different dosing schedules of a single drug could be used to slow the emergence of resistance given toxicity constraints. One of their main conclusions is that drugs slowing the generation of chemoresistant mutants and subsequent evolution are more likely to be successful than those only increasing cell death rates. These and other computational approaches have yet to consider the use of preventive measures to reduce cancer-associated morbidity and mortality whilst limiting resistance. Prevention includes life-style changes and interventions or therapies in the absence of detectable invasive carcinoma (e. g., Etzioni et al., 2003; Lippman and Lee, 2006; William et al., 2009; Hochberg et al., 2013), for example, reduced cigarette consumption (Doll and Peto, 1976) or chemoprevention (Steward and Brown, 2013). In depth consideration of preventive measures and their likely impact on individual risk and epidemiological trends is important given the likelihood that all individuals harbour pre-cancerous lesions, some of which may transform into invasive carcinoma (Bissell and Hines, 2011; Greaves, 2014), and concerns as to whether technological advances will continue to make significant headway in treating clinically detected cancers (Gillies et al., 2012; Vogelstein et al., 2013). Here, we model how continuous, constant measures affect tumour progression and the emergence of resistant lineages. We assume that an individual can contract at most a single cancer, originating from a single lesion. Importantly, we consider cases where the measure may select for the evolution of resistant phenotypes and cases where no resistance is possible. Our approach is to quantify the daily extent to which a growing neoplasm must be arrested in order to either eradicate it or to delay a potentially lethal cancer. Several authors have previously argued how constant or intermittent low toxicity therapies either before or after tumour discovery could be an alternative to MTD chemotherapies (Wu and Lippman, 2011; Hochberg et al., 2013), but to our knowledge, no study has actually quantified based on empirical parameter estimates, the extent to which cancer cell population growth needs to be arrested for such approaches to succeed (see related discussion in Bozic et al., 2010; Gerstung et al., 2011; Bozic et al., 2013). Below we employ the terms ‘treatment’, ‘measure’, and ‘therapy’ interchangeably, all indicating intentional measures to arrest cancer cell population growth. We first derive analytical expressions for the expected total number of cells within a tumour at any given time. We explore dynamics of tumour sizes at given times, and times to detection for given tumour sizes. Specifically, we show that the expected mean tumour size in a population of subjects can be substantially different from the median, since the former is highly influenced by outliers due to tumours of very large size. We then consider constant preventive measures and show that treatment outcome is sensitive to initial conditions, particularly for intermediate-sized tumours. Importantly, we provide approximate conditions for tumour control both analytically and numerically using empirical parameter estimates. We next consider post-diagnostic interventions in which tumour resection either is not complete and leaves residual cells or undetected metastases are present. We contrast these with prevention scenarios where (1) there is no difference in the age at which either prevention or post-diagnostic intervention commences, and (2) prevention and post-diagnostic interventions are alternatives, that is, the former always occurs before the latter. We show as expected that therapeutic outcomes are generally superior under prevention vs post-diagnostic intervention, and that higher impacts on the cancer cell population are usually required for post-diagnostic interventions to achieve a level of control comparable to prevention. Moreover, we find that should resection leave sufficiently large numbers of residual cells (or metastases are not discovered), then a range of the most successful outcomes under prevention is not attainable under post-diagnostic intervention, regardless of potential cell arrest. Finally and importantly, whereas there is little gained in terms of outcomes in post-diagnostic intervention beyond approximately 0. 3% cell arrest per day for both small (10,000) and large (1 million) cancer cell populations, prevention outcomes may achieve continual gains for the latter cell number, up to about 0. 6% cell arrest per day. Previous study has evaluated the effects of deterministic and stochastic processes on tumour growth and the acquisition of chemoresistance (Komarova and Wodarz, 2005; Bozic et al., 2010; Reiter et al., 2013, see review Beerenwinkel et al., 2015). We first consider both processes through exact solutions and numerical simulations of master equations, using the mean field approach (see Appendix 1 for details). A mean field approach assumes a large initial number of cells (Krapivsky et al., 2010) and averages any effects of stochasticity, so that an intermediate state of the system is described by a set of ordinary differential equations (i. e., master equations; Gardiner, 2004). Solutions to these are complex even in the absence of the explicit consideration of both drivers and passengers (Antal and Krapivsky, 2011; Kessler and Levine, 2013). We do not explicitly model the different pre-cancerous or invasive carcinoma states. Rather, our approach follows the dynamics of the relative frequencies of subclones, each composed of identical cells (Baake and Wagner, 2001; Saakian and Hu, 2006). We simulate tumour growth using a discrete time branching process for cell division (Athreya and Ney, 1972; Bozic et al., 2010). For each numerical experiment, we initiate a tumour of a given size and proportion of resistant cells. Briefly, the model framework is as follows. Each cell in a population is described by two characteristics. The first is its resistance status to the measure, which is either ‘not resistant’ (j = 0) or ‘resistant’ (j = 1). The second property is the number of accumulated driver mutations (maximum N) in a given cell line. At each time step of 4 days, cells either divide or die, and when a cell divides, its daughter cell has a probability u of producing a driver mutation and v of producing a resistant mutation. We assume no back mutation, and that cells do not compete for space or limiting resources. The fitness function fij, the difference between the birth and death rates of a cell, is defined by the number of accumulated drivers (i = 0,1, …, N) and resistance status (j = 0,1): a sensitive cancerous cell with a single driver has selective advantage s, and any accumulated driver adds s to fitness, while resistance is associated with a constant cost c. Exposure to a single treatment affects only non-resistant cells (j = 0), incurring a loss σ to their fitness. Thus, the fitness function is: fij=s (i+1) −σ (1−j) −cj. The assumption of driver additivity is a special case of multiplicative fitness, and both are approximately equivalent for very small s. We conducted numerical experiments, each with the same initial states but each using a unique set of randomly generated numbers of a branching process. For each simulation and each time step, the number of cells at time (t + 1) was sampled from a multinomial distribution of cells at time t (see Bozic et al., 2010 for details). Table 1 presents baseline parameter values employed in this study. Hereafter, we refer to σ as the treatment intensity (applied once every cell cycle of 4 days), while the corresponding daily arrest level to non-resistant cells is approximated by σ/4. 10. 7554/eLife. 06266. 024Table 1. Baseline parameter values used in this studyDOI: http: //dx. doi. org/10. 7554/eLife. 06266. 024ParameterVariableValueRangeRef. Time step (cell cycle length) T4 days3–4 days (Bozic et al., 2010) Selective advantages0. 4%0. 1–1. 0% (Bozic et al., 2010) Cost of resistancec0. 1%Mutation rate to acquire an additional driveru3. 4 × 10−510−7–10−2 (Bozic et al., 2010) Mutation rate to acquire resistancev10−610−7–10−2 (Komarova and Wodarz, 2005) Maximal number of additional driversN5 (Figures 1,2) 9 (other figures) 0–9Initial cell populationM0106 cells–Pre-resistance levelκ0. 01%– (Iwasa et al., 2006) Number of replicate numerical simulations (excluding extinctions) –106–Detection thresholdM109 cells107–1011 (Beckman et al., 2012) ‘Range’ is values from previous study and employed in the present study. We first study preventive interventions where a patient has a high risk of developing a cancer and/or a biomarker that indicates the probable presence of a cancer. In either case, so that we can compare and contrast different intervention levels, we assume that the (undetected) tumour contains M0 cells when prevention commences. We examine effects on the mean by considering the distribution of tumour sizes at different times using mean-field dynamics (see Appendix 1). Numerical experiments were then conducted by assuming that tumours initially contained M0 = 106 identical cells (i = 0), of which 0. 01% were resistant. These assumptions are obviously oversimplifications, and we relax some of them below and in the next sections. There is an excellent correspondence between analytical and numerical results for σ varied in range of s (Appendix 1—figure 1A). A more detailed study of the distribution of tumour sizes reveals that the mean diverges considerably from median behaviour in the majority of cases, since the former is strongly influenced by outliers with high-tumour cell numbers (see Appendix 1—figure 1B). Figure 1 shows four examples of numerical experiments. An untreated tumour reaches the assumed detection threshold of 109 cells by about 18 years on average and because it is not subject to strong negative selection (we assume low c), any emerging resistant cell-lines are likely to remain at low frequency (0. 03% at the detection time in the example of Figure 1A). In Figure 1B, low-treatment intensity delays tumour growth and thus time of detection by approximately 16 years, while an increase in dose tends to result in tumours dominated by resistant cells (Figure 1C). Despite being unaffected by treatment, resistant cell populations are sometimes observed to go extinct stemming from stochasticity (Figure 1D), and this tends to occur more at high-treatment levels, because there are fewer sensitive tumour cells to seed new (mutant) resistant cell populations. 10. 7554/eLife. 06266. 003Figure 1. Treatments curb or eliminate tumours. Examples of single patient tumour growth for (A) no treatment. (B) σ = 0. 6%. (C) σ = 1. 0%. (D) σ = 2. 0%. The shaded area shows the change in total tumour size and the hatched area, the resistant part of a tumour. The treatment intensity σ in this and all other figures are represented as cell arrest per day (σ/4). Parameter values as in Table 1. DOI: http: //dx. doi. org/10. 7554/eLife. 06266. 003 We next considered how therapies affected the distribution of tumour detection times in cases where the cancer cell population attained a threshold of 109 cells. The magnitude of the selective advantage s shows that tumour growth is largely driven by its non-resistant part for relatively low-impact treatments σ < 2s (Figure 2A). Importantly, the tumour shifts from being mainly non-resistant to resistant at σ ≈ 2s, which is reflected by the inflection point in the trajectory of the median (indicated by point B in Figure 2A, B). Notice that detection times are also most variable at σ ≈ 2s. The median changes smoothly at high-treatment levels (σ > 2s), tending to a horizontal asymptote. This is explained by the fact that the sensitive part is heavily suppressed at high-treatment levels, meaning that the dynamics are strongly influenced by the actual time point at which the first resistance mutation occurs. 10. 7554/eLife. 06266. 004Figure 2. Treatment level affects both detection time and frequency of resistance. The median (lines) and 90% confidence intervals (shaded areas) of detection times, measured as years beyond the initiation of the preventive measure. Effects of: (A) the selective advantage of each additional driver and (B) the cost of resistance. (C) Samples of the distribution of detection times (in relative frequencies, adjusted for 3-month bins) for corresponding points, indicated in A and B. Dashed black line is the mean and the dashed-and-dotted line is the median. The colour-code indicates the average level of resistance in detected tumours over 3 month intervals (see inset in B). All cells j = 0 at t = 0. Other parameters as in Table 1. Detection time is log-transformed in A and B. DOI: http: //dx. doi. org/10. 7554/eLife. 06266. 00410. 7554/eLife. 06266. 005Figure 2—figure supplement 1. Sensitivity analysis for several key parameters. (A) Maximal number of additionally accumulated drivers. (B) Initial cell number. (C) Level of initial partial resistance of a tumour. (D) Presence or absence of resistant cell-lines. Point colour-codes indicate the average level of resistance in detected tumours over 3 month intervals (see inset in B). For simplicity, only the median is indicated in B and C for the baseline case (blue line). Lines and shading otherwise as in Figure 2. Unless otherwise stated, parameter values as in Table 1. DOI: http: //dx. doi. org/10. 7554/eLife. 06266. 00510. 7554/eLife. 06266. 006Figure 2—figure supplement 2. Effects of initial neoplasm size (A, B) and resistance level (C) on preventive measure success. Success is defined as tumour non-detection by 50 years. Daily effect of treatment on cellular arrest is assumed to be 0. 25%. Unless otherwise stated, parameter values as in Table 1. DOI: http: //dx. doi. org/10. 7554/eLife. 06266. 006 We find, counterintuitively, that early-detected tumours are more likely to be resistant under constant treatments than those detected at later times (A, B, and C in Figure 2C). This is because tumours under treatment that by chance obtain resistance early grow faster than those that do not. By the time of detection, non-resistant tumours usually accumulate up to 4 additional drivers on average, while resistant tumours have fewer. For larger values of cost c, an additional non-regularity emerges at σ ≈ 3s (segment DEF in Figure 2B), and is associated with tumours having a majority of cells with maximum numbers of drivers. This region is also characterized by a different transition to complete resistance (cf. Videos 1,2 for relatively low and high costs of resistance, respectively). For example, at point D, tumours with a majority of non-resistance have less variable detection times than tumours with a majority of resistant cells (points E and F in Figure 2B and corresponding panels in Figure 2C). Treatment levels along the segment DEF result in tumours that are more likely to be resistant as one goes from the centre to the tails of the distribution of detection times. This differs qualitatively from the previous case of a lower cost of resistance, where the tumours are less resistant in the tail of the distribution of detection times (cf segments ABC and DEF in Figure 2B and corresponding panels in Figure 2C). 10. 7554/eLife. 06266. 007Video 1. Treatment level affects both detection time and frequency of resistance. (A) The median (thick line) and 90% confidence intervals (shaded areas with dashed boundaries) for the distribution of detection times. (B) Arbitrary samples of the distribution of detection times and distribution of the mean number of accumulated drivers. The colour-code indicates the average level of resistance in detected tumours over 3 month intervals. Parameters as in Table 1. DOI: http: //dx. doi. org/10. 7554/eLife. 06266. 00710. 7554/eLife. 06266. 008Video 2. Treatment level affects both detection time and frequency of resistance. (A) The median (thick line) and 90% confidence intervals (shaded areas with dashed boundaries) for the distribution of detection times. (B) Arbitrary samples of the distribution of detection times and distribution of the mean number of accumulated drivers. The colour-code indicates the average level of resistance in detected tumours over 3 month intervals. Parameters as in Table 1 except for the cost of resistance c = 0. 4%. DOI: http: //dx. doi. org/10. 7554/eLife. 06266. 008 The inflection point at σ ≈ 2s in Figure 2A is due to the accumulation of additional drivers within tumours and associated increases in the likelihood that the tumour eventually resists treatment. Since the initial population consists of 106 cells, in the absence of treatment, a mutant cell with one additional driver and associated fitness 2s will appear very rapidly. Such a tumour can be suppressed only if σ > 2s. This is supported by additional numerical experiments where we vary the maximal number of additional driver mutations N: the inflection point σ ≈ 2s disappears when N = 0 (Figure 2—figure supplement 1A). The inflection points at σ = 3s, 4s emerge at treatment levels that effectively suppress sensitive subclones with the most drivers before resistance mutations are obtained (cf Figure 2—figure supplement 1A–C with Figure 2—figure supplement 1D and Video 3). Specifically, the peaked distributions, corresponding to better therapeutic outcomes, tend to disappear when resistant subclones emerge. 10. 7554/eLife. 06266. 009Video 3. Treatment level effects on detection times assuming no resistance is possible. (A) The median (thick line) and 90% confidence intervals (shaded areas with dashed boundaries) for the distribution of detection times. (B) Arbitrary samples of the distribution of detection times and the distribution of the mean number of accumulated drivers. The colour-code indicates the average level of resistance in detected tumours over 3 month intervals. The resistance mutation is knocked out (v = 0). Otherwise parameters as in Table 1. DOI: http: //dx. doi. org/10. 7554/eLife. 06266. 009 The initial cancer cell number M0 affects both the median and distribution of detection times (Figure 2—figure supplement 1B). For large initial tumours, growth is deterministic and exponential. As the initial size is decreased from 106 to 105, stochastic effects are increasingly manifested by greater variability in tumour inhibition and an inflection point observed at the 95th percentile. Moreover, we find that a tumour is likely to be eradicated under a range of constant treatments when M0 = 105 or fewer initial cells; in contrast, a tumour is virtually certain to persist regardless of treatment level for M0 = 107 cells or greater (Figure 2—figure supplement 2A, B). In other words, our model indicates that tumours that are c. 1% the size of most clinically detectable, internal cancers will typically be impossible to eradicate by single molecule chemoprevention when resistance is possible. Given the mutation rates assumed here, many tumours with 1 million cells will either already contain or rapidly subsequently acquire resistant cells (Iwasa et al., 2006). It is therefore not surprising that the initial fraction of resistant cells in a tumour has little impact on dynamics (Figure 2—figure supplement 1C). In contrast, another measure of success in control (the fraction of persons with tumours that remain undetected after 50 years of growth) improves substantially with lower numbers of initial resistance mutations, particularly at higher treatment levels (Figure 2—figure supplement 2C). This is because the initial phases of treatment have a major impact on the potential for new resistant mutants: should few be initially present or emerge, they will either go stochastically extinct or will not grow to detection levels (1 billion cells) in the 50 year time frame of these numerical experiments. We conducted further sensitivity analyses by varying accumulation rates u of additional driver mutations. We find that tumours exhibit more or less deterministic growth depending on the initial number of cells M0 and driver mutation rate u whereby the larger the population (Figure 2—figure supplement 1B) or the higher the mutation rate (Appendix 1—figure 4A), the less apparent are stochastic effects. The corresponding analysis is presented in ’Varying mutation rate and initial tumour size‘ in Appendix 1 and Appendix 1—figure 4. Finally, we considered scenarios where the cost of resistance is dose-dependent and specifically situations of drug addiction (Das Thakur et al., 2013). Numerical studies presented in more detail in ’A simple form of drug addiction for resistant cell-lines‘ in Appendix 1 show that under dose-dependent costs, a drug treatment only applied when the number of non-resistant cells exceeds the number of resistant cells (e. g., a metronomic therapy [Fischer et al., 2015]) leads to slower long-term tumour growth than does a constant therapy. We next investigated how a post-diagnostic measure (usually some form of chemotherapy or radiation therapy, but could also involve adjuvants after an initial therapy) affects the probability of treatment success, the distribution of times for tumour relapse, and resistance levels. We assume that a tumour grows from one cell (i = 0, j = 0) and is discovered either at 109 (early) or 1011 (very late) cells, whereupon the primary tumour is removed, leaving a small number (104 or 106) of residual, and/or undetected or inoperable neighbouring micro-metastatic cells, and/or distant metastatic cells. Below, we contrast this with prevention without discriminating the age at which either intervention type commences, whereas in the following section, we consider these as competing alternatives. Figure 3A and Figure 3—figure supplement 1A present the distributions of driver mutations for each scenario. (Recall that in the previous section, we assumed that when a measure commenced, tumours had no additional drivers (i = 0) ). 10. 7554/eLife. 06266. 010Figure 3. Effects of preventive and post-diagnostic interventions against tumours consisting of 1 million cells. (A) The distribution of mean sizes of subclones (hatched bars = before removal and solid bars = post removal). (B) The time distribution of cases in which either intervention type fails to control the tumour below the detection threshold after 50 years (thick line = median, filled area with dashed boundaries = 90% CIs) for different constant treatment intensities. (C) The percentage of cases where the tumour consists of less than 100 resistant cells at 4 years after treatment commences (solid lines), and the percentage of cases where tumour size is below the detection threshold 20 years after the measure begins (dashed-and-dotted lines). (D) The mean number of accumulated drivers within a tumour at the time of detection. Parameter values as in Table 1. DOI: http: //dx. doi. org/10. 7554/eLife. 06266. 01010. 7554/eLife. 06266. 011Figure 3—figure supplement 1. Effects of preventive and post-diagnostic interventions against tumours consisting of 10,000 cells. Same as Figure 3, except interventions against 104 cancer cells. (A) The distribution of mean sizes of subclones for different constant treatment intensities (hatched bars = before removal and solid bars = post removal). (B) The time distribution of cases in which either intervention type fails to control the tumour below the detection threshold after 50 years (thick lines = medians, shaded areas with dashed boundaries = 90% CIs). (C) The percentage of cases when a tumour consists of less than 100 resistant cells at 4 years post-resection (solid lines) and the percentage of cases when tumour sizes are below the detection threshold 20 years after the measure commences (dashed-and-dotted lines). (D) The mean number of accumulated drivers within a tumour at the time of detection. Parameter values as in Table 1. DOI: http: //dx. doi. org/10. 7554/eLife. 06266. 01110. 7554/eLife. 06266. 012Figure 3—figure supplement 2. Time to first discovery as a predictor of post-diagnostic treatment success. Time to tumour relapse following resection as function of the time it takes for the initial cancer cell to attain 109 cells (i. e., the point at which the tumour is discovered, resected, and treatment begins). Each dot represents a numerical simulation from the yellow distribution in Figure 3B (only 1,000 simulation results out of 106are shown). Four different treatment levels are considered. Black solid line is a simple linear regression, and grey area with dashed boundaries indicates extrapolation of high and low bounds accounting for 95% of observations (prediction interval). The fitted linear regression model gives an intercept of 7. 5 years, a slope of 1. 6° and R2 of 0. 024 in (A), 10. 4 years, 2. 2° and R2 of 0. 017 in (B), 12. 9 years, 3. 0° and R2 of 0. 009 in (C), and 13. 1 years, 3. 3° and R2 of 0. 008 in (D). Parameters as in Table 1. DOI: http: //dx. doi. org/10. 7554/eLife. 06266. 01210. 7554/eLife. 06266. 013Figure 3—figure supplement 3. The R2 of regressions from numerical experiments for different treatment levels of time to tumour relapse following resection as function of the mean number of drivers in a resected tumour. Time to tumour discovery is generally more predictive of post-diagnostic therapeutic outcome for lower treatment levels. See Figure 3—figure supplement 2 for details. DOI: http: //dx. doi. org/10. 7554/eLife. 06266. 01310. 7554/eLife. 06266. 014Figure 3—figure supplement 4. Mean number of additionally accumulated drivers in resected tumour as a predictor of post-diagnostic treatment success. The fitted negative exponential regression model y = ae-bx gives a = 13. 5 years, b = 0. 3 and R2 = 0. 696 in (A), 18. 95 years, 0. 3 and R2 = 0. 537 in (B), 23. 0 years, 0. 29 and R2 = 0. 262 in (C), and 23. 9 years, 0. 3 and R2 = 0. 224 in (D). See Figure 3—figure supplement 2 for details. DOI: http: //dx. doi. org/10. 7554/eLife. 06266. 01410. 7554/eLife. 06266. 015Figure 3—figure supplement 5. The R2 of regressions from numerical experiments for different treatment levels of time to tumour relapse following resection as function of the mean number of drivers in a resected tumour. Time to tumour discovery is more predictive of post-diagnostic therapeutic outcome for lower treatment levels. See Figure 3—figure supplement 4 for details. DOI: http: //dx. doi. org/10. 7554/eLife. 06266. 015 First, we examine the case where post-diagnostic resection leaves 106 cells. As suggested by our studies above on prevention, 1 million cells have a high probability of already containing resistant subclones, and deterministic effects dominate subsequent tumour growth dynamics. Comparing the median expectations of years from tumour excision to relapse, early discovery (at 109 cells) yields an additional 3. 4 years compared to late discovery (at 1011 cells) at σ = 1. 5% (medians for low vs high detection thresholds are 14. 8 and 11. 4 years, respectively; Figure 3B). Consider the following example: 20 years after resection and commencing treatment, the probability of tumour non-detection (i. e., the tumour is either eradicated or does not reach the detection threshold) is close to zero, regardless of treatment intensity (Figure 3C). Contrast this with cases of prevention starting at the same cancer cell population size (106 cells) but which fail to control the incipient tumour for the 50 years of the simulation: the detection time of these potentially life-threatening tumours is substantially longer than either of the excision cases (median 25. 5 years for σ = 1. 5%, i. e., 0. 3–0. 4% potential cell arrest per day), and tumours are managed below the detection threshold after 20 years in more than 80% of cases for any σ > 1. 0% (Figure 3C). Now consider a residual population of 1/100th the previous case, that is, 104 cells. Here, stochastic effects play a more important role in dynamics (Figure 3—figure supplement 1A, B). Due to initial heterogeneity (i. e., the co-occurrence of many subclones), when there are 4 and 5 (5 and 6) additional drivers in the dominant subclones of a residual cancer from an excised tumour of 109 (1011) cells, we observe a double peak at 4s and 5s (5s and 6s) (cf Figure 3—figure supplement 1B). These peaks in variability of outcomes are a result of the stochastic nature of the appearance of the first resistance mutations and of additional driver mutations. Interestingly, the secondary detection times (i. e., when residual or metastatic cells grow to form a new tumour) are more variable for small initial tumours compared to larger ones (cf the median 35. 8 years, 90% CIs [17. 0,70. 5] years vs 22. 4, [13. 7,37. 0] years for 109 vs 1011, respectively, with σ = 1. 5%). This effect is due to resistance emergence in more aggressive subclones for larger tumours, such that the tumour relapses more deterministically (i. e., with less variability and faster on average). The probability of tumour non-detection after 20 years and the distribution of the mean number of accumulated drivers within tumours are shown in Figure 3—figure supplement 1C, D, respectively (cf with the previous case, shown in Figure 3C, D). Importantly, for both thresholds of tumour excision, subsequent cancer cell arrest levels beyond approximately σ = 1. 5% make little difference in terms of tumour growth (Figure 3B-D, Figure 3—figure supplement 1B-D), since virtually all of the sensitive cells post-excision will be arrested or killed by the measure beyond this level, leaving uncontrollable resistant cells to grow and repopulate the primary tumour site and/or metastases. (Note that this level is above that found in the previous section. This is because drivers accumulate throughout tumour growth in the results given in Figure 3, whereas tumours were assumed to only start accumulating the first drivers after growth from M0 cells in Figure 2 and Figure 2—figure supplements 1,2). Moreover, we find that for post-diagnostic interventions knowledge about the number of drivers at the time of tumour discovery is a far better predictor of outcome than information about the time from tumour initiation to discovery, and that increases in treatment intensity tend to decrease predictive accuracy (Figure 3—figure supplements 2–5). The above results consider preventive measures and post-diagnostic interventions as independent rather than alternative approaches. Thus, although prevention delays tumour growth for longer times on average than does post-diagnostic intervention, because prevention is always initiated before diagnosis, when considering the relative benefits and risks of each, the actual time gained by the former relative to the latter in terms of cancer-free life will be less than the differences reported in Figure 3B and Figure 3—figure supplement 1B. Figure 4 presents a hypothetical comparative scenario of prevention vs post-diagnostic intervention. Prevention may either succeed without recurrence, or should the measure initially fail and a tumour be clinically detected, the patient has a ‘second chance’ whereby the tumour is resected and treatment continued (assumed at the same treatment intensity σ), either to a further relapse (failure) or non-detection (success) (Figure 4A). Compare this scenario with the more standard post-diagnostic resection followed by treatment, which either results in relapse or detection-free life (Figure 4B). These numerical experiments assume the same starting point (time at which the cancer cell population equals M0, and drivers and resistant subclones are present) for each tumour, and because of a ‘second chance’ following initial failure in prevention, are run for a maximum of 50 years after the starting point (same as the numerical studies in the previous section). We also assume, as before, that potential therapeutic resistance mechanisms to all intervention types are identical. 10. 7554/eLife. 06266. 016Figure 4. Hypothetical process of preventive (with a ‘second chance’) and post-diagnostic measures. A tumour is initiated by one cell and grows to size M0 (either 104 or 106 cells in our numerical studies). Prevention (A) arrests tumour growth at intensity σ (daily level = σ/4). Should the tumour grow to 109 cells, it is diagnosed and resected to M = M0 cells and then treated again at intensity σ. Post-diagnostic intervention (B) does not discover the growing tumour until 109 cells (i. e., σ=σ^=0), whereupon it is resected to M = M0 cells and then treated at intensity σ > 0. Either intervention finally ‘fails’ should the tumour attain 109 cells a second time, no later than 50 years after the initial lesion of size M0. Should the tumour be eradicated or not exceed 109 cells by 50 years after the initial lesion, then the intervention is deemed a ‘success’. DOI: http: //dx. doi. org/10. 7554/eLife. 06266. 016 Figure 5 presents the comparative outcomes (see also Videos 4,5). When prevention starts at (or tumour resection misses) relatively large cancer cell populations (1 million cells), only small comparative gains occur from higher cell arrest in terms of outright treatment success (Figure 5A), whereas interventions starting at much smaller cancer cell numbers (10,000) result in considerably greater outright success (Figure 5B). Looking at situations of relapse only for prevention vs post-diagnostic intervention, the former generally results in superior outcomes in terms of delaying tumour growth, particularly for large residual cell populations (cf Figure 5C, D). In contrast, for lower numbers of residual cells, some post-diagnostic resected tumours in the sample will be initially resistance free (cf Figure 5—figure supplement 1A, B). This, together with fewer accumulated drivers in the highest driver subclones, contributes to improved outcomes should relapse occur (Figure 5D) and overall treatment success at sufficiently high treatment intensities (Figure 5A, B, E, F). Importantly, resected tumours in both the prevention (when it initially fails) and post-diagnostic scenarios may contain numerous resistant cells (example of 0. 25% daily cellular arrest: Figure 5—figure supplements 2,3). Prior selection for resistance in initially failed prevention generally results in larger residual resistant cell populations than pre-therapeutic residual populations in post-diagnostic situations (filled bars, cf captions A and B in Figure 5—figure supplements 2,3), but smaller residual resistant cell populations than treatment failures following post-diagnostic resection (hatched bars, cf captions A and D in Figure 5—figure supplements 2,3). Note that, as expected, secondary failures are associated with larger percentages of resistant subclones and a shift in the distributions towards more drivers (cf captions C and D in Figure 5—figure supplements 2,3). 10. 7554/eLife. 06266. 017Figure 5. Comparison of preventive (blue lines and shading) and post-diagnostic (red lines, yellow shading) interventions. Tumours are either treated at M0 = 106 cells (left panels) or M0 = 104 cells (right panels). (A, B) Probability of treatment success, defined as the proportion of cases where the tumour remains undetected (either extinct or below 109 cells) by 50 years after the initial lesion of M0 cells. (C, D) Distribution of times to relapse for treatment failures. (E, F) Distribution of detection times for all cases including relapsed tumours and tumours remaining undetected prior to and after 50 years (detection times are assigned to 50 years in the latter case). Parameters as in Table 1. See Figure 3 for details. DOI: http: //dx. doi. org/10. 7554/eLife. 06266. 01710. 7554/eLife. 06266. 018Figure 5—figure supplement 1. Resistant cell populations after initial failure. Tumours are either treated at M0 = 106cells (A) or M0 = 104cells (B). Red lines and yellow shading = population following resection. Parameter values as in Table 1. DOI: http: //dx. doi. org/10. 7554/eLife. 06266. 01810. 7554/eLife. 06266. 019Figure 5—figure supplement 2. Distribution of mean sizes of subclones. (A) Cases where a tumour is detected and resected following prevention. (B) Cases where a tumour is detected and resected with no prevention. (C) Cases of relapse following resection and secondary treatment to initially failed prevention. (D) Cases of relapse following resection and primary treatment in cases where there was no prevention. Hatched bars indicate cell numbers in the tumour and solid bar numbers after resection and 106 residual or metastatic cells. Daily arresting level assumed to be 0. 25%. Parameter values as in Table 1. DOI: http: //dx. doi. org/10. 7554/eLife. 06266. 01910. 7554/eLife. 06266. 020Figure 5—figure supplement 3. Distribution of mean sizes of subclones. Same as Figure 5—figure supplement 2, except M0 is 104 cancer cells. DOI: http: //dx. doi. org/10. 7554/eLife. 06266. 02010. 7554/eLife. 06266. 021Video 4. Comparison of preventive (blue lines and shading) and post-diagnostic (red lines, hatched) interventions. Tumours are treated at M0 = 106 cells. (A) The median (thick line) and 90% confidence intervals (shaded areas with dashed boundaries) for the distribution of times to relapse for treatment failures. (B) and (C) Arbitrary samples of the distribution of detection times for preventive and post-diagnostic interventions, respectively. The colour-code indicates the mean number of accumulated drivers over a period of 1 year. The rectangles on the top of B and on the bottom of C show the fifth and 95th percentiles, the blue circle indicates the median, and the red line is the mean. Parameters as in Table 1. DOI: http: //dx. doi. org/10. 7554/eLife. 06266. 02110. 7554/eLife. 06266. 022Video 5. Comparison of preventive (blue lines and shading) and post-diagnostic (red lines, hatched) interventions. Tumours are treated at M0 = 104 cells. (A) The median (thick line) and 90% confidence intervals (shaded areas with dashed boundaries) for the distribution of times to relapse for treatment failures. (B) and (C) Arbitrary samples of the distribution of detection times for preventive and post-diagnostic interventions, respectively. The colour-code indicates the mean number of accumulated drivers over a period of 1 year. The rectangles at the top of B and the bottom of C shows the fifth and 95th percentiles, the blue circle indicates the median, and the red line is the mean. Parameters as in Table 1. DOI: http: //dx. doi. org/10. 7554/eLife. 06266. 022 Figure 5E, F shows the distributions of detection times for all numerical experiments. We see that when both non-relapse (Figure 5A) and relapse (Figure 5C) are taken into account for large cancer cell populations (1 million cells), treating preventively at levels beyond about 0. 3% arrest per day increases median delays in detection times due to outright success (i. e., survival beyond 50 years) but has no effect on the lower 95th percentile (Figure 5E). (Although not shown, arrest beyond approximately 0. 6% per day does not yield further gains). In contrast, post-diagnostic intervention improves only marginally beyond daily arrest levels of about 0. 3% (Figure 5E). Figure 5F shows the corresponding results for smaller cancer cell populations (based on integrating the results in Figure 5B, D), whereby a high median probability of full success is obtained >0. 1% and >0. 3% daily arrest for prevention and post-diagnostic intervention, respectively (Figure 5F). Thus for both cell population levels, prevention generally results in better outcomes compared to post-diagnostic intervention. Whereas primary prevention is becoming an increasingly significant approach in reducing risk of certain cancers (e. g., Colditz and Bohlke, 2014), chemopreventive therapies are uncommon, despite empirical support for their effects (William et al., 2009). Several theoretical and in vitro experimental studies indicate that chemoprevention can reduce risks of life threatening cancers. For example, Silva and colleagues (Silva et al., 2012) parameterized computational models to show how low doses of verapamil and 2-deoxyglucose could be administered adaptively to promote longer tumour progression times. These drugs are thought to increase the costs of resistance and the competitive impacts of sensitive cells on resistant cancer cell subpopulations. However, some of the most promising results have come from studies employing non-steroidal anti-inflammatory drugs (NSAIDs), including experiments (Ibrahim-Hashim et al., 2012), investigations of their molecular effects (Galipeau et al., 2007; Kostadinov et al., 2013), and their use (Cuzick et al., 2015). For example, Ibrahim and co-workers (Ibrahim-Hashim et al., 2012) studied the action of NSAIDs and specifically sodium bicarbonate in reducing prostate tumours in male TRAMP mice (i. e., an animal model of transgenic adenocarcinoma of the mouse prostate). They showed that mice commencing the treatment at 4 weeks of age had significantly smaller tumour masses, and that more survived to the end of the experiment than either the controls or those mice commencing the treatment at an older age. Kostadinov et al. (2013) showed how NSAID use in a sample of people with Barrett' s oesophagus is associated with reductions in somatic genomic abnormalities and their growth to detectable levels. It is noteworthy that it is not known to what extent reductions in cancer progression under NSAIDs are due to either cytotoxic or cytostatic effects or both. Although we do not explicitly model cytotoxic or cytostatic impacts, therapies curbing net growth rates but maintaining them at or above zero could be interpreted as resulting from the action of either cytotoxic and/or cytostatic processes. In contrast, therapies reducing net growth rates substantially below zero necessarily have a cytotoxic component. Our model, or modifications of it to explicitly include cytotoxic and cytostatic effects, could be used in future research to make predictions about optimal dose and start times to achieve acceptable levels of tumour control (or, e. g., the probability of a given tumour size and heterogeneity level by a given age). Decisions whether or not to employ specific chemopreventive therapies carry with them the risk of a poorer outcome than would have been the case had another available strategy (or no treatment at all) been adopted (Esserman et al., 2004). This issue is relevant to situations where alterations in life-style, removal or treatment of pre-cancerous lesions, or medications potentially result in unwanted side effects or induce new invasive neoplasms (e. g., Berrington de Gonzalez et al., 2011). Chemopreventive management prior to clinical detection would be most appropriate for individuals with genetic predispositions, familial histories, elevated levels of specific biomarkers, or risk-associated behaviours or life-styles (Hemminki and Li, 2004; Lippman and Lee, 2006; Sutcliffe et al., 2009; William et al., 2009; Hochberg et al., 2013). Importantly, our approach presupposes that the danger a nascent, growing tumour presents is proportional to its size and (implicitly, all else being equal) a person' s age. Due caution is necessary in interpreting our results, since studies have argued that metastatic potential rather than tumour size may be a better predictor of future survival (Hynes, 2003; Foulkes et al., 2010; Sethi and Kang, 2011). However, given the expectation that prevention typically confronts smaller, less heterogeneous neoplasms, which are less likely to have resistant clones and to have metastasised (Hochberg et al., 2013; Gerlinger et al., 2014), support our basic conclusion that prevention is generally a superior strategy in terms of cancer-free survival compared to post-diagnostic intervention. Over the past decade, several alternative approaches to MTD have been proposed, where the objective is to manage rather than eradicate tumours (e. g., Maley et al., 2004; Komarova and Wodarz, 2005; Gatenby, 2009; Gatenby et al., 2009a, 2009b; Foo and Michor, 2010; Jansen et al., 2015). Tumour management attempts to limit cancer growth, metastasis, and reduce the probability of obtaining resistance mutations through, for example, micro-environmental modification, or competition with non-resistant cancer cell populations or with healthy cells. These approaches usually involve clinically diagnosed cancers: either inoperable tumours or residual or metastatic cancers after tumour excision. In the former situation, tumours are typically large enough in size to contain numerous resistance mutations. In many, if not most, cases, these neoplasms will have metastasized, meaning greater variability both in terms of phenotypes and potential resistance to chemotherapies, and in penetrance of therapeutic molecules to targeted tumour cells (Klein et al., 2002; Byrne et al., 2005). In contrast, the latter situation involves smaller, residual, or metastatic cancer cell populations, composed of high frequencies of resistant variants or dormant cells (Klein et al., 2002). According to our results, both scenarios are likely to involve populations with large numbers of accumulated driver mutations (or, although not considered in our study, fewer driver mutations but each with larger selective effect), which ostensibly contribute to the speed of relapse. Thus, management of clinically detected tumours need not only limit the proliferation and spread of refractory subpopulations but should also aim to control the growth of multi-driver subclones (Figure 5—figure supplements 2,3). In other words, in addition to actual resistance mutations (j = 1), subclones with q drivers will be effectively resistant to therapeutic interventions if q s ≫ σ (Figure 6). We therefore suggest that the frequency distribution of driver mutations and the distribution of resistant subclones within a heterogeneous cancer cell population could be used to instruct decisions of the time course of treatment levels, with the aims of curbing tumour growth, metastasis, and resistance. We found that tumours typically achieve several additional driver mutations by the time they reach detection (Figure 3A; Figure 3—figure supplement 1A; Figure 5—figure supplements 2,3), which approximates certain estimates (Stratton et al., 2009) but falls short of others (Sjoblom et al., 2006). Our results indicate that the two most important variables in determining therapeutic outcome are (1) the size of the initial cancer cell population (i. e., when prevention commences and/or post-diagnosis, following resection), (2) associated tumour heterogeneity in terms of accumulated drivers, and the presence of resistance phenotypes. This highlights the importance of biomarkers as accurate indicators of otherwise undetectable malignancies (Roukos et al., 2007), and the accurate assessment of local or distant metastases (Pantel et al., 1999). We suggest that if order-of-magnitude estimates of cell populations and intra-tumour heterogeneity are possible, then low dose, continuous, constant approaches could be established that lower and possibly minimize risks of the emergence of future, life-threatening cancers. According to our model, such options will generally be superior to more aggressive chemotherapies if therapeutic resistance is a risk factor. The framework proposed here is sufficiently general to portray major events in different types of cancer with emphasis on solid tumours. However, some aspects of cancerous tumour growth are considered only implicitly, and further research is required to formulate more realistic models to include, for example, spatial aspects of tumour growth (Orlando et al., 2013), competition/cooperation between different subclones (Korolev et al., 2014), combinational (multidrug) resistance (Gillet and Gottesman, 2010; Bozic et al., 2013), drug-addiction, observed for example in certain melanomas (Das Thakur et al., 2013), or advantageous resistant mutations, observed in some leukemias (Michor et al., 2005). Moreover, future studies should investigate alternatives to the traditional post-diagnostic therapeutic scenarios considered here (e. g., molecularly targeted therapies [Yap, 2015]). Our study nevertheless predicts that the main hurdle to post-diagnostic MTD interventions remains resistant subclones, since beyond minimal impacts on the order of 0. 3% per day for the larger of the two residual or metastatic cell populations simulated here (which are still very small by clinical diagnostic standards—c 1 mm3), increased therapeutic intensity selects disproportionally for resistance and has negligible benefits in terms of delaying life-threatening cancers.

About one person in every two will get cancer during their lives. Surgery and chemotherapy have long been mainstays of cancer treatment. Both, however, have substantial downsides. Surgery may leave behind undetected cancer cells that can grow into new tumours. Furthermore, in response to chemotherapy drugs, some cancer cells may emerge that resist further treatment. There is therefore interest in whether preventive strategies-including lifestyle changes and medications-could reduce the likelihood of confronting a life-threatening cancer. Now, Akhmetzhanov and Hochberg have developed a mathematical model to help compare the effectiveness of preventive strategies and traditional cancer treatments. The model-which assumes that a person can only develop a single cancer from a single region of pre-cancerous cells-suggests that long-term cancer prevention strategies reduce the risk of a life-threatening cancer by more than traditional treatment that begins after a tumour is discovered. The preventive measures may be less effective in some cases compared to traditional treatments if they initially fail to stop a tumour growing, although on average they still work better than treating the cancer after detection. According to Akhmetzhanov and Hochberg' s model, surgical removal followed by chemotherapy is less likely to be successful than prevention, and when successful, requires larger impacts on the cancer (and therefore creates more side-effects for the patient) to achieve the same level of control as prevention. The model also suggests that even at very low levels of impact on residual cancer cells, chemotherapies are likely to be counterproductive by boosting the subsequent emergence of treatment-resistant tumours. Akhmetzhanov and Hochberg' s model predicts how effective preventive measures need to be in terms of slowing the growth of cancer cells to result in given reductions in the future risk of a life-threatening cancer. Future work should test this model by measuring the effects on tumour growth of prevention and of traditional therapies.

lay_elife

The murine model of experimental cerebral malaria (ECM) has been utilised extensively in recent years to study the pathogenesis of human cerebral malaria (HCM). However, it has been proposed that the aetiologies of ECM and HCM are distinct, and, consequently, no useful mechanistic insights into the pathogenesis of HCM can be obtained from studying the ECM model. Therefore, in order to determine the similarities and differences in the pathology of ECM and HCM, we have performed the first spatial and quantitative histopathological assessment of the ECM syndrome. We demonstrate that the accumulation of parasitised red blood cells (pRBCs) in brain capillaries is a specific feature of ECM that is not observed during mild murine malaria infections. Critically, we show that individual pRBCs appear to occlude murine brain capillaries during ECM. As pRBC-mediated congestion of brain microvessels is a hallmark of HCM, this suggests that the impact of parasite accumulation on cerebral blood flow may ultimately be similar in mice and humans during ECM and HCM, respectively. Additionally, we demonstrate that cerebrovascular CD8+ T-cells appear to co-localise with accumulated pRBCs, an event that corresponds with development of widespread vascular leakage. As in HCM, we show that vascular leakage is not dependent on extensive vascular destruction. Instead, we show that vascular leakage is associated with alterations in transcellular and paracellular transport mechanisms. Finally, as in HCM, we observed axonal injury and demyelination in ECM adjacent to diverse vasculopathies. Collectively, our data therefore shows that, despite very different presentation, and apparently distinct mechanisms, of parasite accumulation, there appear to be a number of comparable features of cerebral pathology in mice and in humans during ECM and HCM, respectively. Thus, when used appropriately, the ECM model may be useful for studying specific pathological features of HCM. Cerebral Malaria (CM), one of the most severe complications of Plasmodium falciparum (Pf) infection, is defined clinically by an unrousable coma in the presence of Pf parasitemia, with no other known cause of neuropathology [1]. Although the syndrome only occurs in 1% of Pf infections, it has a high fatality rate (15–20% of cases), with death typically occurring despite administration of established anti-malarial drug regimens [1,2]. Moreover, whilst CM induced-encephalopathy has historically been considered acute and reversible, recent follow-up studies in individuals post-CM have determined that a significant percentage (10–26%) exhibit long-term neurological sequelae [3]. Individuals with limited prior exposure to parasite are disproportionally susceptible to the syndrome [4]; as a result, the majority of fatal CM cases consist of young children in endemic regions of Africa [5]. Indeed, with an estimated 2–3 million cases of the syndrome annually, CM-associated mortality and neuro-disability imposes a substantial social and economic burden on this region [6,7]. Consequently, there remains an urgent need to understand the pathogenesis of CM, to facilitate the development of more efficacious anti-malarial drugs and/or adjunct therapies for the condition. Neuropathological studies from fatal CM cases have detailed dense sequestration of parasitised erythrocytes (pRBCs) within the cerebral micro-vasculature as a canonical feature of the syndrome [8–11]. Indeed, pRBC sequestration in cerebral capillaries and venules is quantitatively greater in HCM patients, than in individuals who succumb to non-cerebral malarial complications [9,10]. It is believed that pRBC congestion of vessels may impair tissue perfusion by perturbing cerebral flow, and/or lead to local immune-mediated injury via secondary host response (s) to parasite products [11,12]. However, accumulating evidence indicates that CM is a relatively complex neuropathology, with pRBC sequestration typically occurring concomitant with significant intravascular accumulation of mononuclear cells, intracerebral haemorrhage, enhanced blood-brain barrier (BBB) permeability and oedema [5]. Moreover, pathology is not restricted to the cerebral vasculature during CM, and axonal injury and demyelination have also been documented [5,13]. Nevertheless, despite our knowledge of the pathology of fatal CM, restricted access to post-mortem samples for histopathological study has prevented correlation of pathological features with onset of clinical symptoms. Thus, the importance and/or relative contributions of the above observed pathological events to pathogenesis of CM remains incompletely understood. Inaccessibility of the human brain pre-mortem has led to the development and study of the experimental mouse model of cerebral malaria (ECM) [14–20]. Susceptible mice infected with Plasmodium berghei (Pb) ANKA present with similar graded and sequential signs of disease as humans affected with CM (HCM); including ataxia, paralysis, coma and, if untreated, death [21]. Furthermore, mice treated with anti-malarial drugs at the point of neurological dysfunction demonstrate comparable levels of mortality and long-term cognitive dysfunction [22,23]. Such similarities in clinical presentation and long-term consequence between ECM & HCM, suggest the pathophysiological processes underlying the two conditions may be comparable. Indeed, parasite accumulation has been observed in the brains of mice that developed fatal malaria-induced cerebral pathology compared with those that developed asymptomatic infections [24]. Concurrent to parasite accumulation, haemorrhage and BBB disruption have also been observed in the brains of mice experiencing ECM; with the latter perceived as a key feature of the syndrome [25,26]. Additionally, brain-accumulating CD8+ T-Cells have been shown to play a critical role in ECM pathogenesis by promoting BBB disruption via perforin and Granzyme B dependent mechanisms [27–29], potentially following interaction with brain endothelial cells cross-presenting parasite antigen [30,31]. Despite the extensive use of the ECM model, there remains significant debate regarding its validity to study HCM [21,32–35]. In particular, the importance of pRBC sequestration within the brain for the development of ECM has been questioned [36]. Indeed, it is currently unknown whether true pRBC sequestration occurs during ECM, or if pRBCs simply accumulate within intracerebral haemorrhages and/or leukocyte occluded brain vessels [37]. This lack of understanding is because intracerebral parasite accumulation during ECM has previously been studied using spatially insensitive techniques such as RT-PCR or whole body luminescent imaging, rather than through detailed histopathological assessment, such as performed during HCM [24,36–39]. Importantly, the lack of detailed histopathological knowledge of the ECM syndrome means we also currently do not know the spatial relationship between pRBCs and other pathological parameters involved in ECM development, such as CD8+ T-cells, haemorrhage and oedema, or how these pathological events affect different brain regions. Consequently, at present the pathology of ECM has been incompletely characterised and, as such, it is not possible to definitively conclude whether it is a valid model to study all, some, or none of the pathological features of HCM. In this study, to improve our knowledge of the pathology of ECM, we have performed a systematic and quantitative histopathological investigation of ECM using comparable methodologies as utilised in the study of HCM. Crucially, we show that intracapillary parasite accumulation throughout the brain is a canonical feature of ECM, and that a single mature, pRBC seems sufficient to occlude narrow murine capillaries, and thus cause localised haemostasis. Parasite accumulation also appears to mediate the subsequent local intravascular recruitment of low numbers of CD8+ T-cells that, together with parasite, is associated with widespread BBB disruption. Strikingly, BBB disruption appears to occur due to vascular junction remodelling and increased levels of caveolae, rather than through extensive endothelial cell apoptosis. Finally, we detected axonal and myelin injury adjacent to multiple neurovascular pathogenic parameters associated with ECM, indicating two potential common pathways for neurological impairment to occur during malaria-induced cerebral pathology. Collectively, our data indicates that the mechanisms underlying the response of the brain to local parasite accumulation are conserved between humans and mice, and, therefore, supports the use of the ECM model to understand the pathogenesis of HCM. We sought to characterise the pathological features specifically associated with ECM, compared with those that simply occur during uncomplicated malaria infection. Therefore, we utilised two closely related murine plasmodium strains with contrasting infection outcomes. Consistent with our previous studies [26,40,41], C57BL/6 mice infected with Pb ANKA typically developed signs of late stage ECM; including ataxia, convulsions, paralysis and/or coma, on day 7 (p. i.) (Fig 1A). In contrast, mice infected with Pb NK65, despite exhibiting comparable parasitemia (with the notable exception of day 7 p. i.) (Fig 1B) and weight loss (Fig 1C) as mice infected with Pb ANKA, survived the critical window for developing ECM (days 6–12 p. i.) without exhibiting neurological symptoms. Pb NK65 infected mice instead developed hyperparasitemia and succumbed to infection on day 25 (Fig 1A). Thus, this comparative model provides a tractable way to identify host and parasitological events that specifically contribute to the development and progression of malaria-induced cerebral pathology. Although analyses using RT-PCR and luciferase-expressing parasites have shown that ECM is associated with accumulation of parasites in the brain [24,37–39], the compartmentalisation of the pRBCs in the brain during ECM is not known. Consequently, whether parasite sequestration, and subsequent microvascular obstruction, occurs within the brain during ECM development is, at present, unclear. Therefore, we performed a detailed analysis of parasite accumulation in the well-perfused brains of mice infected with Pb ANKA and Pb NK65. Utilising GFP-tagged parasites, which enabled us to perform high resolution histopathological analyses of pRBC location within the intact brain architecture, we observed significantly higher accumulation of pRBCs in all assessed brain regions (S1 Fig) during Pb ANKA infection compared with Pb NK65 infection (we did not detect any innate signal through the GFP channel in the brains of uninfected mice) (Fig 2A and 2B & S2 Fig). We noted differences in the size of parasite GFP signal, and confirmed these differences in expression related to parasite maturity using whole brain homogenate (S3 Fig). To exclude the possibility that differences in GFP expression by Pb ANKA and Pb NK65 parasites were responsible for the observed differences in parasite accumulation, we additionally confirmed that significantly greater intracerebral parasite accumulation occurs during Pb ANKA infection compared to Pb NK65 infection by utilising Pb anti-sera (S4 Fig). Whilst GFP expression is constrained to live parasite, Pb anti-sera visualised all parasite material, and thus the degree of Pb anti-sera immunoreactivity was much higher relative to anti-GFP staining from the same samples (S4 Fig). Importantly, we noted that the >90% of intracerebral pRBCs during ECM were intracapillary, rather than associated with intravascular accumulations of leukocytes and/or haemorrhage (Fig 2C and 2D). Combined, these results demonstrate that global parasite accumulation within cerebral capillaries is a specific event associated with ECM. As pRBC accumulation is principally compartmentalised within the microvasculature during ECM, we theorised that this phenomenon is likely dependent on a form of sequestration. Accordingly, we utilised transmission electron microscopy (TEM) to characterise the precise nature of the interaction between pRBCs and cerebral endothelial cells (ECs) in the brains of mice infected with Pb ANKA. Consistent with our immunofluorescence staining, we observed pRBCs within the capillaries of perfused brains from Pb ANKA mice with late-stage ECM (Fig 3A). Moreover, we detected the occasional electron-dense spot on the surface of some pRBCs adjacent to the EC membrane (Fig 3AI); such events were not evident on the surface of uninfected erythrocytes (Fig 3B). Whilst we noted greater numbers of luminal RBCs compared to luminal pRBCs, importantly, longitudinal sections of capillaries invariably demonstrated that such RBCs accumulated specifically behind vessel-spanning pRBCs (Fig 3C). We further assessed the level and presentation of intravascular pRBC accumulation during ECM by smearing the well-perfused brain tissue of mice infected with Pb ANKA. Brain smears preserve lengthy microvessels, and thus have historically been used in HCM for assessing sequestration [42,43]. We observed several intracapillary trophozoites and schizonts in Romanowsky and H&E stained brain smears (Fig 3D and 3E). Critically, and in agreement with our TEM data, a single pRBC appeared sufficient to occlude a capillary. Consequently, uninfected erythrocytes and/or immature pRBCs could be observed in varying degrees of accumulation behind individual, mature pRBCs within, apparently obstructed, capillaries (Fig 3F–3K). We did not identify any cytoadherent pRBCs in venules or other larger calibre vessels. Conversely, we noted that a number of these larger vessels were distended and enriched with leukocytes, predominantly monocyte/macrophages, often dense with parasitic material (Fig 3L). We did not observe any extravascular pRBCs, though parenchymal macrophages (potentially microglial cells) enriched with parasitic material were observed occasionally (Fig 3M). These observations were validated in H&E stained sections of perfused brain tissue derived from Pb ANKA infected mice; whereby a number of microvessels were seen to be congested with erythrocytes, of which only a minority were parasitised (Fig 3N, 3NII, 3O & 3OIII). Critically, these results indicate that pRBC accumulation during ECM is dependent, or co-dependent, on parasite strain intrinsic capacities to deform within, and/or cytoadhere to, the cerebral microvasculature. Moreover, our results show that a single, pRBC appears sufficient to occlude and cause haemostasis within narrow murine cerebral capillaries. Critically, these observations imply that, despite the lower cerebral parasite biomass noted in ECM compared to HCM, the haemorheological consequences of pRBC accumulation may be similar in both. We next utilised our comparative model to perform a detailed quantitative examination of CD8+ T-cells. Whilst CD8+ T-cells are known to play a critical role in the development of ECM [27], we, and others, are still investigating their role in HCM. Due to an inability to utilise CD8 mAbs in fixed murine tissue [44], T-cells were labelled with CD3. Importantly, the majority of CD3+ T-cells in the brain are also CD8+ during ECM (S5 Fig). T-cells were observed in the cerebral vessels (identified by tomato lectin) of mice infected with either strain of Pb, and absent from the cerebral vessels of uninfected mice (Fig 4A, S6 Fig). Furthermore, whilst the number of T-cells was quantitatively greater in all brain regions from mice infected with Pb ANKA compared to Pb NK65, total T-cell numbers were low (Fig 4B). Indeed, despite ECM being a CD8+ T-cell dependent syndrome, T-cells were, on average, rarer than pRBCs in all corresponding brain regions from mice infected with Pb ANKA (Figs 2B and 4B). T-cells were predominantly found luminal or abluminal to the cerebral microvasculature, or as part of dense intravascular leukocyte accumulations in larger-calibre vessels during ECM (Fig 4C and 4D). Of note, the majority of leukocyte packed vessels contained lectin-labelled monocytes or macrophages, rather than CD3+ T-cells (S7 Fig). There was no evidence of extravasation of T-cells into the brain parenchyma, and the few T-cells observed within the parenchyma were associated with intracerebral haemorrhage (Fig 4C and 4D). As pRBCs and/or parasite material were predominantly found in the same intracerebral compartments as T-cells (Figs 2C and 2D and 4C and 4D), we therefore hypothesised that the two might co-localise within the same subset of vessels. To test this hypothesis, we stained brain smears sampled from mice exhibiting fulminant ECM by H&E, and examined the association between morphologically-identified lymphocytes and parasite. We noted that while arrested pRBCs were often independent of lymphocytes, intracapillary or perivascular lymphocytes were invariably proximal to pRBCs (Fig 4E, S7 Fig). Our results indicate that, in the context of local pRBC accumulation, very few intracerebral T-cells are required for the development of ECM. Moreover, the co-localisation of T-cells with pRBCs implies that pRBCs may promote CD8+ T-cell accumulation. We next employed our comparative model to characterise the neurovascular-pathological events downstream of cerebral T-cell and pRBC accumulation during Pb ANKA infection. H&E staining demonstrated that intracerebral haemorrhage, a commonly described neuropathological feature in HCM and ECM, was quantitatively greater in all brain regions (with the notable exception of the olfactory bulbs) during Pb ANKA infection compared to Pb NK65 infection (Fig 5A and 5B, S8 Fig). Intracerebral haemorrhage was not observed in uninfected brains (S8 Fig). Large amorphous haemorrhages, perivascular bleeding and/or petechiae were all observed in the brains of mice during late-stage ECM (Fig 5C–5F). Whilst thrombosed vessels, with and without extravasated erythrocytes, were evident (Fig 5G and 5H), we saw no evidence of ring haemorrhages; a typical feature of HCM. As opposed to HCM, where haemorrhage occurs predominantly in the white matter [5], haemorrhaging was observed equivalently in the grey and white matter during ECM (Fig 5I and 5J). Our results, therefore, indicate that the frequency of haemorrhaging is increased during ECM, compared to uncomplicated malaria. However, the relative rarity of haemorrhage, in comparison to other pathological events, suggests it may not be the predominant cause of mortality during ECM. Cerebral oedema resulting from enhanced BBB permeability is a common feature of HCM [5,45,46], and is thought to be a critical element of ECM pathogenesis [18,26,47]. However, to date, there has been no attempt to quantitatively assess the nature or presentation of vascular permeability during ECM. We assessed BBB permeability during P. berghei infection by staining for endogenous IgG, a serum protein ordinarily excluded from the cerebral parenchyma by an intact BBB [48]. We observed noticeably higher levels of IgG immunoreactivity, and, correspondingly, significantly increased numbers of permeable vessels, in the brains of mice infected with Pb ANKA compared to those infected with Pb NK65 (Fig 6A and 6B & S9 Fig). Positive IgG staining was not observed in uninfected brains (S10 Fig). Permeable vessels were characterised by a “halo” of IgG (Fig 6C), or dense extravascular depositions of IgG and/or IgG immunoreactive astrocytes (Fig 6D). In some brain regions (in particular the brainstem) from mice infected with Pb ANKA, but not Pb NK65, dense areas of IgG immunoreactive neurons were observed (Fig 6E). Although such neuronal staining has been defined as a historical marker of cerebral oedema [49,50], as it was not possible to relate this parenchymal staining to particular blood vessels we did not quantify this pathological feature in our analysis. Furthermore, we excluded from our analysis any vessels that exclusively exhibited intravascular IgG staining (Fig 6F), as this identified occluded, not permeable, vessels. Extravasation of IgG was typically associated with haemorrhage (Fig 6G), larger calibre leukocyte-occluded vessels (Fig 6H), and/or intracapillary pRBCs (Fig 6I). Interestingly, scattered permeable vessels devoid of pRBCs, leukocytes or haemorrhage were also observed, however, these were typically in the vicinity of vessels exhibiting a specific pathological feature (as described above) (Fig 6J). Consistent with our IgG staining, we also observed clear histological evidence of cerebral oedema during ECM (Fig 6K and 6L), suggesting the severity and/or prevalence leakage must be substantial. Combined, these results, in agreement with the literature [26,51], show that BBB permeability is widespread within the brain during ECM, and that vascular leakage and subsequent oedema is significantly greater during Pb ANKA than during Pb NK65 infection. Importantly, our data also shows that whilst BBB permeability during ECM is typically associated with parasitised microvessels, intravascular accumulations of leukocytes or haemorrhage, permeable vessels devoid of any such associated vascular pathology are also present. Whilst this could indicate that the pathological event triggering the vascular leakage cleared subsequent to analysis, it may also suggest that soluble mediators expressed by the vascular bed at distinct pathological sites may induce diffuse BBB permeability during ECM. We next examined the mechanistic basis for intracerebral vascular permeability during ECM. In particular, as it has been proposed that CD8+ T-cells promote cytolysis of cross-presenting endothelial cells [30,31], we assessed the level of cellular apoptosis in the brains of mice infected Pb ANKA or Pb NK65. Cellular apoptosis (detected by expression of cleaved Caspase 3 (CC3) ) was rarely observed in the brains of mice infected with Pb ANKA, was even less frequent during Pb NK65 infection, and was not observed in the brains of uninfected mice (Fig 7A & S11 Fig). The majority of the apoptotic events during ECM were associated with the vasculature, and were predominantly endothelial cells, leukocytes, or, more infrequently, astrocytes (Fig 7B). Atypical parenchymal cellular apoptosis did not appear to be neuronal, based on morphological criteria (Fig 7B). Notably, cerebral oedema (characterised by uncondensed parenchyma and/or perivascular dilation) was observed proximal to vessels with and without evidence of apoptotic ECs (Fig 7C), suggesting leakage is not dependent on EC loss. Haemorrhages were associated with disrupted vessel staining, but not EC apoptosis; indicating non-apoptotic mechanisms are likely responsible for haemorrhage (Fig 7D). We saw no evidence of conterminous vascular degeneration adjacent to haemorrhage, i. e. endothelial cell apoptosis within the afflicted vascular bed; though we did observe apoptotic leukocytes focal to the lesion (Fig 7D). Importantly, the area and number of vessels was unaltered during Pb ANKA and Pb NK65 infection (S12 Fig). This implies that vascular loss is a limited and stochastic event associated exclusively with haemorrhage, and not a central contributor to the cerebral oedema seen during ECM. Combined, these results indicate that programmed cell death of ECs is highly unlikely to be the major mechanism provoking the widespread vascular leakage that occurs during ECM. As widespread BBB disruption during ECM did not appear to be associated with a loss of cerebral ECs, we subsequently sought to examine whether alterations in transcellular and/or paracellular transport mechanisms could account for enhanced brain vessel permeability. Utilising TEM, we observed extensive pseudopodia, or cytoplasmic extensions, in a number of vessels during ECM. Pseudopodia were seen only rarely on cerebral ECs during Pb NK65 infection and not seen in uninfected samples (Fig 8A & S13 Fig). Caveolae were abundant in cerebral ECs in the brains of mice infected with Pb ANKA compared to Pb NK65 (Fig 8AI and 8AII). In some vessels aggregations of caveolae appeared to form transendothelial pores specifically during ECM (Fig 8B). In addition, large clefts in the cerebral microvascular tight junctions were observed during ECM, but were only rarely seen in uncomplicated malaria infection (Fig 8C and 8D). Interestingly, clefts in tight junctions and accumulations of caveolae were observed proximal to cerebral oedema (Fig 8E and 8F). This suggests that alterations to the transcellular and/or paracellular permeability of the brain microvasculature may be responsible for the vascular leakage observed during ECM. Additionally, we noted an apparent thickening of the basement membrane and luminal contraction consistent with vasospasm in a number of the cerebral microvessels of mice infected with Pb ANKA compared to Pb NK65 (Fig 8A). However, due to the natural range of capillary diameter within the rodent brain [52], definitive assessment of vasospasm was not possible in our analysis. Whilst it is not entirely clear how the cerebral vascular pathology that characterises HCM influences parenchymal brain function to induce coma and death, it has been shown that axonal injury (AI) and myelin loss are common neuropathological features of the syndrome [5,13]. Conversely, there is currently no histopathological data defining the neurological abnormalities that occur during ECM. Using our comparative model, we observed significant evidence of AI specifically during ECM, as shown by β-APP, a protein that accumulates at sites of AI (Fig 9A). Several patterns of β-APP staining were evident: labelling of single axons; diffuse regions; more intense regions; and scattered, intensely-immunoreactive neuronal cell bodies (Fig 9B). AI was noted adjacent to specific vascular pathological features during ECM, including: parasitised capillaries; leukocyte-packed vessels; and haemorrhage (Fig 9C and 9D). In contrast, we observed that the neuronal architecture (defined by NeuN staining) was broadly unaltered during both Pb ANKA and Pb NK65 infection (S14 Fig). Moreover, and consistent with our data in Fig 7, we saw no evidence of apoptotic neurons in the brains of Pb ANKA or Pb NK65 infected mice (S14 Fig). However, we did note neuronal lesions proximal to some haemorrhages in the olfactory bulbs of mice infected with both Pb ANKA and Pb NK65 (Fig 9E). In addition to AI, there was evidence of extensive myelin pathology specifically in the brains of mice infected with Pb ANKA (Fig 9F). We observed discrete regions of gross demyelination, and myelin pallor and fragmentation associated with parasitised capillaries, leukocyte-packed vessels and haemorrhage (Fig 9G and 9H). Collectively, these results suggest the nature of cerebral parenchymal damage is comparable in HCM and ECM, and provide a logical explanation for the clinical similarity in the transient and long-term neurological dysfunction that occurs during and post HCM and ECM. To further improve our understanding of the pathogenesis of ECM, we examined the spatial nature of the defined pathological features within individual ECM-affected brains. Although the magnitude of parasite accumulation varied between cases of ECM, we observed broad trends in regional parasite accumulation within individual cases of ECM (Fig 10A), with parasite load typically greater in the Olb, Ctx, TH, MB and CBX than the other regions (Fig 10A). The number of haemorrhages was typically highest in the Olb (dramatically in some cases), but was of low level and variable in other regions between brains (Fig 10A). Conversely, the spatial nature of T-cell accumulation and permeable vessels was highly consistent in all cases of ECM, with the pathological features showing strong regional overlap (Fig 10A). Thus, although ECM is clearly a graded syndrome where the magnitude of pathological events varies from cases to case, the pathology is not stochastic with specific brain regions consistently more severely affected than others. This implies that architectural or physiological properties may predispose specific brain regions to malaria-induced cerebral pathology. Given the conserved and equivalent regionalisation, we examined the co-dependent relationship (s) between the identified pathological processes and their relative (individually and in combination) contribution in promoting ECM. As expected, there was a significant correlation between regional parasite and T-cell load, which is in agreement with the observation that parasites and T-cells co-localise within the tissue (S15 Fig & Fig 4E). Interestingly, the degree of vascularity did not correlate significantly with the number of parasites, haemorrhages or permeable vessels within brain regions, suggesting that vessel quality, rather than quantity, is more critical in determining regional pathological burden during ECM (S15 Fig). Importantly, through using generalised linear modelling, we found that combinations of histopathological parameters were, generally, better predictors of vascular permeability (which our results indicate is the major pathological event during ECM) within a brain region, compared to any single histopathological parameter (Fig 10B & S1 Table). For example, parasite burden, T-cell load and degree of vascularity combined were a better predictor of the number of permeable vessels within a brain region, than any of these factors in isolation (Fig 10B). Collectively, these data support the assertion that ECM is a multifactorial neuropathology that does not develop in response to a singular, dominant pathological event within any region of the brain. However, parasite accumulation within the brain appears to be a proximal event important for intracerebral T-cell accumulation, localisation and function, which ultimately provokes vascular dysfunction. In this study we have utilised detailed histopathological investigations, analogous to those used in the study of HCM, to definitively assess the relative merit of the ECM model for the study of HCM. Critically, by contrasting cerebral pathology observed during ECM with that during uncomplicated malaria infection, we have also substantially resolved the specific intracerebral events associated with the development of the ECM syndrome. We demonstrated that the global accumulation of pRBCs within the capillaries of the murine brain is a specific and cardinal feature of ECM. The compartmentalisation of pRBCs predominantly within the cerebral microvasculature during ECM, rather than pooled within haemorrhage or entrapped by intravascular leukocyte aggregations, indicates that intracerebral pRBCs likely play a causal role in the late-stages of the murine syndrome. Indeed, the comparable efficacy of anti-malarial drug treatment in reversing ECM and HCM strongly implies that intracerebral pRBCs play an active role in the late-stages of both mouse and human malaria-induced cerebral pathology [2,22]. However, our results also highlight clear differences in the presentation and magnitude of parasite accumulation during ECM compared with HCM. We found that intracerebral pRBCs were typically observed individually and irregularly distributed within brain capillaries during ECM. In contrast, a number of studies have shown that Pf parasitised erythrocytes are densely packed and congest significant lengths of the microvasculature during HCM [8–11,43,53]. Nevertheless, despite these differences, our results imply that some of the consequences of intracerebral pRBC accumulation may be the same in mice and humans. We have shown that in ECM, as in HCM, pRBC-dependent occlusion of brain capillaries and haemostasis are associated features of disease. In ECM, the width of murine cerebral capillaries necessitates the single-file passage of extensively deformed tubular erythrocytes. Consequently, the arrest of a single pRBC appears to be sufficient to occlude a murine brain capillary, and thus cause localised haemostasis. Conversely, in HCM, the available histopathological evidence suggests that mechanical obstruction of brain capillaries and resultant haemostasis depends on the incremental accumulation of large numbers of cytoadherent pRBCs [8–11,43,53] (Fig 11). Indeed, a process whereby uninfected erythrocytes are initially able to squeeze past cytoadhered pRBCs, until a critical threshold of pRBCs is reached within a vessel and mechanical obstruction occurs, is the only logical explanation for the high intracerebral pRBC sequestration indexes observed in HCM (i. e. in one study it was shown that that, on average, 66. 5% of intracerebral RBCs were parasitised, compared to 1. 4% in the peripheral blood [42]). Thus, although the presentation of pRBC accumulation in the cerebrovasculature may be different in ECM and HCM, our results provide a rational potential explanation for the comparable alterations in blood flow observed in vivo during murine and human malaria-induced cerebral pathology [54–59]. Whether the differential natures of pRBC-mediated vascular occlusion during ECM and HCM depend upon established differences in murine and human capillary diameter (average 3um vs 6. 4um) [60–62], or upon the degree to which murine and human cerebral capillaries can mechanically dilate, requires further investigation. The major question, therefore, is how do Pb ANKA parasites accumulate within the brain during ECM? Whilst we did not observe any knob formation on Pb infected erythrocytes (and thus no knob-based cytoadhesion as is observed with Pf [9,11,63,64]), we did observe the occasional electron dense spot on the surface of some parasitised erythrocytes adjacent to the cerebral vasculature during ECM. Such events appear comparable to the knob-independent forms of sequestration demonstrated by Pf infected RBCs in vitro [65], and may reflect the in vitro capacity of Pb ANKA infected RBCs to bind VCAM-1 expressed by brain ECs [66]. However, electron dense spots were not observed consistently on pRBCs within perfused brain microvessels, implying other mechanisms must also contribute to intracapillary pRBC accumulation during ECM. The equivalent propensity of Pb ANKA and Pb NK65 to parasitise larger reticulocytes suggests pRBC size does not determine the capability of different Pb strains to immobilise within cerebral capillaries [67]. However, it may be that some strain intrinsic qualities relating to the rheological properties of pRBCs, including deformability, specify the intracerebrovascular accumulation capacity of Pb infected erythrocytes. Indeed, Pasini et al have previously demonstrated differences in the repertoire of proteins expressed by ECM-inducing and non-ECM inducing Pb strains [68]. Crucially, although the differential expression of these proteins did not directly alter the cytoadherent capabilities of the parasites, it was not assessed whether the repertoire of protein expression influenced pRBC rheology. However, we also noted, consistent with a previous study [55], that vasospasm appeared to be a specific feature of Pb ANKA infection. A narrowing of the vascular lumen would exacerbate any rheological impairment, suggesting variant host responses to different Pb strains may also contribute to haemostasis during ECM. The conclusion that mature Pb ANKA infected RBCs become mechanically trapped within narrow murine brain capillaries during ECM, rather than accumulating as a result of strong cytoadherence, is supported by our failure to detect independent pRBC accumulation in venules or other large calibre vessels. Indeed, we previously failed to observe long-lasting pRBC adherence within the wider pial vessels using intravital microscopy [26]. Moreover, as opposed to observations in HCM [69], we occasionally observed extravascular pRBCs in the perivascular spaces and within haemorrhages (an observation that was relatively more common in the meninges [26]), suggesting Pb ANKA pRBCs are not tightly adhered to the brain endothelium and thus freely liberated from vessels upon necrotic EC loss. Further work will be required to identify the precise factors that dictate the capacity of specific Pb strains to accumulate within the cerebrovasculature during infection to cause ECM. Nevertheless, although our results suggest that ECM is a good model to understand the downstream effects of intracerebral pRBC accumulation and resultant haemostasis, they also imply that it is not a good system to investigate the consequences of direct pRBC cytoadherence to brain ECs. However, relevantly, the importance of direct (parasite sequestration-dependent) compared with indirect (inflammation-driven) activation and dysfunction of human brain ECs in the development of HCM is yet to be definitively identified [70]. Although our data indicates that pRBC-mediated occlusion of the cerebrovasculature appears to occur during ECM, as is observed during HCM, microvascular obstruction alone cannot explain the full repertoire and nature of murine and human malaria-induced cerebral pathology [71]. Beyond ischemia, which does not satisfactorily explain our neuropathological findings, nor the rapidly reversible nature of ECM and HCM, it is unclear how parasitised erythrocytes and subsequent microrheological alterations promote coma and death. Murine studies propose the cross-presentation of merozoite-derived parasitic material by cerebral ECs licences cerebrovascular CD8+ T-cells to promote vascular leakage during ECM [30,31]. Accordingly, we observed significantly greater numbers of intracerebral CD8+ T-cells during Pb ANKA infection compared to Pb NK65 infection. CD8+ T-cells were located within the intra- or perivascular space and, interestingly, were typically proximal to pRBCs or parasite material during ECM. Indeed, there was a strong correlation between parasite and T-cell load within the different brain regions. Thus, our data, in the context of the current literature, suggests intracerebral pRBC accumulation and subsequent microvascular obstruction fulfils three roles vital to the pathogenesis underlying ECM: 1) to provide parasite antigen for cross-presentation; 2) to promote haemostasis, thus ensuring the necessary microenvironment in which cerebral ECs are able to obtain merozoites for cross-presentation, which, under physiological flow conditions, would normally be rapidly cleared; and 3) to instigate signals important for the cerebrovascular localisation of CD8+ T-cells. Notably, despite the importance of CD8+ T-cells in promoting ECM [27], they were observed relatively rarely within the brains of mice infected with Pb ANKA; being less frequent than pRBCs and substantially less populous than macrophages and monocytes. The reasons for the differences in intracerebral macrophage/monocyte and CD8+ T-cell numbers, considering they are similar located in the cerebrovasculature and likely depend on the same EC-derived ligands/integrins, are not clear, but may depend upon temporal differences in recruitment [72]. Nevertheless, the general rarity of CD8+ T-cells within the brain implies that very few are required to promote ECM and, furthermore, may explain the failure, thus far, to consistently locate this cell population in HCM histopathological studies [5,9]. CD8+ T-cell-dependent vascular leakage is currently considered critical to the development of ECM [26,51]. Consistent with this, we observed greater evidence of cerebral oedema during Pb ANKA infection compared to Pb NK65 infection. The causes of oedema during ECM appeared multifactorial in origin as, in accordance with HCM histopathological study [5], we detected increased permeability around haemorrhages, parasitised capillaries, intravascular leukocyte accumulations, and some scattered microvessels devoid of any specific vascular-pathological feature. Indeed, we observed significant correlation between the number of permeable vessels within a brain region and parasite, T-cell or haemorrhage load. These observations additionally support the hypothesis that extensive cerebral oedema may promote the fatal cerebral swelling that occurs during human and murine malaria-induced encephalopathy [25,26,51,73,74]. Our data also provides significant information on the mechanism through which CD8+ T-cells cause vascular leakage during ECM. We showed that, whilst haemorrhage was evidently secondary to EC damage, the majority of vascular leakage occurred independently of EC loss or apoptosis. This suggests, consistent with our previous study [26], and despite the importance of CD8+ T-cell cytolytic functions in promoting BBB dysfunction during ECM [28,29], that vascular leakage occurs without EC loss during murine malaria-induced cerebral pathology. Instead of vascular loss, we demonstrated that significant vascular remodelling occurs specifically during ECM. Clefts within the microvascular tight junctions and increased levels of caveolae were noted within ECs adjacent to cerebral oedema. Moreover, pronounced cytoplasmic extensions, or pseudopodia, were evident in several brain vessels. Such alterations in EC morphology are traditionally viewed as hallmarks of angiogenesis [75]. Consequently, our observations support a model whereby CD8+ T-cells, via Granzyme B and perforin [28,29], promote a vascular stress response in ECs, resulting in the production of angiogenic factors which, although protective against cellular apoptosis, cause lethal alterations to the paracellular and/or transcellular permeability of the cerebrovasculature during ECM (Fig 12). Supporting this hypothesis, in a variation of Theiler’s murine encephalomyelitis model of MS, CD8+ T-cells have been shown to promote the production of VEGF in a perforin-dependent fashion, causing vascular leakage without cerebral EC loss [76]. Importantly, this scenario affords a rational explanation as to why rapid recovery from ECM and restoration of vascular integrity can occur after anti-malarial drug treatment, which would not be possible if vascular leakage were determined by extensive and irreversible EC loss. Intriguingly, not only has vascular leakage in the absence of EC loss been observed in HCM histopathological studies [5], but so has disruption of intercellular tight junctions [12,77]. Our data, in conjunction with the literature, imply the mechanisms underlying vascular dysfunction may be conserved between humans and mice during malaria-induced cerebral pathology. These observations further underline the requirement for highly resolved histopathological studies to be performed to specifically examine the potential importance of CD8+ T cells in the pathogenesis of HCM. Therefore, the final and critical questions are 1) how does the neurovascular pathology characterising ECM influence parenchymal brain function to induce coma and death, and 2) are the pathways to neurological dysfunction conserved between ECM and HCM? We demonstrated that axonal injury (AI) is a significant and specific feature of ECM, as evidenced by positive β-APP staining, a protein that accumulates at sites axonal damage [78]. Axons extend significant distances, and are thus dependent on a huge number of microvessels for the provision of oxygen and glucose to permit their metabolically expensive functions [13]. Indeed, AI has been shown to occur in response to both hypoxia and hypoglycaemia [79,80]. Accordingly, we observed AI proximal to erythrocyte congested vessels, suggesting microvascular obstruction likely accounts for much of the AI observed during ECM. However, we also observed AI adjacent to haemorrhage and intravascular leukocyte accumulation, indicating that axonal dysfunction is potentially a common mechanism through which multiple pathological parameters of ECM impair neurological function. Interestingly, β-APP accumulation within axonal tracts may represent reversible axonal damage, consistent with the rapid neurological recovery observed in ECM after anti-malarial drug treatment [81]. In addition to AI, we frequently observed myelin damage during ECM, with areas of myelin pallor and vacuolation seen proximal to erythrocyte-congested vessels, and physical loss of the myelin sheath detected adjacent to haemorrhage. The progressive accumulation of myelin and axonal damage would explain the graded and sequential neurological dysfunction observed during ECM, including ataxia, fitting and reduced responsivity [82]. Coma may ensue due to a culmination of axonal and myelin pathology (potentially as a programmed neuroprotective response to lower cerebral metabolic demand). Importantly, the nature of AI and myelin damage observed in ECM is highly similar to that reported in HCM [5,13]. Moreover, AI and myelin damage are colocalised to specific, shared vascular-pathological features, including haemorrhage and pRBC occluded microvessels, in both ECM and HCM [5,13] Combined, this implies that the mechanisms responsible for the reversible and/or permanent neurological dysfunction observed during and after an episode of malaria-induced encephalopathy, are very likely conserved between mice and humans. In summary, the results in this manuscript show, in significant detail and for the first time, that mature pRBCs specifically accumulate within the cerebral capillaries during ECM. Although the mechanism and presentation of pRBC accumulation during ECM is significantly different to that observed during HCM, there appears to be overlap in the pathological impact of parasite-induced haemostasis during both syndromes. It is, however, clear that ECM does not appear to be a good model to study the impact and role of pRBC cytoadherence to cerebral blood vessels in the development of malaria-induced cerebral pathology. Nevertheless, in spite of this, a number of pathological features of HCM are observable and appear comparable in nature during ECM, including; BBB disruption, AI and myelin damage. The critical role of CD8+ T cells in initiating the ECM syndrome, when roles for the cells have yet to be revealed in HCM, remains a divisive point. Based upon our data, showing significant similarities in vascular and parenchymal pathology in ECM and HCM, and the fact that so few cerebrovascular CD8+ T cells can dominantly drive cerebral pathology during ECM, detailed investigations of the role of CD8+ T cells during HCM are warranted. Nonetheless, if, after detailed investigation, it is ultimately found that CD8+ T cells cannot be involved in the development and progression of HCM, the observation that many of the pathological features of ECM are similarly found in HCM indicates that convergent signals may ultimately drive the same severe pathological manifestations during ECM and HCM. Thus, when used appropriately (i. e. not solely relying on KO mouse studies to examine pathogenesis or employing treatments before ECM develops), our collective results support the utilisation of the ECM model to understand the pathological events secondary to pRBC accumulation in HCM. In addition, careful utilisation of the ECM model may be useful for the identification of novel adjunct therapies for the repair and resolution of the vascular and parenchymal damage that occurs similarly within the established ECM and HCM syndromes. All animal work was approved following local ethical review by the University of Manchester Animal Procedures and Ethics Committees and was performed in strict accordance with the U. K Home Office Animals (Scientific Procedures) Act 1986 (approved H. O. Project License 70/7293). Female and male 8–10 week old C57BL/6 mice were purchased from Charles River and maintained in individually ventilated cages at the University of Manchester. Cryopreserved P. berghei ANKA GFP [83] and P. berghei NK65 GFP [84] parasites were thawed and passaged once through C57BL/6 mice before being used to infect experimental animals. Animals were infected via intravenous injection of 1x104 parasitised red blood cells (pRBCs). Peripheral parasite burdens of infected mice were followed from day 3 post infection (p. i.) by microscopic examination of giemsa stained thin blood smears and weight loss was monitored. The development of ECM was assessed using a well-established clinical scale [41]: 1 = no signs; 2 = ruffled fur and/or abnormal posture; 3 = lethargy; 4 = reduced responsiveness to stimulation and/or ataxia and/or respiratory distress/hyperventilation; 5 = prostration and/or paralysis and/or convulsions[41]. Stages 4–5 were classified as ECM. P. berghei ANKA infected mice were euthanised (exposure to a rising concentration of CO2) when they reached stage 5 (typically day 7 p. i.) and P. berghei NK65 mice were culled at the equivalent time point. After termination, the hepatic portal vein was severed and mice were transcardially perfused with 10mls of 0. 1M ice cold phosphate buffered saline (PBS) followed by 10mls of ice cold 4% paraformaldehyde (PFA). Brains were dissected out and post-fixed in PFA/20% sucrose for 16-24h at 4°C. Brains were subsequently cryoprotected in PBS/20% sucrose for 48h, snap-frozen in powdered dry ice and stored at -80°C. Brains were serially sectioned at a thickness of 30μm on a freezing sledge microtome (Bright Instruments, Cambridge, UK). Series of coronal sections encompassing 10 spatially-defined anatomical regions of the brain (S1) were stored in cryoprotectant solution (30% ethylene glycol, 20% glycerol in PBS) in the individual wells of a 24 well tissue culture plate (Corning, NY, US) at -20°C until use. Brain sections were stained with direct and indirect immunofluorescent technique for the following: i) anti-GFP (1: 400 rabbit anti-GFP Alexa Fluor 488 conjugated clone A-21311 Life Technologies) for detection of GFP-tagged parasites; ii) CD31 (1: 200 rat monoclonal antibody [mAb] clone MEC 13. 3 BD Pharmingen) for visualisation of vasculature; iii) CD3 (1: 100 rat mAb clone CD3-12 AbD Serotec) for assessment of cerebral T lymphocyte accumulation; iv) Lycopersicon esculentum (tomato) lectin (1: 100 [reconstituted 1mg/ml in PBS] biotin conjugated clone L0651 Sigma-Aldrich) for visualisation of vasculature and activated cerebral macrophage populations; v) cleaved caspase 3 (1: 200 rabbit mAb clone Asp175 5A1E Cell Signalling) for assessment of cellular apoptosis; vi) NeuN (1: 200 mouse mAb biotin conjugated clone A60 Chemicon/Merck Millipore) for demonstration of neuronal architecture; and vii) β-Amyloid Precursor Protein (β-APP 1: 200 rabbit polyclonal antibody [pAb] clone CT695 Zymed/Thermo Fisher Scientific) for detection of axonal injury. The nature of the immunofluorescent staining protocol varied depending on the antibody/epitope pairing. Some stains (i and ii) were performed utilising free-floating protocols. The remaining stains were performed after floating sections were washed in PBS and mounted in distilled water onto Superfrost Plus slides (VWR), before drying vertically overnight at 37°C. All sections were re-hydrated in several changes of PBS before being subjected to heat-mediated antigen retrieval in either Sodium Citrate pH9 buffer (i and ii), Sodium Citrate pH6 buffer (iv, v, vi and vii) or Tris EDTA pH9 buffer (iii, iv and v) pre-heated, respectively, to 80°C, 95°C and 99°C in a water-bath. Sections were subsequently heated for 30 minutes and then allowed to cool at room temperature for 20 minutes. With respect to β-APP (vii), slides were additionally treated with 90% formic acid in distilled water for 15 minutes. All sections were then washed in several changes of wash buffer (0. 1M Tris-HCL pH7. 5,0. 15M NaCl, 0. 05% Tween in distilled water) before being blocked for 1. 5 hours at room temperature in block buffer (0. 1M Tris-HCL pH7. 5,0. 15M NaCl, 1% Bovine Serum Albumin [BSA] 0. 3% Triton X in distilled water). Block was removed and sections were incubated with primary antibody diluted appropriately in block buffer; either at room temperature for 3 hours (iv and v) or 12–20 hours at 4°C (i, ii, iii, iv, v, vi and vii). Sections were rinsed several times in wash buffer, and, for fluorescent detection, sections were incubated for 1. 5 hours at room temperature in excess quantities of secondary antibodies (goat-anti rat 546, goat anti-rat 647, goat anti-rabbit 546, streptavidin 546, and streptavidin 647 Life Technologies/Thermo Fisher Scientific) diluted in block buffer. An intermediate incubation utilising a biotinylated goat anti-rabbit antibody (Vector) was undertaken to amplify signal for adequate fluorescent visualisation of β-APP (vii), whilst a Tyramide Signal Amplification kit (Thermo Fisher Scientific) was utilised as per manufacturer’s instructions in order to enhance detection of CD3 (iii). Sections were finally washed in several changes of wash buffer and counterstained in DAPI (Sigma-Aldrich). Sections were sequentially rinsed in PBS and distilled water, dried overnight in the dark at room temperature and then coverslipped in ProLong Diamond anti-fade Mountant (Life Technologies). Antiserum to Pb ANKA and Pb NK65 infected erythrocytes was prepared as previously described [85]. In brief, mice underwent three rounds of infection and drug cure before whole serum was extracted and IgG purified on Protein-G (HiTrap). Mounted brain sections were blocked with rat serum prior to incubation with anti-Pb ANKA or Pb NK65 IgG for 1 hr at room temperature. Following incubation with anti-PbA IgG, slides were visualised using FITC rat anti-mouse antibody (clone 11-4011-85: E-Bioscience). To assess BBB permeability, brain sections were stained via indirect immunoperoxidase technique for endogenous Immunoglobulin G (IgG 1: 500 Horse pAb biotin conjugated clone BA-2000 Vector). Free-floating sections were washed in PBS and mounted on to Superfrost Plus slides (VWR) in distilled water, then allowed to dry vertically overnight at 37°C. Sections were rehydrated in several changes of PBS and subjected to heat-mediated antigen retrieval in preheated Sodium Citrate pH6 buffer at 95°C for 30 minutes, and then allowed to cool at room temperature for 20 minutes. Slides were rinsed twice in PBS and then endogenous peroxidase activity was blocked by incubation in 3% H2O2 in distilled water at room temperature for 30 minutes. Sections were washed in several changes of PBS and then incubated for 3 hours at room temperature with primary antibody appropriately diluted in PBS, 0. 3% Triton X and 0. 1% BSA. Slides were washed thoroughly in PBS and 0. 1% tween and then incubated for 1. 5 hours at room temperature in ABC solution, as per manufacturer’s instructions (Vector). Colour was developed via a 5 minute incubation in diaminobenzidine tetrahydrochloride (DAB, Merck Millipore). Sections were counterstained with haematoxylin (Vector), dehydrated through alcohol, cleared in two changes of xylene and coverslipped using DPX mounting agent (Sigma-Aldrich). Brain sections were stained via haematoxylin and eosin (H&E) to assess the degree of haemorrhage, parasite sequestration, oedema and white matter disruption. In brief: free-floating sections were washed in PBS and mounted on Superfrost Plus slides (VWR) in distilled water, then allowed to dry vertically overnight at 37°C. Sections were stained using a Thermo Shandon Linstain GLX (Rankin Biomed, US) automated staining machine. Slides were coverslipped using DPX mounting agent (Sigma-Aldrich). Animals were terminated via exposure to a rising concentration of CO2. The hepatic portal vein was severed and mice transcardially perfused with 10mls PBS. Brains were removed and anatomically comparable regions of cortical and cerebellar grey matter, measuring no greater than 1mm in diameter, were excised. Smears were generated between two microscope slides as previously described [43]. For H&E staining, slides were immediately wet-fixed in acetic alcohol at 4°C, and staining performed as previously described for cytological specimens [86]. For Quik-Diff staining, slides were air-dried before subsequent fixation and staining, performed as per manufactures instructions (Baxter). Animals were terminated via exposure to a rising concentration of CO2. The hepatic portal vein was severed and mice transcardially perfused with 10mls PBS. Brains were removed and a single-cell suspension was generated by homogenising tissue through a 70μm cell strainer (BD Falcon) in 5ml of ice-cold PBS. 10μl of cells were pipetted onto a microscope slide and cover-slipped. Images were collected on a Zeiss Axioskop upright microscope or Olympus BX51 upright microscope using a 20x objective and captured using a Coolsnap ES camera (photometrics) through MetaVue software (Molecular Devices). Images were then analysed and processed utilising either ImageJ or Image-Pro Premier software (Media Cybernetics). Under isoflurane anaesthesia, mice were sequentially perfused intracardially with PBS and fixative (2% PFA and 2. 5% glutaraldehyde) at 10ml/minute for 7 minutes. Brains were removed and post-fixed for a further 4 hours before anatomically comparable regions of cortical and cerebellar grey matter, measuring approximately 1mm in width, were excised and post-fixed for a further 20 hours. Tissue was then additionally fixed on ice for 1 hour with 1. 5% potassium ferrocyanide and 2% osmium tetroxide (weight/vol) in 0. 1M cacodylate buffer. This was followed by incubations with 1% thiocarbohydrazide for 20 minutes at room temperature, 2% osmium tetroxide for 30 minutes at room temperature and 1% uranyl acetate at 4°C overnight. The next day, samples were stained with freshly prepared Walton’s lead aspartate (0. 02M in lead nitrate and 0. 03M in aspartic acid, pH 5. 5) for 30 minutes and embedded in Epon 812 (hard forumalation) epoxy resin (Electron Microscopy Science, UK). Resin-embedded samples were subsequently cut at a thickness of 70nm using an ultramicrotome (Leica). Sections were mounted on formvar-coated grids and viewed on an FEI Tecnai 12 Biotwin Transmission Electron Microscope. To assess endothelial cell morphology in each specimen, images of the first 15 capillaries identified were collected digitally using a Gatan Orius SC1000 camera. To examine the interaction between the endothelium and sequestered pRBCs, entire grids were examined at low power to identify regions of interest and then imaged digitally at a high power (Gatan Orius SC1000 camera). Images were analysed and processed utilising ImageJ. Ten spatially-defined anatomical regions of the brain, determined by the Allen reference atlas for the C57BL/6 brain [87], were selected for examination. For quantitative purposes 10 random fields of view per region were captured. Distribution, number and/or area of GFP+ parasites, CD3+ T-cells, haemorrhages, extravascular IgG+ permeable vessels and CD31+ vessels were counted manually in a blinded fashion or via ImagePro Premier’s smart segmentation technology in a semi-automated fashion as previously described [26]. Data are expressed as number or area of objects/mm2 in a given brain region, or, alternatively, as % of a given distribution within the total number of objects within a given brain region. All statistical analyses were performed using GraphPad PRISM (GraphPad Software). Comparison between two groups was made using unpaired t tests with Welch’s correction. Comparison between multiple groups was made using a one-way ANOVA with Tukey’s test for multiple comparisons. Correlation between different variables within individual brain regions was determined using Spearman-Rank test. Generalised linear models were fitted to the data using the lm function of the R statistical language. Linear models were fitted in turn to each measured variable and combinations thereof, within individual brain regions. The quality of different models was compared by computing the R2 value.

Cerebral malaria (HCM) is the most severe complication of malaria infection. Despite this, we have an incomplete understanding of the cause (pathogenesis) of the syndrome. To improve our understanding of HCM pathogenesis, animal models of the syndrome have been developed. The most commonly used model is the murine experimental cerebral malaria (ECM) model. However, to date, there has not been a detailed investigation of the pathology of ECM using the same methodological approaches (histopathology) employed in the study of HCM. Thus, it has been unclear whether ECM is a valid model for HCM. In this histopathological study, we show that, as in HCM, cerebrovascular parasite accumulation is an important feature of ECM. However, unlike HCM, we did not observe large numbers of parasitised red blood cells (pRBCs) attached to the walls of cerebral blood vessels during ECM; instead individual pRBCs were trapped in narrow murine brain capillaries. Nevertheless, despite this, we showed that cerebrovascular parasites were still associated with disturbed blood flow, vascular leakage and impaired neuronal function in ECM, in a similar fashion to that reported in HCM. Therefore, our results define the specific aspects of HCM pathology that can potentially be studied within the ECM model.

lay_plos

Deep sequencing was used to determine complete nucleotide sequences of echovirus 11 (EV11) strains isolated from a chronically infected patient with CVID as well as from cases of acute enterovirus infection. Phylogenetic analysis showed that EV11 strains that circulated in Israel in 1980-90s could be divided into four clades. EV11 strains isolated from a chronically infected individual belonged to one of the four clades and over a period of 4 years accumulated mutations at a relatively constant rate. Extrapolation of mutations accumulation curve into the past suggested that the individual was infected with circulating EV11 in the first half of 1990s. Genomic regions coding for individual viral proteins did not appear to be under strong selective pressure except for protease 3C that was remarkably conserved. This may suggest its important role in maintaining persistent infection. Enteroviruses widely circulate in human populations and only rarely cause clinical disease. Polioviruses were the first enteroviruses to be isolated and the first to have the pattern of changes in their genome during chains of transmission characterized [1,2]. They cause acute flaccid paralysis of limbs and occasionally bulbar paralysis. Paralysis rate in naïve individuals, however, is around one clinical case per 100 to 1000 infections. Other enteroviruses can also cause paralysis but at a lower rate. Enteroviruses cause a variety of other clinical conditions including mild fever, hand-foot-and-mouth disease, herpangina, myocarditis, diabetes, etc. Human echovirus 11 is a member of the human enterovirus B species and is one of the most commonly isolated enteroviruses [3,4]. EV11 viral capsid protein 1 (VP1) sequences segregate into six or more genogroups [5,6]. Data from the Annual Reports of the Central Virology Laboratories, Public Health Service of the Israeli Ministry of Health, Tel Hashomer Israel, indicate that EV11 is endemic in Israel with at least one EV11-positive case being reported on 17 of the 27 years between 1986 and 2012. There was a major peak of 90 EV11-positive stools in 1999 and smaller peaks of in 1991,1993,1995–6,2001,2005 and 2011. Enteroviruses are also able to chronically infect individuals with several kinds of primary immunodeficiency [7–10], and persist for months or years being regularly excreted in stool. This phenomenon is best known for poliovirus, which belongs to species C of human enteroviruses. Chronic poliovirus infection can last for over 30 years [11]. At least 110 immune deficient individuals have been identified who had persistent infections with immunodeficiency-associated vaccine-derived polioviruses (iVDPVs) [12,13]. Additional cases of persistent infection from unidentified individuals have been inferred from environmental surveillance and isolation of ambiguous highly evolved vaccine-derived polioviruses (aVDPVs) [14]. Circulating vaccine-derived polioviruses (cVDPV) represent another kind of VDPV [15]. They have regained virulence and the ability to transmit in human populations indistinguishable from wild polioviruses, and can serve as a model for natural evolution of enteroviruses. VDPVs can cause outbreaks of paralytic disease in susceptible immune competent individuals. The pattern of amino acid substitutions and recombination of iVDPVs and aVDPVs has been shown to differ from cVDPV [16]. Among the EV11 isolates from Israel were 10 referred to hereafter as iEV11s that were obtained from the CSF of an immune deficient individual with Common Variable Immunodeficiency (CVID) who suffered from symptoms of chronic encephalomyelitis between 1995 and 1999. VP1 sequences of these isolates were obtained using Sanger-based sequencing. These sequences were compared with VP1 sequences EV11s isolated from cerebrospinal fluid, stool, rectal and throat swabs of contemporary cases of EV11 infections of non-immune deficient patients (2 in1992,3 in1993,1 each in 1996,1997 and 1998, and 8 in 1999). A comparison of the patterns of differences in the VP1s of the two groups indicated that the iEV11s represented a persistent infection rather than repeated re-infections from contemporary EV11s circulating in the community. Here we report the use of deep sequencing to determine complete genomic sequences of the 10 iEV11 isolates and 16 contemporary cEV11s to confirm that the iEV11s represented a persistent infection and to obtain information on the pattern of its evolution in humans during persistent infections of immune deficient individuals. To our knowledge this is the first analysis of evolution of HEV B during a persistent infection using complete genome sequences. The Ethical Review Board of the Sheba Medical Center, Tel Hashomer approved this study (SMC-7685). The clinical samples from which viruses were isolated are a part of the virus strain collection of the Central Virology Laboratory in Israel. They were received between 1986 and 2014 and results were stripped of all links to personal details pertaining to, or which could be used to identify individual patients. All data were analyzed anonymously. The Ethical Review Board exempted this study from a requirement for obtaining informed consent. The annual incidence of laboratory confirmed EV11-positive stools received by the Central Virology Laboratory in Israel between 1986 and 2014 is presented in S1 Fig. Altogether there were 212 individuals with EV11-positive clinical samples. Among them were eleven EV11 isolates obtained between November 13,1995 and December 21,1999 from a male patient. These isolates are referred to as iEV11s to indicate that they were isolated form an immune deficient individual. The patient, born in 1955, was diagnosed with CVID at age 20 after recurrent bouts of pneumonia, sinusitis, and ear infections along with episodes of aseptic arthritis, as well as leukopenia and alopecia areata advancing to alopecia universalis. His younger brother had died from a bacterial complication known to be associated with CVID. The patient was treated with intravenous immunoglobulins (IViG) between 1975 and 1995, and prophylactically with antibiotics between 1985 and 1995. Following a year or more of poor adherence to IViG treatment, decreasing the infusions to once in 2 months, he presented with decreased hearing, a rash and neurological symptoms of headaches, stroke-like episodes and proximal lower limb weakness. He was diagnosed at the department of Neurology as suffering from chronic enteroviral meningoencephalomyelitis due to EV-11 that did not respond to reintroduction of frequent (bi-monthly) IViG infusions. In 1998 the patient received two one-week courses of daily Pleconaril (400 mg/day), under expanded access in February and then again in October. This treatment did not affect the course of his disease and his lower limb weakness gradually deteriorated along with mild cognitive difficulties. In December 2012 there was sudden worsening of his condition and he lapsed into a coma associated with acute hydrocephalus that did not respond to a ventriculoperitoneal shunt and he succumbed to his illness. Representative EV11 isolates collected prior to and during the isolation of these iEV11 samples that were sent for clinical analysis were chosen for comparison: eight from sporadic cases presenting between 1992 and 1998 and eight collected during a large EV11 outbreak in 1999. Clinical samples and symptoms associated with each isolate are indicated in S1 Table. Virus from the first tissue culture passage (TC1) that had been kept at -20°C were passaged once (TC2) on human human kidney epithelial cells (HuKi) received from Dr. Furin (Department of Health and Welfare, Ottawa, Canada) and RD cells (human rhabdomyosarcoma cell line; ATCC CCL136). Virus from four samples from the CVID patient and seven from among the representative samples were no longer viable by the time of this analysis (“Original TC” in S1 Table). RNA was extracted from the virus in 1 ml of supernatant from the second passage for viable EV11 isolates, or from 1 ml of the original frozen stock for non-viable EV11 using a MagNA Pure LC2. 0 Automatic extractor with MagNA Pure LC Total Nucleic Acid Isolation kit-High Performance (Roche Diagnostics, IN, USA) and eluted into 50 µl of elution buffer according to manufacturer’s instructions. The RNA library was prepared from the total RNA of 27 EV 11 isolates, the NEBNext mRNA Sample Prep Master Mix Set 1 (New England BioLabs, Ipswich, MA) was used according to the manufacturer' s protocol (NEB). Briefly, 0. 5 µg of the total RNA was used for fragmentation by focused ultrasonicator (Covaris) to generate the fragments of optimal sizes (250–300 nt) suitable for Illumina sequencing. cDNA was synthesized using SuperScript III Reverse Transcriptase (Invitrogen) and random primers. The cDNA was converted into double stranded cDNA followed by end repair procedure (Klenow fragment, T4 polynucleotide kinase and T4 polymerase), and was ligated to Illumina paired end (PE) adaptors. Size selection was performed using double AMPure bead selection step (Beckman Coulter), generating DNA libraries ranging from 200 to 500 bp in size. Next the DNA libraries were expanded using 15 cycles of PCR with multiplex indexed primers and purified by magnetic beads (Agencourt AMPure PCR purification system, BeckmanCoulter). Finally, the DNA fragments were analyzed for quality and size distribution by BioAnalyzer using a high sensitivity kit (Agilent Technologies, Inc., Santa Clara, CA). Deep sequencing was performed using MiSeq (Illumina) producing paired end reads of 250 nt long, or HiSeq2000 (Illumina) producing 101 nt long paired-end reads. The sequencing reads were analyzed by in-house' swarm' or High Performance Integrated Virtual Environment (HIVE) software [17,18]. Raw sequence reads were subjected to quality control and reads with phred score below 30 were removed. The reads were aligned to a curated database of 500 reference enteroviruses. Next, aligned sequences were separated into discrete sub-populations [19] and assembled into full length EV11 consensus genomic sequences (see S1 Table). This resulted in complete or near-complete genomic sequences of 25 isolates. For some virus isolates the depth of coverage (S1 Table) at the ends of genome was insufficient for reliable sequence reconstruction. To assemble individual consensuses of viruses present in mixtures a new algorithm was used [19]. The names of virus isolates were shortened to the name of EV11 genogroup followed by an incremental number by incremental order in which they were isolated. Neighbor-joining phylogenetic comparisons were prepared by Clustal X in MEGA v5. 22 [20] or MacVector software using the Tamura-Nei substitution model [21]. Separate analyses were prepared for near-complete sequences with 5' -ends trimmed to the longest sequence common to all sequences in the alignment, the entire open reading frame, the P1 region encoding all four viral capsid proteins, Viral Capsid protein 1 (VP1), the P2 plus P3 coding region encoding all non structural proteins, the 3D RNA-primed RNA polymerase, and the 3’ untranslated region (3’UTR, also partly truncated). The initial alignments of these regions were prepared for MEGA using the Sequencher v5. 2. 2 program (Gencodes, Anne Arbor, MI, USAS). Three-dimensional folding models of Loop V of the internal ribosomal entry site (IRES) in the 5’UTR were prepared using the web-based Unified Nucleic Acid Folding and hybridizing Package [22,23] at the University of Albany NY, USA, (http: //mfold. rna. albany. edu; last accessed November 2014) and the consensus sequence for the Israeli isolates. The three-dimensional folding model for the human enterovirus Z domain in the 3’UTR was taken from Merkel et. al. [24]. Amino acid differences between Israeli EV11 isolates from EV11 genogroup A and the earliest isolate from this genogroup, EV11-A01, were determined using the Sequencher program. Amino acid differences that became fixed during the persistent EV11 infection in the CVID patient were mapped onto the 5 capsomers located at the 5-fold axis of symmetry on the X-ray diffraction crystalline structure of the EV11 capsid (Accession number 1H8T) using the MacPyMol program (The PyMOL Molecular Graphics System, v7. 2. 1, Schrödinger, LLC., [25]. Genomic sequences were deposited in the GenBank/EMBL/DDBJ database (accession numbers KY981557 to KY981581), and the raw Illumina data in sequence read archive (https: //hive. biochemistry. gwu. edu/review/Echovirus11). To determine genomic sequences of EV11 isolates (S1 Table) we have used direct RNA library deep sequencing approach because the amount of live virus in some samples was very low, and the quantity of isolated RNA would not be sufficient for multiple PCR reactions needed for conventional Sanger sequencing. To recover RNA from these samples carrier tRNA was added. RNA was then fragmented by focused ultrasonication, converted to double-stranded cDNA and sequenced using Illumina Miseq or Hiseq technology. This resulted in a variable level of sequencing coverage in most cases ranging from 1,000 to 250,000 with the exception of four samples in which the coverage was lower (See S1 Table). However, even for the sample with the lowest depth of coverage (40) we were able to assemble consensus sequence that had an uninterrupted open reading frame of 2,195 amino acids. The nature of RNA library preparation is such that there is a gradual decline of the number of molecules representing the very ends of the genome. In some cases the low coverage of the terminal regions resulted in consensus sequences that missed few nucleotides from the ends because they could not be reconstructed reliably. The use of this protocol proved to be remarkably effective and the level of sequencing coverage per each nucleotide was sufficient not only for unambiguous reconstruction of consensus sequences but also for determining sequence heterogeneity profiles. The comparison of complete or near-complete genomes the EV11 isolates revealed that they could be separated into four clades or genogroups labeled A through D (Fig 1). Their relationship with other EV11 was determined by comparing with a representative subset of 100 published sequences of virion protein VP1 (Fig 2). On this and subsequent dendrograms the incremental number after the genogroup indicates the temporal order in which they were isolated. Attempts were made previously to classify EV11 isolates into genogroups [5,26]. The comparison of the newly sequenced isolates showed that the closest relative to our genogroup A was 1995 Tunisian isolate (HQ674714), which had 3. 4–3. 7% nucleotide differences with cEV11, and 5. 8–8. 3% with iEV11. This means that our genogroup A matches genogroup IV-4 of Fares. Genogroup B was related to 1991 isolate from Maine (AY121384) with 4. 3–4. 6% difference, placing it into genogroup IV-4 of Fares and D4 of Oberste. Genogroup C was related at 11. 2% to 1994 Tunisian isolate HQ674718 and at 11. 1–11. 5% to 1999 isolate from Kuwait (AY121408), belonging to Fares genogroup IV-3 and Oberste genogroup D3. Finally our genogroup D was very close (1. 1–1. 8%) to 1998 Tunisian isolate HQ674721 (Fares genogroup IV-5, Oberste genogroup A). Enteroviruses frequently recombine with other viruses of the same species. To determine whether the Israeli isolates are also products of recent recombination we have compared them with a reference set of 72 full-length sequences of Species B Enterovirus that contained at least one representative from each serotype, and all 12 full-length genomic sequences of EV11 available in Genbank. Figs 3A, 3B and 3C show the phylogeny of P1, P2, and P3 regions providing no evidence of recombination within the group of isolates from immunodeficient patient. In contrast, analysis of circulating strains revealed potential recombination event between P1 and P2 regions of genogroup D, as evidenced by its “jump” to another branch of the phylogenetic tree. Previous studies found about 30% divergence between different isolates of EV11 [26]. Therefore close similarity between strains described in this study lends support to the notion that all the iEV11s isolated between 1995 and 1999 were sequentially related to four group A isolates from 1992 and 1993. The hypothesis that these 1992 and 1993 isolates were closely related to the iEV11s is strengthened by the observation that cEV11 of genogroup A were closest to the earliest iEV11 isolate (5. 8%), and more distant (8. 3%) from the latest ones. This is illustrated by neighbor-joining phylogenetic tree shown on Fig 4A. It suggests that iEV11 may have evolved from a cEV11 strain close to the Israeli isolates from 1992–1993. Regression lines for the accumulation of nucleotide and amino acid substitutions relative to the closest cEV111 strain shown on Fig 4B can be extrapolated to the early 1990s, a tentative time when the patient became chronically infected with EV11. The first iEV11 strain isolated in 1995 (A5) had a total of 139 nucleotide differences from the closest 1992 cEV11 isolate A1. These mutations never reverted back, and remained in all subsequent iEV11 isolates. An additional 12 “substitutions” that emerged during the same period, reverted back in subsequent iEV11 isolates. Between February 1996 and April 1998 there were 113 additional mutations that emerged and became fixed in all subsequent isolates and 8 more that emerged but then “reverted back” by October 1999. An additional 79 substitutions emerged between April 1998 and October 1999 followed by 2 more between October and December of 1999. Therefore during the four time intervals between 1995,1996,1998, and two dates in 1999 the rates of mutations fixation were: 0. 83*10−2,1. 16*10−2,0. 71*10−2, and 0. 68*10−2 mutations / site / year, a rate close to the molecular clock rate of 1. 03*10−2 reported for circulating poliovirus [1], 1. 51*10−2 for iVDPV [11], and 0. 84*10−2 for EV30 isolated from chronically infected immunodeficient patient [8]. Analysis of the predicted amino acid substitutions that occurred during the iEV11 evolution showed that most (28 of 35, or 80%) of the amino acid substitutions became fixed in the evolving genome (S2 Table). Fixation of 10 amino acid substitutions resulted from 2 nucleotide differences in the codon, one involved 3 nucleotide differences, and one fixed amino acid was represented by a single nucleotide difference followed by a second silent mutation in the six subsequent isolates. Four amino acid differences were unique, e. g., found in only one of the 10 isolates, and one substitution appeared in isolates A06 through A10, but not A11 through A14. The distribution of amino acid changes along the iEV11 genome is shown on Fig 5. It revealed that the entire 3C protein was highly conserved, along with parts of 3D (between amino acids 142 and 300), 3A (amino acids 1–128 and 140–236), 2C (amino acids 2–118), and VP1 (amino acids 157–267). Hot spots of mutations included amino acids 116–144 and 267–292 of VP1, and 145–164 of VP2. The analysis of selective nature of mutations in individual viral proteins was performed by calculating density of nucleotide and amino acid substitutions, Dn and Da, respectively. The values represent the number of nucleotide and amino acid substitutions that have been fixed in at least one isolate, divided by the size of the coding region (nt) or the protein (aa). If the ratio Da/Dn is less than 1, then there is a negative selection against amino acid changes, while the ratio greater than 1 indicates possible positive selection of certain amino acid changes. Fig 6A shows that amino acid substitutions in most viral proteins were largely neutral, except for proteins 3C (protease) and 3D (RNA polymerase) (p-values 0. 0366 and 0. 0332), and marginally significant for 3A (p-value of 0. 0910) that were under negative selective pressure. Amino acid sequence conservation was the strongest for 3C protease, in which only 1 out of 183 amino acids reverted from threonine at position 15 to alanine present in cEV11 strain A01-EV11-5789. In contrast, Dn and Da values for all genes of circulating EV11 differed significantly (p<0. 001), suggesting that the selective pressure at amino acid level was higher and the conservation of proteins was more uniform across the genome (Fig 6B). The predicted amino acid difference among iEV11s relative to the earliest cEV11 isolate A01 from 1992 were mapped onto the three dimensional crystallographic model of the EV11 capsid (Protein Data Bank, PDB ID 1H8T). The fixed amino acid substitutions are shown for the capsid pentamer centered around the five-fold axis of symmetry (Fig 7). Many of the substitutions were on the outer capsid surface and the walls of the canyon, a circular depression surrounding the 5-fold axis of symmetry and important for high affinity binding to host cell receptors [27]. When only iEV11s were compared, most amino acid substitutions accumulated on the northern rim of the canyon next to the 5-fold axis of symmetry. There were 7 amino acids differences in P1 between A01-cEV11-5824 isolated in 1992 and the earliest iEV11, A05-iEV11-7482, isolated in 1995. The same was true for another cEV11 isolate from 1992 A02-cEV11-5789. The amino acids that differed were: N18S, R106C, D143G, E217D, T220A, A223V and H697C. (Notation: amino acid in cEV11 from 1992, the number of the amino acid position in P1, amino acid in iEV11 from 1995.) The 5’-untranslated region of Enterovirus genome contains the internal ribosome entry site (IRES) [28,29]. Analysis of iEV11 sequences revealed that between 1998 and 1999 a 13–14 nucleotide deletion became fixed in the hypervariable region of the 5’-UTR. Domain V in the IRES has been shown to be critical for enterovirus replication and virulence [30]. Stem and loop structures were predicted for RNA sequences of the all Israeli EV11s equivalent to domain V of poliovirus using the m-Fold program. Two highest probability stem-loop structures are shown in Fig 8. Arrows indicate each position where one or more of the Israeli isolates had an alterative nucleotide, and a pentagram or rectangle indicates whether the difference would disrupt or conserve the stem structure, respectively. In the first model one pair was conserved and three disrupted, while in the second model, four pairs were conserved and three disrupted. The overall shape of domain V predicted by the second model is similar to the shape of domain V of polioviruses. A cloverleaf structure at the 5’ end of the 3’-UTR serves as the specific signal for a number of functions including switching from transcription to replication [31]. In HEV B viruses there is an addition Z domain that is well conserved among HEV B viruses and may be important for viral growth in vivo, but not in vitro [24,32]. The resulting structure (Fig 9) was different than that proposed previously [24] for the HEV B consensus motif when nucleotide sequences specific for the iEV11 were mapped into this structure. Nucleotide differences found in one isolate, A13, would have further disrupted this structure in two positions and elongated a stem in another. Normally enteroviruses cause acute infections that are cleared within few days or weeks. However, in rare cases, they can establish persistent infection lasting for years. Most known cases of chronic infection with enteroviruses involve patients with various kinds of primary immunodeficiency affecting B cell functions. However many aspects of this phenomenon remain poorly understood, including the cell and tissue localization, the factors controlling the outcome of virus-host interactions, and the reasons why the infection cannot be resolved. This current study was initiated in attempts to understand the evolution of the virus that persists in a patient chronically infected with EV11 who experienced periodic bouts of encephalitis that eventually led to death. This investigation was conducted within the context of patterns of nucleotide and amino acid differences observed among EV11 viruses isolated from sporadic and clustered EV11 infections in the community during the same time frame. The clinical course of this case differed from persistent poliovirus infections in immune deficient people who are largely asymptomatic. Our goal was to identify the source of infection and to shed some light on the environment the virus replicates in and the selective pressures it is experiencing. We have studied 10 iEV11 strains isolated over a period of almost 4 years from November 1995 till December 1999. To identify the source of the infection we have also sequenced strains of circulating EV11 isolated in Israel between 1992 and 1999. The material that was available to us included primary isolates and a cell culture passage performed in attempt to increase virus concentration. Virus in some samples lost its viability and could not be expanded in cell culture, and therefore the quantity of RNA that was available was insufficient for traditional Sanger sequencing of the entire genome. Therefore we have tried another approach based on deep sequencing using Illumina technology, which proved to be very effective and resulted in reconstruction of complete or near-complete consensus sequences and identification of sequence heterogeneities. This report demonstrates that deep sequencing methodology requires smaller amounts of starting material and is sequence-independent, i. e. does not require specific PCR primers used in traditional Sanger sequencing. Comparison of the 25 genomic sequences generated in this study showed that four different genogroups circulated in Israel in the 1990s and that two of them co-circulated during the 1999 EV11 outbreak. Genogroup A viruses were isolated in 1992–1993, genogroup B in 1996–1997, genogroup C in 1998–1999, and genogroup D only in 1999. Co-circulation of two clusters of EV11 (C and D) in 1999 has been reported previously [5,33]. All iEV11 strains isolated from the chronically infected patient clustered together with four cEV11 isolates of genogroup A. The virus that was the closest relative of the earliest (1995) iEV11 isolate was cEV11 strain isolated in 1992. They had 24 nucleotide differences in VP1-coding sequence (2. 7%). Subsequent isolates gradually accumulated mutations so that the latest 1999 iEV11 isolate contained 51 differences (5. 8%). The pace of this evolution (3. 1% over a period of 3. 8 years) is consistent with the rate of mutants accumulation established for circulating polioviruses [34]. The time plot of the number of nucleotide and amino acid differences in VP1 region was a straight line that could be extrapolated back to the early 1990s, consistent with the isolation date of the closest relative, and suggesting that the patient was infected at around that time with a contemporary cEV11 strain. Comparison of full-length sequences revealed no evidence of recombination with other viruses, and other parts of the viral genome also evolved linearly, albeit at a different pace. Amino acid substitutions that occurred in cEV11 capsid differed from those that accumulated in iEV11. In circulating viruses mutations mostly occurred in the canyon and its southern rim (across from the 5-fold axis of symmetry), while in iEV11 many of them concentrated on the northern rim adjacent to the 5-fold axis. The canyon is the binding site for high affinity receptors that belong to the immunoglobulin superfamily, such as CD155, which is poliovirus receptor [35–37]. In contrast, EV11 and some other enteroviruses use Decay-Accelerating Factor (DAF, or CD55). DAF binds close to the 5-fold axis of symmetry on the surface and not in the canyon [38]. Interaction with DAF is low affinity with high on-off rates compared with canyon binding receptors capable of causing conformational changes of bound viruses [38–41]. For instance, EV11-DAF interaction has a dissociation constant KD = 3µM [40] whereas poliovirus-CD155 has KD = 80–700µM [42]. EV11 infection of polarized epithelial cells uses two different entry routes, directly from apical surface as well as tight junctions, which is dependent on DAF binding [39]. Preferential accumulation of mutations on the northern wall of the canyon and close to 5' -fold axis of symmetry during evolution of iEV11 suggests that the virus may be undergoing adaptation to DAF in specific cell types. This part of the virion surface is also known to harbor neutralizing epitopes, so the changes may also reflect immune evasion by the virus. EV11 were the most sensitive of all enteroviruses to antiviral drug pleconaril [43]. Approximately two years after the onset of chronic infection the immunodeficient patient was treated with pleconaril in an attempt to clear the viral infection. Sequencing results revealed that the strain isolated from the patient at the time contained two mutations in VP1 (V117I and V119I) associated with EV11 resistance to pleconaril [44], explaining why the treatment attempt was unsuccessful. Highly conserved RNA secondary structures rather than sequences are important for replication [32,35]. Changes of nucleotides in essential structures such as domain V of the 5’UTR and the Z motif in the 3’UTR were observed for the iEV11s. Some nucleotide variations between isolates conserved the stem loop structures while others altered them. The rate of accumulation of total nucleotide substitutions (Kt) in capsid regions of iEV11 and replicating poliovirus were similar [1] (0. 76*10−2 and 1. 03*10−2 mutations per site per year, respectively). However, the degree of amino acid sequences conservation was much lower in iEV11 than in poliovirus (Ka values of 0. 72*10−2 and 0. 03*10−2, respectively), suggesting that evolution of iEV11 was taking place under lower selective pressure. Respective values for cEV11 could not be calculated because of the scarcity of data and the unknown evolutionary trajectory of the few known sequences of cEV11. However, this conclusion was confirmed by comparing rates at which nucleotide and amino acid changes are fixed in different parts of viral genome during evolution. There are a total of 61 nucleotide triplets coding for 20 amino acids, therefore if the mutational process is random and there is no selective pressure restricting changes in the protein sequence, there should be 3 times more fixed nucleotide substitutions than fixed amino acid changes. Therefore comparison of densities of mutations in amino acid and nucleotide sequences (Da and Dn, the number of sites at which mutations are present divided by the length of protein and nucleotide sequences respectively) can reveal whether there is a positive or negative selective pressure. The Da/Dn ratio below 1 indicates that protein sequences are conserved, and that a disproportionately bigger number of mutations result in synonymous codons. A ratio greater than 1 may suggest that there is a positive selective pressure favoring accumulation of adaptive mutations. This analysis performed for iEV11 strains showed that in most viral proteins the ratio was close to 1, suggesting that they largely experience a random neutral evolution. The exception was viral 3C protease that was highly conserved and had only one mutation that changed isoleucine at amino acid 6 that is unique for genogroup A strains to methionine present in genogroups B, C, and D. In contrast, the pattern of selective pressure in cEV11 was different, and these viruses experienced higher selective pressure at amino acid level (the average Da/Dn ratio was 0. 79 for iEV11 versus 0. 22 for cEV11). Lower conservation of capsid proteins and different distribution of mutations on the capsid surface in iEV11 compared to cEV11 suggest that the virus may undergo adaptation to replication in different types of cells. Protease 3C that is highly conserved in iEV11 plays an important role in maturation of viral proteins as well as in cleaving several cellular proteins involved in innate antiviral defense [45]. Its conservation in iEV11 isolates may suggest that this function is important for viral persistence. Therefore establishment of chronic EV11 infection involves not only impairment of host immune system (B-cell immunodeficiency), bus also may require virus adaptation while preserving its functions mediating virus-host interactions.

We describe evolution of Echovirus 11 genome in chronically infected immunodeficient patient over a period of several years and compare it with the evolution of circulating echoviruses from which it originated. Ratio of silent to missense mutations in protein coding regions suggests that chronic virus was under lower selective pressure than circulating viruses, except for a region coding for viral protease that may participate in neutralizing host cell anti-viral defense mechanisms. This suggests that adaptation to persistence in immunodeficient host may require maintaining functional viral counter-defense mechanisms.

lay_plos

'I have already told you of the sickness and confusion that comes with time travelling. And this time I was not seated properly in the saddle, but sideways and in an unstable fashion. For an indefinite time I clung to the machine as it swayed and vibrated, quite unheeding how I went, and when I brought myself to look at the dials again I was amazed to find where I had arrived. One dial records days, and another thousands of days, another millions of days, and another thousands of millions. Now, instead of reversing the levers, I had pulled them over so as to go forward with them, and when I came to look at these indicators I found that the thousands hand was sweeping round as fast as the seconds hand of a watch--into futurity. 'As I drove on, a peculiar change crept over the appearance of things. The palpitating greyness grew darker; then--though I was still travelling with prodigious velocity--the blinking succession of day and night, which was usually indicative of a slower pace, returned, and grew more and more marked. This puzzled me very much at first. The alternations of night and day grew slower and slower, and so did the passage of the sun across the sky, until they seemed to stretch through centuries. At last a steady twilight brooded over the earth, a twilight only broken now and then when a comet glared across the darkling sky. The band of light that had indicated the sun had long since disappeared; for the sun had ceased to set--it simply rose and fell in the west, and grew ever broader and more red. All trace of the moon had vanished. The circling of the stars, growing slower and slower, had given place to creeping points of light. At last, some time before I stopped, the sun, red and very large, halted motionless upon the horizon, a vast dome glowing with a dull heat, and now and then suffering a momentary extinction. At one time it had for a little while glowed more brilliantly again, but it speedily reverted to its sullen red heat. I perceived by this slowing down of its rising and setting that the work of the tidal drag was done. The earth had come to rest with one face to the sun, even as in our own time the moon faces the earth. Very cautiously, for I remembered my former headlong fall, I began to reverse my motion. Slower and slower went the circling hands until the thousands one seemed motionless and the daily one was no longer a mere mist upon its scale. Still slower, until the dim outlines of a desolate beach grew visible. 'I stopped very gently and sat upon the Time Machine, looking round. The sky was no longer blue. North-eastward it was inky black, and out of the blackness shone brightly and steadily the pale white stars. Overhead it was a deep Indian red and starless, and south-eastward it grew brighter to a glowing scarlet where, cut by the horizon, lay the huge hull of the sun, red and motionless. The rocks about me were of a harsh reddish colour, and all the trace of life that I could see at first was the intensely green vegetation that covered every projecting point on their south-eastern face. It was the same rich green that one sees on forest moss or on the lichen in caves: plants which like these grow in a perpetual twilight. 'The machine was standing on a sloping beach. The sea stretched away to the south-west, to rise into a sharp bright horizon against the wan sky. There were no breakers and no waves, for not a breath of wind was stirring. Only a slight oily swell rose and fell like a gentle breathing, and showed that the eternal sea was still moving and living. And along the margin where the water sometimes broke was a thick incrustation of salt--pink under the lurid sky. There was a sense of oppression in my head, and I noticed that I was breathing very fast. The sensation reminded me of my only experience of mountaineering, and from that I judged the air to be more rarefied than it is now. 'Far away up the desolate slope I heard a harsh scream, and saw a thing like a huge white butterfly go slanting and fluttering up into the sky and, circling, disappear over some low hillocks beyond. The sound of its voice was so dismal that I shivered and seated myself more firmly upon the machine. Looking round me again, I saw that, quite near, what I had taken to be a reddish mass of rock was moving slowly towards me. Then I saw the thing was really a monstrous crab-like creature. Can you imagine a crab as large as yonder table, with its many legs moving slowly and uncertainly, its big claws swaying, its long antennae, like carters' whips, waving and feeling, and its stalked eyes gleaming at you on either side of its metallic front? Its back was corrugated and ornamented with ungainly bosses, and a greenish incrustation blotched it here and there. I could see the many palps of its complicated mouth flickering and feeling as it moved. 'As I stared at this sinister apparition crawling towards me, I felt a tickling on my cheek as though a fly had lighted there. I tried to brush it away with my hand, but in a moment it returned, and almost immediately came another by my ear. I struck at this, and caught something threadlike. It was drawn swiftly out of my hand. With a frightful qualm, I turned, and I saw that I had grasped the antenna of another monster crab that stood just behind me. Its evil eyes were wriggling on their stalks, its mouth was all alive with appetite, and its vast ungainly claws, smeared with an algal slime, were descending upon me. In a moment my hand was on the lever, and I had placed a month between myself and these monsters. But I was still on the same beach, and I saw them distinctly now as soon as I stopped. Dozens of them seemed to be crawling here and there, in the sombre light, among the foliated sheets of intense green. 'I cannot convey the sense of abominable desolation that hung over the world. The red eastern sky, the northward blackness, the salt Dead Sea, the stony beach crawling with these foul, slow-stirring monsters, the uniform poisonous-looking green of the lichenous plants, the thin air that hurts one's lungs: all contributed to an appalling effect. I moved on a hundred years, and there was the same red sun--a little larger, a little duller--the same dying sea, the same chill air, and the same crowd of earthy crustacea creeping in and out among the green weed and the red rocks. And in the westward sky, I saw a curved pale line like a vast new moon. 'So I travelled, stopping ever and again, in great strides of a thousand years or more, drawn on by the mystery of the earth's fate, watching with a strange fascination the sun grow larger and duller in the westward sky, and the life of the old earth ebb away. At last, more than thirty million years hence, the huge red-hot dome of the sun had come to obscure nearly a tenth part of the darkling heavens. Then I stopped once more, for the crawling multitude of crabs had disappeared, and the red beach, save for its livid green liverworts and lichens, seemed lifeless. And now it was flecked with white. A bitter cold assailed me. Rare white flakes ever and again came eddying down. To the north-eastward, the glare of snow lay under the starlight of the sable sky and I could see an undulating crest of hillocks pinkish white. There were fringes of ice along the sea margin, with drifting masses further out; but the main expanse of that salt ocean, all bloody under the eternal sunset, was still unfrozen. 'I looked about me to see if any traces of animal life remained. A certain indefinable apprehension still kept me in the saddle of the machine. But I saw nothing moving, in earth or sky or sea. The green slime on the rocks alone testified that life was not extinct. A shallow sandbank had appeared in the sea and the water had receded from the beach. I fancied I saw some black object flopping about upon this bank, but it became motionless as I looked at it, and I judged that my eye had been deceived, and that the black object was merely a rock. The stars in the sky were intensely bright and seemed to me to twinkle very little. 'Suddenly I noticed that the circular westward outline of the sun had changed; that a concavity, a bay, had appeared in the curve. I saw this grow larger. For a minute perhaps I stared aghast at this blackness that was creeping over the day, and then I realized that an eclipse was beginning. Either the moon or the planet Mercury was passing across the sun's disk. Naturally, at first I took it to be the moon, but there is much to incline me to believe that what I really saw was the transit of an inner planet passing very near to the earth. 'The darkness grew apace; a cold wind began to blow in freshening gusts from the east, and the showering white flakes in the air increased in number. From the edge of the sea came a ripple and whisper. Beyond these lifeless sounds the world was silent. Silent? It would be hard to convey the stillness of it. All the sounds of man, the bleating of sheep, the cries of birds, the hum of insects, the stir that makes the background of our lives--all that was over. As the darkness thickened, the eddying flakes grew more abundant, dancing before my eyes; and the cold of the air more intense. At last, one by one, swiftly, one after the other, the white peaks of the distant hills vanished into blackness. The breeze rose to a moaning wind. I saw the black central shadow of the eclipse sweeping towards me. In another moment the pale stars alone were visible. All else was rayless obscurity. The sky was absolutely black. 'A horror of this great darkness came on me. The cold, that smote to my marrow, and the pain I felt in breathing, overcame me. I shivered, and a deadly nausea seized me. Then like a red-hot bow in the sky appeared the edge of the sun. I got off the machine to recover myself. I felt giddy and incapable of facing the return journey. As I stood sick and confused I saw again the moving thing upon the shoal--there was no mistake now that it was a moving thing--against the red water of the sea. It was a round thing, the size of a football perhaps, or, it may be, bigger, and tentacles trailed down from it; it seemed black against the weltering blood-red water, and it was hopping fitfully about. Then I felt I was fainting. But a terrible dread of lying helpless in that remote and awful twilight sustained me while I clambered upon the saddle. 'So I came back. For a long time I must have been insensible upon the machine. The blinking succession of the days and nights was resumed, the sun got golden again, the sky blue. I breathed with greater freedom. The fluctuating contours of the land ebbed and flowed. The hands spun backward upon the dials. At last I saw again the dim shadows of houses, the evidences of decadent humanity. These, too, changed and passed, and others came. Presently, when the million dial was at zero, I slackened speed. I began to recognize our own pretty and familiar architecture, the thousands hand ran back to the starting-point, the night and day flapped slower and slower. Then the old walls of the laboratory came round me. Very gently, now, I slowed the mechanism down. 'I saw one little thing that seemed odd to me. I think I have told you that when I set out, before my velocity became very high, Mrs. Watchett had walked across the room, travelling, as it seemed to me, like a rocket. As I returned, I passed again across that minute when she traversed the laboratory. But now her every motion appeared to be the exact inversion of her previous ones. The door at the lower end opened, and she glided quietly up the laboratory, back foremost, and disappeared behind the door by which she had previously entered. Just before that I seemed to see Hillyer for a moment; but he passed like a flash. 'Then I stopped the machine, and saw about me again the old familiar laboratory, my tools, my appliances just as I had left them. I got off the thing very shakily, and sat down upon my bench. For several minutes I trembled violently. Then I became calmer. Around me was my old workshop again, exactly as it had been. I might have slept there, and the whole thing have been a dream. 'And yet, not exactly! The thing had started from the south-east corner of the laboratory. It had come to rest again in the north-west, against the wall where you saw it. That gives you the exact distance from my little lawn to the pedestal of the White Sphinx, into which the Morlocks had carried my machine. 'For a time my brain went stagnant. Presently I got up and came through the passage here, limping, because my heel was still painful, and feeling sorely begrimed. I saw the _Pall Mall Gazette_ on the table by the door. I found the date was indeed to-day, and looking at the timepiece, saw the hour was almost eight o'clock. I heard your voices and the clatter of plates. I hesitated--I felt so sick and weak. Then I sniffed good wholesome meat, and opened the door on you. You know the rest. I washed, and dined, and now I am telling you the story. 'I know,' he said, after a pause, 'that all this will be absolutely incredible to you. To me the one incredible thing is that I am here to-night in this old familiar room looking into your friendly faces and telling you these strange adventures.' He looked at the Medical Man. 'No. I cannot expect you to believe it. Take it as a lie--or a prophecy. Say I dreamed it in the workshop. Consider I have been speculating upon the destinies of our race until I have hatched this fiction. Treat my assertion of its truth as a mere stroke of art to enhance its interest. And taking it as a story, what do you think of it?' He took up his pipe, and began, in his old accustomed manner, to tap with it nervously upon the bars of the grate. There was a momentary stillness. Then chairs began to creak and shoes to scrape upon the carpet. I took my eyes off the Time Traveller's face, and looked round at his audience. They were in the dark, and little spots of colour swam before them. The Medical Man seemed absorbed in the contemplation of our host. The Editor was looking hard at the end of his cigar--the sixth. The Journalist fumbled for his watch. The others, as far as I remember, were motionless. The Editor stood up with a sigh. 'What a pity it is you're not a writer of stories!' he said, putting his hand on the Time Traveller's shoulder. 'You don't believe it?' 'Well----' 'I thought not.' The Time Traveller turned to us. 'Where are the matches?' he said. He lit one and spoke over his pipe, puffing. 'To tell you the truth... I hardly believe it myself.... And yet...' His eye fell with a mute inquiry upon the withered white flowers upon the little table. Then he turned over the hand holding his pipe, and I saw he was looking at some half-healed scars on his knuckles. The Medical Man rose, came to the lamp, and examined the flowers. 'The gynaeceum's odd,' he said. The Psychologist leant forward to see, holding out his hand for a specimen. 'I'm hanged if it isn't a quarter to one,' said the Journalist. 'How shall we get home?' 'Plenty of cabs at the station,' said the Psychologist. 'It's a curious thing,' said the Medical Man; 'but I certainly don't know the natural order of these flowers. May I have them?' The Time Traveller hesitated. Then suddenly: 'Certainly not.' 'Where did you really get them?' said the Medical Man. The Time Traveller put his hand to his head. He spoke like one who was trying to keep hold of an idea that eluded him. 'They were put into my pocket by Weena, when I travelled into Time.' He stared round the room. 'I'm damned if it isn't all going. This room and you and the atmosphere of every day is too much for my memory. Did I ever make a Time Machine, or a model of a Time Machine? Or is it all only a dream? They say life is a dream, a precious poor dream at times--but I can't stand another that won't fit. It's madness. And where did the dream come from?... I must look at that machine. If there is one!' He caught up the lamp swiftly, and carried it, flaring red, through the door into the corridor. We followed him. There in the flickering light of the lamp was the machine sure enough, squat, ugly, and askew; a thing of brass, ebony, ivory, and translucent glimmering quartz. Solid to the touch--for I put out my hand and felt the rail of it--and with brown spots and smears upon the ivory, and bits of grass and moss upon the lower parts, and one rail bent awry. The Time Traveller put the lamp down on the bench, and ran his hand along the damaged rail. 'It's all right now,' he said. 'The story I told you was true. I'm sorry to have brought you out here in the cold.' He took up the lamp, and, in an absolute silence, we returned to the smoking-room. He came into the hall with us and helped the Editor on with his coat. The Medical Man looked into his face and, with a certain hesitation, told him he was suffering from overwork, at which he laughed hugely. I remember him standing in the open doorway, bawling good night. I shared a cab with the Editor. He thought the tale a 'gaudy lie.' For my own part I was unable to come to a conclusion. The story was so fantastic and incredible, the telling so credible and sober. I lay awake most of the night thinking about it. I determined to go next day and see the Time Traveller again. I was told he was in the laboratory, and being on easy terms in the house, I went up to him. The laboratory, however, was empty. I stared for a minute at the Time Machine and put out my hand and touched the lever. At that the squat substantial-looking mass swayed like a bough shaken by the wind. Its instability startled me extremely, and I had a queer reminiscence of the childish days when I used to be forbidden to meddle. I came back through the corridor. The Time Traveller met me in the smoking-room. He was coming from the house. He had a small camera under one arm and a knapsack under the other. He laughed when he saw me, and gave me an elbow to shake. 'I'm frightfully busy,' said he, 'with that thing in there.' 'But is it not some hoax?' I said. 'Do you really travel through time?' 'Really and truly I do.' And he looked frankly into my eyes. He hesitated. His eye wandered about the room. 'I only want half an hour,' he said. 'I know why you came, and it's awfully good of you. There's some magazines here. If you'll stop to lunch I'll prove you this time travelling up to the hilt, specimen and all. If you'll forgive my leaving you now?' I consented, hardly comprehending then the full import of his words, and he nodded and went on down the corridor. I heard the door of the laboratory slam, seated myself in a chair, and took up a daily paper. What was he going to do before lunch-time? Then suddenly I was reminded by an advertisement that I had promised to meet Richardson, the publisher, at two. I looked at my watch, and saw that I could barely save that engagement. I got up and went down the passage to tell the Time Traveller. As I took hold of the handle of the door I heard an exclamation, oddly truncated at the end, and a click and a thud. A gust of air whirled round me as I opened the door, and from within came the sound of broken glass falling on the floor. The Time Traveller was not there. I seemed to see a ghostly, indistinct figure sitting in a whirling mass of black and brass for a moment--a figure so transparent that the bench behind with its sheets of drawings was absolutely distinct; but this phantasm vanished as I rubbed my eyes. The Time Machine had gone. Save for a subsiding stir of dust, the further end of the laboratory was empty. A pane of the skylight had, apparently, just been blown in. I felt an unreasonable amazement. I knew that something strange had happened, and for the moment could not distinguish what the strange thing might be. As I stood staring, the door into the garden opened, and the man-servant appeared. We looked at each other. Then ideas began to come. 'Has Mr. ---- gone out that way?' said I. 'No, sir. No one has come out this way. I was expecting to find him here.' At that I understood. At the risk of disappointing Richardson I stayed on, waiting for the Time Traveller; waiting for the second, perhaps still stranger story, and the specimens and photographs he would bring with him. But I am beginning now to fear that I must wait a lifetime. The Time Traveller vanished three years ago. And, as everybody knows now, he has never returned. EPILOGUE One cannot choose but wonder. Will he ever return? It may be that he swept back into the past, and fell among the blood-drinking, hairy savages of the Age of Unpolished Stone; into the abysses of the Cretaceous Sea; or among the grotesque saurians, the huge reptilian brutes of the Jurassic times. He may even now--if I may use the phrase--be wandering on some plesiosaurus-haunted Oolitic coral reef, or beside the lonely saline lakes of the Triassic Age. Or did he go forward, into one of the nearer ages, in which men are still men, but with the riddles of our own time answered and its wearisome problems solved? Into the manhood of the race: for I, for my own part, cannot think that these latter days of weak experiment, fragmentary theory, and mutual discord are indeed man's culminating time! I say, for my own part. He, I know--for the question had been discussed among us long before the Time Machine was made--thought but cheerlessly of the Advancement of Mankind, and saw in the growing pile of civilization only a foolish heaping that must inevitably fall back upon and destroy its makers in the end. If that is so, it remains for us to live as though it were not so. But to me the future is still black and blank--is a vast ignorance, lit at a few casual places by the memory of his story. And I have by me, for my comfort, two strange white flowers--shrivelled now, and brown and flat and brittle--to witness that even when mind and strength had gone, gratitude and a mutual tenderness still lived on in the heart of man.

The TT notices that, in the confusion of his fight with the Morlocks, he accidentally sent himself into the future, rather than the past. Though he speeds up through time, the alternation of day and night slows down, as does the passage of the sun. Finally, the sun dully ceases to rise and set, and the earth rests with one side facing it, much as one side of the moon faces the earth. The TT slowly reverses the Time Machine until it stops. He observes the reddish landscape and the moss-like vegetation everywhere. There is no wind, and the water of the sea barely moves. The TT has difficulty breathing and believes the air is more rarefied than normal. He spots a large white creature flying away, and then sees a huge, crab-like thing crawling toward him. Another one comes up behind him and touches him with its antennae. The TT quickly pulls his machine's lever and watches more of the giant crabs crawl along the beach as he shoots forward through time. The environment keeps up its deathly appearances as the sun grows larger and duller. After thirty million years, all life save the green vegetation ceases to exist, and it starts to snow. An eclipse turns everything completely dark. After the sun becomes visible again, the TT stops the machine. He feels sick and confused and "incapable of facing the return journey." He sees a black creature crawl out from the sea, and his fear of remaining in this environment compels him to climb back into the Time Machine.

booksum

WASHINGTON, Jan. 13 (UPI) -- Monday's U.S. Supreme Court case on the president's power to temporarily fill high-level posts will bring a ruling that could echo for centuries, a lawyer said. "Rulings like this have implications that last for centuries," Michael Lotito, an employment and labor attorney with law firm Littler Mendelson P.C.'s Workplace Policy Institute, told the Hill newspaper. Presidents since George Washington have made recess appointments of judges and senior federal officials who would normally require Senate confirmation, relying on Article II, Section 2 of the Constitution, which lets presidents temporarily fill vacancies while the Senate is in recess. Recess appointments must be approved by the Senate by the end of the next congressional session, or the position becomes vacant again. But during the partisan struggle between the Obama administration and the Senate over presidential nominations, Obama became the first president to appoint nominees when the Senate was in a "pro forma" session -- a brief call to order and adjournment held every few days at which no formal business was expected to be conducted. The sessions, when only a handful of senators are usually present, have been used to prevent recess appointments. But the Obama administration claimed the Senate was not really available during those sessions to live up to its constitutional responsibility to provide "advice and consent" for presidential appointments, so the administration said Obama's recess appointment power was in effect. "The sham pro forma sessions are nothing more than that," Catholic University law Professor Victor Williams, who filed a brief backing the government's position, told the Hill. Obama temporarily appointed three people to the National Labor Relations Board, which lacked a quorum to conduct business, Jan. 4, 2012. The Senate held a pro forma session the day before, with one senator presiding. A soft drink bottler in Yakima, Wash., later argued the NLRB appointments were unconstitutional after the labor relations board ruled against the bottler in a dispute with the Teamsters union. The NLRB panel that made the ruling included two board members assigned by Obama as recess appointments. The U.S. Court of Appeals for the District of Columbia Circuit agreed with Noel Canning, ruling a president could make recess appointments only during the period between the official end of one congressional session and the start of another. It also ruled a president could use his recess appointment authority only to fill vacancies that arose during the formal recess, not beforehand. The NLRB vacancies arose before the December 2011 end of the 111th Congress and the January 2012 start of the 112th. A Congressional Research Service report found 329 recess appointments since President Ronald Reagan in 1981 would not meet the appeals court's criteria and could therefore be ruled void if the appeals court's ruling is upheld. Upholding the appeals court's decision "would repudiate the constitutional legitimacy of thousands of appointments made by at least 14 presidents since the 1860s," Solicitor General Donald Verrilli wrote in a brief. Verrilli argued the Senate was not permitted to use pro forma sessions to unilaterally hamstring or manipulate the president's recess appointments authority. "Whatever leeway the Senate may enjoy when governing its own affairs, it cannot exploit that leeway to limit the president's constitutional authority," he wrote. But Noel Francisco, a Washington appellate lawyer challenging the recess appointments, argued it was up to the Senate, not the president, to decide when the Senate is in session. "Presidents have discretion to decide whether to make recess appointments, and to select whom they appoint. But they may not also determine when that power is available," Francisco said. "That decision is for the Senate alone." Nothing less than the boundaries of executive power are at stake Monday, as the Supreme Court considers whether President Obama violated the Constitution during his first term. Oral arguments slated for Monday will center on a trio of recess appointments to the National Labor Relations Board (NLRB) that were deemed unconstitutional by lower courts. ADVERTISEMENT If they uphold the decision, experts say the justices could endanger hundreds of NLRB decisions.Even more significant are the ramifications for future presidents, with the court poised either to bolster or blunt the chief executive’s appointment powers.“Rulings like this have implications that last for centuries,” said Michael Lotito, an employment and labor attorney and co-chairman of Littler Mendelson's Workplace Policy Institute.For decades, presidents have used recess appointment powers, when the Senate is away to install judges and fill top federal vacancies that ordinarily would be subject to confirmation proceedings.But with the disputed NLRB appointments, Obama became the first president to appoint nominees, when the Senate was in a “pro-forma” session, when the upper chamber is briefly called to order and adjourned every few days.The sessions are intended to prevent recess appointments, and usually only a handful of senators are present for them. In filling the NLRB posts, the Obama administration claims that the Senate is generally not available to conduct business during the sessions, so the president’s recess appointment power is in effect.“The sham pro-forma sessions are nothing more than that,” said Catholic University law professor Victor Williams, who filed a brief backing the government’s position.The impetus for recess appointments has faded now that Senate Democrats have changed their chamber’s rules to allow for a simple majority vote on presidential nominees. Nevertheless, the case could stunt Obama's and future presidents' authority, when it comes to staffing administrations.The case was brought by Noel Canning, an Oregon-based soft drink bottling and distribution company that challenged the appointments as unconstitutional.In January of last year, the D.C. Circuit Court of Appeals agreed.The appellate court ruling under now review at the Supreme Court found a narrow window for the president to make recess appointments. Under its decision, the president can only make such appointments when the Senate is in recess between sessions of Congress, and only if a vacancy occurred in that same time period.That goes well against protocol adopted by past Democratic and Republican presidents. A Congressional Service Research report found 329 such appointments since 1981 that would not meet that criteria and would be ruled void if the appeals court decision was law.Some see the fight against the labor board as a broader effort in which opponents have sought to stymie the Obama administration’s rules and regulations.“I think the battle against the NLRB over the last few years has been a proxy war about the proper role and scope of government,” said Wilma Liebman, who served as chairwoman of the NLRB from January 2009 to August 2011.The obscure agency has become ground zero in that war, pitting business against labor and Republicans against Democrats.The private sector mobilized a massive lobbying campaign after Obama’s election, fearing a Democratic president might enact a host of policy changes favorable to unions.The effort first targeted legislation that would ease union organizing but shifted to the NLRB nominees and its decisions.“There has been an especially rancorous degree of controversy whipped around the NLRB. It got swept up with the Employee Free Choice Act," Liebman said.The NLRB has had its decisions overturned by the high court before. In 2010, the Supreme Court found the board lacked the authority to make decisions for more than two years because it only had two members — one short of a quorum.About 600 NLRB decisions were made in that time period, and the board was forced to go back through about 100 of them.Liebman said it was a time-consuming process, but one the labor board could do again.“We issued new decisions in a relatively short period of time. We had a process in place and went methodically back through them,” Liebman said. “It took some time that we could have spent doing other things. It wasn't the end of the world.”But having to turn its focus on previous decisions could sidetrack the NLRB from what is seen as an activist agenda at a critical moment, said Lolito, whose firm represents employers.The board is now at full strength for the first time in years and was expected to tackle numerous issues involving union elections.“If this board has to spend the next good couple of years looking backward instead of forward, many in the employer community would say that's good news,” he said.In Monday’s arguments, attorneys with the U.S. Chamber of Commerce will argue on behalf of Noel Canning that the NLRB operated without a quorum for well over a year, causing confusion for both employers and employees.“We look forward to the much needed clarity that the Supreme Court’s decision will bring,” said Lily Fu Claffee, the Chamber's general counsel.The Obama administration’s case hinges on winning three points, according to Georgetown University Law Center’s Nicholas Quinn Rosenkranz, who is arguing against the government.First, it must convince the justices that presidents may make appointments during regular recesses and not, as the appeals court ruled, only during the breaks between numbered sessions on Congress.Next, the court must agree that the appointments may include the filling of vacancies that existed before the recess began, rather than those that occurred during a recess.Finally, it must conclude that the pro-forma sessions do not count as formal sessions of Congress.Proving all three points, particularly the last, would be a tall order, said Rosenkranz, who predicted a 9-0 ruling in favor of Noel Canning.“I don’t think this is a close case,” he said. 4 years ago Washington (CNN) - The U.S. Supreme Court on Monday turned aside Arizona's appeal to reinstate its law banning most abortions after 20 weeks of pregnancy. A federal appeals court last year said the restrictions were unconstitutional. Follow @politicalticker The high court's refusal without comment to intervene now means the provisions passed in 2012 cannot be enforced. Twelve states have similar laws, shortening the period the high court established four decades ago, after it said abortion should be legal until viability, when a fetus is generally believed to survive outside the womb. Current medical science puts that at about 24 weeks into the pregnancy. Legal challenges in other states are working their way through the courts, including in Texas, which passed a variety of measures over access to abortion. The case is Horne v. Isaacson (13-402). The Supreme Court can be seen from near the top of the Capitol Dome on Capitol Hill in Washington. (Susan Walsh/AP) By the numbers, the Supreme Court is headed for a great fallow period. Over the approximately next 100 days, it will hear oral argument in only 25 cases. Despite taking eight new pleas Friday, the court’s workload this term might reach a new low: In its March sitting, it will consider only half its usual number of cases. And yet, as recent days have shown, the court is as central as ever to the national debate. During their holiday break, justices stopped a federal judge’s order allowing same-sex marriages in Utah. They considered anew the Affordable Care Act and whether the Obama administration has made proper accommodations for religiously affiliated groups. The court could decide any day whether to further loosen restrictions on political campaign contributions. Affirmative action is once again on the agenda, as is a separate look at Obamacare that has elements of Citizens United redux — this time about whether corporations are entitled to rights of religious expression. And the justices return from their holiday break Monday to review restrictions on abortion protesters and to referee an unprecedented constitutional conflict between the president and the Congress about the appointment of high-level government officials. “We’re in the middle of a quite remarkable period in the court’s history,” said Kannon Shanmugam, a Washington lawyer who argues before the court. “The court has had several cases implicating major issues of national debate each of the last few years. What that shows is that this is a court that’s not at all shy about tackling hot-button issues.” Of course, the involvement is not always up to the court. For instance, justices might have thought they had done all they wanted to on the subject of same-sex marriage last June. The court struck down the portion of the Defense of Marriage Act that prevented federal recognition of same-sex marriages performed in states where they are legal, and allowed such unions to resume in California without ruling on the basic question of whether states may ban gay marriage. But gay rights advocates around the country went to work trying to convince federal judges that the reasoning of the court’s DOMA decision meant that state bans on same-sex marriage cannot stand, even where voters made them part of state constitutions. That is what happened in Utah, where U.S. District Judge Robert J. Shelby declared that state’s ban unconstitutional. He and the appeals court refused to stay the ruling, so the Supreme Court stepped in to put the marriages on hold while appeals played out. “In the context of same-sex marriage, it seems clear that many of the justices affirmatively wish to delay deciding the question for a number of years,” said Justin Driver, a law professor at the University of Texas. “But it seems equally clear that the justices will in effect be unable to avoid this spotlight for very long.” Ilya Shapiro, senior fellow in constitutional studies at the Cato Institute, agreed that the court is not always seeking a role. “I don’t think that the court is necessarily looking to be ‘in the middle of everything,’ ” he said in an e-mail, repeating a reporter’s wording. “[Chief Justice] John Roberts especially would probably prefer not to be.” But as the court’s docket shrinks, “the high-profile cases stand out more in contrast.” The court has great discretion over its docket in some ways, but when subjects such as same-sex marriage arise or parts of major legislation such as the Affordable Care Act are challenged as unconstitutional, “it sort of has to be” involved, Shapiro said. The marquee case on the court’s agenda as it returns to work is a good example. For 200 years, the court has not had to rule on the meaning of the constitutional provision that allows the president to make “recess appointments” of high-level government officials when the Senate is not in session to provide consent. The clause states that the president “shall have power to fill up all vacancies that may happen during the recess of the Senate.” Nearly every president has used the power, and the executive and legislative branches, no matter which party was in control, have made grudging accommodations. But an extraordinary level of political gridlock between Senate Republicans and President Obama has forced the issue. Republicans used the filibuster to block a vote on Obama appointments to the National Labor Relations Board to the extent that the board could not function. Then, borrowing a procedure pioneered by Senate Democrats when George W. Bush was president, they forced the Senate into pro forma sessions when most senators were out of town to keep Obama from making recess appointments. Obama responded equally brazenly. Unlike Bush, Obama in January 2010 made the appointments anyway. He declared that despite the pro forma sessions, the Senate was not really available to conduct business by voting on his nominees. Lawsuits followed, and a decision by a panel of the U.S. Court of Appeals for the D.C. Circuit ratcheted the dispute, bypassing the question of the pro forma sessions. Under the appellate court’s view, the president may make recess appointments only during the annual breaks between sessions of Congress — sometimes those last only minutes — and the vacancies must occur during those breaks in order for the president to fill them. Such a scenario would virtually eliminate a president’s power to make recess appointments. The Democratic majority in the Senate recently changed the filibuster rule to make it easier for the president to secure a vote for his nominees, diminishing the urgency of the case. But that would change if the Senate and the White House were not controlled by the same political party. If the court had little choice on the recess appointments case, it still in recent years has taken on controversial subjects — affirmative action, the constitutionality of parts of the Voting Rights Act, punishment of juvenile offenders — where it might have declined. “Notwithstanding the reduced docket, I agree that the court has not suddenly become shy about granting certiorari to resolve disputes involving high-visibility issues the public cares about,” said Irving L. Gornstein, director of the Supreme Court Institute at Georgetown Law Center. “It is taking those cases just as much as it ever did, if not more.”

The Supreme Court isn't taking on a lot of cases over the next 100 days-in fact, this March, it will review just half its usual caseload, the Washington Post reports. But the cases it is examining are big ones. Today, for instance, the court is considering just when a president has the right to make political appointments without Senate approval, UPI reports. The case follows President Obama's 2010 appointment of National Labor Relations Board employees when the Senate was holding only pro forma sessions; Obama argued that those sessions were akin to Senate recesses, when the president can make temporary appointments. A lower court strictly limited such "recess appointments"; now the Supreme Court is considering the matter. "Rulings like this have implications that last for centuries," a lawyer tells the Hill; indeed, the court hasn't ruled on the matter in two centuries, the Post notes. With upcoming cases involving affirmative action, campaign finance rules, and ObamaCare and religion, "this is a court that's not at all shy about tackling hot-button issues," notes another lawyer. One case it won't be hearing, however: an appeal by Arizona to bring back a law banning abortions after 20 weeks, CNN reports. That means the law can't be enforced.

multi_news

The War Games By Malcolm Hulke and Terrance Dicks 5:15pm - 5:40pm [SCENE_BREAK] 1, INT: LANDING BAY (Lieutenant Carstairs is pointing his gun squarely at Zoe's chest with an impassive, sightless look on his face.) ZOE: Well what are you doing? It's me, Zoe! CARSTAIRS: You're a spy...German...spy. It's my duty to shoot you. (Carstairs grips the sensitive trigger of his revolver and with a metallic clicking the barrel moves towards a fresh bullet, the hammer beginning to draw back...) SCIENTIST: Carstairs! (The Scientist has appeared behind Carstairs with a squad of guards, evil-looking stun-guns slung over their shoulders.) SCIENTIST: Well done Lieutenant you've captured a dangerous spy! CARSTAIRS: Yes...and this time she won't escape. SCIENTIST: Lieutenant we mustn't kill her. CARSTAIRS: She's a spy, she must die. She...must... (Carstairs relaxes his grip on his weapon.) SCIENTIST: Lieutenant Carstairs, I am your superior officer, you will obey my commands. (Carstairs lowers his weapon and stands to attention.) CARSTAIRS: Yes sir. Prisoner delivered sir. SCIENTIST: Take her away - security section. (The guards take Zoe and march her away.) SCIENTIST: Now Lieutenant, we have work to do. Come with me. (He leads Carstairs away in the opposite direction) [SCENE_BREAK] 2, INT: BARN (Von-Weich is standing in the barn with a gun at his chest held by a member of the resistance. Jamie and Lady Jennifer turn from where they are sitting on two bales of hay.) JAMIE: You realise we have saved your life? (Von-Weich remains stoically silent.) BUCKINGHAM: I don't think he's going to be grateful. We're lucky they didn't kill all three of us. (Jamie sees Harper walk by and calls out to him.) JAMIE: Look, how long are we gonna be kept here? HARPER: Until Russell arrives - he'll decide what to do with you! JAMIE: Well who's Russell? HARPER: He's our Captain, he'll be along. (Harper walks over to Von-Weich.) HARPER: Where is it? (He grabs the officer by the scruff of his neck.) HARPER: Are you gonna tell me where that tunnel is?! BUCKINGHAM: What tunnel? HARPER: Look we've been watching this place, it's where the recruits keep coming from. BUCKINGHAM: But what makes you think there's a tunnel? HARPER: Come on we've seen the recruits come out of here. JAMIE: Look, there's no tunnel! They come out of a big green box, a-a sort of machine! HARPER: A what? Machine? (He glances at Jennifer.) HARPER: Look is he crazy or somethin'? BUCKINGHAM: I know it's difficult to believe, but it is true - I've seen it myself. HARPER: Oh come on, quit kidding about huh? We've got to find that tunnel. JAMIE: But even if you do find out how the troops get here, what good is it going to do you? HARPER: Maybe we can get out of here back to where we came from! BUCKINGHAM: Where do you think you are? HARPER: Look I don't know, all I know is I was brought here and I wanna find my way out, right?! (Spencer, an old weasly-looking redcoat with a beard and a Yorkshire accent strolls in and fiddles with his rifle for a moment. He looks spitefully at Harper.) SPENCER: You found your tunnel yet? HARPER: Oh we're still lookin'. SPENCER: The supplies are loaded, let's get out of here. HARPER: Not until we find that tunnel! SPENCER: There's no tunnel! We've got the supplies, let's go. (Spencer turns and aims his rifle at Von-Weich.) HARPER: Hey do you think you are doing? SPENCER: We can't afford to take prisoners! (Not wanting to let Spencer get the upper hand, Harper grabs the rifle and struggles with him.) HARPER: Oh no you don't! (Harper forces the redcoat down to the floor, the rifle clattering away. Spencer tries to pick up the rifle, but Harper steps on his hand. Spencer counters by butting him in the stomach and punching Harper. While they are fighting there is a moment where Spencer gets the rifle back and almost shoots Harper, but a member of the resistance who are watching the fight in amusement, pulls it away. While the resistance fighters are watching the tussle on the floor, Jamie silently mouths "Now! Come on!..." to Lady Jennifer, and they tiptoe out towards the door... Before they can escape a stocky Boer War soldier strides in cutting off the escape. The soldier withdraws a revolver from his holster and fires two shots into the air causing everyone on the barn to stop what they are doing and look. With a stern expression he rushes towards Harper and Spencer who are kneeling on the floor glaring at each other.) RUSSELL: Get up. (They don't move, so the soldier yanks them up by their collars.) RUSSELL: UP! You're just like a couple of kids aren't ya? I leave you alone and you start fighting amongst yourselves! SPENCER: He started it, you ask any of 'em! HARPER: We were taking prisoners, he wanted to shoot 'em! (Spencer rushes at Harper with a growl, but Russell yanks him back again and holds him by the scruff of his neck.) RUSSELL: Alright Alright! Spencer, take those stores over to our HQ. (Knowing when his is beaten Spencer grumpily obeys and leaves, Russell prods at Harper.) RUSSELL: You ought to know better Harper. HARPER: He wanted to shoot him, I couldn't let him! (Jamie creeps up behind Von-Weich who has silently wandered towards the back of the barn and grabs him.) JAMIE: Look at that! Now do you believe us? (As Jamie pulls Von-Weich back, a secret panel in the wooden wall of the barn falls open. Within the space are the controls and screen of a video link...) [SCENE_BREAK] 3, INT: SECURITY ROOM (A guard leads Zoe through a door into the Security Room, the two sections of the door gliding down and sliding left to close. She is pushed into a white formica bucket seat. The room is weird sixties pulp-sci-fi decor to the very extreme, with more than a touch of Wonkaland to it. The wall and door Zoe was led through floor is decorated in a mass of concentric circles spilling out over the floor. To the sides of the room, the edges of the ceiling and floor have curious diagonal panels which are studded with rows of curious metallic bristles. Towards the back is a railed-off, raised upper level in which there are more bristling panels, and a large black chair, upon which is sat an odd piece of apparatus.) ZOE: Oh what's gonna happen? What are you gonna to do to me? (The guard remains silent.) ZOE: Well you might answer me! (The Security Chief strides towards the rail and looks down. He is a sinister man with round glasses and a nasal voice.) SECURITY-CHIEF: You are the one who will answer the questions. ZOE: Who are you? I won't tell you anything! (The Security Chief picks up the apparatus, a truth-machine which is shaped like a strange kind of helmet, from the chair and descends a set of steps. He places the machine on his head and pulls down the a hinged visor in front of his eyes. He adjusts the power with a knob at the side and a light beams from the end transfixing Zoe.) SECURITY-CHIEF: What is you name? My name is... ZOE: Zoe Herriot. SECURITY-CHIEF: To which resistance group do you belong? When did you join the resistance? ZOE: I did not join the resistance SECURITY-CHIEF: I joined the resistance (Zoe looks confused.) SECURITY-CHIEF: What time do you come from? ZOE: The twenty-first century. SECURITY-CHIEF: Impossible, there is no twenty-first century Time Zone. I was born in... ZOE: I was born in the twenty-first century. SECURITY-CHIEF: Try again. (He turns up power and Zoe begins to show signs of strain.) ZOE: Ooh! SECURITY-CHIEF: I was born in... ZOE: I was born in the twenty-first century. SECURITY-CHIEF: You were first reported in the nineteen-seventeen Time Zone. ZOE: Yes. SECURITY-CHIEF: How did you get there? ZOE: I arrived in the TARDIS. SECURITY-CHIEF: TARDIS? What does that mean? ZOE: Time and relative dimensions in space. It's a space ship. SECURITY-CHIEF: You have a space ship? ZOE: The ship belongs to the Doctor. SECURITY-CHIEF: I'm going to show you some photographs. You will identify anyone know to you, particularly this doctor! (A series of images is superimposed over Zoe's face.) [SCENE_BREAK] 4, INT: PROCESSING ROOM (The Doctor bumbles into the room from the central quadrant without looking where he is going. The processing room is small and doorless. One wall seems to be decorated by some kind of slides, the other wall is made from panels each with a filigree metal pattern. To the rear is a collection of large inflated balls.) SCIENTIST: What are you doing in here? (The Doctor turns to see the Scientist.) DOCTOR: Ah hah-ha yes, I was so interested in your demonstration I-I felt I had to come along and see how you were getting along. SCIENTIST: But you... There's a security alarm out for you! DOCTOR: Really...why? SCIENTIST: The War-Chief, he pointed you out. DOCTOR: Oh no-no-no. Not me, the girl! SCIENTIST: Then why did you run? DOCTOR: Ehm...Well I was, I was trying to catch her of course! Has she been caught? SCIENTIST: Yes. I sent her to the security section for questioning. DOCTOR: Oh good. How're you getting on with him? SCIENTIST: I was just about to reprocess him. DOCTOR: Oh yes, this is interesting. I think you'll find that bit goes just there. (He places a magnetic block onto the control panel.) DOCTOR: Erm, will there be enough of these advanced models ready for our major plan? SCIENTIST: If I knew the precise timing of the plan that would be easier to answer. DOCTOR: Oh but surely you must know, in your position... SCIENTIST: Yes of course, just as you do. But only the War-Chief knows the timing. Now really, I am very busy... DOCTOR: Oh I...I would consider it a great honour if I could simply stand and watch. SCIENTIST: Oh very well. DOCTOR: Thank you. Haha! What are we doing now? SCIENTIST: I am about to de-process him just as you suggested at my lecture. DOCTOR: Oh I see yes, this is intriguing... Ah-ah... Will he remember everything when he wakes up? SCIENTIST: Yes, he'll remember everything. Uh, would you strap him down please? As soon as the de-processing has completed everything will return to normal. DOCTOR: Oh...oh, yes. Certainly. (He pretends to tie Carstairs down. The bleeping of the machine reaches a peak then dies away as the Scientist turns off the machine.) DOCTOR: There we are! Is the de-processing complete? SCIENTIST: Yes. CARSTAIRS: Oh where am I? (Looks around and then sighs reassuredly.) CARSTAIRS: Oh, Doctor! SCIENTIST: You, he recognises you! You're one of... (He chases the Doctor around the couch that Carstairs is lying on.) DOCTOR: Oh no, please argh...hurt him...get it off... (Carstairs springs up and grabs the Scientist.) DOCTOR: No, don't hurt him...put him on there. In the machine quick! (As Carstairs holds the Scientist onto the chair, the Doctor clamps the processing-machine around his head and turns on the power. The Scientist convulses under the power of the machine.) SCIENTIST: Bph! DOCTOR: Haha! Hoist with his own petard! Come along Lieutenant! (Carstairs stands by the machine looking a little dazed.) DOCTOR: Lieutenant? CARSTAIRS: Doctor where are we? DOCTOR: What? Well what do you remember? CARSTAIRS: Well I remember you and Lady Jennifer... And I was captured wasn't I? DOCTOR: Yes that's right, unfortunately. And brought here, to the aliens war centre and brainwashed again, but you're alright now. CARSTAIRS: Oh I see! DOCTOR: I'll explain as we go along, we must find Zoe. (They both leave, then the Doctor slinks back to where the machine is still busily processing the Scientist. He turns down the power a bit and feels the Scientist's forehead.) DOCTOR: Oh...oh. Better leave him on simmer. (He dashes out after Carstairs.) [SCENE_BREAK] 5, INT: BARN (Russell glances at the video link.) RUSSELL: You expect me to believe all this? JAMIE: Yes! They use that thing to talk to each other! (Russell reaches towards the video link.) JAMIE: Ah d-don't touch it! BUCKINGHAM: It works like a telephone. RUSSELL: And you say that something appears in the middle of the barn and hundreds of men get out of it; a box? JAMIE: Aye, we've both seen it! (Von-Weich breaks free from Jamie's grasp and presses a control, Jamie rushes up and grabs him again.) RUSSELL: What's that noise? JAMIE: Don't know. (Von-Weich leers.) VON-WEICH: You'll find out soon enough! [SCENE_BREAK] 6, INT: CENTRAL-CONTROL (A technician sits at a control panel.) TECHNICIAN: This is Central Control, what is your emergency? This is Central Control, what is your emergency? [SCENE_BREAK] 7, INT: SECURITY ROOM (The War-Chief glares coldly at the Security-Chief.) WAR-CHIEF: Did she identify her friends? SECURITY-CHIEF: No, they are not on our files of resistance people. WAR-CHIEF: Then they must be a small independent group. SECURITY-CHIEF: Yes, five of them. First reported in the nineteen-seventeen Time Zone. One has been caught and is being reprocessed, two believed to be still at large in the American Civil War Zone. WAR-CHIEF: And one still at large in this control. SECURITY-CHIEF: He will be caught! WAR-CHIEF: He's evaded your security guards so far, perhaps he will continue to do so? (He grabs Zoe's head in his hands and snaps it up so he can see her face clearly.) SECURITY-CHIEF: From which time does she originate? WAR-CHIEF: She was a nurse like the other girl. Nineteen-Seventeen. SECURITY-CHIEF: And the man? WAR-CHIEF: The same, nineteen-Seventeen. SECURITY-CHIEF: Why did they come here? WAR-CHIEF: They had some confused idea they were getting home. SECURITY-CHIEF: She told you nothing else? Everything quite straightforward? WAR-CHIEF: Yes. The normal pattern of resistance activity - did you expect something else? (The Technician dashes into the room and stands to attention.) TECHNICIAN: Emergency call from the American Civil War Zone sir. WAR-CHIEF: What's wrong? TECHNICIAN: The signal was activated, but there was no message, no contact at all. WAR-CHIEF: Alright, I'll deal with it. (Turns to the Security Chief.) WAR-CHIEF: Come with me. SECURITY-CHIEF: But the girl! WAR-CHIEF: She can wait. [SCENE_BREAK] 8, INT: CENTRAL QUADRANT (The War-Chief strides out of the Security Room and away. The Doctor cautiously moves through a keyhole-shaped arch into the area, Carstairs beside him, and points.) DOCTOR: That must be the Security Room. (They flatten themselves against the wall as the Security-Chief follows the War-Chief, starchily looking forwards.) [SCENE_BREAK] 9, INT: SECURITY ROOM (The Doctor creeps into the room, past a guard and over to Zoe.) DOCTOR: Zoe, what have they done to you? (The guard covers the Doctor with his weapon.) DOCTOR: Don't you point that thing at me! (He walks to the side of the room, the guard turning to keep him covered with the weapon.) DOCTOR: You ought to be ashamed of yourself treating a poor girl like... (Carstairs pulls out his gun and pistol whips the guard into unconsciousness from behind.) DOCTOR: Oh well Done. (The Doctor pulls out a phial of smelling salts and wafts it under Zoe's nose. Gradually she comes to.) DOCTOR: There we are, now are you feeling better? ZOE: Oh my head! What happened? DOCTOR: That's was just the question I was going to ask you - what did happen? ZOE: They questioned me. Someone came in, put on that strange headset and questioned me. DOCTOR: Well let's have a look. (He fetches the truth-machine.) DOCTOR: Oh yes, it's a very interesting little gadget. ZOE: I saw pictures of people. DOCTOR: Really? Oh, well let's try it shall we? (He places the machine on his head.) DOCTOR: Now then... Where does this switch...oh yeah, oh...with a slight a adjustment... (With a twiddling of the knob an image is superimposed that alternates from Russell to Harper.) DOCTOR: There we are, yes. What questions did they ask you about these people, Zoe? ZOE: He was talking about the resistance. At first he seemed to think I was one of them. DOCTOR: Ah yes, the resistance, of course. Yes... (He adjusts the machine a bit more and different images spring forth.) DOCTOR: That's the American Civil War...the nineteen-fourteen/eighteen war, yes it's all recorded here. Scattered groups of resistance. Now if we could organise... (The Doctor replaces the truth-machine where he found it.) DOCTOR: Zoe, could you remember all the details of the resistance members from that thing? Names, faces, Time Zones and so on? ZOE: Yes, of course I can. DOCTOR: Good. Then we can return to the Time Zones and organise the resistance groups into one big army! That way they'd have a better chance. CARSTAIRS: But how do we get back? DOCTOR: Well that's simple - the way we came! Come on. ZOE: Oh Doctor, are you sure? (They all exit.) [SCENE_BREAK] 10, INT: CENTRAL-CONTROL (The War-Chief and the Security-Chief have returned with the technician to his post.) TECHNICIAN: Still no message sir, just the emergency signal. WAR-CHIEF: Well? This is your sphere of responsibility. (He walks away.) SECURITY-CHIEF: Arrange transportation for guards. TECHNICIAN: Central control to Landing Bay, transportation required for security guards. SECURITY-CHIEF: Security squad to report to the Landing Bay immediately. Proceed to American Civil War Zone to investigate the reported emergency. [SCENE_BREAK] 11, INT: CENTRAL QUADRANT (The Doctor, Zoe and Carstairs are walking from the Security room when the squalling alert siren sounds, they rush towards the Landing Bay at an increased pace. Behind them marches a squad of guards, but they turn towards the right and walk away. The Doctor and co. creep back, and he peeks after the guards.) ZOE: What was all that about? DOCTOR: I dunno, but they're going to the Landing Bay. Come on. [SCENE_BREAK] 12, INT: LANDING BAY (The Guards walk down the ramp and arrange themselves in a line. The Doctor, Zoe and Carstairs watch from the shadows as a SIDRAT appears.) [SCENE_BREAK] 13, INT: BARN (Harper walks through the doorway and over to Russell.) HARPER: I've posted sentries all around. If they should send in any troops our outposts will warn us. RUSSELL: Good. BUCKINGHAM: What if they come in one of those things the Doctor went off in? JAMIE: Aye, that's what they'll do! That way they can attack us from inside! RUSSELL: They've got to get inside first! JAMIE: No you don't understand... (There is a sound of materialisation.) JAMIE: Quick, hide! In here Lady Jennifer. (They all scramble for cover as a SIDRAT fades into view. The door scrapes open and two black rubber guards step out, weapons held high, and make for the door. Harper runs from cover and tries to attack them, but they swing around fire their stun-guns at him in unison. Harper is momentarily held up by the terrific rippling force of energy swirling around his body, then as the guards cease firing he slumps to the floor, dead. The rest of the resistance fighters descend on the guards and hold them, removing their weapons.) RUSSELL: Well er... (One of the guards is still struggling.) SOLDIER: Grr..'old it there! JAMIE: Well we dealt with those alright didn't we? (Grabbing one of the guards stun-guns, Russell rushes to Harper's body and examines it.) RUSSELL: There's not a mark on 'im! But he's dead. (He tinkers with the weapon.) RUSSELL: But what are these things, how do they work? (The end of the stun-gun swings round in Jamie's direction as Russell absent-mindedly plays with it.) JAMIE: Hey, careful! RUSSELL: They don't work. JAMIE: Aye, well you probably have to do something very special with them. (Russell stands up, his revolver in his right hand and touches the side of the SIDRAT with the other.) RUSSELL: Your green box machine. JAMIE: Aye! this is just what we need. [SCENE_BREAK] 14, INT: WAR ROOM SECURITY-CHIEF: This emergency call could be resistance activity. There is a large group operating in the American Civil War Zone. WAR-CHIEF: There seem to be large groups operating in most Zones, and now they're even infiltrating here. SECURITY-CHIEF: Only two of them and one of those has already been caught. WAR-CHIEF: Had I not pointed them out you wouldn't have even known they were here! SECURITY-CHIEF: Yes, that has been puzzling me. Just how did you recognise them? WAR-CHIEF: You dare cross-question me?! The security of this entire venture is being threatened by your incompetence, you will find this intruder! SECURITY-CHIEF: The whole base is being searched, it is only a matter of time. If the mental processing were fully effective... (The War-Chief smiles coldly.) WAR-CHIEF: There would be no need for security. When I came to your people I was promised efficiency and cooperation. Without the knowledge I have, this complete venture would have been impossible! SECURITY-CHIEF: We have given you every facility. If you have complaints I suggest you take them to the War-Lord! WAR-CHIEF: I intend to do so! And now I suggest you finish the interrogation of that girl before she escapes as well! (The War-Chief strides out of the room leaving the Security-Chief standing on his own. Silently he crushes one of the tiny model soldiers in his hand) [SCENE_BREAK] 15, INT: SECURITY ROOM (The Security-Chief returns to the Security Room to find the guard unconscious and the prisoner missing. He rushes over to the guard and attempts to shake him into consciousness.) SECURITY-CHIEF: Where is the girl? You fool, where is she?! [SCENE_BREAK] 16, INT: BARN (Jamie is still trying to cajole Russell into taking a trip in a SIDRAT.) JAMIE: Do you not see though? This thing could take us all back to the place where it's controlled from! RUSSELL: How do you know? JAMIE: Oh, where do you think those two came from?! RUSSELL: Well is it true? Answer me! VON-WEICH: Do you think I'd help you? RUSSELL: You'll do as you're told! Take those two away! JAMIE: Now look, you want to find out what's going on don't you? (Russell looks hesitant.) JAMIE: Och, I can't wait for you to make up your mind! (He walks to the open SIDRAT door.) BUCKINGHAM: Oh, wait for me! JAMIE: Ah, now Lady Jennifer, I don't think you should come. BUCKINGHAM: Because I'm a woman? JAMIE: Er, well no...er, well in a way, yes. BUCKINGHAM: That settles it then. I'm certainly coming. You can't go alone! (Lady Jennifer's instant volunteering has shamed Russell.) RUSSELL: No, you're right miss, he can't. I'm going with 'im. But you're staying here. BUCKINGHAM: Oh now look here! RUSSELL: You're a nurse you said? BUCKINGHAM: Yes, but what's that got to do with it? RUSSELL: I've got hundreds of injured men back at my headquarters. They need the help of someone like you. (She sighs.) BUCKINGHAM: Yes I suppose you're right. Tell Lieutenant Carstairs that... Well tell him I'll see him when we all get back. JAMIE: Right. BUCKINGHAM: Well, good luck. (Lady Jennifer offers Jamie her hand and he shakes it.) JAMIE: Oh...thank you. RUSSELL: Take her back to our headquarters. (He salutes Lady Jennifer.) RUSSELL: Thank you Miss. (Pulling out his revolver again he gestures at the guard beside Von-Weich.) RUSSELL: You. Guard him. If he gives you any trouble, shoot 'im! You two, you're coming with me. Come on move! JAMIE OOV: Door's closing! (Russell and his soldiers walk into the machine and with a warm sparkling it vanishes. Von-Weich leers and nods.) [SCENE_BREAK] 17, INT: PROCESSING ROOM (The Security-Chief walks in with two guards to find the Scientist still simmering in the processing-machine. He opens the clamp to release him.) SECURITY-CHIEF: What happened? (The Scientist groggily looks at the Security-Chief.) SCIENTIST: Wha...that young officer got away. SECURITY-CHIEF: How? SCIENTIST: Oh the one in the lecture who asked all the questions he tricked me. I must report this to the War-Chief. SECURITY-CHIEF: No! (He glances at the guards.) SECURITY-CHIEF: You two, wait outside. (They leave.) SCIENTIST: But he must be informed. SECURITY-CHIEF: These people are no ordinary resistance group. SCIENTIST: The girl said they have the secret of space/time travel. SECURITY-CHIEF: That's impossible. SCIENTIST: She was under the truth-machine. SECURITY-CHIEF: But the War-Chief... He's the only one who understands space/time travel. SCIENTIST: And his people. SECURITY-CHIEF: What are you suggesting? SCIENTIST: He is not one of our race; who knows where his true loyalties lie? SECURITY-CHIEF: But he came to us of his own accord. SCIENTIST: He is a traitor to his own people. How can we be sure he is not a traitor to us? (The Scientist slowly nods) [SCENE_BREAK] 18, INT: WAR ROOM (The technician sits monitoring the communications.) TECHNICIAN: Transportation returning from the American Civil War Zone sir. WAR-CHIEF: Good. TECHNICIAN: But they're not following the routine sir, they haven't called in. WAR-CHIEF: Have you tried to make contact? TECHNICIAN: Yes sir. WAR-CHIEF: Alright inform the Security-Chief... (He thinks for a moment then puts an arm out to stop the technician.) WAR-CHIEF: No! No, I'll handle this myself. (He moves to a station and activates the communicator.) WAR-CHIEF: This is the War Chief to security section. All guards to Landing Bay, the following procedure to be carried out... [SCENE_BREAK] 19, INT: LANDING BAY (The Doctor, Zoe and Carstairs crouch in a dark corner and watch as a technician adjusts a complex set of magnetic blocks and bars on the main control panel.) ZOE: How long have we got to wait here Doctor? DOCTOR: Until one of those things arrives. If I can get inside I'm-I'm sure I can control it. (The alarm squawks through the Landing Bay.) CARSTAIRS: The alarm signal, they've probably found that guard. DOCTOR: The light's flashing, that means an arrival. (A line of guards file down and take up positions on the ramp, their stun-guns raised.) ZOE: It looks as though somebody important's coming. (As the sound of an arriving SIDRAT echoes through the Landing Bay the guards drop their weapons from the raised position. The door to the green box scrapes open and Jamie, Russell and the resistance soldiers cautiously wander out into the Landing Bay not seeing the guards on the ramp. Zoe turns to the Doctor and whispers.) ZOE: It's an ambush! (But they can do nothing but watch as the guards discharge their stun-guns with deadly accuracy into the unsuspecting crowd of resistance fighters. One by one they all drop, and the last to fall under the lethal onslaught is Jamie.)

The Doctor attempts to rescue Zoe and Carstairs while Jamie and Lady Jennifer try to convince the resistance of the true nature of Von Weich and the others.

summ_screen_fd

Highly active antiretroviral therapy (HAART) suppresses human immunodeficiency virus (HIV) replication to undetectable levels but cannot fully eradicate the virus because a small reservoir of CD4+ T cells remains latently infected. Since HIV efficiently infects only activated CD4+ T cells and since latent HIV primarily resides in resting CD4+ T cells, it is generally assumed that latency is established when a productively infected cell recycles to a resting state, trapping the virus in a latent state. In this study, we use a dual reporter virus—HIV Duo-Fluo I, which identifies latently infected cells immediately after infection—to investigate how T cell activation affects the estab-lishment of HIV latency. We show that HIV latency can arise from the direct infection of both resting and activated CD4+ T cells. Importantly, returning productively infected cells to a resting state is not associated with a significant silencing of the integrated HIV. We further show that resting CD4+ T cells from human lymphoid tissue (tonsil, spleen) show increased latency after infection when compared to peripheral blood. Our findings raise significant questions regarding the most commonly accepted model for the establishment of latent HIV and suggest that infection of both resting and activated primary CD4+ T cells produce latency. Once highly active antiretroviral therapy (HAART) became available in 1995, HIV infection was transformed from a deadly disease into a chronic lifelong condition [1]. The antiretroviral drugs used in HAART target multiple stages of the viral lifecycle, which can reduce patient viremia to undetectable levels [2–4]. However, HAART cannot eradicate HIV [5] because infected individuals harbor a small reservoir of latently infected cells that contain a transcriptionally silent but reactivatable provirus [6]. Because this latent reservoir prevents viral eradication, there is an urgent need to study and better understand the mechanisms of latency. HIV infection primarily targets CD4+ T cells, and the most extensively studied latent reservoir resides within resting CD4+ T cells [7–9]. During infection, HIV enters a target cell and reverse-transcribes its genomic viral RNA into a double-stranded cDNA that then enters the nucleus and integrates into the host genome, where it becomes controlled by the host transcriptional machinery. In most cases, integration of the viral genome leads to productive infection, in which viral genes are transcribed followed by virion production. However, in rare instances, latency occurs instead of productive infection and is characterized by a provirus that produces little-to-no viral transcripts [10]. Because the latently infected cell is not producing viral proteins, it escapes the viral cytopathic effects and is ignored by the immune system. Furthermore, since antiretroviral drugs only target active viral replication, they are ineffective against latent proviruses. Latent HIV is primarily found within memory CD4+ T cells, which have a long half-life in vivo [11,12], allowing latent virus to persist within infected individuals for decades [13]. However, when latently infected memory CD4+ T cells encounter an antigen or are exposed to specific cytokines or chemokines, proviral transcription is activated, leading to productive infection [8,14]. This “reactivation” is likely the cause of viral rebound after a patient stops HAART, and it explains why infected individuals must take antiretroviral drugs for life. HIV latency has proven difficult to study because latently infected cells are very rare in vivo (~1 in 1 × 106 cells) [11], and they cannot be distinguished from uninfected cells [15]. Despite these challenges, several in vitro latency models exist, which have led to important observations about how latently infected cells are maintained and reactivated (reviewed in references [16,17]). However, it is not clear how the latent reservoir is established because current technologies only quantify latently infected cells by reactivating them from latency. We recently developed a dual reporter virus, HIV Duo-Fluo I, that can distinguish between cells that are productively infected, latently infected, or uninfected, and allows us to purify each population [18]. Using this new reporter virus, we can study the kinetics of HIV latency immediately after infection by employing two separate fluorescent markers: an LTR-driven eGFP marker (productive infection) and an LTR-independent mCherry marker (latent infection) driven by an EF1α promoter (Fig 1A). It should be noted that we use the term “productive infection” here and throughout the manuscript to indicate an infection resulting in the expression of the LTR-driven GFP reporter. Since the virus used in this manuscript is env-deficient, these infections are not truly productive. However, they are behaving like a productive infection in terms of virus expression levels. Using this dual reporter virus, we have studied how HIV latency is established with a unique focus on the role of T cell activation. Based primarily on in vitro evidence, it is generally accepted that HIV predominantly replicates in activated CD4+ T cells [19–22]. Conversely, resting CD4+ T cells present several barriers to HIV infection (reviewed in reference [23]), as they do not support efficient nuclear import [24] or integration of the viral cDNA [22,25]. However, the most notable obstacle to infection of resting CD4+ T cells occurs at the stage of reverse transcription [26,27]. Resting CD4+ T cells do not support reverse transcription nearly as efficiently as activated cells because, at least in part, they contain the restriction factor SAMHD1 [28,29]. Additionally, in vivo, most HIV-infected resting CD4+ T cells exhibit a memory phenotype, suggesting that they arose from the infection of previously activated CD4+ T cells. Based on this evidence, a leading theory postulates that latency is established from infected activated CD4+ T cells that revert back to a resting memory state. According to this model, the transition to a resting memory state is associated with a decrease in NFκB and pTEFb activity, two critical factors for HIV transcription, and with a concomitant silencing of the HIV genome [30]. However, for this type of latency to occur, the infected cell would have to survive the virus-induced cytopathic effects and the host immune response that usually kill productively infected cells very quickly (cells survive ~1 day) [31,32]. Another possibility is that infection occurs at a “sweet spot” in the trajectory that activated CD4+ T cells taken from full activation to a fully rested state. This sweet spot would be characterized by permissivity for HIV reverse transcription and integration but not for HIV transcription [33]. Interestingly, previous studies have reported that resting CD4+ T cells can be directly infected, with the strongest evidence coming from in vivo and ex vivo studies of both SIV and HIV infection [34–39]. Most studies that show resting CD4+ T cells can be directly infected have been performed with cells isolated from primary lymphoid tissues. In vivo studies have found that resting CD4+ T cells in lymphoid tissue harbor viral RNA [35], and ex vivo studies have shown that directly infecting resting CD4+ T cells from lymphoid tissue results in productive infection [40]. Strikingly, a subsequent study found that resting CD4+ T cells in ex vivo lymphoid cells isolated from tonsillar tissue can support HIV infection, but purified CD4+ T cells isolated from that same lymphoid tissue could not [41], suggesting that the lymphoid tissue microenvironment is critical for rendering resting CD4+ T cells permissive to HIV infection. Indeed, several lymphoid tissue–associated factors, including cytokines [42], chemokines [43], extracellular matrixes [44], and cell surface markers [45], enhance HIV infection in resting CD4+ T cells. Therefore, HIV latency may be established by the direct infection of resting CD4+ T cells when they are exposed to soluble factors that do not induce classic T cell activation. In this study, we use the dual reporter virus, HIV Duo-Fluo I, to investigate the role of T cell activation on the establishment of HIV latency in primary CD4+ T cells. We also use HIV Duo-Fluo I to explore the theories of how HIV latency is established; namely, whether it occurs through infection of activated CD4+ T cells that return to a resting state or through the direct infection of resting CD4+ T cells. We find that both resting and activated primary CD4+ T cells can support both productive and latent infection. In the case of activated T cells, the latent state is established very early in the infection and is not significantly influenced by the return of that activated cell to a resting state. We further observed that the fraction of cells that become latent (latent/productive) is higher in resting CD4+ T cells than in activated CD4+ T cells. The literature is replete with conflicting reports on whether resting CD4+ T cells can be infected by HIV, either productively or latently [23]. Many studies indicate that resting CD4+ T cells are refractory to productive HIV infection but can become permissive to infection after treatment with certain cytokines or chemokines that do not induce classic T cell activation [46,47]. To test the permissivity of resting CD4+ T cells to our HIV Duo-Fluo I virus, we isolated total CD4+ T cells from peripheral blood of uninfected donors via depletion of all non-CD4+ T cells (negative selection). These cells did not express CD69 or CD25 (Fig 1B) and can therefore be considered resting. These cells were either left untreated or were treated with the cytokine IL-7 or the chemokine CCL19 for 72 h prior to infection. Stimulation with IL-7 or CCL19 slightly increased CD25 expression, such that 0. 58% and 1. 38% of cells expressed CD25, respectively (Fig 1B). As a positive control, resting CD4+ T cells were stimulated with αCD3/αCD28 activating beads in the presence of IL-2 for 72 h prior to infection, which led to significant expression of both CD69 and CD25 activation markers (Fig 1B). Both untreated and treated cells were spinoculated with HIV Duo-Fluo I for 2 h at 37°C and then returned to culture in the presence of IL-2. Productive infection (GFP+ and mCherry+/GFP+) and latent infection (GFP-/mCherry+) were monitored daily by flow cytometry for 6 days following infection (S1 Fig). Compared to αCD3/αCD28-stimulated cells at 6 days postinfection, untreated resting CD4+ T cells showed significantly lower levels of HIV infection but, nonetheless, permitted both productive and latent infection (Fig 1C). Importantly, productive and latent infection of resting CD4+ T cells over the 6-day time-course was not the result of replication-competent virus being present in our HIV Duo-Fluo I viral stocks (S2 Fig) [48]. Despite minimally affecting T cell activation, both IL-7 and CCL19 treatment led to an increase in HIV infection compared to untreated resting CD4+ T cells (Fig 1C and 1D), suggesting that the permissibility of resting CD4+ T cells can be enhanced without undergoing classic T cell activation, which agrees with previous studies [47,49]. However, we show how productive and latent infection is distributed within resting CD4+ T cells after such treatments (Fig 1D). Treating resting CD4+ T cells with CCL19 increased productive infection 2-fold over untreated cells, while IL-7 treatment produced a 5-fold increase in productive infection. Latent infection increased by 3-fold after treatment with either CCL19 or IL-7. In addition to cytokine and chemokine treatment, we also investigated the role of the human protein SAMHD1 in restricting HIV infection within resting CD4+ T cells. To do this, we infected resting CD4+ T cells with HIV Duo-Fluo I containing Vpx, which was provided in trans as a fusion protein with Vpr. Vpx is a lentiviral accessory protein encoded by HIV-2 that degrades SAMHD1 and thereby allows the virus to infect many cell types—resting CD4+ T cells [28], dendritic cells, monocytes, and macrophages [50,51]—that are usually off limits to HIV-1 because of a SAMHD1-imposed post-entry block. Infecting untreated resting CD4+ T cells with Vpx-containing HIV Duo-Fluo I increased their infection levels over untreated cells infected with HIV Duo-Fluo I alone (Fig 1C and 1D). This increase in infection correlates with SAMHD1 protein down-regulation mediated by Vpx (S3A Fig) and has little to do with T cell activation (S3B Fig). As such, levels of productive infection increased significantly and were almost comparable to those of activated CD4+ T cells, whereas levels of latent infection were more than 2-fold greater than in resting untreated cells infected with HIV Duo-Fluo I alone. Overall, this increase in infection suggests that knocking down SAMHD1 in resting CD4+ T cells biases the cells toward productive infection, an observation that is confirmed by calculating the ratio of latently infected to productively infected cells (Fig 1E). Based on the ratio of latently infected to productively infected cells, IL-7–treated resting CD4+ T cells and activated CD4+ T cells also support more productive infection than latent infection. Conversely, untreated and CCL19-treated resting CD4+ T cells support more latent infection than productive infection. To ensure that infecting resting CD4+ T with HIV Duo-Fluo I did not lead to any silent infection events—in which viral integration occurred but failed to produce expression of either fluorescent marker—we sorted the uninfected populations (GFP-/mCherry-) of both untreated and treated CD4+ T cells by FACS at 6 days post-infection. These cells were then stimulated with αCD3/αCD28 activating beads in the presence or absence of the integrase inhibitor, raltegravir, to distinguish between the reactivation of any pre-integration latent virus and post-integration latent provirus that might be present (S4 Fig). As a control, we isolated (via FACS) latently infected cells (GFP-/mCherry+) from infected cells that were pretreated with αCD3/αCD28 activating beads and subjected them to the same treatments as the uninfected cells. We analyzed reactivation of the latent virus by flow cytometry 48 h after stimulation. All cell populations contained some reactivatable pre-integration latent virus (Fig 1F); the highest levels were observed in CCL19- and IL-7-treated populations and in the untreated population infected with Vpx-containing HIV Duo-Fluo I. Untreated resting CD4+ T cells infected with the HIV Duo-Fluo I virus alone showed the lowest levels of reactivatable pre-integration latency, followed closely by activated CD4+ T cells. By analyzing the reactivatable post-integration latent provirus, we found that the uninfected cell population isolated from activated CD4+ T cells contains very little reactivatable provirus (0. 26%, Fig 1F) compared to the initial latent population identified in activated CD4+ T cells after infection (1. 01%, Fig 1D). These findings suggest that HIV Duo-Fluo I can efficiently identify latently infected cells within activated CD4+ T cells. Conversely, uninfected cells isolated from untreated resting CD4+ T cells contained 0. 40% reactivatable provirus, but only 0. 14% latently infected cells were identified in this cell population after the initial infection. This finding suggests that HIV Duo-Fluo I identifies only a fraction of latently infected cells within resting CD4+ T cells. Similarly, IL-7–treated resting CD4+ T cells and untreated resting CD4+ T cells infected with the Vpx-containing HIV Duo-Fluo I both contained over 1% reactivatable provirus within their isolated uninfected cell populations, which was more than twice the size of the latent cell populations identified in these respective populations after the initial infection (Fig 1D). This suggests that IL-7 treatment and SAMHD1 knockdown lead to silent infection events in resting CD4+ T cells, and, thus, an underestimation of the latently infected cell population. Interestingly, CCL19 treatment of resting CD4+ T cells produced 0. 2% reactivatable provirus from the isolated uninfected cell population, while the initial latent cell population after infection was 0. 47%. Overall, these data suggest that HIV Duo-Fluo I can be used to identify latently infected cells within activated CD4+ T cells, but may underestimate the percentage of latently infected cells within resting CD4+ T cells. Because HIV replicates most efficiently in activated CD4+ T cells [23], and the largest in vivo latent reservoir is within resting memory CD4+ T cells, we next investigated whether HIV latency is preferentially established in CD4+ T cells that become infected as they transition from an activated to a resting state. To do this, we isolated total CD4+ T cells from peripheral blood of uninfected donors and stimulated the cells with αCD3/αCD28 activating beads in the presence of IL-2 for 3 days (Fig 2A). We then removed the αCD3/αCD28 activating beads and allowed the cells to return to a resting state in the presence of IL-2 for 20 days. We infected the CD4+ T cells with HIV Duo-Fluo I at peak activation (day 4) and every 5 days thereafter as they transitioned back to resting. As indicated by expression of the activation markers CD69 and CD25, the cells transitioned from active to resting over the 20 days and remained >60% viable (Fig 2B). Maximal activation occurred at day 4 with 79% of cells expressing both CD69 and CD25 (Fig 2C). By day 9, however, 31% of cells no longer expressed CD69 or CD25, and by day 24,92% of cells no longer expressed either activation marker. Despite most cells losing CD69 expression by day 14 (<1% CD69+), a small fraction of cells continued to express CD25 throughout the experiment—with 8% of cells still CD25+ at day 24—suggesting that while most CD4+ T cells had returned to a resting state by day 24, a small population was still transitioning back to a resting state. Others have observed similar expression profiles while trying to return activated CD4+ T cells to a resting state [52,53]. We analyzed productive infection and latent infection by flow cytometry at 3 days postinfection for each time point (Fig 2D), and we determined the average of each cell population from three donors (Fig 2E). Infection at day 4, when the CD4+ T cells were maximally activated, produced the highest levels of both productive and latent infection. As the cells returned to a resting state, the levels of both productive infection and latent infection decreased, suggesting that HIV most effectively infects CD4+ T cells when they are at their highest activation state; as CD4+ T cells stop expressing the activation markers CD69 and CD25, they become less permissive. However, the ratio of latent infection to productive infection steadily increased from 0. 010 at day 4 when the cells where most active to 0. 025 at day 24 when the cells exhibited a more resting phenotype (Fig 2F). Therefore, while activated CD4+ T cells support the most robust infection, latent infection is more likely to occur relative to productive infection in cells that are resting or are transitioning back to a resting state. To explore another possible way latency is established, we next investigated whether productively infected activated primary CD4+ T cells can return to a resting state and contribute to the latent reservoir. We isolated CD4+ T cells from uninfected donor blood and activated them with αCD3/αCD28 activating beads in the presence of IL-2 for 3 days. At this point, we spinoculated the cells with HIV Duo-Fluo I for 2 h at 37°C (Fig 3A). After infection, cells were kept in an activated state by returning them to culture in the presence of activating beads and IL-2. Four days postinfection, CD4+ T cells were sorted to isolate three distinct populations: uninfected (GFP-/mCherry-), productively infected (GFP+/mCherry- & GFP+/mCherry+), and latently infected (GFP-/mCherry+) cells. After sorting, a small fraction of each population was used to measure HIV integration via Alu-gag PCR (S5 Fig), while the majority of each population was cultured with IL-2 and allowed to return to a resting state over an 11-day period. The activation state of each cell population was monitored by the expression of the activation markers CD69 and CD25 (Fig 3B), as well as changes in cell size (S6 Fig). The activation markers CD69 and CD25 were maximally expressed at day 4, when the cells were infected with HIV Duo-Fluo I (Fig 3B). After sorting, each cell population began to lose both CD69 and CD25 expression, and the cell size of each population began to shrink (S6 Fig). However, none of the distinct cell populations fully returned to resting during the 11-day period. The uninfected cell population contained 21. 6% of CD69 and CD25 double-negative cells at 11 days post-activation compared to 16. 6% for the latently infected population and just 9. 03% for the productively infected population. This indicates that the uninfected population is returning to a resting state more quickly than either of the infected cell populations. Additionally, the productively infected cell population contained 12. 2% CD69 and CD25 double positive cells at 11 days post-activation compared to 5. 58% for the latently infected cell population and just 2. 44% for the uninfected cell population, indicating that the productively infected CD4+ T cells are returning to a resting state at a slower rate than either latently infected or uninfected CD4+ T cells. As such, latently infected cells are less activated than productively infected cells. While activated cells were returning to a resting state over the 11-day period, they experienced changes in their infection profiles (productive or latent infection) (Fig 3C). A small percentage of the uninfected cell population became infected, which most likely reflects pre-integration latency [18]. More interestingly, 98. 8% of the productively infected cell population continued to express GFP throughout the 11 days, with 1. 18% of the cells no longer expressing GFP. Based on these data, only a small fraction of productively infected cells have returned to a resting state over the course of the 11 days, and potentially contribute to the latent reservoir. Over the course of the 11 days, the latently infected cell population began to display two distinct phenotypes. First, over 6% of the latently infected cell population spontaneously reactivated as exhibited by their GFP expression (Fig 3C). Second, over 60% of the latently infected cells lost expression of mCherry, in addition to not expressing GFP. By analyzing the activation marker expression profile for these distinct populations (S7 Fig), we found that those cells that lost mCherry expression had higher levels of CD69/CD25 double-negative cells (24. 9%) than the latently infected cells that continued to express mCherry (12. 8%) and those that spontaneously reactivated (3. 23%). Taken together, these data demonstrate that the majority of activated CD4+ T cells that become latently infected revert back to a resting state, while also silencing the EF1-α-driven mCherry expression. After allowing the sorted cell populations to return to a resting state for 11 days, we stimulated each population with αCD3/αCD28 activating beads to reactivate any latent provirus (Fig 3D). In the uninfected cell population, stimulation produced a small amount of reactivatable provirus which, again, is likely due to pre-integration latency [18]. Interestingly, stimulation of the productively infected cell population did not lead to reactivation of the ~1% of cells that no longer expressed GFP. Lastly, stimulation of the latently infected cell population produced only a small amount of reactivatable provirus (~2%). However, we observed a large shift from latently infected cells that no longer express mCherry to latently infected cells that do express mCherry, as evidenced by a 20% increase in mCherry+ cells after αCD3/αCD28 stimulation (Fig 3D). These results indicate that latently infected cells that lose mCherry expression over time are not dying but instead returning to a resting state. Lastly, based on evidence that resting CD4+ T cells within lymphoid tissue can support HIV replication [34–39], we wanted to investigate how latency is established within ex vivo lymphoid tissue using our HIV Duo-Fluo I virus. We isolated CD4+ T cells from tonsillar and splenic tissues, as well as from peripheral blood from uninfected donors. Because lymphoid organs contain over 98% of the body’s CD4+ T cells, and are the primary sites of HIV replication, we also isolated total lymphoid cells from tonsillar and splenic tissues from uninfected donors in the form of human lymphoid aggregated cultures (HLACs), which closely mimic the conditions encountered by HIV, in vivo [36]. In addition, we isolated total peripheral blood mononuclear cells (PBMCs) from uninfected donors. CD4+ T cells, PBMCs, and HLACs were either left untreated or stimulated with αCD3/αCD28 activating beads in the presence of IL-2 for 3 days and then spinoculated with HIV Duo-Fluo I for 2 h at 37°C. We assessed expression of the activation markers CD69 and CD25 before and after stimulation (Fig 4A). PBMCs and CD4+ T cells isolated from peripheral blood expressed very little CD69 or CD25 and were thus considered resting cells. However, CD4+ T cells isolated from tonsillar and splenic tissues, as well as HLACs from these tissues, highly expressed the early activation marker CD69 (38% and 42%, respectively) but expressed low levels of the intermediate activation marker CD25. Thus, CD4+ T cells isolated from lymphoid tissue are not resting cells, but they are also not fully activated. After stimulation with αCD3/αCD28 activating beads in the presence of IL-2 for 3 days, CD4+ T cells from all three tissues expressed high levels of both activation markers, reflecting classic T cell activation. However, upon stimulation, CD4+ T cells from peripheral blood achieved higher activation levels than CD4+ T cells isolated from either lymphoid tissue. Lastly, expression of CD69 and CD25 among αCD3/αCD28-stimulated PBMCs and HLACs was consistently lower than in purified CD4+ T cells, and may reflect the size of the CD4+ T cell population within each culture. Levels of productive and latent infection were analyzed by flow cytometry 72 h post-infection (Fig 4B and 4C). Untreated CD4+ T cells from peripheral blood, which expressed no activation markers, produced very little productive infection (0. 47%). Despite expressing moderately high levels of CD69, untreated CD4+ T cells isolated from tonsillar tissue did not give rise to significantly higher levels of productive infection (0. 68%) compared to CD4+ T cells from peripheral blood. However, untreated CD4+ T cells from splenic tissue—which expressed CD69 at levels comparable to those of CD4+ T cells isolated from tonsillar tissue—did show an increase in productive infection (1. 8%) as compared to untreated CD4+ T cells from peripheral blood. Levels of latent infection in untreated CD4+ T cells isolated from both lymphoid tissues were at least 2-fold greater than those observed in untreated CD4+ T cells from peripheral blood (Fig 4C), suggesting that CD4+ T cells within lymphoid tissue are more likely to become latently infected. Overall, untreated PBMCs and untreated HLACs from both lymphoid tissues displayed lower levels of productive infection than untreated CD4+ T cells isolated from each tissue (Fig 4C). Infection of untreated PBMCs resulted in a 3. 5-fold decrease in productive infection as compared to untreated CD4+ T cells from the blood, while infection of untreated HLACs from tonsil resulted in a 4-fold decrease in productive infection as compared to untreated CD4+ T cells from the same tissue. Untreated HLACs from the spleen resulted in an 11-fold decrease in productive infection as compared to untreated CD4+ T cells from the same tissue. In contrast, the levels of latent infection did not change between untreated total lymphoid cells and untreated CD4+ T cells from each tissue (Fig 4C). In addition, despite differences in activation levels, infection of untreated HLACs from both lymphoid tissues did not result in an increase of either productive or latent infection as compared to untreated PBMCs. When analyzing the ratio of latent infection to productive infection, we found that HIV Duo-Fluo I infection of all untreated cells from the three different tissues was at least 5-fold more likely to result in latent infection than their αCD3/αCD28-stimulated counterparts (Fig 4D). Thus, activated cells exhibit a higher propensity for productive infection, while resting cells exhibit a higher propensity for latent infection. Additionally, infecting untreated total lymphoid cell populations results in more latent infection than when infecting purified untreated CD4+ T cells from the same tissue, suggesting that co-culture of CD4+ T cells with other lymphoid cells promotes latent infection. The role that T cell activation plays in establishing HIV latency within CD4+ T cells is still not fully understood. HIV replication is clearly most efficient in activated CD4+ T cells [19–22], and the largest in vivo latent reservoir is within memory CD4+ T cells [11,12]. This evidence suggests that HIV latency is established in one of two ways: 1) Activated CD4+ T cells become productively infected but survive viral cytopathic effects and evade elimination by the immune system long enough for the cell to transition to a resting memory state; or 2) CD4+ T cells that are transitioning from an activated to a resting memory state are infected by HIV while the cellular environment can still support integration of viral cDNA but cannot support proviral transcription. However, studies have shown that both naive and memory CD4+ T cells contain integrated viral DNA [34], and that direct infection of resting CD4+ T cells in lymphoid tissue results in productive infection [40]. These findings suggest that HIV latency can also be established in another way: direct infection of resting CD4+ T cells. In this study, we show that all three scenarios can produce latent HIV infection. We further show that HIV latency can be established in activated CD4+ T cells without them first returning to a resting state. Additionally, infecting activated CD4+ T cells is more likely to result in productive infection, while infecting resting CD4+ T cells is more likely to result in latent infection. Finally, HIV latency is more likely to occur in resting lymphoid cell aggregates than in resting CD4+ T cells cultured alone. Using primary CD4+ T cells isolated from the blood of uninfected donors, we demonstrate that infecting resting and activated CD4+ T cells with our HIV Duo-Fluo I virus causes both productive and latent infection in the two populations. In activated CD4+ T cells, HIV latency is established within the first few days of infection and does not require the cell to return to a resting state. We showed this previously [18], as did another group that developed a similar dual-reporter virus [54]. That construct uses a different LTR-independent promoter (CMV) than our EF1α promoter, and it places the LTR-driven eGFP cassette in the Gag region, while ours replaces the Nef open reading frame. Despite these differences, both dual-reporter viruses can detect latent infection events in activated CD4+ T cells early after the initial infection, and these latently infected cells can be reactivated by different stimuli. Additionally, we sorted these latently infected cells and showed that they still express significant amounts of both CD69 and CD25 activation markers; the cells only stop expressing these markers as they are allowed to return to a resting state. As they return to a resting state, latently infected CD4+ T cells also stop expressing the EF1α-driven mCherry marker, suggesting that as these cells return to resting, both promoters become silenced, perhaps by packaging into heterochromatin [55]. This means that HIV latency may be established after activated CD4+ T cells are initially infected, and it is these cells, potentially, that survive and return to a resting memory state, thus significantly contributing to the latent pool. How HIV latency is established in activated CD4+ T cells immediately after infection is still unknown, but it may arise from stochastic viral gene expression [56–59]. Our studies also suggest that activated CD4+ T cells that become productively infected can contribute to the latent pool as they return to a resting state. In our studies, these cells did not return to a completely resting state because so many of the cells died (S6 Fig), a likely consequence of viral cytopathic effects. However, the data clearly indicate that a small population of productively infected cells starts to return to a resting state and as they do, they lose expression of the LTR-driven GFP marker. However, when these cells were then stimulated with αCD3/αCD28, they failed to express GFP, suggesting that they could not be reactivated by CD3/CD28 stimulation, though it is possible that other reactivating agents could work. Finally, it is important to note that these productively infected CD4+ T cells that did eventually shut down LTR-driven GFP expression, did so in a culture dish. It remains to be seen, in vivo, if productively infected CD4+ T cells can survive long enough to return to a resting state and contribute to the latent pool, or if activated CD4+ T cells that become latently infected immediately after infection are the major contributors. Finally, infecting activated CD4+ T cells produces more productively infected cells than latently infected cells, while infecting resting CD4+ T cells produces more latently infected cells. These results reflect that HIV replicates more efficiently in activated CD4+ T cells, but they also show that resting CD4+ T cells can support HIV infection, at least up to the point of viral integration. In resting primary CD4+ T cells, we show that both productive and latent HIV infection can be achieved, though at levels much lower than those seen in activated CD4+ T cells. The infection kinetics in resting CD4+ T cells seem to be slower than in activated cells, since peak infection was not reached until 6 days after infection, while activated cells reached peak infection 4 days after infection (S1 Fig). These results agree with other’s findings [22,60]. Also in agreement with previous findings, resting CD4+ T cells were made more permissive to HIV infection when exposed to the chemokine CCL19, which increases the ability of resting CD4+ T cells to support latent infection [47]. However, our data demonstrate that CCL19 also increases permissibility to productive infection, although its overall effect on resting CD4+ T cells increases latent infection. Interestingly, the cytokine, IL-7, which increases permissibility of resting CD4+ T cells to productive HIV infection, also increased both productive and latent infection in resting CD4+ T cells in our study. Lastly, infecting untreated resting CD4+ T cells with a Vpx-containing virus significantly increased productive infection but only modestly increased latent infection. Interestingly, infecting resting CD4+ T cells with our HIV Duo-Fluo I virus produced a significant amount of silent infection events, in which expression of both fluorescent proteins was silenced, camouflaging latently infected cells within our uninfected population. In fact, the isolated uninfected population of resting CD4+ T cells contained more silently infected cells than the number of latently infected cells that were identified via the mCherry fluorescent marker after the initial infection. This was true for all untreated and treated resting CD4+ T cells, except CCL19-treated cells, and was highest in IL-7-treated cells and untreated resting CD4+ T cells infected with a Vpx-containing virus. The reasons for this are unclear. Within resting CD4+ T cells, viral integration occurs in regions of the host genome that are unfavorable for viral gene expression [61], and studies also suggest that latently infected cells are more likely to contain provirus in or near heterochromatin [62,63]. Integration into such regions would be unfavorable not only for LTR-driven gene expression but also for EF1α-driven gene expression. In the presence of Vpx, SAMHD1 cannot inhibit HIV reverse transcription, allowing the virus to bypass one of the major obstacles to replication in resting CD4+ T cells. Therefore, integration of the viral cDNA may occur more readily in these unfavorable heterochromatic regions. Treating cells with IL-7, which signals through the JAK/STAT pathway [64], may produce a similar situation. Lastly, previous studies have reported that resting CD4+ T cells can only be infected by HIV in the context of total lymphoid cell aggregates [41]. However, our results show that infecting untreated resting CD4+ T cells (alone) and untreated resting total lymphoid cells from peripheral blood and lymphoid tissue all produced productive and latent populations. Although, we did find that latent infection is more likely to occur in total resting lymphoid cell aggregates than in resting CD4+ T cells alone. The reasons for this are still unclear, but recent studies have shown that co-culture of resting CD4+ T cells with myeloid dendritic cells [65], or co-culture of resting CD4+ T cells with endothelial cells [66], enhances HIV latency, further proving that the lymphoid environment plays an important role in how HIV latency is established within resting CD4+ T cells. Overall, our studies show that HIV infection can occur in both resting and activated CD4+ T cells, such that infection of resting cells more often results in latent infection and infection of activated cells more often results in productive infection. Based on our data, we now have a better understanding of the contribution that each infected cell type makes to the latent reservoir. Our study underscores why we must consider both resting and activated CD4+ T cells when investigating how HIV latency occurs. Pseudotyped HIV Duo-Fluo I viral stocks were generated by co-transfecting (using the standard calcium phosphate transfection method) HEK293T cells with a plasmid encoding HIV Duo-Fluo I and a plasmid encoding HIV-1 dual-tropic envelope (pSVIII-92HT593. 1). We generated a Vpx-containing HIV Duo-Fluo I pseudotyped virus by co-transfecting HEK293T cells with the HIV Duo-Fluo I plasmid, the pSVIII-92HT593. 1 plasmid, and a plasmid encoding a Vpr-Vpx fusion protein (pSIV3+, generously donated by Warner Greene). Supernatants were collected after 72 h and filtered through a 0. 45 μM membrane to clear cell debris, and were then concentrated by ultracentrifugation (76,755 x g) for 2 h at 4°C. Concentrated virions were resuspended in complete media and stored at -80°C. Virus concentration was estimated by p24 titration (HIV-1 alliance p24 ELISA kit, Perkin-Elmer). Primary CD4+ T cells and peripheral blood mononuclear cells (PBMCs) were purified from healthy donor blood (Blood Centers of the Pacific, San Francisco, CA, USA and Stanford Blood Center). CD4+ T cells were isolated by negative selection using the RosetteSep Human CD4+ T Cell Enrichment Cocktail (StemCell Technologies). PBMCs were purified by Histopaque-1077 density gradient. Purified resting CD4+ T cells and PBMCs from peripheral blood were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), L-glutamine (2 mM), penicillin (50 U/ml), and streptomycin (50 mg/ml). Human lymphoid aggregate cultures (HLACs) were purified using tonsillar or splenic tissue from uninfected donors (Cooperative Human Tissue Network) as previously described [67]. CD4+ T cells were isolated from HLACs by negative selection using the EasySep Human CD4+ T Cell Enrichment Kit (StemCell Technologies). HLACs and CD4+ T cells isolated from splenic and tonsillar tissues were cultured in RPMI 1640 supplemented with 20% heat-inactivated FBS, 100 mg/ml gentamicin, 200 mg/ml ampicillin, 1 mM sodium pyruvate, 1% nonessential amino acids (Mediatech, Manassas, VA, USA), 2 mM L-glutamine, and 1% fungizone (Invitrogen, Indianapolis, IN, USA) Purified resting CD4+ T cells were either left untreated or treated for 3 days with 20 ng/ml IL-7 (R&D Systems) or 100 μM CCL19 (R&D Systems). Purified CD4+ T cells isolated from peripheral blood and tonsillar and splenic tissues, as well as PBMCs and HLACs, were stimulated with αCD3/αCD28 activating beads (Life Technologies) at a concentration of 1 bead/cell in the presence of 30 U/ml IL-2 (PeproTech) for 3 days. All cells were spinoculated with either HIV Duo-Fluo I alone or Vpx-containing HIV Duo-Fluo I at a concentration of 100 ng of p24 per 1 × 106 cells for 2 h at 1,200 × g at 37°C. After spinoculation, all cells were returned to culture in the presence of 30 U/ml IL-2, except for CD4+ T cells pre-stimulated with αCD3/αCD28 activating beads, which were placed back in culture with the αCD3/αCD28 activating beads and 30 U/ml IL-2. Uninfected cells were stained in fluorescence-activated cell sorting (FACS) buffer (phosphate buffered saline supplemented with 2 mM EDTA and 2% FBS) with αCD69-PE and αCD25-APC (eBioscience) and fixed in 1% paraformaldehyde. Infected cells were stained in FACS buffer with αCD69-V450 and αCD25-APC/Cy7 (BD Biosciences) and fixed in 1% paraformaldehyde. Data were collected on a FACS Caliber and a FACS LSRII (BD Biosciences), and analyses were performed with FlowJo software (TreeStar). Untreated and treated CD4+ T cells from Figs 1F and S4 were sorted with a FACS AriaII (BD Biosciences) based on their GFP and mCherry fluorescence at 6 days post-infection, and they were placed back in culture with or without 30 μM Raltegravir (National AIDS Reagent Program). CD4+ T cells stimulated with αCD3/αCD28 activating beads in the presence of 30 U/ml IL-2 from Fig 3 were sorted based on their GFP and mCherry fluorescence at 4 days post-infection. Untreated resting primary CD4+ T cells infected with either HIV-Duo-Fluo I alone or Vpx-containing HIV Duo-Fluo I were lysed 6 days post-infection in radioimmunoprecipitation assay buffer (150 mm NaCl, 1% Nonidet P-40 (vol/vol), 0. 5% AB-deoxycholate (vol/vol), 0. 1% sodium dodecyl sulfate (SDS) (vol/vol), 50 mm Tris-HCl (pH 8), 1 mm DTT, and EDTA-free Protease Inhibitor (Calbiochem). Cell lysates were used for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) immunoblotting analysis. The primary antibodies used were rabbit polyclonal anti-SAMHD1 (Sigma-Aldrich, Cat# SAB2102077) and monoclonal anti-β-actin (A5316, Sigma-Aldrich). DNA was prepared after cell sorting of uninfected, productively infected and latently infected cell populations using the DNeasy Kit (Qiagen). Real-time PCR was used to detect total HIV DNA, β-globin, and integrated HIV DNA as previously described [68].

The study of HIV latency has been hindered because there are few latently infected cells in vivo, and we cannot distinguish latently infected cells from uninfected cells prior to reactivation of the latent provirus. In general, HIV latency is quantitatively studied by reactivating latently infected cells after latency has been established. However, this practice limits the investigation of how latency is established and how latent provirus can be reactivated. Our recently developed dual reporter virus, HIV Duo-Fluo I, can identify latently infected cells early after infection. In this study, we use HIV Duo-Fluo I to investigate how T cell activation affects the outcome of HIV infection.

lay_plos

RELATED U.S. APPLICATIONS [0001] Not applicable. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0002] Not applicable. REFERENCE TO MICROFICHE APPENDIX [0003] Not applicable. FIELD OF THE INVENTION [0004] Therapeutic peptide vaccine complex designed for the prevention and treatment of conditions in mammals and in particular, in humans, canines, felidae, and equidae whose protective immunity depends on the stimulation of T lymphocytes of the Th1 type and notably of a state of hypersensitivity of the retarded type. [0005] Therapeutic peptide vaccine complex also designed for diagnosis of cell-mediated immunity depending on the T lymphocytes of the Th1 type using various “in vivo” and “in vitro” methods. BACKGROUND OF THE INVENTION [0006] During the immune response, the T lymphocyte comprises an important source of cytokines. Their production is induced after stimulation in a specific manner in the presence of antigens or in a non-specific manner in the presence of mitogens (concanaviline A, phytohemagglutinin). [0007] If it is clearly established that the T lymphocytes, and among them, especially the CD4+ lymphocytes, represent the main source of cytokines, the T sub-populations involved in this phenomenon are varied and seem to vary as a function of the stimulus. [0008] It is possible to divide, in a very simplified way, the immune responses into two large qualitatively distinct categories, humoral responses which bring into play the production of antibodies by the B lymphocytes, and cellular responses (retarded hypersensitivity reaction, cytotoxic reaction), for which the effective cells are the T lymphocytes. It seems that in the majority of experimental models and clinical situations studied, the cytokines having a lymphocytary origin are essentially produced by the auxiliary T cells CD4+ (or helpers), whose role is to modulate or regulate the humoral and cellular immunity. These auxiliary T cells recognize the antigen in combination with the class II molecules of the major histocompatibility complex. A major concept emerged in 1985 when T. Mosmann and R. Coffman proposed that the T lymphocytary cells CD4+ expressing auxiliary functions were in fact heterogens. Thus, using a study of the lymphocytary clones T CD4+ of mice, cultivated over the long term, the authors described the existence of two major sub-populations that can be distinguished by their profile of secretion of cytokines, i.e. Th 1 cells (for T helpers of type 1) and Th 2 cells (for T helpers of type 2). [0009] The example of leishmaniasis will be used, which is a parasitic infection endemic, or even epidemic, in tropical and subtropical regions of the world. The leishmania, flagellate protozoa of the family of trypsanosomatidae and of the genus Leishmania, are the pathogenic agents responsible for these diseases. [0010] Numerous studies concerning the immune responses during experimental murine leishmaniases have led to the demonstration of the predominant role of cell-mediated immunity and the existence of a duality of the immunological response. There are fundamentally two types of responses against leishmanias: one is described by the “sensitivity”, the other is described by the “resistance”. The different subpopulations of T lymphocytes (CD4+) limit or exacerbate the infection by means of the lymphokines they secrete. It has thus been demonstrated that the subpopulation of auxiliary T lymphocytes of the Th1 type (producer of interferon gamma and interleukine 2) was capable of eliminating the amastigote intracellular forms by means of the activation of macrophages (Reiner S. L et al., Annu Rev Immunol, 1995, 13, 151-177. Review). Conversely, the subpopulation of auxiliary T lymphocytes of the Th2 type (producer of interleukine 4) is responsible for exacerbating the disease. [0011] In humans, certain facts are comparable by nature. In the dog (natural “reservoir” receptive host in the life cycle of L. infantum ), the duality of the immunological response is likely. Only one study led by Pinelli et al. (Infect. immun., 62:229, 1994) on experimental animals naturally infected by L. infantum, made it possible to show that the asymptomatism of the dog (clinical state frequently encountered) is accompanied by the absence of a humoral response and by the development of a cell-mediated immunity of the Th1 type with a hypersensitivity reaction of the positive retarded type and elevated rates of interleukine 2 and TNF-α circulating in the biological liquids. [0012] A good vaccine candidate must thus match one or more strongly immunogenic parasitic antigens capable either of blocking the differentiation of the Th2 lymphocytes (Gurunathan S et al., J. Exp Med, 1997 Oct. 6, 186, 1137-1147) (mode of intervention comparable to “desensitization” treatments currently practiced in cases of allergy), or promoting the emergence of the Th1 lymphocytes ensuring the implementation of a protective immunity. [0013] Planning to vaccinate against leishmanias is still problematic today. The attempts have been numerous, but the results are weak and/or contradictory. It can be cited that the use of living parasites, irradiated parasites, and completely killed parasites (Moreau Y et coll., 1994, Médecine et Armées, 22, 1, 89-93) which have given variable levels of protection among mice and humans. [0014] In the 1980s, purified extracts of parasitic antigens were used in dogs in inducing an exacerbation of the disease: LIF2 fraction and anti-idiotypical vaccine from Dr. Montjour&#39;s team. (CHAUVY, J “Immunotherapy trials on a canine population in an endemic zone of leishmania ” thesis no. 36.1993-OGUNKOLADE B.W. et coll. Vet Parasitol.), GP63 and lipophosphoglucane (MOREAU Y et coll, Médecine et Armées, 1994, 22, 1, 89-93) have not produced a satisfactory result. Currently, several molecules are in trials and a final result is pending. It can be cited that the heat shock protein HSP83 of Leishmania major which stimulates the Th1 method and the protein DP72 (JAFFE. C et al, J of Immunol, 1990, 144, 699-706). However, none of the current immunization protocols make it possible to obtain a sufficient level of protection or in any case, it is not reproducible. [0015] To date, no work has been done with synthetic peptides. BRIEF SUMMARY OF THE INVENTION [0016] The present invention comprises an immunomodulator complex that uses one or two peptides with an adjuvant inducing either an immunostimulation of the lymphocytary system T of the type Th1 in a reproducible manner, or an immunomodulation of lymphocytes of the type Th2 into a type Th1. [0017] It is important to emphasize two essential facts. Firstly, the Th1 and Th2 cells exert, by means of certain cytokines that they produce, a reciprocal counter-regulation effect, which explains why in the majority of these cases, an alternation of the cell-mediated and humoral responses is observed. Thus, the immune responses that favor a reaction of the T lymphocytes of the Th1 type, i.e. with organisms inducing an immunity depending on the T lymphocytes, are frequently associated with a low humoral response; this is the case, for example, in mycobacterial infections. [0018] Conversely, a response that favors the production of antibodies will be most often associated either with the participation of the T lymphocytes of the Th2 type or with a relative deficiency of the specific cellular immunity; this is the case of visceral leishmaniasis. The second fundamental circumstance is that lymphocytes fully differentiated into cells of the Th1 and Th2 type are not pre-existing in the non-sensitized simple host. During a physiological immune response, after which more often than not the antigens are rapidly eliminated, the activated CD4+ cells, rather than being expressed either by a Th1 type phenotype or by a Th2 type phenotype, usually secrete cytokines specific to the Th1 and Th2 type. This explains why in humans, the demonstration of the existence of a Th1/Th2 dichotomy necessitated the study of the cytokinic system coming from lymphocytes not from normal subjects normally immunized, but from patients having chronic diseases such as parasitic infections, allergies, or auto-immune diseases. [0019] The polarization of the immune responses into a Th1 or Th2 phenotype has been associated with numerous pathological situations. [0020] For infections by leishmania, Typanozoma, Candida, and other intracellular organisms such as Mycobacterium and Listeria, a Th1 response correlates to resistance to the pathogen. On the other hand, certain conditions such as canine atopic dermatitis, allergies and asthma lead to the exacerbation of a response of the Th2 type. The recourse to immunostimulants which permit the passage from a Th2 response to a Th1 response, thus the passage from a state of immediate hypersensitivity to a state of hypersensitivity of the retarded type, must lead to a recovery. [0021] It can be considered that the example of asthma, for which the medical community is worrying about the increasing number of individuals who suffer from it: approximately 200 million people in the world, and according to the World Health Organization, 180,000 of them died in 1997. In France, 2,000 people die from asthma every year. [0022] Asthma is a chronic inflammatory condition of the respiratory tracts where numerous cells of the immune system are intervening. [0023] The disease becomes chronic because of the quasi-permanent accumulation of inflammation mediators, as well as the quasi-incessant recruitment of destructive polynuclear eosinophils: these cells accumulate in the bronchial mucous membrane and release basic proteins that destroy the bronchial epithelium. The damage caused by the basic proteins leads to a hyperactivity of the bronchial tubes: the nerve endings that innervate the bronchial tubes are exposed and are no longer protected from outside attacks. [0024] It can be recalled that the activated T lymphocytes release notably interleukine 4, which starts the production of the immunoglobulins E. This “allergic orientation”, again called method Th2, conflicts with the anti-infectious reactions of the Th1 method: in the context of the fight against a bacteria (the bacillis of tuberculosis, for example), the activated T lymphocytes release interleukine 2 and gamma interferon. The gamma interferon activates the cells which have the antigen, and which, in turn, produce interleukine 12. Thus, interleukine 4 orients the immune reactions towards the allergic reactions, while gamma interferon seems to promote the reactions of the defensive type. [0025] And yet this orientation of the immune system will be set very early, in the very young infant. In a newborn baby exposed to a pathogenic agent, the immune system becomes oriented to the defensive method: it is the T lymphocytes, producers of interleukine 2 and gamma interferon, which are activated first and will stay. These children will have less allergic risks. [0026] On the other hand, newborn babies raised in a sterile setting have risks of being confronted with an environment high in allergens and low in pathogenic micro-organisms. [0027] If this is the case, the still immature immune system will activate the T lymphocytes which secrete interleukine 4, giving the immune system an allergic orientation, which favors asthma. This hypothesis, coming from the work of Tim Mosman and Robert Coffann, from the Institute of Molecular and Cellular Biology DNAX, in Palo Alto, would explain the increase in the prevalence of the asthmatic illness in industrial countries. [0028] A reorientation of the immune reactions of sensitive persons, by limiting the allergic Th2 method of immune response, to an activation of the Th1 method would be a possibility for the treatment of asthma. [0029] It can be extended that this phenomenon to other forms of immediate hypersensitivity. [0030] Immediate hypersensitivity is the most frequent form of allergy and results from the synthesis of immunoglobulins E (IgE), antibodies specific to the allergens from the environment. These immunoglobulins E become attached to the cells whose mastocytes are the key elements. The contact of the allergen and the immunoglobulins E activates the mastocytes which release the mediators of the inflammation: this is degranulation of the mastocytes. Grouped under the name of anaphylaxia and atopic diseases are all clinical manifestations linked to the phenomena of immediate hypersensitivity which result from the production of immunoglobulins E, the great culprits of the allergy. However, anaphylaxia corresponds to a physiopathological mechanism independent of any hereditary factor (it is, for example, the anaphylactic shock due to hymenopteran venoms), while atopia is the expression of a particular genetic aptitude to produce immunoglobulins E in excess directed against various natural substances of the atmospheric environment (pollens, molds, pollutants), household environment (dust mites, cockroaches, mammals) or professional environment, and also against foods; this results in respiratory allergies (asthma, rhinites), cutaneous allergies (urticaria, atopic eczema), ophthalmic allergies and digestive allergies. [0031] In these pathologies, two points must be mentioned: 1—the series of events initiated by the attachment of the allergen complex—IgE on different cellular populations and notably on mastocytes 2—the role of the cytokines which, by the intermediary of the synthesis of the IgE, participate in the sensitization phase, on the one hand, and in the perpetuation of the allergic reaction on the other hand. [0034] Today, treatments that confine the syndromes having an allergic origin comprise a part of the anti-inflammatory arsenal (anti-histamine and corticoids). The predominant place taken by the cytokines in the fundamental mechanisms of immediate hypersensitivity opens prospects for new targeted therapies: it is hoped notably to make the Th1/Th2 equilibrium swing in favor of the Th1 method and to modulate the cellular signalization induced by the cytokines when they become attached to their receptors located on the lymphocytes, but also on the mastocytes and on the eosinophils. DETAILED DESCRIPTION OF THE INVENTION [0035] The present invention relates to a vaccine complex comprising 2 peptides named A16E and A16G and/or their derivatives, specific to the parasite Leishmania, combined with an adjuvant. The invention also involves the use of these fragments and the adjuvant as diagnostic reagents in vitro and in vivo, and as reagents that induce a stimulation of the T lymphocytes of the Th1 type ensuring an inhibition of the development of the lymphocytes of the Th2 type, notably for the prevention and the treatment of conditions linked to a condition of immediate hypersensitivity, i.e. an immune condition of the Th2 type. [0036] The two peptides A16E and A16G have the following sequences of amino acids: A16E: (16 amino acids): A-A-R-S-A-R-S-R-E-G-Y-S-L-T-D-E A16G: (16 amino acids): A-A-S-S-T-P-S-P-G-S-G-C-E-V-D-G [0037] In the peptide compound A16E, L can be replaced by I, and S by C. [0038] In the peptide compound A16G, C can be replaced by S, and S by C. [0039] The peptide compound according to the invention can also be comprised of possible derivatives of the peptides A16E and A16G, these derivatives comprising at least five adjoining amino acids taken in the sequences A16E or A16G. In this case, the derivatives used will be, preferably, the derivative E-G-Y-S-L taken in the sequence A16E, and the derivative S-G-C-E-V taken in the sequence A16G. The other possible derivatives are: A-A-R-S-A A-A-R-C-A A-R-S-A-R A-R-C-A-R R-S-A-R-S R-C-A-R-C R-C-A-R-S R-S-A-R-C S-A-R-S-R C-A-R-C-R C-A-R-S-R S-A-R-C-R A-R-S-R-E A-R-C-R-E R-S-R-E-G R-C-R-E-G S-R-E-G-Y C-R-E-G-Y R-E-G-Y-S R-E-G-Y-C E-G-Y-S-I E-G-Y-C-I E-G-Y-C-L G-Y-S-L-T G-Y-C-L-T G-Y-S-I-T G-Y-C-I-T Y-S-L-T-D Y-C-L-T-D Y-S-I-T-D Y-C-I-T-D S-L-T-D-E C-L-T-D-E S-I-T-D-E C-I-T-D-E A-A-S-S-T A-A-C-C-T A-A-C-S-T A-A-S-C-T A-C-C-T-P A-C-S-T-P A-S-S-T-P A-S-C-T-P S-S-T-P-S C-C-T-P-C C-C-T-P-S C-S-T-P-C C-S-T-P-S S-C-T-P-C S-C-T-P-S S-S-T-P-C S-T-P-S-P S-T-P-C-P C-T-P-S-P C-T-P-C-P T-P-S-P-G T-P-C-P-G P-S-P-G-S P-C-P-G-S P-S-P-G-C P-C-P-G-C S-P-G-S-G S-P-G-C-G C-P-G-S-G C-P-G-C-G P-G-S-G-C P-G-C-G-C P-G-C-G-S P-G-S-G-S G-S-G-C-E G-C-G-C-E G-C-G-S-E G-S-G-S-E C-G-C-E-V C-G-S-E-V S-G-S-E-V G-C-E-V-D G-S-E-V-D C-E-V-D-G S-E-V-D-G [0040] The peptide compound according to the invention can also be included in any polypeptide. [0041] The peptides obtained by synthesis and their derivatives are constructed in octopus or are made immunogenic by connections to the carriers (large molecules of the KLH type), and are administered to mammals in the presence of an adjuvant, preferably muramyl dipeptide (MDP): this combination comprises the therapeutic peptide vaccine complex. [0042] In a preferred manner, the protein/adjuvant ratio is comprised between 1/0.1 and 1/6. [0043] In the case of an infection by leishmaniasis, the studies done in dogs have made it possible to determine the optimal vaccine dose to be 100 μg of muramyl dipeptide for 50 μg of injected proteins. However, the beginning of a response is observed starting at 30 μg of injected proteins. [0044] The mechanism of specific action of the peptide complex obtained according to the invention is verified using traditional methods that allow the dosage of peptides, their identification, and using more specific methods that show that the innovative peptide complex takes effect either by immunostimulation of the lymphocyte system of the Th1 type, or by immunomodulation of a Th2 type towards a Th1 type. [0045] For each mammal studied (dog, for example), a serologic analysis is done with the peptides A16E and/or A16G. [0046] A parasitological exam was done from the sample taken directly from the candidate studied, a dog, for example. [0047] A smear of a puncture of the bone marrow is made on a slide. This smear, fixed once by methanol, is stained with May-Grünwald-Geimsa and observed by an immersion microscope (x 1000). [0048] Samples of the bone marrow are brought under cultivation in the biphase cultivation medium NNN (Novy and Mac Neal, 1904, 4. Infec. Dis., 1:1-30), of which RPMI 1640 supplemented with 20% decomplemented fetal calf serum constitutes the liquid phase. The blind subculturings were done every four to six days. The cultures are regularly observed in a photon microscope (×400) for 20 min. [0049] The parasitemy was quantified as follows: +/− elongated refractive immobile forms; + 1 to 5 mobile promastigote forms/field; ++ &gt;5 mobile promastigote forms/field; +++ culture at confluence. [0050] Evidence of the involvement of a cell-mediated immunity of the Th1 type: [0051] Leishmanias of the promastigote forms are cultivated in the standard culture media. Parasites are harvested from the end of the exponential phase (6-7 days). The parasitic residue is washed three times by centrifugation (2500 g, 15 nm, 4° C.) in a PBS buffer. [0052] After having verified the viability of the parasites using a vital stain (Trypan Blue), a suspension containing 2×10′ parasites per ml is inactivated in a PBS buffer containing 0.01% merthiolate (Pinelli et al., 1994, Infect. Immun., 62: 229-235). This constitutes the leishmanias for the intradermoreaction test (IDR). [0053] The study of the immune response of the Th1 type that follows was performed on dogs. [0054] Dogs are placed in lateral decubitus and a delicate and non-irritating shearing is done on the thoracic zone approximately 5 cm by 10 cm behind the elbow. Four circles 10 mm in diameter are marked using a felt-tip pen. [0055] Into the center of the circles, 0.1 ml of solution is injected in an intradermo-injection. Two circles receive the solution of leishmanias and the 2 other circles receive the saline methiolated solution in the negative control. The reading of the Intra Dermo Reaction (IDR) is done 48 hours later using an allergologic gauge. [0056] The test is considered positive if the mean of the two observed induration diameters is greater than or equal to 5 mm. The observation of an erythema without induration will be considered to be a negative test (Pinelli et al., 1994, Infect. Immun., 62: 229-235; Marty et al., 1994, Trans. Roy. Soc. Trop. Med. Hyg., 88, 658-659). [0057] The IDRs can also be performed with a solution of peptides A16E and A16G or their derivatives and MDP, for example, in proportions of 10 μg of peptides for 20 μg of MDP. [0058] The present invention thus also involves a product for in vivo diagnosis which reveals a retarded hypersensitivity immune condition of the Th1 type using peptides A16E and A16G and their derivatives in an intra-dermo reaction in mammals. [0059] One can also carry out dosage of nitrogen monoxide (NO) to know the destructive activity of the monocytes against the Leishmanias. The synthesis of NO by the monocytes is in fact a sign of the destruction of the leishmanias by the monocytes having been activated by the cytokines of the interferon gamma type (IFNγ). [0060] NO has a high chemical reactivity. In the presence of water and oxygen, this molecule is rapidly oxidized in a stochiometric manner and thus forms the nitrites (NO2-) according to the reaction: 4NO°+O 2 +2H 2 O-4NO 2 − +4H +. [0061] The nitrites accumulate in the media and are easily detectable chemically by the Griess method. [0062] To 50 μl of supernatant to be tested, 60 μl of 60 μl of Griess B (N-(1-napthtyl)ethyl-enediamine 0.3%) is added. The colorimetric reaction develops in the dark for 2 minutes. The optical densities obtained at 540 nm are corrected by the subtraction of the OD obtained on the wells containing only the culture medium. [0063] The values obtained are recorded on a calibration curve (OD=f(NO 2 ) made from the known concentrations of NO 2 −. [0064] For this test, the monocytes and lymphocytes are isolated from the venous blood of dogs. The monocytes are brought under cultivation for 3 days at the rate of 10 5 cells per well in the culture chambers (Labtek) in a medium RPMI 1640 complete (containing 25 mM HEPES, 2 mM L-glutamine, 100 U penicillin per ml) at 37° C. in a humid atmosphere containing 5% CO 2. After 3 days of cultivation, the macrophages are washed in RPMI complete medium, supplemented with fresh medium. The cells are put in incubation either alone, or in the presence of 5 μg of peptides, or in the presence of autolog lymphocytes. [0065] When they are used, the lymphocytes cultivated separately are washed, counted, and added to the macrophages in the ratio of 2 lymphocytes per macrophage. [0066] Next, a test is done on the lymphocyte proliferation. [0067] The peripheral blood mononuclear cells (PBMC) of dogs are separated on the Ficoll gradient (density 1.078) by centrifugation at 800 g for 20 mn at ambient temperature. These cells are brought under cultivation on a plate having 96 wells at a concentration of 2.10 5 cells per well in the presence of 2 μg per ml of Concanavalin A (Sigma), 5 μg per ml of ES P (excretion—secretion promastigotes) or 20 ml of supernatants of culture harvested in the stationary phase of growth of promastigotes (SP) per well, and in the absence of any additive in a volume of 200 ml of the medium RPMI 1640 containing 5% decomplemented fetal calf serum, 2 mM of L-glutamine, 100 U of penicillin per ml, 100 mg of streptomycin per ml. The optimal antigen and mitogen concentrations have been determined in prior experiments. The PBMCs are incubated for 72 hours in a humid atmosphere at 37° C. in the presence of 5% CO 2 then for 20 hours with 0.5 μCi of 3 H thymidine. The cells are harvested over a filter and the incorporation of the radioactivity is determined by counting in a scintillating liquid (β-counter). All of the tests are done in triplicate. [0068] A more rapid and more sensitive immunohistochemical method using BrdU (5-bromo-2′-desoxyuridine), a structural analog of thymidine, is also used to measure the cellular proliferation (BrdU, cell proliferation detection kit III, Boehringer Mannheim, Germany). In our experiments, the BrdU is added for 18 hours after 72 hours of incubation. The cells which have incorporated the BrdU in their ADN are easily detectable in the presence of a monoclonal antibody directed against the BrdU. [0069] The proliferative responses are expressed in stimulation indices that represent the ratio of the average proliferation after stimulation to the mean proliferation in the absence of antigen. [0070] The lymphocytary proliferation has also been estimated by visual readings in a photon microscope (−: negative; +/−: slight proliferation; +: little proliferation less than 5 points per microscopic field; ++: mean proliferation greater than 5 points; +++: strong proliferation). [0071] In parallel to the study of the specific activation of the T lymphocytes the Th1 type by intradermoreaction, dosage of the NO (NO is synthesized by monocytes activated by the cytokines of the T cells of the Th1 type) and lymphocyte proliferation, a serological monitoring by traditional immunofluorescence using strips coated with the promastigotes (serological reference method for canine leishmaniasis) is done. [0072] For the procedure of the studies, other specific techniques were used. [0073] Method of Infectious Examination: [0074] The infectious examination consists in intravenously injecting 106 treated promastigotes in metacyclic phase in the complement of a healthy dog and 5.106 peritoneal macrophages of a healthy dog, infected in vitro by the amastigotes. [0075] The promastigotes and infected macrophages are diluted in sterile physiological serum for a final volume of 1.5 ml. This mixture is made just prior to injection. [0076] Detection of immunoglobulins of the isotype IgG2 of dogs, specifically of the peptides A16E and A16G and their derivatives: [0077] This detection is done by the ELISA method according to the microtitration technique of Kweider et al (J. Immunol. 1987, 138, 299) using a bonded anti IgG2. For this method, the peptides are biotinylated prior to coating on microplates. This connection to the large molecules of the biotin type notably has the effect of making the peptides A16E or A16G or their derivatives more antigenic. [0078] The peptides can also be linked with glutaraldehyde or combined with the bodies of polylysine (presentation of the OCTOPUS type, for example). [0079] The innovative character of the peptide complex according to the invention does not reside solely in the induction of a specific cellular response of the Th1 type but also in the production of low rates of specific immunoglobulins of the isotype IgG such as the IgG2 in dogs. These specific IgGs can be detected by various in vitro methods, for example: ELISA, DOT BLOT, WESTERN BLOT, IMMUNOCHROMATOGRAPHY, LATEX and any other in vitro method that involves intervention of a conjugate system or other systems for display of the Ag-Ac reaction. For example, an ELISA system can be used on a plastic support, and a WESTERN BLOT system can be used on nitrocellulose membranes or other polymers involving the intervention of a enzyme conjugate. Latex supports can also be used. The A16E, A16G peptides and their derivatives can also be bonded, for example, to radio-isotopes, fluorescent molecules, luminescent molecules, or colored particles. [0080] In fact, certain preliminary work among humans (KAWANO. P et al, Parasite Immunol, 195, 17, 451-458) and in dogs (NIETO C. G et al, Vet Immunol and Immunopathology, 1999, 67, 117-130) shows that the IgG isotypes would be markers of the immunitary dichotomy Th1/Th2. More specifically, a dog suffering from leishmaniasis with the conclusive clinical signs has a high level of antibodies mainly of the isotype IgG1, while an asymptomatic dog has antibodies specific to the isotype IgG2. The dogs that received our peptide complex have low levels of IgG2 specific to the A16E and/or A16G peptides, which is in keeping with the preferential expansion of T lymphocytes of the Th1 type. [0081] The detection of the presence of IgG isotypes specific to the A16E and A16G peptides and their derivatives makes it possible, notably: to show a humoral response depending on the Th 1 lymphocytes and thus to show an immune state of the Th1 type to show a state of retarded hypersensitivity, to monitor the immune response in vaccinated or treated mammals, to monitor the effectiveness of a chemiotherapeutic and/or immunotherapeutic treatment. [0086] The peptide complex comprising the A16E and A16G peptides and their derivatives and the adjuvant, according to the invention, can be administered in various ways. However, it is administered in a preferred manner in four ways: either by subcutaneous injection or by intradermal injection or by intramuscular injection or orally. [0091] Other administration methods can be used, like the parenteral or intravenous method. [0092] In a general manner, a vaccine appears in injectable form comprised of a lyophilizated fraction that is combined with a liquid fraction or diluent. The doses used for prevention and immunotherapy are different, and are also different depending on the mode of injection: sub-cutaneous and intramuscular method: injection of a dose (50 μg of peptides and 100 μg of adjuvant) in dogs regardless of breed and sex for a preventative effect). injection of half doses (25 μg of peptides and 50 μg of adjuvant) for immunotherapy of leishmanian dogs. intradermo method: injection of a half dose in leishmanian dogs for a preventative effect injection of a quarter dose in leishmanian dogs for a therapeutic effect. [0099] The methods of injections are continued in the examples of immunotherapy and vaccination. [0100] Immunotherapy Results [0101] According to specialists such as PINELLI (PINELLI. E et al, Infect Immun, 1994, 62, 229-235), leishmanian dogs correspond to the activation of the lymphocytary system of the Th2 type having a high antibody response. [0102] This increased production of antibodies corresponds to hyper-proteinemia and induces the appearance of immune complexes that cause a renal problem (increase in the creatinine and blood urea). [0103] Thus, it was attempted to modulate towards a Th1 state by administering doses of the peptide complex by the intra-dermo method to the totally leishmanian dogs. The monitoring of the immune state and the clinical observation were done before and after treatment. EXAMPLE 1 MANON, the Dog [0104] A female dog of the British spaniel breed, age 4 years old, belonging to Mr. P, has numerous cutaneous lesions accompanied with a general state of fatigue and a thin appearance, all reminiscent of a leishmaniasic canine. [0105] The veterinarian, Dr. L M, diagnoses leishmaniasis. This diagnosis is confirmed by a direct observation in a microscope of leishmanias from a cutaneous tracing and a serological analysis which gives a titer by immunofluorescence leishmaniasis positive at {fraction (1/3200)}. [0106] The analysis of the immunitary state prior to any injection makes it possible to confirm that the dog was indeed in an immunitary state of the Th2 type with a high antibody titer as well as negative IDR tests and NO dosages. We established an immunotherapy which consisted in making 2 intradermo injections of 25 μg of peptides (½ A16E, ½ A16G) and 100 μg of muramyl dipeptide adjuvant, each injection being 3 weeks apart. [0107] One week after the second injection, MANON the dog regained her appetite and a certain vitality. Dr. LM began to observe a slight cutaneous improvement. [0108] One month after the last injection, MANON regained a normal clinical appearance with notably an increase in weight of 1 kg and a disappearance of 80% of all cutaneous lesions. Analysis of the immunitary state makes it possible to confirm a reduction in the anti- leishmania antibody titer which dropped to {fraction (1/400)} by immunofluorescence. In parallel, the IDR (IDR performed with leishmaniasis and also with the A16E and A16G peptides), the NO dosage and the lymphoblastic proliferation were positive. In addition, after treatment, MANON the dog has IgG2 specific to the 2 peptides, IgG2 dosed by the ELISA method and Western Blot. These IgG2 specific were absent prior to any treatment. [0109] A study of the parasites by cultivation on the NNN medium turned out to be negative. 8 months after treatment, MANON the dog did not show any modifications. The biological analyses make it possible to confirm that MANON is still in an immunitary state Th1. EXAMPLE 2 PEPPONE the Dog [0110] A male dog of the Griffon breed, named PEPPONE, age 5 years old, belonging to Mr. B, has clinical signs specific to leishmaniasis. According to Dr. GH, presence of numerous shiny squama, right periocular hair loss, ulcerous lesions at the level of the 2 front elbows, and a pronounced state of fatigue. Biological analyses with notably a positive leishmaniasis serology at {fraction (1/400)} by immunofluorescence confirms the clinical diagnostic. [0111] An immunotherapy was established, which consisted in making 3 intradermo injections of 25 μg of peptide complex (½ A16E, ½ A16G), and 100 μg of muramyl dipeptide adjuvant, each injection being 10 days apart. The analysis of the immune state prior to any injection showed that PEPPONE the dog had developed an immune system of the Th2 type with a greatly positive parasitemy from the bone marrow. [0112] One month after the last injection, the leishmaniasic clinical signs of PEPPONE had retroceded with notably a healing of the ulcerous lesions, a sizeable disappearance of the squama and an almost non-existent periocular hair loss. The serology still has a titer by immunofluorescence equal to {fraction (1/400)}. On the contrary, the analysis of the cellular response makes it possible to confirm that PEPPONE has an active Th1 state with an IDR (IDR done with leishmanines and also with peptides A16E and A16G), positive dosage of NO and lymphoblastic proliferation. [0113] In parallel, the parasitemy is negative (cultivation of the bone marrow in a NNN medium). [0114] At the humoral level, PEPPONE the dog has IgG2 specific to the A16E and A16G peptides after treatment, IgG2 dosed with ELISA and Western Blot. [0115] The present invention thus indeed consists of a therapeutic peptide complex that induces the passage from an immune state of the Th2 type, with sizeable production of antibodies that exacerbate the clinical manifestations, to an immune state of the Th1 type that leads to healing. [0116] The peptide complex composed of two peptides A16E and A16G and the muramyl dipeptide adjuvant thus comprises a therapeutic compound. [0117] Vaccination Results [0118] In order to evaluate the efficacy of the peptide vaccine complex according to the invention, the vaccine complex was tested on 5 perfectly healthy dogs. These 5 dogs have a negative leishmaniasic serology, a negative parasitemy as well as fully negative cellular response tests specific to Leishmania. In addition, none of the 5 dogs has IgG2 specific to one or the 2 peptides A16E and/or A16G. [0119] These 5 dogs live in a place free from any phlebotomes. We define 3 groups of dogs. 1—Control group (placebos) Negative control: the dog named LILI, Pointer breed, female, age: 3 years Sole adjuvant control: the dog named MIMI, British spaniel breed, female. Age: 6 years 2—Group of dogs vaccinated with peptides only (50 μg) and muramyl dipeptide adjuvant (100 μg) The dog named MAMA, Weimaraner breed, female. Age: 2 and a halfyears old =&gt;peptide A16E The dog named NUNU, Pointer breed, male. Age: 2 and a half years old =&gt;peptide A16G 3—Group of dogs vaccinated with peptides A16E and A16G (25 μgof each peptide) and muramyl dipeptide adjuvant (100 μg) The dog named LEON, British spaniel breed, male. Age: 4 years. [0128] The vaccine injection scheme is as follows: [0129] A clinical monitoring of the 5 dogs was done every two weeks. The biological analyses were scheduled as follows: [0130] The biological analyses consist of: biochemical analyses: urea, creatine, transaminases hematological analyses: count, formula serology leishmaniasis: quantitative anti- Leishmania immunofluorescence, dosage by the ELISA method of IgG2s specific to the peptides A16E and/or A16G cellular response tests: IDR test (IntraDermoReaction) done with leishmanines and also with A16E and A16G peptides, dosage of NO and lymphoblastic proliferation. [0135] To these analyses must be added the search for Leishmania by direct observation in a microscope and cultivation on NNN medium from bone marrow after the infectious test. [0136] Results: [0137] Clinical Monitoring: [0138] No significant clinical manifestation appeared during all of this study. A slight weight loss and the appearance of some squama in the dog LILI must be noted, 4 months after the infectious test. [0139] Biological Monitoring: 1—The biochemical and hematological parameters stayed normal all during this study. 2—Leishmaniasis serology and parasitemy. [0142] Prior to any injection, the 5 dogs have negative serologies and parasitemies. The following table shows the serological responses obtained during our experiments and the monitoring of the parasitemy (analyses made 2 months and 12 months after the infectious test). SEROLOGY Cultivation IF ELISA PARASITEMY on NNN Dogs Quantitative IgG2 Direct Exam Medium Dogs LILI − − + − Control MIMI − − + 0 Dogs MAMA (A16E) − +(0.700) − − Immunized NUNU (A16G) − +(0.520) − − LEON (A16E + − +(0.780) − − A16G) Key: IF: Immunofluorescence (considered positive if the titer is ≧1/100) ELISA: Cut off = 0.300 OD (optical density) Parasitemy: cultivation on medium NNN − = absence ++ = more than 5 mobile promastigote forms/field [0143] Only the immunized dogs have specific antibodies of IgG2 (ELISA towards the corresponding peptides) and negative parasitemies. A slight appearance of total antibodies ({fraction (1/200)} in IF) must be noted in all of the dogs after the infectious test. [0144] Only the control dogs (LILI and MIMI) have positive parasitemies and an absence of specific anti-peptide antibodies IgG2. [0145] Cell-Mediated Response [0146] Before any injection, the 5 dogs have a fully negative cell-mediated response to Leishmania infantum. According to the following table, only the immunized dogs have positive lymphoblastic proliferation tests, IDRs linked to the production of NO by the monocytes. [0147] The following table shows the cellular type responses obtained (analyses made 2 months after the infectious test). Dosage Lymphoblastic IDR of NO Proliferation Dogs Leishmanines A16E A16G (in μM) Test Dogs LILI + − − 0, 3 −1, 1 (3) Control MIMI − − − 0, 2 −1, 2 (3, 1) Dogs MAMA (A16E) + + Limit 2, 6 +2, 1 (3, 1) Immunized NUNU (A16G) + Limit + 2, 8 ++3, 1 (3, 6) LEON (A16E + + + + 4, 2 +++3, 7 (3, 8) A16G) Key: IDR: The Intra Dermo Reaction test is considered positive (+) if the induration is ≧5 mm 48 h. after intradermoinjection Dosage of NO: Lymphoblastic proliferation test: The results are expressed by a reading in a photon microscope and in stimulation indices (between parentheses, stimulation index + Concanavaline A) [0148] From this analysis, the peptide compounds with the adjuvant do indeed induce a cell-mediated immunity of the Th1 protector type, to which it is necessary to add an induction of the antibodies of isotype IgG2. The mixture of the two peptides at an equal concentration thus gives a more pronounced cellular response (high rate of NO synthesized by activated monocytes). [0149] The peptide complex comprised of the two peptides A16E and A16G and the muramyl dipeptide adjuvant constitutes a good vaccine complex.

A therapeutic vaccine peptide complex for preventing or treating disorders in mammals and particularly in humans, in Canidae, Felidae and Equidae whose protective immunity depends on the stimulation of type Th1 lymphocytes and in particular a delayed state of hypersensitivity. The vaccine peptide complex contains the following amino acid sequence (A16E): A-A-R-S-A-R-S-R-E-G-Y-S-L-T-D-E sequence wherein L can be substituted by I, and S by C, and/or the following amino acid sequence (A16G): A-A-S-S-T-P-S-P-G-S-G-C-E-V-D-G sequence wherein C can be substituted by S, and S by C, and an adjuvant which preferably induces a cell-mediated response.

big_patent

FRONTIER IN SPACE BY: MALCOLM HULKE 5:50pm - 6:15pm [SCENE_BREAK] 1: SPACE (A large battle cruiser circles the floating cargo ship, turning in order that it can approach its airlock.) GARDINER: (OOV: Over radio.) Earth Battle Cruiser to Earth cargo ship number C982. We are now approaching you. Do you read me? Over. [SCENE_BREAK] 2: INT. CARGO SHIP. BRIDGE (GARDINER appears on the monitor on the deserted bridge. He is a young man in military uniform with a headgear that covers the ears but leaves the top of his head bare.) GARDINER: (On monitor.) Earth Battle Cruiser to Earth cargo ship number C982. We are now approaching you. Do you read me? Do you read me? (The DOCTOR runs on the bridge and sits in STEWART'S chair.) GARDINER: (On monitor.) Prepare to be boarded. Repeat: prepare to be boarded. Do you read me? Over. DOCTOR: (Into microphone.) Hello battle cruiser, battle cruiser. This is the cargo ship. Over. GARDINER: (On monitor.) What is your situation? DOCTOR: (Into microphone.) The ship has been attacked and the cargo stolen. Over. GARDINER: (On monitor.) Do you have casualties? DOCTOR: (Into microphone.) Yes, the crew are stunned, but otherwise they're unharmed. Over. GARDINER: (On monitor.) We shall lock on...five seconds from now. (The DOCTOR checks his watch, switches off the microphone and leaves the bridge.) [SCENE_BREAK] 3: SPACE (The nozzle of the battlecruiser docks with the cargo ship.) [SCENE_BREAK] 4: INT. CARGO SHIP. AIRLOCK AREA (The echoing sound of the docking reaches the airlock area. JO is helping STEWART drink from a small flask as the DOCTOR returns.) DOCTOR: It's alright, Jo. We're being rescued. STEWART: What happened? DOCTOR: Well, don't worry, old chap. You'll be all right now. I think... (STEWART hears airlock lock opening as the cabin pressurises.) STEWART: Draconians! They're boarding! (GARDINER enters through the airlock, accompanied KEMP by some heavily armed soldiers.) GARDINER: Stewart? (He points his gun at the DOCTOR.) GARDINER: Who are you? DOCTOR: Passengers. GARDINER: I see. Having a fancy dress party? (To STEWART.) What happened? STEWART: Dragons - attacked us. GARDINER: Did they get the cargo? STEWART: I don't know. JO: Yes, they took everything. DOCTOR: Including some rather valuable property of mine. GARDINER: Well, tough luck. DOCTOR: Oh, thank you very much. (HARDY gets to his feet.) HARDY: (Groggily.) Dragons...they attacked us. (GARDINER goes to help him.) GARDINER: Yes, we know. (To the DOCTOR.) You say you're passengers. Isn't that a little unusual on a cargo ship? DOCTOR: Well, we're here, aren't we? GARDINER: (To STEWART.) Where did you pick these two up? STEWART: I don't know. I can't seem to remember... GARDINER: (Harshly.) Pull yourself together! How did they get on board? (STEWART thinks desperately for an answer.) STEWART: Stowaways...that's it - they were stowaways. They were sending messages! HARDY: That's right - they were helping the Dragons! They're traitors! (The soldiers all raise their blasters to cover the DOCTOR and JO.) JO: But that's absolute nonsense! Look, we didn't want to be on this ship - it was an accident! GARDINER: You said you were passengers? DOCTOR: I was merely trying to avoid a lot of tiresome explanations, old chap. GARDINER: Stewart, I'll leave Kemp on board to take your ship back to Earth. STEWART: What about these two? GARDINER: Lock them in the hold. Put a guard on them. JO: But we haven't done anything! GARDINER: They say you have! Take them away. DOCTOR: Now, if you'll only listen to me... GARDINER: You can talk to Earth Security. They'll listen - it's their business. Put 'em in the starboard cubicle. KEMP: Alright, get moving. (At KEMP'S gunpoint, the DOCTOR and JO are led away into the hold area.) [SCENE_BREAK] 5: INT. CARGO SHIP. HOLD (...and back towards their previous 'cell'.) KEMP: In there. [SCENE_BREAK] 6: INT. CARGO SHIP. STARBOARD CUBICLE (JO enters and then the DOCTOR who is roughly pushed in by KEMP. He collides with JO as the door is slammed shut behind them.) JO: Woops! No need to push! [SCENE_BREAK] 7: INT. CARGO SHIP. HOLD (The door is secured and the soldiers move off.) [SCENE_BREAK] 8: INT. CARGO SHIP. STARBOARD CUBICLE DOCTOR: Listen! (They hear echoing noises through the ship.) DOCTOR: The ships - they're unlocking. [SCENE_BREAK] 9: SPACE (And sure enough, the battlecruiser moves off from the cargo ship.) [SCENE_BREAK] 10: INT. CARGO SHIP. HOLD (JO looks out of the grille and sees KEMP stood on guard nearby.) [SCENE_BREAK] 11: INT. CARGO SHIP. STARBOARD CUBICLE JO: Still watching the door. DOCTOR: That's what he's there for, isn't it? (The DOCTOR sits calmly on the bench while JO starts to pace the room, thinking furiously. Suddenly, her face lights up.) JO: Right! We'll give it a few minutes, then I'll start groaning and pretending I'm ill. When he comes in, you can use your Venusian karate! DOCTOR: And then what? JO: Then, we'll take his gun, go to the flight deck and make somebody take us back to Earth. DOCTOR: (Quietly.) Jo? This ship's already going back to Earth. (JO'S face falls.) JO: Oh... Oh, dear, mm. (She puts a finger to her lip, paces, thinks once more and again has a eureka moment...) JO: Hey, I've got a terrific idea! I saw this film once, you see, and there were these two gangsters, big fellers they were, with sort of cauliflower ears... (The DOCTOR sighs...) DOCTOR: Look, Jo... JO: And one of them... DOCTOR: Will you stop pacing up and down like a perishing panda! Now come and sit down. Let me think, will you? (JO sits quietly.) DOCTOR: That's better. (But not for long...) JO: Doctor? (The DOCTOR sighs once more.) JO: Well, now the Ogrons have gone, why don't the crewmen remember what really happened? DOCTOR: Because the true facts have been erased from their minds, that's why. JO: Well, yes, but why do they keep telling lies about us? DOCTOR: Well, they don't know that they're lying, Jo. They're desperately trying to fit us into their version of things. JO: Well, what are we going to do then? DOCTOR: Well, when we get back to Earth, we've got to reach someone in authority whose mind isn't already closed. JO: Closed to what? DOCTOR: Jo, look, these people believe that the Draconians are attacking their spaceships, right? JO: Right. DOCTOR: And we know that they're wrong, don't we? JO: Well, yes, it was Ogrons. DOCTOR: Well, we also know that the Ogrons couldn't possibly have created that hallucinatory device that makes the Earthmen think they're Draconians. JO: Oh... Oh, well that's simple then. I mean, all we've got to do is: find out what's going on, who's behind the Ogrons, where they've taken the TARDIS, go and get it back and then we can all go home - right? DOCTOR: Right. JO: Oh. I don't know what I've been worrying about... [SCENE_BREAK] 12: INT. EARTH. PRESIDENT'S OFFICE (KEMP appears on a small black and white videoscreen on the desk in the PRESIDENT'S office, watched by the PRESIDENT herself and GENERAL WILLIAMS.) KEMP: (On monitor.) We are now in space port ten. The ship will land in...fifteen minutes. The crew are safe. Also aboard - two human stowaways, origins unknown. GENERAL WILLIAMS: I want a cordon around the landing area the minute that ship touches down. Nobody on, nobody off till I get there. KEMP: (On monitor.) Sir. (GENERAL WILLIAMS switches the screen off.) GENERAL WILLIAMS: I'd better get down there. I want to handle the preliminary interrogations myself. PRESIDENT OF EARTH: Good. (He heads for the door.) PRESIDENT OF EARTH: General Williams. (He stops.) PRESIDENT OF EARTH: Whatever you find out, you'll report directly to me. GENERAL WILLIAMS: (Dryly.) Naturally. And now if you'll excuse me? (He heads out. A ghost of a smile flickers over the PRESIDENT'S face.) [SCENE_BREAK] 13: INT. CARGO SHIP. STARBOARD CUBICLE (The DOCTOR and JO wait in their room on the cargoship. JO is once more pacing as the DOCTOR remains sat calmly.) JO: What do they think they're doing? We've been landed for simply ages. DOCTOR: Well, twelve minutes, to be exact, Jo. JO: Shh! (They can hear footsteps approaching.) JO: Someone's coming. (The door opens and KEMP enters, his blaster raised. More soldiers are outside the door.) KEMP: Outside. JO: We want to see somebody in authority. KEMP: You're going to - outside. (He pushes her outside the door. The DOCTOR follows, taking his cloak with him.) [SCENE_BREAK] 14: INT. CARGO SHIP. HOLD (GENERAL WILLIAMS walks up at that moment.) GENERAL WILLIAMS: Now then, what's all this about? [SCENE_BREAK] 15: INT. EARTH. PRESIDENT'S OFFICE (Having heard the version of events from the DOCTOR and JO, WILLIAMS has returned to the PRESIDENT.) PRESIDENT OF EARTH: Draconian agents? Are you sure? GENERAL WILLIAMS: What else can they be? Their story is obvious nonsense. PRESIDENT OF EARTH: But why? We... Why would the Draconians leave them on board the ship? GENERAL WILLIAMS: (Firmly.) The Draconians are preparing for war. PRESIDENT OF EARTH: You're still only suspecting then? There is no proof. GENERAL WILLIAMS: Then they need to plant human agents, traitors, on this planet to sabotage our war effort. PRESIDENT OF EARTH: But we are bound to suspect them. They must have realised? GENERAL WILLIAMS: The Draconians probably thought the crew was dead. We could easily have accepted those two as passengers - two unfortunate refugees from a Draconian attack, ideally placed for espionage. PRESIDENT OF EARTH: If you are right, the sooner we confront the Draconians with this, the better. (She presses the intercom on her desk.) PRESIDENT OF EARTH: (Into intercom.) Bring the prisoners in. (To GENERAL WILLIAMS.) We'll bring them face to face with the Draconian ambassador. [SCENE_BREAK] 16: INT. SECURITY BUILDING. CORRIDOR OUTSIDE CELL (GARDINER leads the DOCTOR and JO down a heavily guarded gloomy passage in the security building. The passage is roofed by heavy bars which create shadows of bars across the walls and floors. He points to a doorway.) GARDINER: In there. [SCENE_BREAK] 17: INT. SECURITY BUILDING. CELL (With some reluctance, the DOCTOR and JO enter a stark cell. There is one padded bench and a small table affixed to a wall. The walls are a scruffy white. The DOCTOR starts to divest himself of his cloak. GARDINER follows them in, his tone and manner slightly softer...) GARDINER: When did you last eat? JO: Quite some while ago. GARDINER: Well, I'll see they send you some food. DOCTOR: I'd like to get a message through to your president. GARDINER: Not a chance. I'm going straight back to my ship. Don't want to get mixed up with security. It's not healthy. (He looks at the two unusual prisoners.) GARDINER: Look, I'll give you a piece of advice. DOCTOR: Yes? GARDINER: Sooner or later, you're going to tell them everything. They'll use the mind probe. You talk to them now. You'll save yourselves a lot of trouble. (He walks out of the cell and presses a sensor on the wall. A metal door hums and slides closed. JO jumps up with worry.) JO: A mind probe?! (The DOCTOR sits calmly.) DOCTOR: Oh, you don't want to worry about those things, Jo. As long as you tell 'em the truth, they can't do you any harm. JO: They can't? DOCTOR: No, of course not. Well, they're only sort of computers with a few extra knobs on. And you know how stupid computers can be, don't you? Now come and sit down, stop worrying. (JO sighs with worry.) DOCTOR: Come on. Sit down. (He takes her hand and she sits.) DOCTOR: Did I ever tell you the story about how I was once captured by the Medusoids? JO: What are they? DOCTOR: Medusoids? JO: Mmm. DOCTOR: How can I describe them to you? Well, they're a hairy jellyfish with claws, teeth and a leg. JO: Eurgh! DOCTOR: Anyway, they put me under one of these mind probes things, you see, and tried to get me to tell them where I was going. So, I said I was on my way to meet a giant rabbit, a pink elephant and a purple horse with yellow spots. (JO giggles.) JO: What happened? DOCTOR: Well, the poor old machine just couldn't believe it - had a nervous breakdown. JO: And then what happened? DOCTOR: Well, they put me under another one of these mind probe things and the same thing happened. JO: But you weren't telling the truth - I mean, you weren't really going to meet a giant rabbit, a pink elephant and a...? What was it? DOCTOR: A purple horse with yellow spots. Yes, I was. You see, they were all delegates for the third Intergalactic Peace Conference. JO: How did you get away from these things? DOCTOR: Well they had to turn me loose eventually. JO: Why? DOCTOR: They ran out of mind probes! (JO bursts into laughter and the DOCTOR joins in.) [SCENE_BREAK] 18: INT. EARTH. PRESIDENT'S OFFICE (HARDY and STEWART stand in front of the PRESIDENT'S desk while she questions them with GENERAL WILLIAMS. The DRACONIAN PRINCE and his similar looking First Secretary stand behind the two spacemen, listening impassively.) PRESIDENT OF EARTH: And you are quite sure it was a Draconian battle cruiser. STEWART: Of course, Madam. HARDY: They locked on, and boarded us. We saw them. They were Draconians. PRESIDENT OF EARTH: Thank you. That will be all. I hope you will soon recover from your ordeal. (The two men nod and leave.) PRESIDENT OF EARTH: (To the PRINCE.) Well, your Highness? DRACONIAN PRINCE: These men are your servants. They are saying what they have been ordered to say. GENERAL WILLIAMS: On this occasion, we have more than our servants to confront you with. We have captured two of your human agents. DRACONIAN PRINCE: We have no human agents. Subversion and espionage are expressly forbidden by the treaty of peace between our two empires. GENERAL WILLIAMS: A treaty which you have flagrantly broken. DRACONIAN PRINCE: (Annoyed.) I shall return to my embassy. (He turns to leave.) PRESIDENT OF EARTH: (Urgently.) Just a moment please, your Highness. (Into intercom.) Bring them in. (The door hums open and the DOCTOR and JO and led in by a guard at blaster point.) GENERAL WILLIAMS: Now these people stowed away on the cargo ship. They transmitted signals which enabled your battlecruiser to home in on its prey. DRACONIAN PRINCE: I know nothing of this. PRESIDENT OF EARTH: Perhaps you do not, but some servant of the Draconian empire employed them. DOCTOR: Madam? I can assure that I've never been employed by anybody! Least of all by the Draconians. DRACONIAN PRINCE: (Smiles.) Your servants should have been better rehearsed in their lies. GENERAL WILLIAMS: Or they're showing a misguided loyalty to their Draconian masters. DOCTOR: If we really were working for the Draconians, why did they leave us in your ship? GENERAL WILLIAMS: To act as spies when you were brought back to Earth. DOCTOR: Allow me to congratulate you, sir. You have the most totally closed mind that I've ever encountered! (To the PRESIDENT.) Madam, I beg of you to listen to me. Some third party is trying to provoke war between Earth and Draconia. You are both being duped. GENERAL WILLIAMS: Take them away. (The guard steps forward.) PRESIDENT OF EARTH: Just a minute, please. (To the DOCTOR.) Why should a third party wish to do this? DOCTOR: I've no idea, Madam, but believe me, that is what is happening. GENERAL WILLIAMS: (Impatiently.) Take them away. (The DOCTOR and JO are grabbed by two soldiers and hustled out. The DOCTOR yells back as he is led away.) DOCTOR: Now please listen to me! If you don't you'll be involved in a war that could cause the death millions! (The DRACONIAN PRINCE walks over to the PRESIDENT'S desk and looks haughtily down at her.) DRACONIAN PRINCE: Is this the evidence upon which you accuse me? PRESIDENT OF EARTH: I must ask you to convey a formal protest to your Emperor. (He strides towards the door...) DRACONIAN PRINCE: I shall inform him of this latest insult to the honour of the Draconian empire. (He and his secretary leave. The PRESIDENT sits with her head in her hands.) GENERAL WILLIAMS: We should have used the mind probe before we saw the ambassador. If we'd had a full confession...! PRESIDENT OF EARTH: Has it occurred to you that they might have been speaking the truth? GENERAL WILLIAMS: Huh! Is it likely? Mysterious alien giants that can change their form? A pocket spaceship that appear inside another? PRESIDENT OF EARTH: I suppose you're right. It is nonsensical. GENERAL WILLIAMS: Just you leave them to me. I'll get the truth out of them - eventually. [SCENE_BREAK] 19: EXT. SECURITY BUILDING (The security headquarters is an ugly futuristic concrete construction, made up of blocks of buildings, ramps and walkways. All of these are patrolled by guards. Two of them lead the DOCTOR and JO up a couple of steps and along one of the walkways between guards posted at regular intervals. They are led up to a doorway in a higher point of the building.) [SCENE_BREAK] 20: INT. SECURITY BUILDING. CORRIDOR OUTSIDE CELL (They are then led along the corridor that leads back to their cell.) [SCENE_BREAK] 21: INT. SECURITY BUILDING. CELL (They enter their cell and the door hums closed. JO sits.) JO: Well, we've seen the President. DOCTOR: I've got to get them to listen to me - for their sakes as well as ours. JO: Well, why their sakes? DOCTOR: Well, that was a Draconian with the President, you know? Obviously an ambassador of some kind. JO: So? DOCTOR: Well, clearly Earth and Draconia are on the brink of war, and they both believe it's the others fault. JO: Well never mind about that. What about getting us out of here? DOCTOR: Well I can but try. (He takes out his sonic screwdriver and, after a smile at JO, examines the door.) DOCTOR: Yeah, seems a fairly conventional type of electronic lock - rather old-fashioned really. Here goes. (He switches the screwdriver on. A whine starts to build up, followed by a blaring alarm. The DOCTOR gives up with a rueful look.) DOCTOR: Yes, well, I think we'll sit here for a while after all. (The two sit quietly down as the alarm continues to blare...) [SCENE_BREAK] 22: INT. EARTH. DRACONIAN EMBASSY. AMBASSADOR'S OFFICE (The office of the DRACONIAN ambassador is plain and functional. There is one white desk and a single white visitor's chair. A light sculpture hangs from the ceiling and a full length window in the corner overlooks a terrace and pleasant garden. The double doors open and the PRINCE and the First Secretary enter.) DRACONIAN PRINCE: But why? Why should they produce such an elaborate lie? DRACONIAN FIRST SECRETARY: The ways of the Earthmen are devious. They're an inscrutable species. DRACONIAN PRINCE: Obviously they are preparing the next stage of their plan. First, the attacks on our ships and now - this. DRACONIAN FIRST SECRETARY: Is it possible, your Highness, that for once the Earthmen spoke the truth? Some plan of the Emperor of which your Highness has not been informed? DRACONIAN PRINCE: The Emperor would not possibly contemplate such a plan. We do not break the treaty of peace. DRACONIAN FIRST SECRETARY: Shall I prepare your Excellency's report to the Emperor on this meeting? DRACONIAN PRINCE: I must have more information. DRACONIAN FIRST SECRETARY: It would be useful to question the humans who were found on the ship. DRACONIAN PRINCE: They are supposed to be prisoners. DRACONIAN FIRST SECRETARY: (Meaningfully.) Prisoners have been known to escape, your Highness. DRACONIAN PRINCE: (Also meaningfully.) Not without help. And that would be a grave act of hostility. I could not possibly countenance such a plan. DRACONIAN FIRST SECRETARY: But...should two escaping prisoners...seek sanctuary in this embassy, it would be uncivilised to turn them away. (The two aliens look at each other. The PRINCE smiles.) DRACONIAN PRINCE: I must not detain you longer. No doubt you have "duties" demanding your attention? DRACONIAN FIRST SECRETARY: (Bows.) Your Highness. (He leaves.) [SCENE_BREAK] 23: INT. EARTH. PRESIDENT'S OFFICE (The PRESIDENT stands in her office, looking into space and thinking. The desk monitor buzzes.) PRESIDENT OF EARTH: Yes? (Her SECRETARY appears on the screen.) SECRETARY: (On monitor.) The first secretary from the Draconian embassy wishes to speak you, Madam President. PRESIDENT OF EARTH: Very well. (The picture changes to that of the First Secretary.) DRACONIAN FIRST SECRETARY: (On monitor.) I'm honoured that you consent to speak to me, Madam President. PRESIDENT OF EARTH: What is it that you wish to say? DRACONIAN FIRST SECRETARY: (On monitor.) It concerns the two Earthmen who were found on board your cargo ship. PRESIDENT OF EARTH: Well? DRACONIAN FIRST SECRETARY: (On monitor.) His Highness would like to question them - in your presence, of course, Madam President. PRESIDENT OF EARTH: For what purpose? DRACONIAN FIRST SECRETARY: (On monitor.) His Highness feels that such an interrogation would help to convince you that they are not agents of Draconia. PRESIDENT OF EARTH: I shall have them brought here immediately. I suggest his Highness joins me here. We shall question them together. [SCENE_BREAK] 24: INT. SECURITY BUILDING. CORRIDOR OUTSIDE CELL (Three guards, blasters ready, approach the DOCTOR and JO'S cell. The lead one presses the wall sensor and the door opens.) [SCENE_BREAK] 25: INT. SECURITY BUILDING. CELL (He enters. The DOCTOR and JO are still sat down on the bench.) CELL GUARD: On your feet. DOCTOR: Mmm, why? CELL GUARD: You heard me - move. DOCTOR: Not unless you give me some good reason. CELL GUARD: The President wants you. Is that good enough? JO: Perhaps she believed you? (The DOCTOR gets up.) DOCTOR: Are you sure it's the President? Not another ridiculous interrogation. CELL GUARD: I said move! DOCTOR: Come on, Jo, Perhaps we can convince them after all. (The two leave.) [SCENE_BREAK] 26: EXT. SECURITY BUILDING (The two are led out into the sunlight that manages to permeate between the heavily guarded concrete blocks. Up above them, a blaster with sights fixed, is raised. It fires and the guard in front of the two prisoners falls to the ground. The DOCTOR pushes JO to the safety of one side. He looks up and sees that their attacker is a Draconian soldier. Another shot rings out and another Earth soldier falls.) GUARD: Move, ... ! (The guards start to return the fire but there are several Draconians and they have the advantage of surprise.) DOCTOR: Get up! Right! (The DOCTOR tries to lead JO away but they are quickly surrounded by the Draconians who have now dealt with most of the guards who lie dead around them. The DOCTOR tries to fight off the Draconians but a blaster is put to his neck. Nearby, JO runs up a flight of steps, pursued by three guards.) JO: Quickly - the Doctor! (At the top of the flight of steps, JO runs into a fourth guard. The other three run up and re-take her.) JO: But you don't understand! It's the Draconians - they've got the Doctor! [SCENE_BREAK] 27: INT. EARTH. PRESIDENT'S OFFICE GENERAL WILLIAMS: We must demand the immediate withdrawal of the Draconian embassy. PRESIDENT OF EARTH: Break off diplomatic relations completely? GENERAL WILLIAMS: (Angrily.) What diplomatic relations? The embassy staff have behaved like criminals. PRESIDENT OF EARTH: (Angrily.) We don't know that the ambassador was behind this! GENERAL WILLIAMS: The attackers were Draconians. They were clearly seen. The Draconian embassy tricked you into moving the prisoners, then mounted an armed attack in order to rescue their agents. PRESIDENT OF EARTH: Is the girl here? GENERAL WILLIAMS: She's outside now. (She presses the intercom.) PRESIDENT OF EARTH: (Into intercom.) Bring the girl in. I wish to question her. GENERAL WILLIAMS: And the closing of the Draconian embassy. PRESIDENT OF EARTH: I do not intend to break off diplomatic relations! GENERAL WILLIAMS: But surely there's no alternative once this news gets out? PRESIDENT OF EARTH: (Firmly.) Then it mustn't, General Williams. It is your responsibility to ensure a complete security blackout on this incident. GENERAL WILLIAMS: (Surly.) Very well, Madam. Under protest. (The door hums open and JO is led in by a guard.) PRESIDENT OF EARTH: (To the guard.) You can go. (The guard turns and leaves.) PRESIDENT OF EARTH: (To JO.) Come forward, my dear. (JO nervously approaches her.) PRESIDENT OF EARTH: You realise that the escape of your colleague has left you in a very serious position? JO: But he didn't escape - he was kidnapped! GENERAL WILLIAMS: (Coldly.) He was rescued - by your Draconian paymasters. PRESIDENT OF EARTH: The wisest course you can take now is to make a full confession. Remember your colleague has left you to your fate. JO: Oh, but you've got it all wrong! You see, the Doctor was pleased when you sent for him because he wanted to talk to you. And... GENERAL WILLIAMS: (Interrupts.) We have eye witness reports on what occurred. PRESIDENT OF EARTH: We need to know how you came to work for the Draconians. GENERAL WILLIAMS: When were you recruited? How many agents do they have on Earth? What are their plans? PRESIDENT OF EARTH: If you tell us everything, I'll promise I'll see to it personally that you will be treated leniently. JO: (Desperately.) But I don't know what you're talking about! Look, we told you the truth when we first came here! We're not working for the Draconians. GENERAL WILLIAMS: We're wasting time. I suggest you let me apply deft interrogation techniques without delay. JO: Look, you can use your mind probe or whatever - I'm telling you the truth. [SCENE_BREAK] 28: INT. EARTH. DRACONIAN EMBASSY. AMBASSADOR'S OFFICE (The DOCTOR is sat in a chair in the middle of the floor by two Draconian guards. The PRINCE and the First Secretary are there.) DOCTOR: Well, I must say it's very nice of you gentlemen to invite me here. And where is Miss Grant? DRACONIAN FIRST SECRETARY: Your companion is still with your fellow Earthmen. DOCTOR: Don't you realise what you've done? You've now finally convinced them that we're both Draconian agents. DRACONIAN PRINCE: There is no need to maintain this pretence. We know that you are both agents of the Earth government. DRACONIAN FIRST SECRETARY: You're part of a plot against the Draconian empire! DOCTOR: My dear chap, I've already been through all this with the President of Earth. She thinks I'm working for you. DRACONIAN PRINCE: You are working for General Williams. DOCTOR: I'm what? DRACONIAN PRINCE: General Williams hates out people. Once before, he caused war between us and the Earthmen. Now he plans to do so again. DRACONIAN FIRST SECRETARY: Such a war would be madness since both empires would be destroyed. DOCTOR: Yes, I couldn't agree with you more. That's exactly what I've been trying to say to you. The Earth cargo ship was not attacked by Draconians. DRACONIAN FIRST SECRETARY: It was not attacked at all. The whole story is a lie! DOCTOR: I can assure you that it was attacked - but by Ogrons. DRACONIAN PRINCE: Ogrons? DOCTOR: Yes. Some third party's employing them trying to make trouble between Earth and Draconia. (The PRINCE and the First Secretary look at each other.) DRACONIAN PRINCE: (To the DOCTOR.) If you tell us the details of General Williams' plan, we shall be able to expose him to your President. There will still be a chance for peace. DOCTOR: My dear chap, nobody is more devoted to the cause of peace than I am. But I cannot tell you something that I don't know. DRACONIAN FIRST SECRETARY: (Icily.) We have mind probing techniques just as efficient as those employed by you Earthmen. If you do not speak now, we shall force you to confess! (The DOCTOR slams his chair arm in anger and impatience.) DOCTOR: Don't you realise you're completely on the wrong tack? There is a plot - yes - but the Earthmen are not behind it, any more than you are. (The First Secretary gestures to the two guards.) DRACONIAN FIRST SECRETARY: Take him away! (They approach the chair. The DOCTOR stands, grabs the two guards and throws them forward towards the Secretary. He then does a backward flip on his chair as one of the guards goes for his blaster.) DRACONIAN PRINCE: No! (The DOCTOR runs through one of the open windows and onto the terrace followed by the Secretary and the guards.) [SCENE_BREAK] 29: EXT. EARTH. DRACONIAN EMBASSY (The exterior of the embassy is another futuristic building but this time of a warmer brick. The DOCTOR looks over the terrace edge and then climbs over it. He runs down to a lower terrace. One of the guards above yells down...) FIRST GUARD: Stop him! (There are two more guards on patrol below. They react as the DOCTOR runs down the planted terrace and raise their blasters.) SECOND GUARD: Halt! (The DOCTOR karate chops this guard and easily pushes down the next who tries to stop him. He jumps out onto a lawn and thus out of the embassy area. He is almost instantly surrounded by Earth guards.) [SCENE_BREAK] 30: INT. SECURITY BUILDING. CELL (JO sits alone and concerned in the cell. The door hums open and the DOCTOR stands there at gunpoint. He waves.) DOCTOR: Hello, Jo. (He walks in and JO rushes up to him.) JO: Doctor, where've you been? (The door closes.) DOCTOR: I've been paying a brief but unwilling visit to the Draconian embassy. And I'm afraid I had to leave in rather a hurry and found myself back here. JO: Well, what happened? DOCTOR: Well, believe it or believe it not, Jo, they think we're working for General Williams and trying to provoke a war. JO: Oh no! (She slumps down on the bench disconsolately.) DOCTOR: Yes, Earth is blaming Draconia and Draconia is blaming Earth. JO: And both sides are blaming us. DOCTOR: That's about it. (Suddenly, JO hears the same pulsating signal that she heard on the cargo ship. She jumps to her feet with a look of fear on her face.) DOCTOR: What's the matter? JO: Well, that sound...the one I heard on the cargo ship. I think I can hear it again. [SCENE_BREAK] 31: EXT. SECURITY BUILDING (Outside, the guards on the walkways also hear the sound. They look round for the source. One of them looks down from a ramp and sees the strange form of an Ogron below. He pulls out his blaster and sees the ape-like creature change into that of a Draconian - who promptly shoots the guard. A second running battle breaks out in the area, but this time between the far stronger Ogrons and the Earth guards. One Ogron is a casualty but they easily break through the cordon of guards and run up the stairways to the entrance door. There the sole guard sees the Ogrons as Draconians before they shoot him down. They then rush at the door...) [SCENE_BREAK] 32: INT. SECURITY BUILDING. CORRIDOR OUTSIDE CELL (...smashing it down with little finesse. They shoot down the two guards who approach them and then shoot the lock of the DOCTOR and JO'S cell.) [SCENE_BREAK] 33: INT. SECURITY BUILDING. CELL (The door hums open and they enter, blasters pointed.) JO: Ogrons! OGRON: You - come!

The Doctor and Jo are taken to Earth, where both humans and Draconians believe them to be a spy for the other side.

summ_screen_fd

Vibrio cholerae expresses two primary virulence factors, cholera toxin (CT) and the toxin-coregulated pilus (TCP). CT causes profuse watery diarrhea, and TCP (composed of repeating copies of the major pilin TcpA) is required for intestinal colonization by V. cholerae. Antibodies to CT or TcpA can protect against cholera in animal models. We developed a TcpA holotoxin-like chimera (TcpA-A2-CTB) to elicit both anti-TcpA and anti-CTB antibodies and evaluated its immunogenicity and protective efficacy in the infant mouse model of cholera. Adult female CD-1 mice were immunized intraperitoneally three times with the TcpA-A2-CTB chimera and compared with similar groups immunized with a TcpA+CTB mixture, TcpA alone, TcpA with Salmonella typhimurium flagellin subunit FliC as adjuvant, or CTB alone. Blood and fecal samples were analyzed for antigen-specific IgG or IgA, respectively, using quantitative ELISA. Immunized females were mated; their reared offspring were challenged orogastrically with 10 or 20 LD50 of V. cholerae El Tor N16961; and vaccine efficacy was assessed by survival of the challenged pups at 48 hrs. All pups from dams immunized with the TcpA-A2-CTB chimera or the TcpA+CTB mixture survived at both challenge doses. In contrast, no pups from dams immunized with TcpA+FliC or CTB alone survived at the 20 LD50 challenge dose, although the anti-TcpA or anti-CTB antibody level elicited by these immunizations was comparable to the corresponding antibody level achieved by immunization with TcpA-A2-CTB or TcpA+CTB. Taken together, these findings comprise strong preliminary evidence for synergistic action between anti-TcpA and anti-CTB antibodies in protecting mice against cholera. Weight loss analysis showed that only immunization of dams with TcpA-A2-CTB chimera or TcpA+CTB mixture protected their pups against excess weight loss from severe diarrhea. These data support the concept of including both TcpA and CTB as immunogens in development of an effective multivalent subunit vaccine against V. cholerae. Cholera is an intestinal infection that is associated with acute watery diarrhea and is caused by the Gram-negative bacillus Vibrio cholerae. Cholera is spread by the ingestion of contaminated food and water. An estimated 3–5 million people are infected yearly with cholera, resulting in approximately 100,000 deaths [1]. Cholera is endemic in over 50 countries in the developing world where risk factors such as over-crowding, lack of clean food and water, and poor sanitation allow for its persistence in the environment [1], [2]. Cholera can cause severe life-threatening dehydration, and stool outputs as high as 500–1000 ml/hr can rapidly lead to death in untreated patients [2]. The most effective treatment for cholera is rehydration therapy, and if treatment is started early enough the case fatality rate (CFR) is below 1% [3]. However, it is often difficult for poor and impoverished patients to have access to medical treatment. Cholera can be prevented by vaccination. In 2011, a review of published studies on five variants of an oral whole-cell killed (WCK) cholera vaccine showed that their overall protective efficacy after two years was 62% in adults, they were less effective in children under 5 years of age, and they were unlikely to provide protection beyond three years [4]. In 2013, a study of a re-formulated WCK oral cholera vaccine in Kolkata, India, showed a 5-year cumulative protective efficacy of 65% in all individuals over 1 year of age, but a lower 5-year protective efficacy of 42% in children from 1 to 5 years of age [5]. Nevertheless, a recent critical analysis concludes that current WCK cholera vaccines are poorly suited to control endemic or epidemic cholera because of limited efficacy in young children, requirements for multiple doses, a cold chain, and complex delivery logistics, and costs that are high for resource-poor regions [6]. Finding solutions for such issues is an important goal for developing improved cholera vaccines. We are investigating development of safe and effective subunit vaccines against cholera. Subunit vaccines can present important virulence determinants such as colonization factors and toxins that might not be present or highly immunogenic in a WCK or living attenuated vaccine. For Vibrio cholerae, two essential virulence factors are cholera toxin (CT) and the toxin-coregulated pilus (TCP). CT is an AB5 toxin that is primarily responsible for diarrhea in cholera. CT consists of a monomeric A subunit (CTA) and a homopentameric B subunit (CTB) [7], binds to monosialosyl ganglioside GM1 receptors on enterocytes [8], enters them by endocytosis, trafficks to the endoplasmic reticulum, and releases its CTA1 fragment for retrotranslocation into the cytosol [9]. In the cytosol, CTA1 ADP ribosylates the α subunit of Gs (Gsα), resulting in activation of adenylate cyclase, accumulation of intracellular adenosine-3,5-cyclic monophosphate (cAMP), and downstream events including an efflux of ions and water into the intestinal lumen that presents clinically as diarrhea [10], [11]. TCP is a type IV pilus composed of repeating subunits of the major pilin subunit TcpA [12]. TCP functions in vivo by mediating bacterium-bacterium interactions that are essential for the formation of microcolonies on the surface of enterocytes in the small intestine [13], [14]. In recent infant mouse experiments, TCP has also been demonstrated to mediate attachment of V. cholerae to epithelial cells and to form TCP matrices that engulf the bacteria and may help to protect them from antimicrobial agents [14]. The importance of CT and TCP for V. cholerae virulence has been demonstrated both in animals and in humans, as strains of V. cholerae that fail to produce either CT or TcpA are severely attenuated [12], [15]–[17]. Immunization with CT or non-toxic derivatives of CT has been shown to elicit protective immunity in animal models but not in humans [18]–[21]. Passive orogastric administration of anti-TCP antibodies can provide excellent protection in the infant mouse model of cholera [22], [23], but immunization of humans with intact TCP or with TcpA subunits has not yet been investigated. In the study reported here, we tested recombinant forms of TcpA and CTB (either alone, in combination, or as a holotoxin-like chimera) as candidate cholera vaccines in the infant mouse model of cholera. All procedures involving experimental animals were approved by the University of Colorado Denver (UCD) Animal Care and Use Committee. These procedures were done in compliance with all institutional and governmental requirements and regulations regarding the appropriate ethical use of animals in research. UCD is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, International (file number 00235). All genes were PCR amplified using genomic DNA from V. cholerae El Tor strain N16961 and for FliC from genomic DNA from Salmonella typhimurium strain 14028s. The TcpA-A2-CTB chimera dual promoter expression plasmid pGAP31-2XT7 was constructed in several steps. First, the a2 gene fragment encoding CTA2 was amplified by PCR using the forward primer oA2-Fnot and the reverse primer oA2-Rxho containing the NotI and XhoI restriction sites respectively (Table 1; restriction sites shown in bold). The primer oA2-Fnot contained a point mutation in the a2 coding sequence to change a cysteine to a serine (Table 1; point mutation underlined). Second, the tcpA gene fragment encoding residues 29–199 of the mature TcpA polypeptide was PCR amplified using the forward primer oTcpAn16961-Fmsc and the reverse primer oTcpAn16961-Rnot containing the MscI and NotI restriction sites respectively (Table 1). Previous studies demonstrated this polypeptide to be soluble, surface-exposed, and immunogenic [24], [25]. Third, the a2 and tcpA genes were subcloned into an altered pET22b (+) [EMB Biosciences, Gibbstown, NJ] expression plasmid in which the ampicillin resistance marker was replaced with a kanamycin resistance marker. The kanamycin resistance marker was obtained from pET28b (+) [EMD Biosciences, Gibbstown, NJ] which was cut with EcoRI and PpuMI, and the isolated restriction fragment was then ligated into pET22b (+). The insertion of the tcpA and a2 gene fragments was downstream and in frame with the pelB signal sequence. Fourth, a second T7 promoter containing the mature ctb gene in frame with the pelB signal sequence was PCR amplified from the expression plasmid pGAP20K [26] using the forward primer oT7-FppuMI and the reverse primer oCTB-RpshAI. Finally, the t7-pelB-ctb gene product was subcloned into the PshAI and PpuMI sites of the TcpA-A2 expression plasmid generated in step 3 above, thereby creating the dual T7 promoter expression plasmid pGAP31-2XT7 (Fig. 1). The CTB expression plasmid pGAP20K, which encodes the ctxB allele from V. cholerae El Tor strain N16961, was constructed as previously described [26]. The N-terminal 6-histidine tagged-TcpA expression plasmid was created by PCR amplifying the tcpA gene fragment encoding residues 29–199 of the mature TcpA polypeptide (Fig. 1) using the forward primer oTcpAn16961-Fnde and the reverse primer oTcpAn16961-Rxho (Table 1). This was inserted into pET28b (+) using the NdeI and XhoI restriction sites, downstream and in frame of an N-terminal 6-his tag, creating the expression plasmid pGAP33. The N-terminal 6-histidine-tagged-FliC expression plasmid was created by PCR amplifying the Salmonella typhimurium fliC gene using the forward primer oFliC-Fnde and the reverse primer oFliC-Rxho (Table 1). This was inserted into pET28b (+) using the NdeI and XhoI restriction sites, downstream and in frame with the N-terminal 6-his tag, creating the expression plasmid pGAP32. The TcpA-A2-CTB chimera was produced in Escherichia coli BL-21 (DE3) Star™ cells (Invitrogen, Grand Island, NY). Half-liter cultures were grown in NZTCYM medium pH 7. 5 (1% N-Z-amine AS [Sigma, St. Louis, MO], tryptone 1%, NaCl 0. 5%, yeast extract 0. 5%, casamino acids 0. 1%, MgSO4 0. 2%) and 100 µg/ml kanamycin at 37°C, 250 rpm until cultures reached an OD600 of ∼3. 0. The cultures were then placed at 16°C and 250 rpm for 30 minutes to acclimate to the new temperature then induced with 0. 2 mM IPTG and grown overnight at 16°C for ∼16–18 hrs. 6His-TcpA (29–199) was produced in SHuffle™ T7 Express E. coli (NEB, Ipswich, MA) in half liter cultures of TCYM media pH 7. 5 (tryptone 1%, NaCl 0. 5%, yeast extract 0. 5%, casamino acids 0. 1%, MgSO4 0. 2%) and 100 µg/ml kanamycin at 37°C, 250 rpm until cultures reached an OD600 of ∼3. 0. The cultures were then placed at 16°C, 250 rpm for 30 minutes, and then induced with 0. 1 mM IPTG and grown overnight as above. Cultures of E. coli BL-21 (DE3) cells producing 6His-FliC were grown in half liter cultures of TCYM pH 7. 5 at 37°C 250 rpm until they reached an OD600 of ∼2. 0–3. 0. After acclimating to 30°C with shaking at 250 rpm for 30 minutes, the cultures were then induced with 0. 5 mM IPTG and incubated for 4 hrs. CTB was grown and induced as described previously [26]. Preparation of the bacterial extracts and primary metal affinity purification of all proteins was performed as described previously [26]. A secondary purification step on the strong cation-exchange resin POROS 20 HS (Applied Biosystems, Carlsbad, CA) was performed for TcpA-A2-CTB, 6His-TcpA (29–199), and CTB. Both the TcpA-A2-CTB chimera and 6His-TcpA (29–199) were purified under the same conditions. Each was dialyzed overnight at 4°C in 25 mM potassium phosphate buffer pH 6. 8. Soluble and filtered protein was loaded onto a POROS 20 HS (Applied Biosystems, Carlsbad, CA) column, and eluted with a linear 0 to 0. 5 M gradient of NaCl in 25 mM potassium phosphate buffer at pH 6. 8. This purified, soluble, recombinant 6His-TcpA (29–199) protein is subsequently called TcpA. For CTB, the protein was first desalted using Zeba™ Desalt Spin Columns (Thermo Fisher Scientific, Rockford, IL) following the manufacturer' s protocol. CTB was desalted into potassium phosphate buffer pH 6. 6 and then filtered through a 0. 45 µM syringe filter to remove precipitated material. An ion-exchange purification step was then conducted using POROS 20 HS (Applied Biosystems, Carlsbad, CA) resin. The bound protein was eluted using a linear 0 to 0. 5 M gradient of NaCl in 25 mM potassium phosphate buffer pH 6. 6. 6His-FliC was dialyzed overnight against 20 mM Tris-Cl pH 8. 0. An ion-exchange purification step was then conducted using the strong anion-exchange resin POROS 20 HQ (Applied Biosystems, Carlsbad, CA). The bound protein was eluted from the resin using a linear gradient of 0 to 0. 5 M NaCl in 20 mM Tris-Cl buffer at pH 8. 0. This purified, soluble, recombinant 6His-FliC protein is subsequently called FliC. Following purification TcpA, CTB, and FliC were dialyzed overnight at 4°C against 1× PBS and stored at −80°C. The TcpA-A2-CTB chimera was dialyzed overnight at 4°C against 50 mM Tris buffer containing 200 mM NaCl and 1 mM EDTA at pH 7. 5. Female CD-1 mice, 6–8 weeks old, were purchased from Charles River Labs and given food and water ad libitum. Groups of 7–10 mice were immunized three times IP at 14 day intervals. The group that was immunized with the TcpA-A2-CTB chimera received 50 µg/dose. All other groups received amounts of each antigen that were equimolar with the amount of the corresponding antigenic component in a 50-µg dose of the chimera. For the groups immunized with TcpA combined with FliC or FliC alone, the dose of FliC administered for the first or second immunization was 5 µg, and a 2 µg dose was administered for the final immunization. Blood and fecal samples were collected one day prior to the initial immunization (Day −1) and on days 21 and 42. Sample collection and processing was performed as previously described [26]. To obtain blood samples from infant mice, the pups were first asphyxiated with CO2, and then a scalpel was used to sever the cervical spinal cord. A heparinized capillary tube was used to collect blood that seeped from the incision. One or two pups were used per dam, and blood was collected on the same day that siblings were challenged with V. cholerae. Blood was pooled if two siblings were used to obtain sera. To measure antigen-specific antibody amounts in serum and fecal extracts, we used quantitative ELISAs as described previously [26]. The concentration or amount of antigen-specific IgG or IgA antibodies in unknown samples was determined by interpolation from a standard curve using KC4 v3. 4 software (Bio-Tek. Winooski, VT). GM1 ganglioside ELISAs were performed as previously described [26] using sera from rabbits immunized with recombinant TcpA or CTB. The infant mouse challenges were performed as previously described [21], [26]. All pups were six days old at the time of inoculation. The pups were monitored for survival over the course of 48 hrs. Pup weights were recorded immediately prior to inoculation and at 24 and 48 hours post-infection. For pups that died prior to 24 hours, their carcass weights were measured and included with the group at 24 hours. For pups that died between 24 and 48 hours, their carcass weights were measured and included with the group at 48-hours. All statistical comparisons were performed using GraphPad PRISM 4 (La Jolla, CA). ANOVA analysis using the Tukey-Kramer post-test was used to determine statistical significance between immunization groups for antigen-specific antibody concentration differences and for weight loss differences between immunization groups for the infant mouse challenge. Within-group statistical differences for antigen-specific antibody amounts at days 21 and 42 were analyzed using a paired two-tailed t-test. Survival curves were generated using Kaplan-Meier method, and statistical differences between experimental groups were determined using the log-rank (Mantel-Cox) test. P values less than 0. 05 were considered significant. Both gene and protein sequences for the antigens used in this study can be found in the National Center for Biotechnology Information (NCBI) database. The genes (accession numbers) are as follows: ctxB (NC_002505), tcpA (AF536868), and fliC (NC_016856). The protein sequences (accession numbers) are as follows: CTB (NP_231099), TcpA (AAN15109) and FliC (YP_005237927). The TcpA-A2-CTB chimera was expressed in E. coli using the dual T7 promoter plasmid pGAP31-2XT7 (Fig. 1), and it was purified using sequential metal affinity chromatography and ion-exchange chromatography. Upon heating and denaturation, the purified chimera separated into the TcpA-A2 fusion protein (∼23 kDa) and monomeric CTB (∼11. 5 kDa; Fig. 2). TcpA-A2 migrated more slowly than TcpA, reflecting the greater molecular mass of the fusion protein due to the presence of CTA2 at its carboxyl-terminus (Fig. 2). We used GM1 ganglioside ELISAs to demonstrate immunoreactivity of the TcpA-A2 moiety and both immunoreactivity and ganglioside GM1 receptor-binding activity of the pentameric CTB moiety of the purified TcpA-A2-CTB chimera (Fig. 3). Solutions containing equimolar amounts of the TcpA-A2-CTB chimera, or TcpA alone, or CTB alone, were serially diluted and added to ELISA plates that had previously been coated with ganglioside GM1 and then blocked to prevent nonspecific binding of the test antigens. Subsequently the plates were washed and then probed with either anti-CTB rabbit antiserum or anti-TcpA rabbit antiserum. The TcpA-A2-CTB chimera and CTB (but not TcpA) bound avidly to the plates coated with GM1 ganglioside. Bound TcpA-A2-CTB chimera and bound CTB were both detected with anti-CTB antiserum (Fig. 3, top), but only the bound chimera was detected with anti-TcpA antiserum (Fig. 3, bottom). None of these antigens bound above background levels to control plates that were blocked but not coated with ganglioside GM1. Taken together, these results showed that the TcpA-A2-CTB chimera is a bi-functional oligomeric complex that exhibits TcpA immunoreactivity associated with its TcpA-A2 fusion polypeptide and both CTB immunoreactivity and ganglioside GM1 binding activity associated with its pentameric CTB subunit. To compare the immunogenicity of the TcpA-A2-CTB chimera with non-chimeric forms of TcpA and CTB, we immunized groups of 7 to 10 female CD-1 mice three times by the IP route according to the immunization timeline shown in figure 4. In an attempt to show whether inherent immunogenicity of recombinant TcpA protein could be enhanced by use of an adjuvant, we included separate groups of mice immunized either with TcpA alone or with TcpA plus the recombinant flagellin subunit protein FliC from Salmonella typhimurium. FliC is a toll receptor 5 (TLR5) agonist and has been demonstrated previously to act as an adjuvant for co-administered antigens [27]. Serum and fecal antigen-specific antibody responses were measured using quantitative ELISA for the samples collected on days 21 and 42 (Fig. 4). We found that immunization with the TcpA-A2-CTB chimera elicited a significantly higher mean concentration of serum anti-TcpA IgG on day 21 compared with all other immunization groups (P<0. 001, Fig. 5A). However by day 42,14 days following the third and final immunization, the mean serum anti-TcpA IgG concentrations for the groups immunized with the TcpA-A2-CTB chimera, the TcpA+CTB mixture, and the TcpA+FliC mixture were comparable (P>0. 05), but all were significantly greater than the mean serum anti-TcpA IgG concentration after immunization with TcpA alone (P<0. 01). These results demonstrated that either incorporating the TcpA-A2 fusion protein into the TcpA-A2-CTB chimera or administering TcpA together with CTB or FliC enhanced the immunogenicity of TcpA, and the results for the samples collected at day 21 suggest that the TcpA-A2-CTB chimera presented the TcpA moiety in its most immunogenic form. Serum anti-CTB IgG responses were robust in all groups that received CTB, either as CTB alone, as TcpA+CTB, or as the TcpA-A2-CTB chimera (Fig. 5B). There were no significant differences in mean anti-CTB IgG concentrations between these groups either at day 21 (P>0. 05) or at day 42 (P>0. 05), although the mean titers were significantly higher at day 42 than at day 21 (P<0. 0001). As expected, control mice immunized with PBS did not develop any detectable anti-TcpA or anti-CTB antibodies. The amounts of antigen-specific IgA antibody and total IgA immunoglobulin were measured in each fecal extract. In figure 6, the amount of antigen-specific IgA antibody is shown as a percentage of total IgA for each fecal extract. We normalized the data in this way to minimize differences that might result from mouse-to-mouse variations in production of fecal IgA or sample-to-sample variations in recovery of IgA from the fecal specimens. Immunization with either the TcpA-A2-CTB chimera or the TcpA+FliC mixture elicited a significantly greater mean fecal anti-TcpA IgA response on day 21 than immunization with any of the other antigens (Fig. 6A, P<0. 05). Interestingly, at day 42 the mean fecal anti-TcpA IgA response to immunization with the TcpA-A2-CTB chimera was less than at day 21, but the mean fecal anti-TcpA IgA response to immunization with TcpA+FliC was greater than at day 21. However, neither of these pairwise differences in mean antibody amounts between days 21 and 42 was significant (P>0. 05). On day 42 the mean fecal anti-TcpA IgA response to immunization with TcpA+CTB had increased dramatically to a level that was comparable to the TcpA+FliC immunization group (P>0. 05), and both were significantly greater than mean values for all other immunization groups at day 42 (P<0. 05) (Fig. 6A). As with the serum CTB-specific IgG responses, the fecal anti-CTB IgA responses at day 42 were not significantly different in any of the groups that received CTB as an immunogen, either as CTB alone, as TcpA+CTB, or as the TcpA-A2-CTB chimera (Fig. 6B; P>0. 05). In contrast to the increases in the serum CTB IgG concentrations that occurred from day 21 to day 42, however, the fecal CTB-specific IgA percentages on day 21 and day 42 were comparable (P>0. 05). Finally, for each group that was immunized both with TcpA and CTB (e. g., immunized with either TcpA-A2-CTB or TcpA+CTB), the mean percentage of fecal anti-CTB IgA was greater than the mean percentage of fecal anti-TcpA IgA in the same group, both at day 21 and at day 42 (compare results and note differences in scales for the Y axes for Figs. 6A and 6B). To compare the protective efficacies of selected vaccine regimens, immunized dams were mated (see timeline in Fig. 4) and groups of their reared 6-day old pups were challenged orogastrically with 10 LD50 of V. cholerae El Tor strain N16961 and monitored for survival at 24 and 48 hrs (Fig. 7A). All pups from dams immunized either with TcpA-A2-CTB chimera (n = 20) or TcpA+CTB (n = 20), and all sham-infected pups (n = 20) survived for 48 hrs. All pups from dams immunized with CTB alone (n = 20) survived for 24 hrs, and 70% survived for 48 hrs (P = 0. 0087 vs. pups immunized with TcpA-A2-CTB or TcpA+CTB). In contrast, pups from dams immunized with TcpA+FliC (n = 20) or FliC alone (n = 10), like pups from PBS immunized dams (n = 20), experienced 77. 5–80% mortality by 24 hrs and 100% mortality by 48 hrs (P<0. 0001 vs. pups immunized with TcpA-A2-CTB or TcpA+CTB). We did not challenge pups from dams immunized with TcpA alone, because those dams were previously shown to have much lower serum and fecal anti-TcpA antibody levels than dams immunized with TcpA+FliC (Figs. 5A and 6A). To investigate under more stringent conditions the contributions of anti-TcpA and anti-CTB antibodies in protecting infant mice against cholera, we challenged additional pups from the immunized dams with a higher 20 LD50 challenge dose of V. cholerae El Tor strain N16961 (Fig. 7B). All pups from dams immunized with TcpA-A2-CTB chimera (n = 19) or TcpA+CTB (n = 20), and all sham-infected pups (n = 20), survived for 48 hrs. In contrast, no pups from dams immunized with CTB alone (n = 20), TcpA+FliC (n = 20), or PBS (n = 20), survived for 48 hrs (P<0. 001 vs each of the three previous groups). The mean concentrations of serum anti-TcpA IgG at day 42 did not differ significantly in dams immunized with TcpA-A2-CTB chimera, TcpA+CTB, or TcpA+FliC (Fig. 5A, P>0. 05), and the mean percentages of fecal anti-TcpA IgA also did not differ significantly in the dams immunized with TcpA+CTB or TcpA+FliC (Fig. 6A, P>0. 05). Similarly, the mean concentrations of serum anti-CTB IgG at day 42 did not differ significantly in dams immunized with TcpA-A2-CTB chimera, TcpA+CTB, or CTB alone (Fig. 5B), and the mean percentages of fecal anti-CTB IgA did not differ significantly among dams in these immunization groups (Fig. 6B). Published studies show that transfer of maternal antibodies to pups (which can occur in utero, by suckling, or by both pathways) is the primary mechanism by which immunization of dams confers immunologically specific protection to their pups [28]–[30]. Therefore, the complete protection achieved in pups from dams immunized with the TcpA-A2-CTB chimera or the TcpA+CTB mixture, versus the lack of any protection in pups from dams immunized with TcpA+FliC or CTB alone, cannot be explained either by poorer serum or fecal anti-TcpA or anti-CTB antibody responses, respectively, vs. the comparable antigen-specific antibody responses in the mice immunized with the TcpA-A2-CTB chimera or the TcpA+CTB mixture. Sham-infected pups experienced about 2% weight loss at 24 hrs and 10% weight loss at 48 hrs because they were separated from their dams since 3 hrs before challenge (Fig. 8). In addition, mouse pups develop diarrhea if they are not fully protected against V. cholerae infection by active or passive immunity [31]. At the 10 LD50 challenge dose of V. cholerae N16961, pups from dams immunized with the TcpA-A2-CTB chimera or TcpA+CTB mixture did not lose significantly more weight at 24 or 48 hrs than sham-infected pups (Figs. 8A and 8B; P>0. 05), and all survived for 48 hrs (Fig. 7A). In contrast, pups from PBS immunized dams or dams immunized with TcpA+FliC or FliC alone did experience much greater weight losses than sham-infected pups both at 24 hrs (Fig. 8A; P<0. 001) and at 48 hrs (Fig. 8B; P<0. 05), and all died by 48 hrs (Figs. 7A and 7B). The severity and timing of their excess weight losses and their death within 48 hrs reflected the onset of severe diarrhea. Pups from dams immunized with CTB experienced less-dramatic excess weight losses at 24 hours than pups from dams immunized with PBS, TcpA+FliC, or FliC (Fig. 8A; P<0. 001), but they experienced greater weight losses than sham-infected pups at 24 and 48 hrs (Figs. 8A and 8B; P<0. 001). This resulted in a 70% survival rate of CTB immunized pups at 48 hrs. These findings indicate that pups from dams immunized with CTB were partially protected against challenge with 10 LD50 of V. cholerae N16961, and they experienced less severe diarrhea than PBS immunized pups (Figs. 7A and 7B). At the 20 LD50 challenge dose, pups from dams immunized with the TcpA-A2-CTB chimera exhibited significantly greater weight losses at 24 hrs than sham-infected control pups (Fig. 8C; P<0. 05), and pups from dams immunized either with the TcpA-A2-CTB chimera or with TcpA+CTB exhibited significantly greater weight losses at 48 hrs than sham-infected control pups (Fig. 8D, P<0. 05) although all pups in both groups survived for 48 hrs. The excess weight losses among pups in these two groups indicate that they experienced mild diarrhea at the 20 LD50 challenge dose. The challenged pups from dams immunized with TcpA+FliC or CTB alone experienced much greater weight losses than the sham-infected controls (P<0. 001; Figs. 8C+8D), and all of them died before 48 hrs, indicating that they experienced severe diarrhea at the 20 LD50 challenge dose. One or two pups from each dam in the immunization groups included in the suckling mouse challenge studies described above were sacrificed at the same time that their siblings were challenged to obtain serum samples for measurements of antigen-specific IgG concentrations by quantitative ELISA. As shown in Fig. 9, the mean anti-TcpA IgG serum antibody concentrations from the pups were statistically equivalent regardless of whether their dams were immunized with TcpA-A2-CTB chimera, TcpA+CTB, or Tcp+FliC (P>0. 05). The mean anti-CTB IgG serum antibody concentrations from the pups were also statistically equivalent regardless of whether their dams were immunized with TcpA-A2-CTB chimera, TcpA+CTB, or CTB (P>0. 05). Consistent with results shown previously for serum antibodies at day 42 in immunized dams, the mean concentrations of antigenic-specific serum IgG antibodies from these pups were much greater for the anti-CTB antibodies than for anti-TcpA antibodies. None of the sera from pups had detectable anti-TcpA-specific IgA antibodies, and only one serum from a pup born to a dam immunized with TcpA-A2-CTB chimera had detectable anti-CTB-specific IgA antibodies. Early studies showed that pups from non-immunized dams survived large orogastric challenge doses of V. cholerae (500–2000 LD50) when the bacteria were pre-mixed with anti-CT or anti-TCP antiserum [23], [32], but pups from dams immunized against TcpA or CTB survived only when challenge doses were much smaller (1–15 LD50) [21], [26], [33]. Titers of serum anti-TcpA IgG1 and IgA antibodies in dams correlated with survival rates of their challenged pups [33]. Survival rates of challenged pups from immunized dams fell more rapidly as the dam' s log10 anti-TcpA IgG1 titers decreased than did survival rates of pups from unimmunized dams with comparable decreases in anti-TCP antiserum doses [33]. Pups given anti-TCP antiserum intraperitoneally also survived V. cholerae challenges given 24 hrs later [23]. Taken together, these findings show that intestinal anti-TcpA or anti-CTB antibodies protect infant mice from potentially lethal V. cholerae challenges and indicate that maternal antibodies are delivered into the intestines of infant mice either actively by suckling or passively by transudation from internal body fluids. In the studies reported here, we investigated whether immunizing dams with TcpA-A2-CTB chimera or TcpA+CTB protected their pups more effectively than immunizing dams with TcpA or CTB alone in the infant mouse model of cholera. We challenged separate groups of pups with 10 LD50 and 20 LD50 doses of V. cholerae El Tor N16961 to assess protection under stringent conditions. Few previous studies compared protective efficacy of immunization with TcpA+CT (or CTB) vs. TcpA or CT (or CTB) alone in animal models of cholera. Transcutaneous immunization (TCI) of dams with CT+TcpA protected pups better against a 1 LD50 challenge with V. cholerae N16961 (69% survival) than did TCI with CT alone (36% survival), but TCI with TcpA alone induced no detectable anti-TcpA antibodies in dams and their pups were not challenged [25]. Although that study and our study used different methods to measure antigen-specific antibodies, the more robust protection of pups at higher challenge doses that we observed likely indicates higher serum anti-TcpA and anti-CTB antibody levels in our immunized dams. Other investigators used the ligated ileal segment model in adult rabbits to compare protection conferred by intranasal immunization with TcpA+CTB, TcpA alone, or CTB alone [34]. Fluid accumulation in ligated ileal segments decreased by 41. 1% vs. unimmunized controls in rabbits immunized with TcpA and by 70. 5% in rabbits immunized with CTB, but no fluid accumulated in ligated ileal segments of rabbits immunized with TcpA+CTB [34]. The immunized rabbits also developed intestinal sIgA antibodies against the TcpA and/or CTB antigens that they received [34]. In our studies (see Fig. 7), all pups from dams immunized with TcpA-A2-CTB chimera or TcpA+CTB survived 48 hrs after a 10 or 20 LD50 challenge dose of V. cholerae El Tor N16961; no pups from dams immunized with TcpA+FliC survived 48 hrs at either challenge dose; and pups from dams immunized with CTB had 70% 48-hr survival at the 10 LD50 challenge dose a 0% 48-hr survival at the 20 LD50 dose. At each challenge dose, the 100% survival rate for pups with both anti-TcpA and anti-CTB antibodies significantly exceeded the sum of the survival rates for pups with only anti-TcpA antibodies and for pups with only anti-CTB antibodies [e. g., (0%+70%) = 70% cumulative survival at 10 LD50 and (0%+0%) = 0% cumulative survival at 20 LD50 among pups with only anti-TcpA antibodies plus pups with only anti-CTB antibodies]. Because these differences in survival could not be explained by significant differences in mean values of TcpA-specific or CTB-specific serum IgG or fecal IgA antibodies among groups of pups from dams immunized with vaccine formulations that contained any form of TcpA or CTB, respectively (see Figs. 5 and 6), our results constitute strong preliminary evidence that anti-TcpA and anti-CTB antibodies act synergistically rather than additively to prevent death in the infant mouse model of cholera. In humans, cholera is caused either by V. cholerae serogroup O1 (with classical and El Tor biotypes and Inaba and Ogawa serotypes) or V. cholerae serogroup O139 (first recognized in 1992–1993) [2], [3]. Early clinical isolates of V. cholerae O139 were closely related to V. cholerae O1 El Tor (but with different genes at the O antigen locus), but later V. cholerae O139 isolates belong to multiple lineages derived from different V. cholerae progenitors [35], [36]. The O1 and O139 lipopolysaccharides are essential for virulence of V. cholerae O1 and O139 in humans, are important protective antigens, and elicit serogroup-specific antibodies that do not cross-react with each other [36]–[38]. CT and TcpA are also essential for virulence of V. cholerae O1 and O139 and are immunogenic in humans [16], [17], [39]–[41], but analyzing their roles in protective immunity is complicated by the existence of multiple antigenically cross-reacting variants of each protein among classical, El Tor and “hybrid El Tor” isolates of V. cholerae O1 and V. cholerae O139 [22], [23], [26], [42]–[44]. Early studies in human volunteers suggested that immunity against cholera was mediated primarily by antibacterial rather than antitoxic mechanisms [20], and for decades the best serological (but non-mechanistic) correlate of protection among patients and volunteers who recovered from a previous episode of cholera was the titer of complement-dependent vibriocidal antibodies [45]. More recent studies in humans showed that serum IgA (but not IgG) antibodies against CTB, LPS, or TcpA also correlate with protection against cholera [3], [46], [47]. However, because recovery confers protection against a future episode of cholera that persists much longer than titers of vibriocidal, anti-CTB, anti-LPS, or anti-TcpA antibodies remain elevated, prompt anamnestic antibody responses following exposure to V. cholerae are believed to be important for long-term immunity against cholera. Consistent with this view, patients who recover from cholera have been found to develop IgG and IgA memory B (BM) cells specific for LPS, TcpA, and CTB as well as effector memory T (TEM) cells specific for CTB [45]–[48]. The mechanisms by which intestinal anti-CTB and anti-TcpA antibodies protect against cholera (e. g., blocking CT-mediated toxicity and TCP-mediated contributions to colonization) are believed to be similar in humans and in infant mice. Our results provide proof-of-principle that immunizing dams with TcpA-A2-CTB chimera or TcpA+CTB can protect 100% of pups against challenges with up to 20 LD50 of V. cholerae El Tor N16961, which produces CTB and TcpA variants homologous to those used for immunization. To the best of our knowledge, no other reported immunization regimen for dams protects pups so well against such a stringent challenge in the infant mouse model of cholera. Further studies will be needed: 1) to assess the relative protective efficacy of current TcpA-A2-CTB and TcpA+CTB vaccines against challenges with V. cholerae O1 classical or El Tor or V. cholerae O139 strains that produce homologous or heterologous variants of CTB and TcpA; and 2) to determine whether immunization with at least two variants each of TcpA and CTB can provide broader protection than immunization with one variant of each protein against V. cholerae challenge strains that produce several different CTB and/or TcpA variants. Using a TcpA-A2-CTB chimera instead of a TcpA+CTB mixture in a vaccine formulation has several potential advantages, since the chimera is a chemically defined, highly immunogenic, macromolecular complex that can be assembled spontaneously in E. coli and purified as a single entity. If necessary, different variants of TcpA and CTB could be incorporated into different chimeras, which could then be combined to create a vaccine formulation containing multiple variants of CTB and TcpA. The WCK oral cholera vaccine that provided significant protection for 5 years in an endemic region does not contain CTB, and it is unclear whether the heat and formalin treatments used to inactivate V. cholerae during preparation of that vaccine cause any damage to the immunogenicity of TcpA or other protein protective antigens of the bacteria [5], [6]. Our results show clearly that TcpA and CTB can be used successfully as protective subunit immunogens against cholera in the infant mouse model. Extending to humans the potential value of incorporating TcpA and CTB into effective vaccines against cholera will require additional studies to address the need: 1) to elicit production of antigen-specific sIgA antibodies in the human intestine; 2) to achieve long-term memory for protective intestinal immune responses; and 3) to develop vaccine formulations, adjuvants, routes of delivery, and immunization regimens to accomplish these goals.

Vibrio cholerae is the bacterium that causes cholera, a pandemic diarrheal disease transmitted by ingestion of contaminated food or water. We developed a novel vaccine containing two protective antigens of V. cholerae, TcpA and CTB, incorporated into a defined oligomeric protein chimera. CTB is the non-toxic binding domain of cholera toxin, the protein that causes profuse watery diarrhea in cholera patients. TcpA is the subunit of the toxin-coregulated pilus, a V. cholerae surface structure that is required for intestinal colonization and disease. Intraperitoneal immunization of adult female mice with this TcpA-A2-CTB chimera elicited stronger early anti-TcpA responses and equivalent anti-CTB responses compared to immunizing with a TcpA+CTB mixture. Furthermore, all reared infant mice from females immunized with the chimera or TcpA+CTB were protected against a large challenge dose of V. cholerae that was sufficient to kill all infant mice from non-immunized control and TcpA- or CTB-immunized adults. Our study supports the concept of including both TcpA and CTB as antigens in development of a safe and effective subunit vaccine against cholera.

lay_plos

The ability of pathogens to cause disease depends on their aptitude to escape the immune system. Type IV pili are extracellular filamentous virulence factors composed of pilin monomers and frequently expressed by bacterial pathogens. As such they are major targets for the host immune system. In the human pathogen Neisseria meningitidis, strains expressing class I pilins contain a genetic recombination system that promotes variation of the pilin sequence and is thought to aid immune escape. However, numerous hypervirulent clinical isolates express class II pilins that lack this property. This raises the question of how they evade immunity targeting type IV pili. As glycosylation is a possible source of antigenic variation it was investigated using top-down mass spectrometry to provide the highest molecular precision on the modified proteins. Unlike class I pilins that carry a single glycan, we found that class II pilins display up to 5 glycosylation sites per monomer on the pilus surface. Swapping of pilin class and genetic background shows that the pilin primary structure determines multisite glycosylation while the genetic background determines the nature of the glycans. Absence of glycosylation in class II pilins affects pilus biogenesis or enhances pilus-dependent aggregation in a strain specific fashion highlighting the extensive functional impact of multisite glycosylation. Finally, molecular modeling shows that glycans cover the surface of class II pilins and strongly decrease antibody access to the polypeptide chain. This strongly supports a model where strains expressing class II pilins evade the immune system by changing their sugar structure rather than pilin primary structure. Overall these results show that sequence invariable class II pilins are cloaked in glycans with extensive functional and immunological consequences. Members of the Neisseria genus are Gram-negative proteobacteria that include several commensals such as N. sicca, N. lactamica or N. elongata and two human pathogens, N. gonorrheae and N. meningitidis. Both of these are highly adapted for interaction with humans, their unique host. N. gonorrheae colonizes the human urogenital tract and is responsible for a sexually transmitted infection characterized by a massive inflammatory response and purulent discharge. Neisseria meningitis is responsible for devastating sepsis and meningitis [1]. N. meningitidis proliferates on the surface of epithelial cells lining the nasopharynx in approximately 5 to 30% of the total human population. Pathogenesis is initiated when bacteria access the bloodstream from the throat, survive and multiply in the blood. Systemic infection and perturbation of vascular function lead to sepsis, the most severe form of the disease associated with organ dysfunction, limb necrosis and death in certain cases. N. meningitidis can also cross the blood-brain barrier and access the cerebrospinal fluid, leading to meningitis. Type IV pili (Tfp) are extracellular filamentous organelles that can be found on a large number of bacterial species [2]. In the case of Neisseria spp. they are key virulence factors. These abundant structures are 6–8 nm wide, can measure several microns in length and are expressed by all pathogenic Neisseria spp. strains. Type IV pili are primarily composed of a single protein or major pilin, called PilE in Neisseria spp., which is assembled in a polymeric helical fiber. Neisseria type IV pilins have been grouped in two classes (class I and class II) based on the recognition of the SM1 antibody. This antibody reacts with the linear epitope E49YYLN53, which is specific to class I pilins [3]. It was later recognized that the genomic location of the class I and II pilin genes are also different [4,5]. Type IV pili provide several properties to the bacteria: auto-aggregation, adhesion to host cells, intracellular signaling, competence and a form of motility called twitching motility [6]. The importance of this structure during N. gonorrheae infection has been demonstrated in human volunteers [7]. Male volunteers inoculated with a type IV pili deficient strain only developed a watery urethral discharge or none at all. More recently, using mice grafted with human skin, Melican et al. showed that type IV pili mediated adhesion of N. meningitidis is a determining factor in vascular damage observed during purpura fulminans [8]. As a countermeasure against this virulence factor the immune system produces antibodies against type IV pili [9]. The efficacy of N. meningitidis to proliferate in the throat and in blood during productive infection thus depends on its ability to evade type IV pili specific antibodies. The amino acid sequence of class I pilins can vary by a process called antigenic variation [10]. Beside the expression locus of the major pilins a variable number of non-expressed (silent) pilS loci with different but homologous sequences are present in Neisseria spp. genomes. Pilin antigenic variation results from a gene conversion, which transfers DNA from the silent cassettes to the expression locus. Thus, the pilin sequence can change generating multiple different antigens. Surprisingly, it was recently recognized that pilins belonging to class II lack this antigenic variation in Neisseria meningitidis [11,12]. Strains with sequence invariable pilE genes are frequently isolated worldwide independently of serogroup, year or country of isolation [5]. Interestingly class II pilin genes are restricted to certain clonal complexes, and all pilin genes from clonal complexes cc1, cc5, cc8, cc11 and cc174 are class II. Importantly, these clonal complexes display among the highest disease to carriage ratio, in other words they are hypervirulent [13]. Another interesting feature of these clonal complexes is the association with epidemic meningococcal disease (cc1, cc5 and cc11). Countries in the “meningitis belt” in sub-Saharan Africa have the highest burden of meningococcal disease with both large seasonal epidemics, and much higher incidence rates compared to other areas of the world where outbreaks are small and sporadic. These studies therefore raise the question of how, in absence of primary structure variation, do class II expressing strains evade immunity targeted against type IV pili? Another potential source of surface variation is post translational modification and in particular glycosylation. Pilin glycosylation has been identified in strains expressing class I pilins that display a single glycosylation site on Ser63 [14–17] but has never been studied in class II pilin expressing strains. Importantly, genes involved in the glycosylation of surface structures (pgl genes) are submitted to phase variation. As a consequence, oligosaccharides present on the bacterial surface vary between strains and change for one strain during the course of nasopharynx colonization and infection. The Neisseria spp. glycosylation pathway starts with the synthesis of an undecaprenyl diphosphate (Undpp) monosaccharide in the cytoplasm. Three enzymes are involved in this step, PglB, C and D. In strains expressing the pglB1 allele these enzymes synthesize an undecaprenyl-DATDH (diacetamido trideoxyhexose) and in strains expressing the pglB2 allele a GATDH (glyceramido trideoxyhexose) core [18]. The Unpp-monosaccharide can then be modified with additional sugars by three glycosyltransferases pglH, pglA and pglE, the latter two being submitted to phase variation. When in the ON-phase, pglA leads to the addition of a galactosyl residue on GATDH or DATDH [19]. PglH adds a glycosyl residue on the same site [20]. Recently a pglH2 allele was described whose product adds an N-acetyl glucosamine residue on the first sugar [21]. When a disaccharide is formed a third sugar can be added by the PglE transferase. PglF is then responsible for the translocation of this structure to the periplasmic side of the inner membrane [22]. Finally, the PglO/L oligosaccharide transferase adds the sugar chain onto the pilin [23,24]. Given the clinical importance of strains expressing class II pilins and the invariable nature of their sequences, we decided to explore how such strains could evade immunity directed against type IV pili. Since pilin glycosylation is a potential source of surface structure variation we determined the nature of class II pilin glycosylation and show that this could provide immune escape in the absence of primary structure variation. The prototypical strain expressing a class II pilin is the FAM18 strain that was isolated from the cerebrospinal fluid of a patient in North Carolina USA in the 1980s. Its genome has been sequenced and is publicly available [25]. We used a Nalidixic acid resistant variant of this strain called FAM20 to characterize the posttranslational modifications of this representative class II pilin [26]. Type IV pili were purified and characterized using a combination of high-resolution mass profiling and top-down mass spectrometry [27]. Mass profiling of the FAM20 strain produced an exceptionally complex spectrum with over 20 different proteoforms [28] clearly distinguishable (Fig 1A, FAM20). Pilin masses ranged from 15967 Da to over 16850 Da while the molecular mass predicted from the genome is only 14524 Da strongly indicating that numerous post translational modifications (PTMs) were present. Pilin sequences from individual clones were identical indicating that differences in mass were not due to recombination at the pilin locus as expected from previous studies [12]. In order to identify the different PTMs on this pilin we proceeded to simplify the spectral pattern using specific mutants deficient for genes involved in PTMs. Certain proteoforms of the FAM20 major pilin were separated by 124 Da suggesting that phosphoethanolamine (PE) was present. As pptA is responsible for PE modification a pptA deletion mutant was generated (Fig 1A, pptA) and type IV pili were purified from this strain [29,30]. The overall pattern of pilin purified from the FAM20pptA strain was shifted towards lower masses and the number of major proteoforms was reduced to about 12. Peaks with differences of mass corresponding to one hexose were also frequently observed in the spectra (162 Da). Since FAM18 PglH was found to add a glucose residue onto DATDH (glycosyl transferase) we generated and tested a pglH mutant [20]. The complexity of the spectrum obtained with the pglH strain was also greatly reduced confirming the activity of this enzyme (Fig 1A, pglH). To combine the effects of each mutation a double mutant was made. The pglHpptA double mutant generated pili with only 3 major proteoforms (15693,15967 and 16241 Da). Strikingly, the mass difference between the 3 peaks corresponded to one GATDH moiety (274 Da, Fig 1, pglHpptA). This result is a strong indication that the FAM20 pilin is glycosylated at least at 3 sites in contrast with previously analyzed strains that showed only one glycosylation site [18,23]. The reduced complexity of the pptApglH double mutant allowed us to identify the PTMs found on the 3 proteoforms using top-down mass spectrometry (Fig 1B, S1 Fig and S1 Table). Different charge states corresponding to each proteoform were submitted to Electron Transfer Dissociation (ETD) fragmentation. As expected, the 3 proteoforms were found to be modified with the usual pilin modifications: N-terminal methylation, disulfide bond between the two cysteine residues and two phosphoglycerol moieties on Ser69 and Ser97 (Table 1). Proteoform 1 displayed two glycosylation sites with GATDH at Ser63 and Ser90. Proteoform 2 harbored three glycosylation sites at Ser63, Ser87 and Ser98. Proteoform 3 harbored a fourth glycosylation site at Ser90 in addition to those found on proteoform 2. The FAM20 strain can therefore harbor up to 4 different glycans on the same pilin monomer. This detailed top-down analysis of the mutant strain allowed us to precisely assign specific PTMs to the 23 different proteoforms found in the wild type FAM20 highlighting the tremendous diversity of structures found on the pilin of this strain (Fig 2). The presence of a strikingly high number of pilin glycosylation sites in FAM20 raised the question of whether this is a particularity of FAM20 or a common feature of strains expressing class II pilins. We therefore collected several class II pilin expressing strains, isolated during a 2003–2006 period from patients suffering from sepsis and meningitis at the Limoges university hospital in France and analyzed their PTM (Table 2). Strains were selected to be part of different serogroups and sequence types to represent a diverse panel with the common feature of expressing class II pilins. To allow detailed genetic analysis the entire genomic sequence of two of these clinical strains was established and genes involved in pilin production and its glycosylation characterized (Fig 3A and 3B, LIM534 and LIM707). In both cases sequences of the major pilin are closely related to the FAM20 type II sequence (S2 Fig) and the pilin gene is also located between the katA and prlC genes as expected for class II expressing strains (Fig 3A). Type IV pili were purified from these strains and submitted to high resolution intact mass profiling. Overall, spectra were less complex than the FAM20 strain with 3–6 major proteoforms (Fig 3C). Nevertheless pilin purified from LIM534, LIM712, LIM675 and LIM707, consistently displayed evidence of multiple glycosylation sites (Fig 3C). The difference in mass between major proteoforms could be explained by the sequential addition of several DATDH/GATDH or DATDH-Hex/GATDH-Hex glycans depending on the strain. Top-down MS analysis of these different proteoforms demonstrated that proteoforms contained 2–5 glycosylation sites (Fig 3D). These results show that strains expressing class II pilins from different clonal complexes isolated from different continents and in different time periods share the common feature of carrying multiple glycosylation sites. This strongly suggests that such multisite pilin glycosylation is a general feature of class II expressing strains. The observation that class II pilin-carrying strains bear multiple glycosylation sites as opposed to class I strains that carry only one, could be explained by two non-exclusive hypotheses. First, the particular primary structure of class II pilins may itself be more favorable to glycosylation due to a larger number of accessible serine residues. Second, this difference is due to the genetic background and in particular to the pgl genes expressed by these strains. As a first attempt to address this question the genomic regions carrying the pgl genes were analyzed in two of the isolated class II pilin expressing strains, LIM534 and LIM707 but this did not reveal any obvious explanation for the number of glycosylation sites. For instance, the PglO/L oligosaccharide transferase was highly conserved between the class I (8013) and class II (FAM18, LIM534 and LIM707) pilin-expressing strains with identity scores between 98 and 100%. As in the class I pilin-expressing strains the pglBCDFH genes are localized between the ribD and avtA genes apart from the pglA and pglE genes which are located on a separate region (Fig 3B). The LIM707 strain carries a split pglB2 gene (GATDH) previously found to maintain functionality and an insertion containing orf2 and pglH between the pglF and pglB genes [20,31]. The LIM534 strain expresses a pglB1 (DATDH) gene and displays an insertion containing the orf2 and pglH genes but, interestingly, the pglH gene is interrupted by a transposase explaining why only a monosaccharide is found on the pilin. To address the potential role of the pilin sequence in determining the number of glycosylation sites we generated two “class swap” mutant strains, the first with a class II pilin in a class I pilin-expressing genetic background and the reciprocal strain with a class I pilin in a class II background. In the first case, a class II LIM707 pilin was expressed in the context of the 8013 background (8013pilELIM707, Fig 4A). Pilin from the 8013 strain normally harbors GATDH at a single glycosylation site [18]. Expression of the class II LIM707 pilin in the 8013 background strain led to a pilin modified with up to 3 GATDH moieties (Fig 4B and 4C). The glycosylation sites were the same as in the original LIM707 strain. This result indicates that the pgl genes from the 8013 strain are capable of modifying the pilin at multiple sites and that the number of glycosylation sites is determined by the pilin sequence itself rather than the pgl genes. To confirm this result the reverse situation was generated and the 8013 pilin (class I) was expressed in the FAM20 background (FAM20pilE8013, Fig 4D). High resolution MS analysis showed that pilins purified from this strain comprised of a more complex array of proteoforms due to modification with PE, PC, di and trisaccharides but the vast majority contained a single glycosylation site at Ser63 (Fig 4E and 4F). Interestingly, in this case about 10% of the pilin also contained a second glycosylation. Taken together these results show that the genetic environment of the strain determines the type of sugar added, DATDH or GATDH, mono, di or trisaccharide but the presence of multiple glycosylation sites in class II pilins is largely determined by the primary structure of this class of pilins. Neisseria meningitidis class I pilin glycosylation has been shown to contribute to adhesion by interacting with the platelet activating factor (PAF) receptor on the surface of human airway cells [32]. In contrast, in Neisseria gonorrhoeae strains deficient for pilin glycosylation exhibited an early hyper-adhesive phenotype but were attenuated in their ability to invade primary cervical epithelial cells [33]. The multiple glycosylation sites found on the class II pilins raised the question of their function more acutely. To explore the function of glycosylation FAM20, LIM707, LIM534 and 8013 strains deleted for the pglC and pglD genes were generated. Surprisingly, however, despite repeated attempts we were unable to purify pili from the FAM20pglC and FAM20pglD strains. Electron microscopy observation showed that these two strains do not display any type IV pili on their surface (S2 Fig). Complementation of mutant strains with the corresponding genes restored piliation. In the case of the FAM20 strain, glycosylation appears to be necessary for efficient pilus assembly. This result was unique to the FAM20 strain as the other two class II pilin expressing strains showed normal piliation in absence of glycosylation. The impact of the loss of glycosylation on the typical pilus properties of adhesion to endothelial cells and auto-aggregation was then determined (Fig 5A–5D). As expected from the absence of pili, the FAM20pglD strain showed very low adhesive capacity indistinguishable from the non-piliated mutant (Fig 5A). In contrast, adhesion by the LIM707, LIM534 and 8013 strains were unaffected by the absence of pilin glycosylation. Similar results were found on pulmonary epithelial cells (S3 Fig). Bacterial aggregation was evaluated as a second type IV pilus-dependent property (Fig 5B–5D). As expected, the FAM20pglC and pglD mutants did not show any aggregation. Bacterial aggregation of LIM534 and LIM707 was higher in the absence of glycosylation. Interestingly, instead of being spherical, bacterial aggregates formed by the unglycosylated LIM707 and LIM534 strains displayed unusual heterogeneous shapes. Such an aggregation phenotype characterized by more aggregation and polymorphous aggregates is reminiscent of strains deficient for the PilT retraction ATPase [34]. To evaluate whether the absence of glycosylation was altering pilus retraction the motility of these strains was evaluated (S4 Fig). Twitching motility depends on cycles of pilus extension and retraction that drag the bacteria on a surface. The glycosylation mutants of the LIM534 and LIM707 strains did not show any defect in motility indicating normal retraction on individual bacteria (S4 Fig). This suggests that class II pilin glycosylation destabilizes pilus-pilus contacts allowing for dynamic interactions between pili in the context of aggregates. Taken together these results show that the absence of glycosylation on class II pilins leads to strong functional changes, and such changes vary depending on the strain. In the most dramatic situation pili were not expressed on the surface. In other cases, pilus-pilus interactions were stabilized leading to enhanced aggregation. The important functional impact of the multisite glycosylation displayed by class II pilins described above suggests that sugars occupy a large percentage of the pilus surface. To explore this hypothesis the structures of pilin fibers were modeled using the N. gonorrhoeae MS11 pilin as a template [35] and taking into account glycan PTM. Three glycans per monomer were included in the model as it represents the dominant and average proteoform. Pilus assembly was performed as previously described [36] and corresponding sugars were built by energy minimization and added onto the pilus fiber. Organization of the whole structure was then refined first in vacuo and then in water. Glycosylated pilus structures formed of class II pilins consistently show global coverage of the fibers by sugars (Fig 6A). Higher magnification of the FAM20 pilus glycosylated on 3 sites per monomer, the most abundant proteoform, shows extensive coverage of the pilus surface (Fig 6B). Glycosylation therefore strongly changes the structure of the pilus fiber. These results also suggest that glycosylation will perturb antibody recognition of the pilus fibers. In particular, antibodies directed against the pilus structures would have limited direct access to the protein backbone. As this could compensate for the absence of sequence variation in the class II pilins we decided to investigate this point further. All atoms of the pilus in cylindric coordinates were projected on a plane according to their height and angular coordinate, in order to obtain a flat representation of the pilus surface on a 2D grid (Fig 6C). The surface at the tip of antibodies, typically involved in antigen binding, is roughly circular in shape with a diameter in the order of 5 nm. Since interaction with the antigen does not require the whole surface we approximated the antibody-antigen binding site by using a 2 nm diameter disc [37]. The disc was tested against each position along a grid covering the pilus surface and for each position the presence of sugar was evaluated. The percentage of positions where antibody binding was not affected by sugars was then determined (Fig 6D). For the 8013 class I expressing strain that displays a single glycosylation site the presence of sugars decreased antibody accessibility to 65% of the surface. In the case of the class II pilins that carry multiple glycosylation sites, antibody accessibility was reduced to 15%, 11% and 9% of the surface in the FAM20, LIM534 and LIM707 strains respectively. These results show that the surface-accessible amino-acid residues of class II pilins are largely masked by glycosylation sites. Our results show that, unlike in class I pilins, a large portion of the pilus surface is coated with sugars in class II pilins. Over the years pilin glycosylation of class I pilins has been studied from 3 different Neisseria meningitidis strains demonstrating a single conserved glycosylation site at Ser63 (Table 3). Strain C311#3 displays a Gal (β1–4) Gal (α1–3) 2,4-DATDH [16], strain 8013 one GATDH residue [14] and NIID280 a DATDH residue [17]. In addition, N. gonorrhoeae strain N400 presents a hexose residue linked to a DATDH on its class I pilin also at Ser63 [15]. In this single study using top-down mass spectrometry, we describe for the first time the glycosylation pattern of 5 different strains expressing class II pilins including the FAM18 reference strain. Pilins from all of these 5 strains display 3 to 5 glycosylation sites (Table 3). Independently of the serogroup, clonal complex, geographic site or temporal period of isolation of the strains (Table 2), class I pilins show a single site of glycosylation while class II pilins have multiple sites of glycosylation. Molecular modeling reveals that multisite glycosylation of the pilin monomer leads to the coverage of the pilus surface. This could have important consequences in terms of adaptation of the bacteria to the host immune response. More specifically, this could explain why amino-acid sequence variation is not required in class II strains because the polypeptide chain is not exposed to the extracellular milieu and thus not submitted to pressure by the immune system. That is not to say that antibodies cannot recognize glycosylated class II pilins: indeed glycopeptides from type IV pili are immunogenic [38,39]. Rather, these results suggest that the immune escape scenario would then be different between class I and II pilins. In the case of class I pilins, after throat colonization by a given strain the IgAs specific for the pilin primary sequence will be produced and lead to killing of the initial strain, but variants arising from recombination at the pilin genetic locus will survive until a second adaptation of the immune system. This cycle can potentially repeat itself numerous times. In the case of class II strains the primary structure is cloaked in oligosaccharides, and only antibodies targeted to epitopes that include sugar moieties will efficiently lead to bacterial killing. In this case variants in the sugar structure will survive. Type IV pili have been considered as potential vaccine antigens against Neisseria gonorrhoeae infections but sequence variation in class I pilins has hampered these attempts [40,41]. In the absence of sequence variation in class II pilins it could be tempting to use such proteins as vaccine antigens but our results show that glycosylation would complicate this approach. A number of arguments support the idea that sugar structure does change during infection of individuals and during epidemics. It has been shown that sera from infected patients during acute and convalescent stage meningococcal disease recognize the major pilin [9] and thus establishes that type IV pili are indeed a target of the immune system and its pressure. It is also well documented that certain pgl genes such as pglA and pglE are submitted to phase variation [42]. This implies that the functionality of these genes and thus the nature of pilin glycosylation can vary at rates between 10−2 to 10−6 per cell per generation giving the bacteria the opportunity to evade antibody response against type IV pili [43,44]. Beyond phase variation, pgl genes appear to be the site of rapid changes including at the epidemic scale. Lamelas et al. performed a longitudinal study in Northern Ghana between 2001 and 2009 [45] where they collected and sequenced the genomes of 100 strains in order to identify evolutionary changes during these epidemic waves. This revealed that the pgl genes were the site of no less than 5 successive recombination events during this period. Importantly, all of the strains in this study display class II pilins (Gerd Pluschke, personal communication). Our work also provides evidence of changes in pilin glycosylation. In the case of the LIM534 strain the pglH gene is interrupted by an insertion sequence. This insertion event is a molecular signature of changes in the nature of the sugars coating a class II pilin. Taken together these studies underline the high level of variation undergone by class II pilin glycosylation likely to evade the immune response directed against pili. Our results also provide an explanation for the molecular mechanism that leads to multisite glycosylation in class II pilins. We showed that expression of a class II pilin in a strain normally expressing a class I pilin leads to glycosylation on multiple sites on the pilin backbone. This result shows that the pilin amino-acid sequence is a determining factor for multisite glycosylation. Alignment of the pilin sequences from class I and II strains suggests two scenarios. Certain glycosylated serines present on class II pilins are simply absent in class I pilins (e. g. serines at alignment positions 88,91 or 118 in class II are absent in class I). Alternatively the serine is present on class I pilins but the local sequence is different (e. g. serines at alignment position 68 and 99) and this would likely affect glycosyltransferase recognition. Predominance of pilin sequence in the determination of glycosylation sites is confirmed by the reciprocal situation. When a class I sequence is expressed in the class II expressing background, the vast majority of the pilin contains only one sugar. It is noteworthy however that the genetic background, and most probably the pgl genes, also plays a partial role in determining the number of glycosylation sites. Indeed, when the class II pilin is expressed in its normal background the main proteoform contains 3 glycans per monomer whereas when it is expressed in the class I expressing background the main proteoform contains only 2. Furthermore, when the class I sequence is expressed in the class II expressing background the main proteoform contains one sugar but 10% of pilins also contain 2 sugars. It is therefore likely that pilins of different classes have co-evolved with their respective pgl systems and the glycosylation systems in class II pilin expressing strains are more efficient. Independently of the number of glycosylation sites, the nature of the sugars on the pilin is determined by the pgl genes. The FAM20 strain expresses a pglB2 allele, pglA and pglE genes are in the OFF phase and the insertion with the ORF2 and pglH genes is present. Accordingly, pilins from this strain are modified with a GATDH core, determined by the pglB2 allele, and between 1 and 2 hexose residues likely being a glucose transferred by PglH. Interestingly, the PglH transferase expressed by this strain is partially functional in the sense that certain sites display a GATDH-hexose while others a GATDH monosaccharide. This specificity of the pglH allele contributes to the complexity of the pattern found on pilins expressed by the FAM20 strain. The LIM707 strain contains the same expression pattern of pgl genes as the FAM20 (pglB2 allele, pglAOFF, pglEOFF, pglH present) leading to the production of a GATDH-Hexose type of sugar. In contrast to the FAM20 strain, all sugars on the LIM707 pilin are disaccharides. For the LIM534 strain, we observe the pglB1 allele, pglA and pglE alleles are in the OFF phase and the pglH gene is present but interrupted by an insertion sequence. In this case, only DATDH is present as predicted by the genomic data. When pilin sequences are introduced into a different genetic background the nature of sugar changes with the genetic background of the strain. When the pilin gene from the LIM707 strain is introduced in the 8013 background, the pilin becomes modified with a GATDH residue as found in the 8013 strain rather than with a GATDH-Hexose disaccharide. Similarly, introduction of the 8013 pilin gene into the FAM20 strain leads to the expression of pilin modified with GATDH-hexose. Overall these results show that the pilin sequence determines the number of glycosylation sites and the pgl gene pattern the nature of the sugar. An intriguing result of this study is the difference in functional consequence of the lack of glycosylation in the different class II pilin expressing strains. The most striking phenotype is in the FAM20 strain where type IV pili are simply not expressed on the surface of the bacteria in absence of glycosylation. This phenotype is specific to the FAM20 strain as the LIM707 or LIM534 strains still adhere to host cells via their type IV pili despite inactivation of the pgl genes. Another specificity of the FAM20 strain is the high number of proteoforms expressed. In addition to the 2–4 glycosylation sites displayed by this strain, phosphoglycerol (2) phosphoethanolamine (1–3) and phosphocholine (2) modifications are also present. It is possible that in absence of sugar these numerous modifications generate a specific structural environment that is incompatible with pilus expression. It is also possible that the piliation machinery has co-evolved with the glycosylation of this strain and that specific interactions with the machinery such as with the PilQ secretin require glycosylation. Further work is required to elucidate at which step glycosylation is necessary for piliation in the FAM20 strain. For instance, identification of the point at which pilus biogenesis is blocked in the FAM20pglC/D strains will yield useful information to understand the mechanisms of pilus biogenesis. A role for glycosylation in the assembly and function of pili in other organisms has been described. In Neisseria gonorrhoeae, pilin (class I) glycosylation has subtle effects on pilus dynamics [46]. Perhaps a similar but more prevalent mechanism is at play in the FAM20 strain. Glycosylation of the Pseudomonas aeruginosa major pilin is also necessary for efficient piliation [47]. In Archea, the archaellum, a swimming organelle closely related to type IV pili bears glycosylation sites that are required for assembly [48] Surprisingly, in strains LIM707 and LIM534 absence of glycosylation of their class II pilins did not affect adhesion to either endothelial or epithelial cells. This is in contrast with previous studies on strains expressing class I pillins that reported a decrease of adhesion in strains lacking pilin glycosylation due to direct interactions of the sugars with cellular receptors [33]. Recently, the recombinant non-glycosylated class I pilin from the 8013 strain was shown to interact with the surface protein CD147 [49]. In principle class II pilins could mediate interactions with cellular receptors through the polypeptidic chain but modeling shows that its accessibility is limited by the numerous surface exposed glycans. Intriguingly, our results thus make a direct interaction between the major pilin and a cellular receptor difficult to imagine at the structural level. Further structural work is required to clarify this point. In contrast, the absence of glycosylation of the pilins expressed by LIM707 and LIM534 led to enhanced aggregation and to an unusual aggregation behavior likely due to stabilized pilus-pilus interactions. These results are consistent with the idea that multisite class II pilin glycosylation leads to changes in surface properties of type IV pili. These results also point out that the functional consequences of pilin glycosylation could be different in class I and class II pilins. Overall, our work revises the current view of pilin glycosylation. Starting from a single modification per pilin in class I strains we now realize that a large proportion of N. meningitidis strains express class II pilins and carry multiple glycosylation sites. Our study first reveals the profound implications in terms of pilus biogenesis, structure and function. The results presented here also have important implications in terms of immunity against type IV pili, vaccine design and how N. meningitidis manages to escape the immune system. In particular the presence of multiple glycosylation sites provides a simple explanation for the absence of pilin sequence variation in class II pilins and suggests that variations in sugar structure are the main motor for immune evasion in these strains. The worldwide distribution, hypervirulence and association with epidemic forms of the disease of strains carrying class II pilins underscore the importance of these results to understand Neisseria meningitidis infections. In the more global context of infectious diseases our study highlights the wealth of strategies exploited by pathogens to escape the immune system and the key role played by glycosylation. N. meningitidis strains were grown on GC agar base plates (Conda Laboratorios, Spain) containing Kellogg' s supplements [7] and, when required, 5 μg/ml chloramphenicol at 37°C in moist atmosphere containing 5% CO2. Escherichia coli transformants were grown in liquid or solid Luria-Bertani medium (Difco) containing 100 μg/ml ampicillin. Neisseria meningitidis strains used in this study are described in Table 4.

During infection pathogens and their host engage in a series of measures and counter-measures to promote their own survival: pathogens express virulence factors, the immune system targets these surface structures and pathogens modify them to evade detection. Like numerous bacterial pathogens, Neisseria meningitidis express type IV pili, long filamentous adhesive structures composed of pilins. Intriguingly the amino acid sequences of pilins from most hypervirulent strains do not vary, raising the question of how they evade the immune system. This study shows that the pilus structure is completely coated with sugars thus limiting access of antibodies to the pilin polypeptide chain. We propose that multisite glycosylation and thus variation in the type of sugar mediates immune evasion in these strains.

lay_plos

Myosin Ic is thought to be the principal constituent of the motor that adjusts mechanical responsiveness during adaptation to prolonged stimuli by hair cells, the sensory receptors of the inner ear. In this context myosin molecules operate neither as filaments, as occurs in muscles, nor as single or few molecules, as characterizes intracellular transport. Instead, myosin Ic molecules occur in a complex cluster in which they may exhibit cooperative properties. To better understand the motor’s remarkable function, we introduce a theoretical description of myosin Ic’s chemomechanical cycle based on experimental data from recent single-molecule studies. The cycle consists of distinct chemical states that the myosin molecule stochastically occupies. We explicitly calculate the probabilities of the occupancy of these states and show their dependence on the external force, the availability of actin, and the nucleotide concentrations as required by thermodynamic constraints. This analysis highlights that the strong binding of myosin Ic to actin is dominated by the ADP state for small external forces and by the ATP state for large forces. Our approach shows how specific parameter values of the chemomechanical cycle for myosin Ic result in behaviors distinct from those of other members of the myosin family. Integrating this single-molecule cycle into a simplified ensemble description, we predict that the average number of bound myosin heads is regulated by the external force and nucleotide concentrations. The elastic properties of such an ensemble are determined by the average number of myosin cross-bridges. Changing the binding probabilities and myosin’s stiffness under a constant force results in a mechanical relaxation which is large enough to account for fast adaptation in hair cells. The myosin family includes at least 20 structurally and functionally distinct classes [1,2]. Although they all exhibit a common chemomechanical cycle, myosin molecules have remarkably diverse functions-including intracellular transport, force production in muscles, and cellular migration-as well as important roles in sensory systems [3]. To understand the emergence of these different functions, it is necessary to characterize the biophysical details of the chemomechanical cycle for each myosin class. Myosin molecules transduce chemical energy into mechanical energy through the hydrolysis of adenosine triphosphate (ATP). The hydrolysis reaction and the subsequent release of inorganic phosphate (Pi) and adenosine diphosphate (ADP) induce structural changes that result in a power stroke and generate forces. The biochemical reaction rates and the response to external forces determine the specific function of each myosin [3]. On the basis of their biochemical and mechanical properties, myosins have been classified into four groups: (i) fast movers, (ii) slow but efficient force holders, (iii) strain sensors, and (iv) gates [4]. Although single-molecule experiments and structural studies have vastly advanced our understanding of force-producing molecules, we still lack a consistent description that quantitatively relates cellular functions to the molecular details. One prominent case is myosin Ic, which has been identified as a component of the adaptation motor of the inner ear [5]. Hair cells in the inner ear transduce mechanical stimuli resulting from sound waves or accelerations into electrical signals. On the upper surface of each hair cell stands a hair bundle comprising dozens to hundred of actin-filled protrusions called stereocilia. Cadherin-based tip links connect the tip of each stereocilium to the side of the longest adjacent one. When a mechanical force deflects the bundle, the resultant shearing motion raises the tension in the tip links. This tension increases the open probability of transduction channels and allows ions to diffuse into the stereocilia, depolarizing the hair cell. To retain sensitivity, a hair cell adapts to a prolonged stimulus by changing the tension in the tip links. This adaptation has a fast component lasting a millisecond or less and a slow component of a few tens of milliseconds, the molecular details of which remain uncertain. To explain slow adaptation, it has been proposed that an ensemble of myosin Ic molecules alternately step up or slide down the actin filaments inside the stereocilia to regulate the tension in the tip links. Sliding of myosin is triggered by a locally elevated Ca2+ concentration. This picture has been quantitatively supported by experimental studies on hair cells and complemented by mathematical descriptions [6–9]. Fast adaptation describes the rapid reclosure of transduction channels after abrupt stimulation of the hair bundle. This process is poorly understood and several possible explanations at a molecular level are debated [6,10]. One promising mechanism is the release model, in which a component of the transduction apparatus becomes more flexible and abruptly releases some of the tension in the tip links, allowing the channels to close rapidly [11,12]. Although myosin Ic has been implicated in both slow and fast adaptation and an ensemble of myosin Ic molecules is a good candidate for the element that releases [10], the precise role of myosin Ic in adaptation has yet to be elucidated. The rapid response of the transduction channels to a displacement of the hair bundle suggests a direct mechanical activation through the transformation of the deflection into a force by a spring [6,13]. This mechanism underlies the gating-spring hypothesis that is the prevailing explanation for mechanotransduction by hair cells. The elastic property of the gating spring is the most important parameter in setting the precise relation between the deflection of a hair bundle and the open probability of the ion channels. Despite numerous studies of the molecular components of the hair bundle and their biophysical properties, we remain uncertain of the identity of the gating spring [14–18]. Every molecule that lies in series with the tip link could in principle influence the elastic properties, including the ensemble of myosin Ic molecules. These molecules bind and unbind from actin filaments and thereby change the elasticity dynamically. In order to fully explain mechanotransduction by hair cells, it is important to understand how the dynamics of single myosin Ic molecules determines the elastic properties of an ensemble and how it is regulated. Over the past few years, the biophysical properties of individual myosin Ic molecules have been characterized in optical traps, biochemical assays, and structural studies [19–24]. Like other myosin isoforms, myosin Ic displays catch-bond behavior, a prolonged attachment to an actin filament in response to increased external force [19,25]. The force-sensitive step in myosin Ic’s cycle is the isomerization following ATP binding, however, and not ADP release as in other slow myosins [19,20]. To understand how this behavior relates to the molecule’s physiological function, we introduce a consistent mathematical description of myosin Ic’s cross-bridge cycle. After the introduction of the basic framework by Huxley and Huxley, cross-bridge models have been widely used to describe the dynamics of myosin motors [2,26–35]. However, these models often assume irreversible transitions at fixed nucleotide concentrations that determine the input of chemical energy. In a seminal work, T. L. Hill showed how to couple a description of an enzymatic cycle to free-energy transduction in a thermodynamically consistent manner, an approach that has been applied to study muscle myosin [36–39]. We build our cross-bridge cycle for myosin Ic on these concepts and furthermore include the catch-bond behavior. Our description allows a quantitative analysis of the differences between in vitro and in vivo conditions, of Ca2+ regulation, and of cooperativity between force-producing molecules. Here we introduce a thermodynamically consistent description of myosin Ic based on single-molecule data and focus on the responses to external force, to different nucleotide concentrations, and to the availability of actin. We use this description to predict the elastic properties of an ensemble of myosin molecules and highlight the potential implication for the release model of fast adaptation. As a functional description of myosin Ic we introduce a chemomechanical cycle consisting of five states: one state in which myosin is unbound from actin and four actin-bound states. Because we primarily focus on the force-producing states, we consider only a single, effective unbound state that combines the actin-detached ADP⋅Pi and ATP states. Each of the actin-bound states is associated with the nucleotide occupancy of the binding pocket of the myosin head (Fig 1). Myosin Ic performs its main, 5. 8 nm power stroke upon phosphate release; a smaller power stroke of 2 nm follows ADP release. To account for the work done by these power strokes, we include a force dependence of the associated transition rates. We consider an effectively one-dimensional description in which the force acts along the coordinate of the power stroke: a positive force is oriented in a direction opposite to the power stroke. The nucleotide-binding rates depend linearly on the nucleotide concentrations and the actin-binding rates increase linearly with the actin concentration. By cycling through the five states, myosin performs work whose magnitude is bounded by the free-energy input associated with the nucleotide concentrations. We base our description on the free-energy transduction of enzymes and thus ensure thermodynamic consistency. To incorporate myosin Ic’s unique force sensitivity, we include a simple force dependence of the rate of unbinding from the filament of myosin in the ATP state. Under high forces, we expect myosin Ic to be trapped in the ATP state. Therefore we consider the ADP state (3), the nucleotide-free state (4), and the ATP state (5) as strongly bound. The remaining states are weakly bound or unbound (Fig 1). Our description, which captures many of the characteristics of myosin Ic, incorporates as free variables the experimentally controllable quantities external force, nucleotide concentrations, and actin concentration. This approach allows us to obtain analytic expressions for quantities that have been measured in experiments, then to use that information to determine the unknown parameter values of the model. An overview of the parameters is given in Table 1. A mathematical description of the cross-bridge cycle and details of the estimation of parameter values are presented in the Methods section. In a single-molecule experiment using an isometric optical clamp, the lifetime of the myosin Ic-actin bond was measured for different external forces and two sets of nucleotide concentrations [20]. Because a rapid transit into and out of the weakly bound state (2) could not be resolved experimentally, this bound lifetime must be interpreted as the average time tsb that myosin Ic spends in the strongly bound states. We determined an analytic expression for the unbinding rate t sb - 1 from the strongly bound states (Eq 53) as functions of force and nucleotide concentrations and fit this function simultaneously to two sets of experimental data acquired for distinct nucleotide concentrations. This unbinding rate is independent of the transition rate ω15 and of the actin concentration. Both quantities determine how often the molecule binds to the filament rather than how long it remains bound. From the average time that myosin Ic resides in the weakly bound states we estimate the binding rate ω15 for an actin concentration of 100 μM appropriate for the experiments. A detailed explanation for the fitting procedure is given in the Methods section. Fits of the unbinding rate t sb - 1 from the strongly bound states describe the experimental data well, indicating that our description is able to capture the force sensitivity of myosin Ic (Fig 2a). Although none of the transition rates can account individually for the plateau around zero force, their combined effect in the cycle clearly displays such a behavior, which is characteristic of myosin Ic. The numerical values obtained in this way for the transition rates ω21 ≃ 164 s−1 and ω 51 0 ≃ 314 s - 1 suggest that in the absence of force, state (2) and state (5) are both configurations from which the myosin head rapidly detaches. The force-distribution factors (δ) indicate that phosphate release is only weakly dependent on force (δ1 ≃ 0. 12) and ADP release not at all (δ1 ≃ 0). The concentrations of nucleotides in cells differ from those in single-molecule experiments. We can use our description to predict the behavior of myosin molecules for different nucleotide concentrations. Although in single-molecule experiments the phosphate concentration usually remains low, the phosphate concentration in vivo is on the order of 1 mM [2]. In cells the ATP concentration is also near 1 mM and the ADP concentration is around 10 μM [2]. In the remainder of this study we refer to these numbers as the physiological nucleotide concentrations. The unbinding rate does not significantly change for higher phosphate concentrations (Fig 2a and 2b). The main reason for this robust behavior is the very low rate constant for phosphate binding (Eq 38). Even for a millimolar phosphate concentration the phosphate-binding rate ω32 is very small compared to the other transition rates in the cycle. In contrast, increasing the ADP concentration decreases the overall binding rate because the molecule spends more time in the ADP state. This effect can be counteracted by an increase in the ATP concentration (Fig 2b). Using the formulation given in the Methods section with the explicit solutions in Eqs 45–49, we can determine the steady-state probability distribution for the cross-bridge cycle at different nucleotide and actin concentrations (Fig 3). For physiological nucleotide concentrations and 100 μM of actin, myosin is trapped in the ATP state (5) under forces exceeding 2 pN (Fig 3a). Comparing only the strongly bound states, the molecule predominantly occupies the ADP state (3) for forces smaller than 1. 5 pN. According to our description, myosin Ic’s cycle through the strongly bound states is limited by ADP release for forces smaller than 1. 5 pN and by ATP release for forces larger than 1. 5 pN. This result is consistent with experimental findings [19,20]. In the stereocilium of a hair cell, myosin Ic is thought to extend between the crosslinked actin filaments of the cytoskeleton and the insertional plaque to which the tip link is anchored [5,6, 40]. To analyze the implications of an environment with a high concentration of actin, we determined the probability distribution for an actin concentration of 10 mM (Fig 3b). Because of the increased binding probability, the unbound state (1) is depopulated. The weakly bound ADP⋅Pi state (2) dominates for forces smaller than 2 pN and the ATP state (5) for larger forces. An increased ADP concentration of 250 μM traps the myosin head in the ADP state for forces smaller than 2 pN and larger than 4 pN (Fig 3c). In the intervening regime the ATP state predominates. In our stochastic description without irreversible transitions, we define myosin’s effective velocity as the average number of forward power strokes minus the average number of reverse power strokes per time. We refer to this definition as an effective velocity to emphasize that this quantity is neither the gliding velocity of an actin filament nor the ensemble velocity of several myosin Ic heads cooperating to produce a continuous movement. Every time the myosin head traverses the states (2) → (3) → (4) it performs a net power stroke of size Δx1 + Δx2. In contrast, the reverse pathway (4) → (3) → (2) is associated with a reverse power stroke of size − (Δx1 + Δx2). The effective velocity v is accordingly given in terms of the combined local excess fluxes ΔJij (Eq 26) as v ≡ Δ x 1 Δ J 23 + Δ x 2 Δ J 34. (1) An increasing actin concentration enhances the binding of myosin and therefore decreases its cycling time, which leads to a higher effective velocity (Fig 4). The velocity saturates for an actin concentration above 1 mM. For large forces the effective velocity decreases until it becomes negative for forces larger than the stall force. According to our thermodynamic description the stall force F s = k B T Δ x 1 + Δ x 2 ln [ ATP ] K eq [ ADP ] [ P i ] (2) arises directly from Δμ = Eme, the equality of the Gibbs free energy for the hydrolysis reaction and the mechanical output. This relation reflects an implicit assumption that all of the chemical energy can be converted into mechanical energy. To account for mechanical inefficiency, the description could be extended with a loss parameter. Because we restrict our analysis to forces smaller than 6 pN, for which power strokes have been observed experimentally, we ignore the precise behavior for larger forces and consider the stall force for myosin Ic as an unknown quantity. A widely accepted definition of the duty ratio is the fraction of the total duration of an ATPase cycle that myosin spends in the strongly bound states [3,41–43]. Ignoring the weakly bound, actin-attached states or combining them into other states, the duty ratio is often defined as the fraction of the total cycle time during which myosin is attached to an actin filament [2,44–46]. Because the initiation of myosin Ic’s power stroke is limited by phosphate release, myosin Ic can bind to actin in the ADP⋅Pi state but detach without proceeding through the cycle if it detaches prior to Pi release. Such an event contributes to the attachment to the filament but not to the time that the molecule spends in the strongly bound states. The time that the molecule spends in the strongly bound states therefore differs from that spent attached to the filament. The probability Psb of occupying the strongly bound states accordingly differs from the probability Pon of being attached to actin. Our complete cycle description allows us to explicitly calculate both probabilities and to compare them. We determine Psb in terms of the fraction of the cycle that the molecule spends in the strongly bound states as P sb ≡ t sb t sb + t wb = ∑ i = 3 5 P i, (3) in which tsb is the average time spent in the strongly bound states, twb is the average time spent in the weakly bound and detached states, and Pi is the steady-state probability (Eqs 45–49). Similarly, we obtain Pon from the fraction of the total cycle time during which the myosin molecule is attached to the filament as P on ≡ t on t on + t off = ∑ i = 2 5 P i, (4) in which ton is the average time that myosin is attached to the filament, toff the average time that myosin is detached, and Pi is again the steady-state probability (Eqs 45–49). Whereas the former quantity is closely related to the duty ratio, the later quantity is important for estimation of the number of bound molecules in an ensemble. The probabilities of being attached to actin and of occupying the strongly bound states depend on the ADP concentration, on the available actin, and on the external force (Fig 5). In general, because of the catch-bond behavior an increasing force enhances the probability of attachment to actin. An elevated ADP concentration likewise traps myosin Ic in the strongly bound ADP state and increases both probabilities (Fig 5a and 5c). An increased accessibility of actin enhances the binding of the myosin head, which results in a high-almost unity-probability of being bound to the filament at high actin concentrations (Fig 5d). In contrast, the probability of occupying the strongly bound states saturates at a high actin concentration, for entering these states is limited by phosphate release (Fig 5b). Although in vestibular hair cells myosin Ic activity is required for fast adaptation, the precise molecular details remain unknown [10]. Here we focus on two aspects that might contribute to the mechanism: the cooperative unbinding of an ensemble of myosin heads under force and a qualitative Ca2+ dependence that changes the binding probability and the elasticity of individual myosin Ic molecules [23,24]. In particular, we determine how these properties influence the overall elasticity of an ensemble. The myosin heads contribute to the rigidity of the adaptation motor by crosslinking the insertional plaque to the actin cytoskeleton. We think of each myosin head as a linear spring, arranged in parallel to the others, such that the overall stiffness is given by the sum of the actin-attached myosin heads multiplied by the stiffness of each myosin molecule. Because the binding and unbinding of the heads depend on the force and the nucleotide and actin concentrations, these quantities also influence the overall elastic properties of the ensemble. In general the binding process could be very complicated because of the geometry and possible steric interactions between the heads. Furthermore the helical structure of the actin filaments provides binding sites with an appropriate orientation only about every 37 nm [5]. These constraints change the number of myosin molecules that can potentially interact with actin. In our description, the total number of myosin heads is thus an effective number of molecules that can potentially bind to actin. To estimate the average number of bound myosin molecules in an ensemble, we use the attachment and detachment rates determined from our description of the chemomechanical cycle. We assume that each myosin head can bind to the filament with a binding rate kon and unbind with an unbinding rate koff. Both rates stem directly from our description, kon = ω12 + ω15 and koff from Eq 58. Because of the stochastic binding and unbinding, the number n of bound molecules fluctuates. To describe the system as a Markov chain, we introduce a state space (Fig 6a) associated with the number of bound myosin heads [47]. The effective transition rates between these states are k on n ≡ (N - n) k on, (5) and k off n ≡ n k off. (6) Here kon depends on the actin concentration and koff on the nucleotide concentrations and on the force f per myosin molecule. We assume that an external force F applied to the ensemble is distributed equally among the attached myosin molecules, resulting in the effective force f = F/n per attached head. If one head releases from the filament then the force is redistributed among the remaining bound heads and the force per myosin molecule accordingly increases, which changes the unbinding rate koff. In general this mechanism leads to cooperative effects because the unbinding rate depends on the number n of attached myosin heads. In the case in which the myosin heads act independently, the transition rates of a single head are independent of the number of attached myosin molecules. We determine the average number of bound myosin molecules from the linear Markov chain as explained in the Methods section, n = ∑ n = 0 N n 1 + ∑ l = 0 N - 1 ∏ i = 0 l k on i k off i + 1 - 1 ∏ j = 0 n - 1 k on j k off j + 1. (7) For the cooperative case in which koff = koff (F/n), we evaluate this equation. In the independent case, in which the unbinding rate koff is independent of the number of bound myosin heads, we can simplify this expression to n = N 1 + k off / k on = N t on t on + t off = N P on. (8) Note that Pon = Pon (f) is a function of the force acting on a single myosin head. For the independent case, we estimate this force by f = F/N. However, in this way we underestimate the magnitude of the force per molecule because we expect that N > n. For a better approximation, we distribute the external force between the mean number of bound motors, f = F/〈n〉, an approach that leads to an implicit equation for 〈n〉 that is not easy to solve. For physiological nucleotide concentrations and for 100 μM actin, we notice in Fig 5d that 〈Pon〉 ≈ 0. 5. Using this value, we estimate that in a group of 30 molecules about 〈 n ˜ 〉 ≃ 15 of them are bound on average. We then approximate the average force on a myosin molecule as f = F / 〈 n ˜ 〉 for the independent case. Note that in the independent case the force per myosin head does not depend on the number of bound heads, in contrast to the cooperative case. The mean number of bound myosin heads is influenced by the cooperative release of the molecules and the three approaches are different for intermediate forces (Fig 6b). We calculate the average number of bound myosin heads as a function of force for different total numbers of myosin molecules (Fig 7a). In small ensembles, the force per head is higher and therefore more heads are bound as a result of the catch-bond behavior. Increasing the concentration of available actin causes more myosin heads to attach to the filament (Fig 7b). To validate our effective description, we compare our analytic results to Monte Carlo simulations as detailed in the Methods. In these simulations, each myosin head is represented as a spring that is attached to a rigid common structure. At each time step of the simulation the extensions of all springs are calculated by solving Newton’s law of force balance. In this way, we obtain for each myosin head a force that determines the transition rates of the chemomechanical cycle of that molecule. There are important differences from the analytic approach. Whereas in the simulation a myosin head proceeds stochastically through the five-state chemomechanical cycle, the heads only bind and unbind in the analytic description. As a consequence the myosin molecules step stochastically and exert fluctuating forces on each other, which in turn influences their dynamics. In our analytic model, the myosin heads are only indirectly coupled through the number of bound motors and not through an elastic interaction. The simulations show reasonable agreement with the analytic results (Figs 6b and 7). An increased coupling stiffness increases the forces between the myosin heads, which in turn result in a longer attachment because of the catch-bond behavior (Fig 6b). Especially for a high actin concentration, the agreement between the simulations and the analytic description is very good. In the following, we will focus on this particular case and therefore consider only the analytic description. These results show that the average number of bound myosin heads depends on the external force, the total number of myosin molecules, the actin concentration, and-not shown here-the nucleotide concentrations. We expect that the mechanical properties of a cellular structure including myosin Ic molecules also depend on these quantities. To investigate the elastic properties of an ensemble of myosin Ic heads, we determine the force-extension relation F = n κ x, (9) in which κ is the spring constant of a single myosin head. The underlying assumption of this approach is a linear force-extension relation of the individual myosin heads, for which we take the value of κ = 500 μN/m [21]. Applying forces below 20 pN to the ensemble leads to an extension smaller than 5 nm (Fig 8a). A reduced total number N of myosin molecules increases the extension because the force per myosin head is larger and stretches it farther. To test whether a mechanical release of myosin Ic molecules is related to fast adaptation, we investigate two qualitative effects of Ca2+. First, Ca2+ could decrease the binding probabilities of the myosin head to actin [23]. Second, it could change the stiffness of myosin by initiating the dissociation of one or more calmodulin molecules from the light chains, allowing the myosin molecules to attain a more flexible conformation [24]. We next consider the mechanical release owing to Ca2+ binding, Δ x ≡ F 1 κ Ca 2 + n Ca 2 + - 1 κ n. (10) We first study the effect of a reduced binding probability on the mean number of bound myosin molecules and maintain their stiffness before and after Ca2+ binding, κ Ca 2 + = κ = 500 μ N/m. We reduce the binding probability by the factor β and determine the resulting release for N = 10 or N = 20 myosin molecules (Fig 8b). A large decrease of the binding probability leads to fewer bound molecules and a larger release. The release for a group of 10 myosin molecules exceeds that for an ensemble of 20 molecules: the force on each individual myosin head is higher and stretches the molecule farther. However, the overall distance for forces smaller than 20 pN is still less than 20 nm. When we add to the 100-fold decrease of the binding probability a tenfold decrease of myosin’s elasticity and determine the resulting release for different total numbers of myosin molecules (Fig 8c), the displacement is of the order of several tens of nanometers and becomes almost insensitive to force for a group of 50 myosins. An important goal of biology is understanding how the structures and interactions of molecules result in measurable functions of cells and organisms. By combining findings on different spatial scales in a consistent manner, mathematical descriptions help us understand how physiologically relevant function is determined by the interplay of molecular components. We have constructed a quantitative description of myosin Ic’s chemomechanical cycle and studied the resulting properties at both a single-molecule and an ensemble level, which allows us to discuss important implications on the physiological function of hair cells at the whole-cell level. On the single-molecule level, it is important to understand how different members of the large myosin family display distinct biophysical properties despite a common general structure of the chemomechanical cycle. To describe myosin Ic, we constructed such a cycle and chose as control parameter the nucleotide concentrations and the external force, both of which are experimentally accessible and biologically relevant. Our simplified, one-cycle description reproduces many of the characteristic features of myosin Ic, especially the force-dependent exit from the strongly bound states. The probabilities of occupying the different states indicate that myosin Ic’s strongly bound states are dominated by the ADP state for forces below 1. 5 pN and by the ATP state for larger forces (Fig 3). Although this behavior is in contrast to previous models in which the ADP state is the only force-sensitive state, it is nevertheless consistent with the role of myosin Ic in adaptation [5,21]. Increasing the ADP concentration traps the myosin heads in the ADP state, bound to actin filaments (Figs 3 and 2b). This effect can be reversed by increasing the ATP concentration (Fig 2b). Such a behavior accords with recordings of transduction currents in hair cells isolated from the bullfrog: changing nucleotide concentrations alters the relative occupancy of the states in the cross-bridge cycle and thus the number of bound myosin molecules, which in turn controls the tension on the mechanically sensitive ion channels. Indeed, in the presence of an ADP analog, adaptation disappears and the tension on the channels increases. Both effects can be reversed by increasing the concentration of ATP [48]. This qualitative agreement constitutes direct evidence that the model, although constructed from single-molecule measurements in vitro, captures important aspects of the behavior of living cells. Our description suggests a low effective velocity for myosin Ic. Although velocities of only tens of nanometers per second have been reported from motility experiments in vitro [10,49–51], larger values have been discussed [5]. In motility assays, multiple myosin molecules work together to create motion. How the velocity measured in motility experiments is related to the effective rate of a cross-bridge cycle and to other biophysical parameters of the molecules is an open question [52–56]. However, our stochastic simulations suggest that 200 elastically coupled myosin Ic molecules, each described by the five-state chemomechanical cycle, display a motility rate of 25 nm⋅s-1 which is in good agreement with the experimental values of 16–22 nm⋅s-1 [51]. In these experiments the myosin molecules where coupled through a membrane. Greater speeds of 60 nm⋅s-1 have been reported in gliding assays, but the data were acquired at a temperature of 37°C [22], whereas the numerical values of the biochemical rates of our model stemmed from experiments conducted at 20°C. We conclude that our description of myosin Ic constrained by single-molecule data accords with the experimental data on a larger scale. Speeds of tens of nanometers per second are too low to be consistent with rates estimated for the adaptation motor in the inner ear, which has been associated with the function of myosin Ic [5,10]. Depending on the species, the velocity of the adaptation motor ranges from several hundred to a few thousand nanometers per second [5,57,58]. The discrepancy between the velocities in vivo and in vitro might stem from several factors. It is still unknown to what extent these rates relate to the speed of myosin Ic molecules and to relaxations of other elastic elements. It has been suggested that the recoil of an elastic element located parallel to the myosin heads, the extent spring, contributes to the dynamics [59,60]. Furthermore the reaction rates of the myosin cycle could be different in vivo and in vitro. In particular, the complex composition of the cytosol and molecular modifications could lead to differences in the energy barriers between the states [61]. Another possibility is that myosin Ic, which has been shown to constitute the adaptation motor of young mice [10], might be replaced during subsequent development by the closely related paralog myosin Ih, which has been identified as a hair-bundle protein [62]. Myosin Ih’s molecular properties have yet to be characterized and it might operate more swiftly. In hair cells the deflection by a stimulus is communicated to the transduction channel by an elastic element, the gating spring. Of uncertain origin, this elasticity displays complex behavior with implications for sensory coding [63]. Our model suggests that a cluster of myosin Ic molecules contributes to this elasticity and additionally provide the regulatory function to explain fast adaptation. If we assume that Ca2+ reduces the binding probability of myosin by a hundredfold and its stiffness by tenfold, the resultant release on the order of 40 nm accords with measurements from frog hair bundles [10]. For displacements exceeding 400 nm the extent of fast adaptation is independent of the stimulus [10]. We speculate that the insensitivity of the release to the external force for an ensemble of 50 myosin molecules is related to this experimental observation (Fig 8c). Although biochemical studies have suggested that stereocilia contain around 100–200 myosin Ic molecules, the number of actively engaged molecules in an adaptation motor is probably lower [5]. Although myosin Ic’s cycle is slow, binding of Ca2+ could rapidly change the relative occupancy of specific states. Under force, most of the myosin heads are trapped in the ATP state. The binding of Ca2+ to a myosin Ic molecule triggers the release of calmodulin from the IQ domains and increases the molecule’s flexibility, as recently shown in a structural study [24,64]. A sudden increase of flexibility would release the myosin head from any force until all elastic elements have relaxed to a new equilibrium state. In the absence of force, our description predicts a transition rate for unbinding from the ATP state as large as ω 51 0 ≃ 314 s - 1. This value is so great that the head would unbind immediately, probably before the forces could be redistributed among the bound myosin molecules. Although the load-free biochemical rates have been reported to be rather insensitive to Ca2+, this mechanism might explain a possible Ca2+-induced unbinding of myosin molecules from the actin filament under force [23]. Such a fast disengagement of the myosin molecules is necessary for the adaptation motor to slide down the stereociliary actin and thus to relax the tension in the tip links in order to accomplish adaptation to an abrupt stimulus. Because the transduction channels have been localized at the lower end of the tip links, Ca2+ regulation of the adaptation motor is effective only at the next lower insertional plaque [65]. Because the tallest stereocilia lack transduction channels through which Ca2+ could enter, the forces between different rows of stereocilia are differently regulated. Inner hair cells consisting of three rows of stereocilia might therefore display less Ca2+-regulated slow adaptation [14]. How much of the hair cell’s function is impeded by the reduced regulation is an open question. Hair-bundle models based on detailed descriptions of the relevant molecular mechanisms, such as myosin Ic’s chemomechanical cycle, could provide more insight. Biochemical experiments and studies of single-molecule motility are ordinarily conducted under chemostatic conditions in which energy sources such as ATP and products such as ADP and Pi are maintained at nearly constant concentrations. In the present study, however, we have endeavored in two ways to model the behavior of myosin Ic under more lifelike conditions. First, we have imposed a thermodynamic constraint that requires the modeled reaction cycles to respect energy balance. And second, we have examined an extensive range of concentrations for the relevant nucleotides and their products. A typical stereocilium, which is about 3 μm in length and 0. 2 μm in diameter, has a volume of only 100 aL. Even a substance found at a high concentration in the cytoplasm, such as ATP at 1 mM, can be depleted rapidly in such a small volume. When transduction channels open, for example, the plasma-membrane Ca2+ ATPase in a stereocilium confronts a flood of Ca2+ that could exhaust the available ATP in only milliseconds! It is thus important to understand the operation of myosin Ic-based motors under realistic and potentially fluctuating conditions. A final feature of the adaptation motors that remains to be investigated is the noise associated with their activity. By pulling directly on a tip link, each motor influences the opening and closing of the transduction channel or channels at the link’s opposite end. In conjunction with thermal bombardment of the bundle as a whole and stochastic clattering of the transduction channels, the adaptation motors in a hair bundle thus contribute to the mechanical noise that interferes with the detection of faint sounds and weak accelerations [66]. It will be interesting to learn whether the activation mechanism of the myosin molecules in adaptation motors or perhaps their cooperative behavior has been optimized to mitigate this source of noise. Our study has provided new insights into biological mechanisms. The chemomechanical cycle suggests that the force-dependent unbinding rate is rather robust even under physiological nucleotide concentrations. Although the force-sensitive state is the ATP-bound state, an increased ADP concentration reduces the unbinding rate and the myosin Ic molecules are strongly bound to actin filaments. The elastic properties of an ensemble of myosin Ic molecules can be regulated by an external force and by the actin and nucleotide concentrations. Although the reaction rates of actin-bound myosin Ic are largely insensitive to Ca2+ [23], we have shown that in an ensemble of myosin Ic molecules a possible reduction of the binding rate and elasticity could nevertheless account for fast adaptation by hair cells. The cross-bridge cycle of a myosin molecule consists of distinct states associated with different biochemical compositions and molecular conformations. The transitions between these states involve myosin’s head binding to and unbinding from the actin filament, nucleotide binding and release, and conformational changes. We simplify the cross-bridge cycle and describe myosin’s dynamics with one state (1) in which the head is detached from actin and four states (2) − (5), in which the head is attached (Fig 1). The four actin-bound states correspond to distinct occupancies of the nucleotide-binding pocket: in state (2) ADP and Pi are bound, whereas in state (3) only ADP is bound. State (4) is the nucleotide-free state and state (5) refers to the ATP-bound state. We represent the cross-bridge cycle as a time-continuous Markov process for which we must specify the transition rates between the states. Although all transition rates could be force- and nucleotide-dependent, it is reasonable to assume that the main effect of the nucleotide concentrations is exerted on the nucleotide-binding rates. Before introducing those transition rates, we discuss the force dependencies of the mechanical transitions. The transition rates associated with a mechanical power stroke decrease with an increase in the opposing force. Experimental data indicate that myosin Ic performs its power stroke in two steps: the lever arm is remodeled by a distance of Δx1 ≃ 5. 8 nm upon phosphate release and then by Δx2 ≃ 2 nm upon ADP release [20]. Assuming local equilibrium, we associate the ratio of the forward and backward transition rates for the power stroke upon phosphate release with a Boltzmann factor as ω 23 ω 32 = exp (- (Δ G 23 + F Δ x 1) / k B T). (11) Here ΔG23 is the Gibbs free-energy difference between the states, FΔx1 is the mechanical work performed by the power stroke of distance Δx1 against the opposing load force F, and kB T is the Boltzmann constant times the temperature [67]. The equation above relies on an assumption of local equilibrium that does not indicate how the individual transition rates depend on the force. Therefore, we use the following general forms for the individual transition rates: ω 23 ≡ ω 23 0 exp (- δ 1 F Δ x 1 / k B T), (12) ω 32 ≡ ω 32 0 exp ( (1 - δ 1) F Δ x 1 / k B T), (13) in which we introduce the force-free rate constants ω 23 0, ω 32 0 and the force-distribution factor δ1 ∈ [0,1]. The restriction on the numerical values for the force-distribution factor is a consequence of the assumption that a force opposing the power stroke diminishes the corresponding transition rate [67,68]. For an effective description based on a projection of a high-dimensional free-energy landscape on to a single reaction coordinate, the force-distribution factor is not restricted [69]. Using the same argument as for the release of phosphate, the general forms of the forward and backward transition rates associated with the power stroke upon ADP release are ω 34 ≡ ω 34 0 exp (- δ 2 F Δ x 2 / k B T), (14) ω 43 ≡ ω 43 0 exp ( (1 - δ 2) F Δ x 2 / k B T). (15) To account for the force-dependent behavior of myosin Ic, we must include the force sensitivity of the isomerization following ATP binding [19]. In our simplified state space this sensitivity effectively changes the unbinding rate ω51 from the ATP state. We therefore introduce a force-dependent factor g (F) that modifies the unbinding rate ω 51 ≡ g (F) ω 51 0. (16) We require that for zero force g (F = 0) = 1 and for large force g (F ≫ 1) = ωoff saturates and use g (F) ≡ 2 (1 - ω off) 1 + exp (ξ F / k B T) + ω off, (17) in which ξ is a characteristic length scale. In general the binding interface between the head of a molecular motor and its filament is more complicated than the idealized receptor-ligand bond considered by Bell [70]. The bond interface consists of multiple partial charges that lead to complex unbinding pathways through multiple states in the free-energy landscape [71–74]. To capture the characteristic behavior, we use the force factor of Eq 17 that has been used previously to describe the chemomechanical cycle of kinesin-1 and myosin V [75–79]. In the following we give an intuitive justification of the force factor given in Eq 17. In our description the ATP state (5) comprises several sub-states including the binding and isomerization of ATP. The unbinding rate ω51, which must be considered as an effective rate for proceeding through all the sub-states, therefore includes the force dependence of the isomerization step. As a first approximation, we consider that isomerization is not associated with a conformational change that would result in a displacement of an applied load. The free-energy between the state (A) before isomerization and the state (B) after isomerization accordingly does not depend on the applied force. We assume that an applied force increases the free-energy barrier between those two states without changing the difference of the energy between the states. Motivated by Kramers rate theory [80], we use the force dependence ω A B = ω A B 0 exp (- F ξ / k B T) for the forward transition rate and the same force dependence for the reverse transition rate ω B A = ω B A 0 exp (- F ξ / k B T). We consider the main forward pathway through these sub-states, → (A) ⇌ ω A B ω B A (B) → ω B, (18) which implies the effective transition rate ω eff = ω B 1 + ω B A 0 ω A B 0 + ω B ω A B 0 e F ξ / kB T. (19) This approach leads to a force dependence similar to that in Eq 17. Using the same argument for the reverse pathway, we find that the transition rate ω54 has a similar force dependence. As described later in Eq 43, the force dependence of the transition rate ω54 is imposed naturally by thermodynamic consistency. To capture the dependence on the nucleotide concentrations, we consider the nucleotide-binding steps as first-order reactions that are independent of force, leading to ω 32 0 ≡ ω ^ 32 0 [ P i ], (20) ω 43 0 ≡ ω ^ 43 0 [ ADP ], (21) ω 45 ≡ ω ^ 45 0 [ ATP ]. (22) Note that the units of the rate constants with a caret are M−1s−1. Such a linear dependence of the transition rates on the reactants is motivated by macroscopic chemical-reaction laws and is widely used to describe chemomechanical cycles [2,67]. In a similar way, we assume a linear dependence of the actin-binding rates on the actin concentration, ω 12 ≡ ω ^ 12 [ actin ], (23) ω 15 ≡ ω ^ 15 [ actin ]. (24) The remaining transition rate ω54 is determined by a balance condition obtained from thermodynamic consistency. We have specified above the general forms of the transition rates of our theoretical description of myosin Ic. We next introduce the dynamics and the thermodynamic constraints. We consider the stochastic dynamics of the myosin head as a continuous-time Markov process [81]. The probability Pi (t) of finding myosin in state (i) therefore evolves in time t according to the master equation d d t P i (t) = - ∑ j Δ J i j (t), (25) with a local net flux between the states (i) and (j) given by Δ J i j (t) ≡ P i (t) ω i j - P j (t) ω j i. (26) A thermodynamically consistent description, which ensures that myosin does not produce more mechanical energy than the chemical energy provided by the nucleotide concentrations, implies a relation between the mechanical energy and the chemical energy. This relation provides a constraint on the transition rates of the cycle that is obtained by incorporating free-energy transduction [37]. We can express the change in the Gibbs free energy of the hydrolysis reaction for a dilute solution by Δ μ ≡ k B T ln [ ATP ] [ ADP ] [ P i ] K eq, (27) in which Keq is the equilibrium constant for the reaction, here with the numerical value Keq ≃ 4. 9 ⋅ 105 M [2]. Note that at the equilibrium concentration of the nucleotides the change in the free energy Δμ vanishes. The mechanical energy, which is the work done by the protein against an external force F, is given by E me ≡ (Δ x 1 + Δ x 2) F. (28) This mechanical energy is produced when the protein passes through a forward cross-bridge cycle, which in our description represents directed transitions through the states (1), (2), (3), (4), (5), and finally (1). In contrast, the backward cycle is associated with a path traversed in the opposite direction. Thermodynamically consistent coupling of the energy conversion by the protein to the hydrolysis reaction then imposes the constraint ω 12 ω 23 ω 34 ω 45 ω 51 ω 21 ω 32 ω 43 ω 54 ω 15 = exp ( (Δ μ - E me) / k B T), (29) which can further be related to the entropy production [37,77]. This equation has an intuitive interpretation [37]: the left side is the ratio of the average number of complete forward cycles to the average number of complete backward cycles, whereas the right side is the exponential of the difference between the chemical input energy and the mechanical output energy. At equilibrium this difference vanishes and the right side is equal to one, which requires the completion of identical numbers of forward and backward cycles. This constraint ensures that the average net cycling of the protein is thermodynamically consistent with the energy input. In our simple approach each hydrolysis reaction produces a power stroke, meaning that there are no futile cycles and therefore the chemistry is tightly coupled to the mechanics. We incorporate into our description of myosin Ic as many experimental data as possible. Some transition rates and parameter values have been reported [19,20]; an overview of these is given in Table 1. The force-dependent lifetime of an actin-myosin bond has been determined with an optical trap [20]. Because of the finite time resolution of the experimental apparatus, it is reasonable to assume that the strongly bound states rather than the weakly bound ones dominate the lifetime. We accordingly interpret the reported lifetime as the time that myosin is attached to actin in the strongly bound states. As a consequence, the reported duty ratio r ≃ 0. 11 and force-free binding time in the strongly bound states tsb ≃ 0. 213 s provide an estimate of the time that the myosin molecule resides in the weakly bound states, t wb = t sb 1 - r r ≃ 1. 72 s. (30) The rate constant, ω 23 0 for phosphate release has been reported for human myosin-IC [82] as ω 23 0 ≃ 1. 5 s - 1. (31) Because of the opaque nomenclature of myosin-I isoforms, human myosin-IC is instead myosin Ie in the nomenclature of the Human Genome Organization [83]. This value is reasonable if the rate-limiting step is phosphate release and the order of magnitude is in agreement with the considerations for myosin Ic given in [19]. We next discuss the binding probability and the free-energy difference associated with the power stroke. Because there are to our knowledge no direct measurements for myosin Ic, we use values reported for myosin II. To estimate the probability π2 that the head binds in state (2), we refer to the cycle for rabbit skeletal muscle [2]. The reported numbers suggest a probability π 2 ≃ 0. 998, (32) which implies that myosin starts its cycle predominantly in state (2). Using the definition of this binding probability π 2 = ω 12 ω 12 + ω 15, (33) we can relate the binding rates to each other as ω 12 = π 2 ω 15 1 - π 2. (34) Because both transition rates depend linearly on the actin concentration (Eqs 23 and 24), the actin concentration cancels and ω ^ 12 = π 2 ω ^ 15 1 - π 2. (35) Several studies suggest that a large free-energy difference is associated with the main power stroke [2,28,32–34,38]. Using the value ΔG23 ≃ −15 kBT inferred from fitting a model of the myosin II cycle to experimental data acquired with frog muscle [28], we obtain the ratio ω 23 0 ω ^ 32 0 [ P i ] = exp (- Δ G 23 / k B T), (36) which provides the phosphate-binding rate constant ω ^ 32 0 = ω 23 0 [ P i ] exp (Δ G 23 / k B T). (37) Assuming a phosphate concentration of [Pi] = 1 mM in frog muscle and using Eq 31, we determine that ω ^ 32 0 ≃ 4. 5 · 10 - 10 s - 1 μ M - 1. (38) A complementary approach is to use the values for the rabbit muscle cycle [2]; we then obtain ω ^ 32 0 ≃ 4. 6 · 10 - 9 s - 1 μ M - 1, a value one order of magnitude larger. Because both rate constants are very small compared to the other transition rates of our description, they make no significant difference in our results. In a one-cycle description, the effects of changing the ATP and ADP concentrations are tightly coupled and determined by the magnitudes of the rate constants. If we assume a very fast and irreversible unbinding from the ATP state, ω51 ≫ 1 and ω15 = 0, and an irreversible Pi release, ω32 = 0, the average time in the strongly bound states reads t sb = ω 34 + ω 43 + ω 45 ω 34 ω 45. (39) In the force-free case the ADP and ATP binding rates are given in Eqs 21 and 22. Using the equilibrium binding constant KADP to estimate the rate constant for ADP binding as ω ^ 43 0 = ω 34 0 K ADP, (40) we rewrite Eq 39 as t sb = ω 34 0 (1 + [ ADP ] / K ADP) + ω ^ 45 0 [ ATP ] ω 34 0 ω ^ 45 0 [ ATP ]. (41) The two rate constants and the equilibrium constant have been determined experimentally as ω 34 0 ≃ 3. 9 s - 1, ω ^ 45 0 ≃ 0. 26 s - 1 μ M - 1 and KADP ≃ 0. 22 μM [20]. Because of the low ATP-binding rate constant and the small equilibrium constant KADP for ADP release, the lifetime of the strongly bound states is very sensitive to elevated ADP concentrations. Because the experimental findings differ, we resolve this problem in our one-cycle description by using the higher value of KADP ≃ 1. 8 μM for the equilibrium constant for ADP release [22,23]. Although another possibility would be to introduce a multi-cycle description, that strategy increases complexity and the number of unknown parameters. To satisfy thermodynamic consistency, we express the transition rate for ATP release in terms of all the other transition rates as given by the balance condition of Eq 29, ω 54 = ω 12 ω 23 ω 34 ω 45 ω 51 ω 21 ω 32 ω 43 ω 15 exp (- (Δ μ - (Δ x 1 + Δ x 2) F) / k B T). (42) This transition rate is dependent on force in the same way as ω51 but independent of the nucleotide concentrations, as can be concluded by applying eqs (12) – (16), (20) – (22) and (27), resulting in ω 54 = ω 12 ω 23 0 ω 34 0 ω ^ 45 0 g (F) ω 51 0 ω 21 ω ^ 32 0 ω ^ 43 0 ω 15 K eq ≡ ω 54 0 g (F). (43) We are left with seven unknown parameter values: the unbinding rates ω51 and ω21, the binding rate ω15, the force distribution factors δ1 and δ2, the characteristic length ξ, and the offset rate ωoff. To estimate these values, we use analytic expressions of the average time spent in the weakly bound states and the effective unbinding rate from the strongly bound states and fit these functions to the experimental data. We derive these analytic expressions in the following section. The probability Pi of finding myosin in one of the states of the cycle is given by the solution to the steady-state master equation 0 = - ω 12 - ω 15 ω 21 0 0 ω 51 ω 12 - ω 21 - ω 23 ω 32 0 0 0 ω 23 - ω 32 - ω 34 ω 43 0 0 0 ω 34 - ω 43 - ω 45 ω 54 ω 15 0 0 ω 45 - ω 54 - ω 51 P 1 P 2 P 3 P 4 P 5, (44) and read P 1 ≡ (ω 23 ω 34 ω 45 ω 51 + ω 21 (ω 34 ω 45 ω 51 + ω 32 (ω 43 + ω 45) ω 51 + ω 32 ω 43 ω 54) / N, (45) P 2 ≡ (ω 12 (ω 34 ω 45 + ω 32 (ω 43 + ω 45) ) ω 51 + (ω 12 + ω 15) ω 32 ω 43 ω 54) / N, (46) P 3 ≡ (ω 15 (ω 21 + ω 23) ω 43 ω 54 + ω 12 ω 23 (ω 45 ω 51 + ω 43 (ω 51 + ω 54) ) ) / N, (47) P 4 ≡ (ω 15 (ω 23 ω 34 + ω 21 (ω 32 + ω 34) ) ω 54 + ω 12 ω 23 ω 34 (ω 51 + ω 54) ) / N, (48) P 5 ≡ (ω 12 ω 23 ω 34 ω 45 + ω 15 (ω 21 ω 32 ω 43 + ω 23 ω 34 ω 45 + ω 21 (ω 32 + ω 34) ω 45) ) / N, (49) in which N is determined by the normalization ∑i Pi = 1. To determine the effective unbinding rate from the strongly bound states, we calculate the average attachment time in the strongly bound states using a framework introduced by Hill [84,85]. We promote the detached state (1) and weakly bound state (2) to absorbing states by setting the transition rates ω15, ω23, ω12, and ω21 to zero (Fig 9a). The associated effective unbinding rate then becomes the inverse of the average time to absorption for the appropriate initial condition. The basic idea is to use an ensemble average instead of a time average. The dynamics of the correct ensemble is described by a closed diagram in which the absorbing state is eliminated by redirecting the transitions into that state to the starting states weighted with the appropriate starting probabilities [86]. For example, the transition from state (3) to state (2) is redirected with the weight 1 − π3 to state (5) and with weight π3 to state (3). The latter transition, a self loop, cancels in a master equation and can therefore be disregarded. This procedure creates a closed diagram (Fig 9b) for which the steady-state probability pi of being in state (i) is determined from the master equation 0 = - (1 - π 3) ω 32 - ω 34 ω 43 π 3 ω 51 ω 34 - ω 43 - ω 45 ω 54 (1 - π 3) ω 32 ω 45 - ω 54 - π 3 ω 51 p 3 p 4 p 5, (50) together with the normalization condition ∑pi = 1. This probability distribution provides the average state occupancy before absorption. The average rate of arrivals at either of the absorbing states is therefore given by the probability current, which is identical to the effective unbinding rate from the strongly bound states t sb - 1 ≡ ω 32 p 3 + ω 51 p 5. (51) The probability of starting in state (3) and not in state (5) is given by the relative probability current into state (3) of the complete cycle as π 3 ≡ ω 23 P 2 ω 23 P 2 + ω 15 P 1, (52) in which P1 and P2 are given in Eqs 45 and 46, respectively. For the sake of completeness we give the rather cumbersome expression for the effective unbinding rate from the strongly bound states, t sb − 1 = ( ( (ω 15 ω 21 + ω 12 ω 23) ω 32 ω 43 + (ω 12 ω 23 (ω 32 + ω 34) + ω 15 (ω 21 ω 32 + (ω 21 + ω 23) ω 34) ) ω 45) ω 51 + (ω 15 ω 21 + (ω 12 + ω 15) ω 23) ω 32 ω 43 ω 54) / H, (53) in which H ≡ ω 15 (ω 21 (ω 32 (ω 43 + ω 45) + ω 34 ω 45) + ω 23 ω 34 ω 45 + (ω 23 (ω 34 + ω 43) + ω 21 (ω 32 + ω 34 + ω 43) ) ω 54) + ω 12 ω 23 ( (ω 43 + ω 45) ω 51 + ω 43 ω 54 + ω 34 (ω 45 + ω 51 + ω 54) ). (54) The time that myosin spends in the weakly bound states can be obtained from Eq 3 as t wb = t sb 1 ∑ i = 3 5 P i - 1, (55) in which Pi are the steady-state probabilities given in Eqs 47–49. To determine the time during which myosin is attached to the filament, we promote state (1) to an absorbing state. The corresponding closed diagram is obtained by redirecting the transition from state (2) to state (1) with weight 1 − π2 to state (5) and the transition from state (5) to state (1) with weight π2 to state (2). The steady-state probability si of being in state (i) for this closed diagram is the solution of the master equation 0 = - ω 23 - (1 - π 2) ω 21 ω 32 0 π 2 ω 51 ω 23 - ω 32 - ω 34 ω 43 0 0 ω 34 - ω 43 - ω 45 ω 54 (1 - π 2) ω 21 0 ω 45 - ω 54 - π 2 ω 51 s 2 s 3 s 4 s 5, (56) together with the normalization condition ∑si = 1. This probability distribution gives the probability current into state (1), which is identical to the unbinding rate from the actin filament k off ≡ ω 21 s 2 + ω 51 s 5. (57) Again, for completeness, we give the unwieldy expression for the unbinding rate from the filament, k off = (ω 23 ω 34 ω 45 + ω 21 (ω 34 ω 45 + ω 32 (ω 43 + ω 45) ) ) ω 51 + ω 21 ω 32 ω 43 ω 54 A + B + C, (58) in which A ≡ π 2 (ω 34 ω 45 + ω 32 (ω 43 + ω 45) ) ω 51 + ω 32 ω 43 ω 54, (59) B ≡ (1 - π 2) ω 21 (ω 34 ω 45 + (ω 34 + ω 43) ω 54 + ω 32 (ω 43 + ω 45 + ω 54) ), (60) C ≡ ω 23 (π 2 (ω 43 + ω 45) ω 51 + ω 43 ω 54 + ω 34 (ω 45 + π 2 ω 51 + ω 54) ), (61) π 2 ≡ ω 12 / (ω 12 + ω 15). (62) Note that π2 is independent of the time toff during which myosin is detached from the filament, and therefore independent of the actin concentration. As a consequence the unbinding rate koff is also independent of the actin concentration. To describe the ensemble of myosins as a Markov chain, we introduce a state space (Fig 6a) associated with the number of bound myosin heads [47]. Assuming that the heads bind and unbind independently of one another, transitions between these states can be expressed in terms of the individual binding and unbinding rates kon and koff. A transition from state (n), in which n myosins are bound, to state (n + 1) is associated with the binding rate k on n ≡ (N - n) k on. (63) The reverse transition is described by the unbinding rate k off n ≡ n k off. (64) We next incorporate an external force F into the description. As a first approximation, we assume that the myosin heads share this load equally, resulting in an effective force F/n exerted on each of the bound myosin heads and in a modified transition rate k off n ≡ n k off (F / n). (65) With this specific choice of the transition rates we determine the probability Sn of being in state (n) from a master equation. The solution for such a finite linear Markov chain is a standard result in stochastic dynamics and can be obtained recursively or by standard methods [47,81]. The probability of being in state (n) is S n = S 0 ∏ i = 0 n - 1 k on i k off i + 1, (66) in which S0 is determined from the normalization ∑Si = 1 as S 0 = 1 + ∑ n = 0 N - 1 ∏ i = 0 n k on i k off i + 1 - 1. (67) From this probability distribution we obtain the average number of bound myosin heads as n = ∑ n = 0 N n S n. (68) We describe the dynamics of a myosin head with the five state chemomechanical cycle that we developed in this study. Each myosin head is coupled with a spring to a rigid common structure. The extension of this spring determines the force that is exerted on the myosin molecule and thus all transition rates of its cycle. We assume that a myosin head binds without tension to the actin filament and when it proceeds through its cycle the power-stroke transitions stretches the spring. At each time step we determine the extensions of all myosin springs from Newton’s law of force balance and adjust all transition rates accordingly. For the Monte Carlo simulations we use a Gillespie algorithm which is a standard method to simulate multi-component stochastic reactions and used for elastically coupled motor molecules [35,78,79,88]. After disregarding the transient behavior of our simulations, we determine the average number of myosins bound to actin from a time average. We consider all myosin molecules in state (2) to (5) bound to actin.

Myosin molecules are biological nanomachines that transduce chemical energy into mechanical work and thus produce directed motion in living cells. These molecules proceed through cyclic reactions in which they change their conformational states upon the binding and release of nucleotides while attaching to and detaching from filaments. The myosin family consists of many distinct members with diverse functions such as muscle contraction, cargo transport, cell migration, and sensory adaptation. How these functions emerge from the biophysical properties of the individual molecules is an open question. We present an approach that integrates recent findings from single-molecule experiments into a thermodynamically consistent description of myosin Ic and demonstrate how the specific parameter values of the cycle result in a distinct function. The free variables of our description are the chemical input and external force, both of which are experimentally accessible and define the cellular environment in which these proteins function. We use this description to predict the elastic properties of an ensemble of molecules and discuss the implications for myosin Ic's function in the inner ear as a tension regulator mediating adaptation, a hallmark of biological sensory systems. In this situation myosin molecules cooperate in an intermediate regime, neither as a large ensemble as in muscle nor as a single or a few molecules as in intracellular transport.

lay_plos

Taenia solium is known to cause human cysticercosis while T. saginata does not. Comparative in vitro and in vivo studies on the oncosphere and the postoncospheral (PO) forms of T. solium and T. saginata may help to elucidate why cysticercosis can occur from one and not the other. The aim of this study was to use in vitro culture assays and in vivo models to study the differences in the development of the T. solium and T. saginata oncosphere. Furthermore, this study aimed to evaluate the expression of cytokines and metalloproteinases (MMPs) in human peripheral blood mononuclear cells (PBMCs), which were stimulated by these oncospheres and PO antigens. T. solium and T. saginata activated oncospheres (AO) were cultured in INT-407 and HCT-8 intestinal cells for 180 days. The T. solium began to die while the T. saginata grew for 180 days and developed to cysticerci in INT-407 cells. Rats were inoculated intracranially with AO and PO forms of either T. saginata or T. solium. Rats infected with T. solium AO and PO forms developed neurocysticercosis (NCC), while those infected with the T. saginata did not. Human PMBCs were stimulated with antigens of AO and PO forms of both species, and the production of cytokines and metalloproteinases (MMPs) was measured. The T. solium AO antigen stimulated a higher production of IL-4, IL-5, IL-13, IFN-γ, and IL-2 cytokines compared to T. saginata AO. In the PO form, the T. saginata PO antigen increased the production of IL-4, IL-5, IL-13, IFN-γ, IL-1β, IL-6, IL-10, TNF-α and IL-12 cytokines compared to T. solium, suggesting that this global immune response stimulated by different forms could permit survival or destruction of the parasite depending of their life-cycle stage. Regarding MMPs, T. solium AO antigen stimulated a higher production of MMP-9 compared to T. saginata AO antigen, which may be responsible for altering the permeability of intestinal cells and facilitating breakdown of the blood-brain barrier during the process of invasion of host tissue. Taenia solium and T. saginata are two taeniid cestodes that cause the diseases taeniasis and cysticercosis [1]. These are zoonotic diseases, and swine and bovine act as intermediate hosts, causing porcine and bovine cysticercosis, respectively. Humans act as the definitive hosts in both T. solium and T. saginata infection leading to taeniasis. In the case of T. solium, humans can also act as accidental intermediate hosts causing human cysticercosis [2]. However, only T. solium causes human cysticercosis, while T. saginata does not [3]. When cysticercosis involves the central nervous system in humans, it is called neurocysticercosis (NCC). NCC is common throughout Latin America, sub-Saharan Africa, most of Asia, and parts of Oceania. Human NCC is believed to be the leading cause of acquired epilepsy worldwide [4,5]. The eggs of T. solium and T. saginata contain a six-hooked larva called the oncosphere [6]. When the eggs hatch, this oncosphere is released into the intestine. Intestinal fluid dissolves the oncospheral membrane, releasing and activating the oncosphere. The T. solium activated oncosphere can then penetrate the intestinal wall. Once in the tissue, usually in the muscle or the central nervous system, the oncosphere can transform into a postoncospheral form, and completely develop into cysticerci—a larval stage that consists of a fluid-filled sac containing an invaginated scolex. When this happens, the parasite produces a variety of molecules, which modulate the host immune response in order to evade parasite destruction [7]. The postoncospheral (PO) form is an intermediate stage between an oncosphere and a fully developed cysticercus in tissue [8]. The PO form of T. solium and T. saginata can be obtained in vitro by co-culture of oncospheres with a monolayer of mammalian feeder cells [9,10]. T. solium oncosphere and in-vitro generated PO forms can develop into cysticercus in rats causing NCC [9,11]. However, little is known regarding the in vitro development of the oncosphere to PO form of T. solium and T. saginata, specifically the immunological events that occur at the host/parasite interface. Also, it is not known if the development of the T. saginata oncosphere to PO form could cause NCC in the rat model as T. solium does. In vitro and in vivo models could lead to a better understanding of host-parasite relationships. Host immune cells such as macrophages, lymphocytes, and polymorphonuclear leukocytes can produce cytokines and metalloproteinases (MMPs) in order to prevent the development of the parasite [12,13]. Because of this, the parasite has developed mechanisms to evade or modulate the host immune response. Comparative studies on the oncosphere and the PO form of T. solium and T. saginata are limited. This study focused on the in vitro development of the oncosphere to the PO form for T. solium and T. saginata. These in vitro-developed larvae were then tested for infectivity in rats. Later, we obtained antigens from the T. solium and T. saginata oncosphere and PO forms to stimulate the production of cytokines and MMPs in healthy human peripheral blood mononuclear cells (PBMCs). HCT-8 and INT-407 cells, obtained from the American Tissue Culture Collection (ATCC, Manassas, VA), were used to obtain T. solium and T. saginata PO forms. Cells were incubated at 37°C in 5% CO2 and grown in a specific medium as recommended by ATCC (EMEM media for INT-407, and RPMI for HCT-8; all medium was supplemented with 10% fetal bovine serum). The medium was changed every two days. Once cell confluency was obtained, cells were harvested using trypsin-EDTA (Sigma Chemical Co). Cells were placed into 24-well plates (1x105 cells per well) for maturation assay. The assays described below were performed when cells formed a monolayer. Tapeworms were collected after medical treatment of newly diagnosed patients, as described by Jeri et al [14]. Hatching of eggs and oncosphere activation were performed, as described by Verastegui et al [15]. The eggs were obtained from gravid proglottids of adult tapeworms by gentle homogenization in a 2. 5% potassium dichromate solution (Sigma, St. Louis, Missouri). Eggs were then washed three times in distilled water with centrifugation steps to collect the eggs between washes in 2500g for 5 minutes. The eggs were hatched, and the oncospheres were released using a solution of 0. 75% sodium hypochlorite in water for 10 minutes (Mallinckrodt Baker, Inc, Phillipsburg, NJ). Oncospheres were then washed three times in RPMI medium (Sigma, St. Louis, Missouri), and activated by incubation at 37°C for 45 minutes (in the case of T. solium) or 90 minutes (in the case of T. saginata) with artificial intestinal fluid (1 g pancreatin (Sigma Chemical Co., St. Louis, MO), 200 mg Na2CO3, and 1 ml of fresh porcine bile (for T. solium), or 1 ml of fresh bovine bile (for T. saginata), with enough RPMI 1640 medium (pH 8. 04) to make 100mL). After activation, the oncospheres were washed three times with RPMI medium and counted using a Neubauer chamber. Parallel in vitro maturation assays with INT-407 and HCT-8 monolayer cells, using T. solium and T. saginata activated oncospheres, were conducted in order to compare the morphological characteristics during development of each species using the methodology reported by Chile et al [9]. Ten thousand activated oncospheres were cultured in confluent INT-407 and HCT-8 monolayer cells for two weeks. During that time, the medium was changed every three days. At day 15 of culture, the postoncospheral forms were collected and rinsed twice with fresh medium, then transferred to another well containing a confluent of monolayer cells. This process was repeated every three days for up to six months to allow the postoncospheral forms to continue to develop. Cultures were inspected daily using an inverted microscope (Leitz labovert FS). Parasites were collected at 15,30,60,120, and 180 days of incubation. To determine if T. saginata AO and PO forms can develop into viable cysts in vivo, 15-day old Holtzman rats, purchased from Universidad Peruana Cayetano Heredia, Lima, Peru, were infected intracranially (in the bregma) with oncospheres and 15-day old PO forms from either species following the methodology reported by Verastegui et al., 2015 [11]. Rats were anaesthetized with ketamine (100 mg/kg body weight) and xylazine (5 mg/kg body weight) before infection. Six rats were inoculated with 180 T. solium AO in 100 μL of saline solution; seven rats were inoculated with 180 T. saginata AO in 100 μL of saline solution. The negative control was 2 rats inoculated with saline solution. Eight rats were inoculated with ten T. solium 15-day PO forms in 100 μL of saline solution, eight rats were inoculated with ten T. saginata 15-day PO forms in 100 μL of saline solution, and five rats were inoculated with 100 μL of saline solution as a control. A 24-gauge syringe needle was used. After four months, necropsy was performed. Rats were anaesthetized with ketamine (100 mg/kg body weight) and xylazine (5 mg/kg body weight). Anaesthetized rats were perfused with 200 ml of PBS and then with 100 ml of 4% paraformaldehyde in PBS. Brains were carefully removed, post-fixed for 24 hours at 4°C with 4% paraformaldehyde in PBS, and stored in 70% ethanol. Brains were observed macroscopically to identify extraparenchymal cysticerci. Five millimeter coronal brain sections were cut until the intraparenchymal cysticerci were observed. Antigens were obtained from AO and 30 day-PO forms of both parasites. AO and PO were obtained as described above. Parasites were rinsed three times with PBS buffer, sonicated, and centrifuged at 10,000g for 15 min at 4°C. The supernatant (total soluble antigens) was separated, and proteins were quantified using Bradford Protein Assay (Bio-Rad) and stored at -70°C until ready for use. Healthy volunteers (n = 13) with negative serology for NCC were invited to participate in this study. After the volunteers signed an informed consent form, 10 mL of venous blood was collected from each non-infected donor. PBMCs were collected from 10 ml EDTA blood. The blood samples were centrifuged at 400g on a Ficoll-Hypaque gradient (Ficoll–Paque TM PLUS, GE Healthcare) for 10 minutes at room temperature for the separation of mononuclear cells. Cells were again suspended in RPMI medium plus 5% of inactivated human serum. Cell viability was evaluated with trypan blue and counted in a Neubauer chamber. Each well contained 2x105 group of cells was cultured in a 96-well plate at 37°C with 5% CO2 for 48 hours in an RPMI medium containing 5% of inactivated human serum. Cells were stimulated with 5 μg/ml of phytohemaglutinin (PHA) as the positive control, 20 μg/ml of AO antigens (both species, separately), and 20 μg/ml of antigens from PO forms at 30 days of maturation (both species, separately). At the end of the incubation period, PBMCs were harvested and centrifuged for 10 min at 400g, and supernatants were collected and stored at −70°C until tested for cytokine and metalloproteinase content by multiplex analysis. MILLIPLEX MAP kit High Sensitivity Human Cytokine Magnetic Bead Panel (Millipore) was used to measure cytokines (IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13 and TNF-α), and Fluorokine MAP (R&D system, Minneapolis USA) was used to measure MMP (MMP-2 and MMP-9) in the supernatant of stimulated PBMCs following the manufacturer’s instructions. The cytokines and MMP were detected by the Bio-plex 200 system (Bio-Rad Laboratories, Hercules, CA) using Luminex xMAP technology. Experiments were done according to the Guide for care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics Animals of the Universidad Peruana Cayetano Heredia, Lima, Peru (Permit Numbers: 61242). For cytokines and MMP assays, the median fluorescent intensities of the beads with the cytokines or MMP now bound were converted to concentrations (pg/ml), using a five-parameter logistic model using the Bio-Plex Manager™ 6. 0 software (Bio-Rad Laboratories, Hercules, CA). Each MMP´s or cytokine’s concentration was normalized by subtracting the data of medium alone (control). The Mann Whitney test was used to compare two unpaired groups, and ANOVA followed by Tukey’s post-test was used for three or more group using the Prism V 6. 0 statistical program (GraphPad). A P-value of <0. 05 was considered statistically significant. T. saginata and T. solium developed from the oncosphere to the PO form in both cell lines, INT-407 and HCT-8 cells (Table 1). In HCT-8 cells, both species of PO forms developed until 60 days of culture, and then began to die. However, after 60 days in INT-407 cells, the T. saginata PO forms continued to develop to cysticerci, while the T. solium PO forms began to die. At 180 days of culture 0. 25% of T. saginata oncospheres developed to cysticerci. The morphological characteristics of the T. saginata and T. solium PO forms obtained from culture in INT-407 monolayer cells were compared. At 15 days of incubation, both species were morphologically similar (Fig 1A), ranging between 60 to 250 μm in size. Both had an oval form without hooks and shared characteristic movement. For both species at 30 days of incubation, PO forms increased in size (between 200 to 1000 μm), maintained their oval form, and formed a protuberance at one end of the body (Fig 1B). At day 60 of culture, the T. solium PO form had a spherical shape, similar to a cyst without a scolex, and cells accumulated in the protuberance of one end of the body (Fig 1C). The T. saginata PO form had similar characteristics but also showed a neck-like prolongation at one end of the body (Fig 1C), reaching up to 3000 μm. Additionally, at day 60, T. solium PO forms began to die while T. saginata PO forms continued growing until 180 days of culture. At day 120 of culture, T. saginata developed into a cysticercus with a spherical form and an evaginated pre-scolex containing four characteristic suckers and measuring 4 mm in diameter (Fig 2A). At day 180 of culture, T. saginata cysticerci increased in size compared to 120 day, reaching up to 6 mm and also displayed a well-defined scolex and suckers (Fig 2B). We infected rats with T. saginata oncosphere and PO forms in order to evaluate if T. saginata can develop into cysticercus in vivo, as T. solium does. Table 2 shows rats that were infected with T. solium oncosphere or 15-days PO form developed NCC, while rats that were infected with T. saginata oncosphere or 15-days PO form did not develop NCC. Given that T. saginata does not develop to the cysticerci form in humans, we investigated the differences in host immune response to infection by T. solium and T. saginata. To determine this, we evaluated the production of cytokines during stimulation with AO and 30-day PO antigens of both species. When compared by stages (AO and PO form) species, both T. solium and T. saginata PO forms stimulated a higher production of the majority of cytokines evaluated than the AO forms (S1 Fig). Nevertheless, when compared by species stages (AO and PO form), we observed a higher production of IL-4, IL-5, IL-13, IFN-γ and IL-2 cytokines stimulated by the T. solium AO form (Fig 3A and 3B) compared to the T. saginata AO; but in PO form, the T. saginata 30-day PO form stimulated a higher production of a variety of cytokines, including, IL-4, IL-5, IL-13, IFN-γ, IL-1β, IL-6, IL-10, TNF-α and IL-12 compared to the T. solium 30-day PO form (Fig 4A, 4B and 4C). To evaluate whether MMPs are involved in the host immune response against the parasites, we stimulated PBMCs with T. solium and T. saginata forms. The T. solium AO stimulated higher MMP-9 production compared to the T. saginata AO, while in PO form there was no significant difference between both species (Fig 5). There was no significant difference in the production of MMP-2 by PBMCs stimulated with different antigens of both parasites (S2 Fig). The present study demonstrates that T. saginata activated oncospheres can develop into cysticerci in vitro in a human cell line, while T. solium AO do not. In contrast, in the rat model, the T. saginata oncosphere and PO forms did not develop to cysticerci in vivo as T. solium does. We suspect the differences in development between the two species in the in vitro model are due in large part to the environment of the growth media and the specific cell line, whereas in vivo it is the host immune response that plays a predominant role in regulating parasite development and survival. For instance, Heath and Smyth noted that serum used for in vitro culture contains factors unique to each host-parasite system, which can stimulate development of the parasite [16]. In our study, both species were cultured in media containing fetal bovine serum (FBS). FBS is known to contain large amounts of α- and β-globulin, proteins which have been shown to stimulate the development of the T. saginata oncosphere but not the development of other parasites like T. taeniaformis [17]. Additionally, the cell line used for culture likely plays an important role as T. saginata is able to develop to a cysticercus in vitro using cell line INT-407, while T. solium does not. On the other hand, T. saginata and T. solium do not develop into cysticercus using the HCT-8 cell line. INT-407 cells originate from the duodenum while the HCT-8 cell line originates from the colon. Furthermore, it is known that INT-407 cells are contaminated with HeLa cells that express different surface molecules that could promote the development of T. saginata. To our knowledge, this is the first study to achieve in vitro development of T. saginata PO forms to cysticerci. In the in vivo, T. solium oncosphere and PO forms developed to cysticerci in brain while T. saginata forms did not. This finding is likely due to T. solium’s ability to evade the host immune response, either through binding of host plasma proteins or by synthesizing surface proteins that are antigenically similar to those of the rat [18]. Previous work has demonstrated that T. taeniaeformis oncospheres are able to initiate PO development after acquiring a component of rat serum on their surface, a component that presumably protects the oncosphere in the newly invaded host from recognition as a foreign antigen [17]. In the same study, T. saginata failed to bind this component and was subsequently attacked by the host immune system. A similar observation has been noted in pigs after infection with T. saginata eggs [19]. Another explanation may be genetic factors that play a role in the rat’s resistance to infection from T. saginata [20]. Although the rat is not a natural host of T. solium, we observed in previous studies that the cysticerci that develop in the rat brain are morphologically equal to those that develop in humans, as is the observed inflammatory response and subsequent pathology [11,21]. Therefore, we believe the rat, like the human, has molecules that prevent the development of T. saginata. The cytokines response plays an important role in survival of the oncosphere at the time of initial infection. We suspect T. solium stimulates a different cytokines profile than T. saginata allowing the T. solium oncosphere to survive, while T. saginata is destroyed by the host. To probe this hypothesis, we stimulated cytokine and MMP production in healthy human PBMCs using antigens of oncosphere and PO forms from both species. We observed that T. solium oncospheres stimulate a higher production of IL-4, IL-5, IL-13, IFN- γ and IL-2 compared to T. saginata oncospheres. The IL-4, IL-5, IL-13, are cytokines typically associated with a T helper 2 (TH2) response, the predominant protective immune response against helminthic infections [22]. On the other hand, IFN- γ and IL-2 are associated with a T helper 1 (TH1) response, which is thought to be responsible for destruction of the parasite [23]. The TH2 response is important in protecting against extracellular helminthic parasites through suppression of the TH1 response, neutralization of toxins, and defense of the host against damage [22,24]. Inflammatory reactions are dependent on a delicate balance between TH1 and TH2 type responses. In the case of the T. solium oncosphere, it appears a mix immune reaction of TH1/ TH2 type response that aids survival of the parasite. Perhaps in doing so, T. solium oncospheres are able to migrate from the vasculature to the brain, where they develop into the PO form. Similar mixed TH1/TH2 phenotypes have been observed in patients with NCC [7,13] whereas a predominantly TH2-type response is associated with asymptomatic disease [25,26]. In both species, the 30-day PO form generated an overall greater inflammatory response than the AO form. Levels cytokines increased when exposed to equal concentrations of PO vs AO antigen, suggesting that antigen composition changes as the parasite is maturing [9]. However, the T. saginata 30-day PO form stimulated a profile of pro-inflammatory cytokines (IL-1β, IL-6, IL-12, TNF-α), plus a mix of TH1 and TH2 related cytokines (IL-4, IL-5, IL-13 and IFN-γ) that was stronger than the response produced by the T. solium 30-day PO form. IL-6, a pro-inflammatory cytokine, plays a role in the death of microorganisms by stimulating the behavior of neutrophils [27]; and TNF-α is strongly expressed at the sites of parasite and cell destruction [28]. Together with the overproduction of IL-4, IL-5, and IL-13, these cytokines may mount a response that could destroy the T. saginata PO form and prevents development of the cyst in vivo. Although these cytokines do have the suggested properties mentioned, they also could be involved in complex networks with mixed effects with respect to inflammation, for example IL-6 has been shown to exhibit anti-inflammatory properties [29]. T. solium oncospheres stimulated increased MMP-9 production in PBMCs compared to production by T. saginata oncospheres. MMP-9 is an endopeptidase produced by neutrophils, macrophages, monocytes, and intestinal epithelial cells [30,31]. It can degrade components of the blood brain barrier (BBB) as well as the extracellular matrix, increasing intestinal epithelial permeability [30,32,33]. MMP-9 has been associated with the breakdown of the BBB in a murine model of NCC [34,35] and is present in high concentrations in sera of symptomatic NCC patients [36]. We hypothesize that T. solium stimulates the production of MMP-9 as a means of enhancing epithelial permeability in order to pass restrictive biological barriers like the intestine and the BBB during early stages of infection. In conclusion, we have found novel evidence to suggest that T. saginata PO forms are capable of developing into cysticerci in the human cell line INT-407 while not in HCT-8 cells. In vivo, T. saginata fails to develop into cysticerci in the rat brain, suggesting there are factors in the host immune system (that are not present in the in vitro culture) that destroy the parasite. In the oncosphere stage, T. solium stimulated a strong mix of TH1 and TH2-related cytokines and MMP-9 production in healthy PBMCs, which may mediate the inflammatory response and promote oncosphere survival in the vasculature, aiding the entrance of oncospheres into the brain. In the PO form, T. saginata stimulated a strong pro-inflammatory and mix of TH1/TH2-related cytokines, responses that could be causing destruction of the parasite in the tissue. These differences between both species of Taenia found in vitro and in vivo could explain why the larval stage of T. saginata does not develop in the human host, while T. solium does, despite having similar life cycles.

Taenia solium and Taenia saginata are two parasites that cause the tissue infection cysticercosis in their intermediate hosts, pigs and cows, respectively. One major difference between them is that T. solium can also cause neurocysticercosis in the human brain, while T. saginata cannot. Neurocysticercosis is thought to be the major cause of adult-onset seizures in developing countries. It is not well understood why only T. solium can survive in human tissue; however, the host inflammatory response likely plays an important role. The authors found that human immune cells stimulated with T. solium in the early stages of the parasite life cycle produced a more robust cytokine response than T. saginata. However, in the mature stage, which occurs once T. solium reaches the brain, T. solium antigens stimulated a lower inflammatory response compared to T. saginata, suggesting the parasite is able to manipulate the host immune response in some way to evade destruction. These findings may support the differences in growth observed by the authors when rat brains were inoculated with either parasite species. This study provides new insights into the different ways T. solium and T. saginata activate the immune response to survive and develop within the host.

lay_plos

Judge Brett Kavanaugh’s opening statement today, a fiery, scorched-earth speech that may well have salvaged his bid for the Supreme Court, was not only aimed at the Senate Judiciary Committee. It carried an even bigger goal: to keep President Donald Trump from losing faith in his nomination. Kavanaugh was being urged by some — including old friends from Bush world — to take a softer approach today. But in the end, he tuned out the advice, and sat down to write a speech with a sole aide. An overarching goal: please Trump and, in turn, circle the conservative wagons behind him, an official close to the process said. Two White House officials said that while the President never seriously considered asking Kavanaugh to withdraw, he did discuss the idea — publicly and privately — that he would quickly announce another nominee and use the rejection of Kavanaugh to rally conservatives in the mid-term elections. It didn’t come to that, but there were “incredibly tense moments” in the White House after Christine Blasey Ford finished her testimony. A White House official said the president found Ford “compelling” and “very credible” and had serious questions about how Kavanaugh would perform. “He didn’t disappoint. He exceeded every expectation,” a White House official said. The President was publicly silent throughout the day, but his hand was guiding nearly every pivotal move – from Sen. Lindsey Graham’s boisterous speech to the decision to shut down the Republican-selected special prosecutor. READ: Christine Blasey Ford's Opening Statement For Senate Hearing Enlarge this image toggle caption J. Scott Applewhite/AP J. Scott Applewhite/AP Christine Blasey Ford has accused Supreme Court nominee Judge Brett Kavanaugh of sexually assaulting her when they were in high school in the early 1980s. On Thursday the psychology professor is testifying before the Senate Judiciary Committee. Read her opening statement below. Chairman Grassley, Ranking Member Feinstein, Members of the Committee. My name is Christine Blasey Ford. I am a Professor of Psychology at Palo Alto University and a Research Psychologist at the Stanford University School of Medicine. I was an undergraduate at the University of North Carolina and earned my degree in Experimental Psychology in 1988. I received a Master's degree in 1991 in Clinical Psychology from Pepperdine University. In 1996, I received a PhD in Educational Psychology from the University of Southern California. I earned a Master's degree in Epidemiology from the Stanford University School of Medicine in 2009. I have been married to Russell Ford since 2002 and we have two children. I am here today not because I want to be. I am terrified. I am here because I believe it is my civic duty to tell you what happened to me while Brett Kavanaugh and I were in high school. I have described the events publicly before. I summarized them in my letter to Ranking Member Feinstein, and again in my letter to Chairman Grassley. I understand and appreciate the importance of your hearing from me directly about what happened to me and the impact it has had on my life and on my family. I grew up in the suburbs of Washington, D.C. I attended the Holton-Arms School in Bethesda, Maryland, from 1980 to 1984. Holton-Arms is an all-girls school that opened in 1901. During my time at the school, girls at Holton-Arms frequently met and became friendly with boys from all-boys schools in the area, including Landon School, Georgetown Prep, Gonzaga High School, country clubs, and other places where kids and their families socialized. This is how I met Brett Kavanaugh, the boy who sexually assaulted me. In my freshman and sophomore school years, when I was 14 and 15 years old, my group of friends intersected with Brett and his friends for a short period of time. I had been friendly with a classmate of Brett's for a short time during my freshman year, and it was through that connection that I attended a number of parties that Brett also attended. We did not know each other well, but I knew him and he knew me. In the summer of 1982, like most summers, I spent almost every day at the Columbia Country Club in Chevy Chase, Maryland swimming and practicing diving. One evening that summer, after a day of swimming at the club, I attended a small gathering at a house in the Chevy Chase/Bethesda area. There were four boys I remember being there: Brett Kavanaugh, Mark Judge, P.J. Smyth, and one other boy whose name I cannot recall. I remember my friend Leland Ingham attending. I do not remember all of the details of how that gathering came together, but like many that summer, it was almost surely a spur of the moment gathering. I truly wish I could provide detailed answers to all of the questions that have been and will be asked about how I got to the party, where it took place, and so forth. I don't have all the answers, and I don't remember as much as I would like to. But the details about that night that bring me here today are ones I will never forget. They have been seared into my memory and have haunted me episodically as an adult. When I got to the small gathering, people were drinking beer in a small living room on the first floor of the house. I drank one beer that evening. Brett and Mark were visibly drunk. Early in the evening, I went up a narrow set of stairs leading from the living room to a second floor to use the bathroom. When I got to the top of the stairs, I was pushed from behind into a bedroom. I couldn't see who pushed me. Brett and Mark came into the bedroom and locked the door behind them. There was music already playing in the bedroom. It was turned up louder by either Brett or Mark once we were in the room. I was pushed onto the bed and Brett got on top of me. He began running his hands over my body and grinding his hips into me. I yelled, hoping someone downstairs might hear me, and tried to get away from him, but his weight was heavy. Brett groped me and tried to take off my clothes. He had a hard time because he was so drunk, and because I was wearing a one-piece bathing suit under my clothes. I believed he was going to rape me. I tried to yell for help. When I did, Brett put his hand over my mouth to stop me from screaming. This was what terrified me the most, and has had the most lasting impact on my life. It was hard for me to breathe, and I thought that Brett was accidentally going to kill me. Both Brett and Mark were drunkenly laughing during the attack. They both seemed to be having a good time. Mark was urging Brett on, although at times he told Brett to stop. A couple of times I made eye contact with Mark and thought he might try to help me, but he did not. During this assault, Mark came over and jumped on the bed twice while Brett was on top of me. The last time he did this, we toppled over and Brett was no longer on top of me. I was able to get up and run out of the room. Directly across from the bedroom was a small bathroom. I ran inside the bathroom and locked the door. I heard Brett and Mark leave the bedroom laughing and loudly walk down the narrow stairs, pin-balling off the walls on the way down. I waited and when I did not hear them come back up the stairs, I left the bathroom, ran down the stairs, through the living room, and left the house. I remember being on the street and feeling an enormous sense of relief that I had escaped from the house and that Brett and Mark were not coming after me. Brett's assault on me drastically altered my life. For a very long time, I was too afraid and ashamed to tell anyone the details. I did not want to tell my parents that I, at age 15, was in a house without any parents present, drinking beer with boys. I tried to convince myself that because Brett did not rape me, I should be able to move on and just pretend that it had never happened. Over the years, I told very few friends that I had this traumatic experience. I told my husband before we were married that I had experienced a sexual assault. I had never told the details to anyone until May 2012, during a couples counseling session. The reason this came up in counseling is that my husband and I had completed an extensive remodel of our home, and I insisted on a second front door, an idea that he and others disagreed with and could not understand. In explaining why I wanted to have a second front door, I described the assault in detail. I recall saying that the boy who assaulted me could someday be on the U.S. Supreme Court and spoke a bit about his background. My husband recalls that I named my attacker as Brett Kavanaugh. After that May 2012 therapy session, I did my best to suppress memories of the assault because recounting the details caused me to relive the experience, and caused panic attacks and anxiety. Occasionally I would discuss the assault in individual therapy, but talking about it caused me to relive the trauma, so I tried not to think about it or discuss it. But over the years, I went through periods where I thought about Brett's attack. I confided in some close friends that I had an experience with sexual assault. Occasionally I stated that my assailant was a prominent lawyer or judge but I did not use his name. I do not recall each person I spoke to about Brett's assault, and some friends have reminded me of these conversations since the publication of The Washington Post story on September 16, 2018. But until July 2018, I had never named Mr. Kavanaugh as my attacker outside of therapy. This all changed in early July 2018. I saw press reports stating that Brett Kavanaugh was on the "short list" of potential Supreme Court nominees. I thought it was my civic duty to relay the information I had about Mr. Kavanaugh's conduct so that those considering his potential nomination would know about the assault. On July 6, 2018, I had a sense of urgency to relay the information to the Senate and the President as soon as possible before a nominee was selected. I called my congressional representative and let her receptionist know that someone on the President's shortlist had attacked me. I also sent a message to The Washington Post's confidential tip line. I did not use my name, but I provided the names of Brett Kavanaugh and Mark Judge. I stated that Mr. Kavanaugh had assaulted me in the 1980s in Maryland. This was an extremely hard thing for me to do, but I felt I couldn't NOT do it. Over the next two days, I told a couple of close friends on the beach in California that Mr. Kavanaugh had sexually assaulted me. I was conflicted about whether to speak out. On July 9, 2018, I received a call from the office of Congresswoman Anna Eshoo after Mr. Kavanaugh had become the nominee. I met with her staff on July 11 and with her on July 13, describing the assault and discussing my fear about coming forward. Later, we discussed the possibility of sending a letter to Ranking Member Feinstein, who is one of my state's Senators, describing what occurred. My understanding is that Representative Eshoo's office delivered a copy of my letter to Senator Feinstein's office on July 30, 2018. The letter included my name, but requested that the letter be kept confidential. My hope was that providing the information confidentially would be sufficient to allow the Senate to consider Mr. Kavanaugh's serious misconduct without having to make myself, my family, or anyone's family vulnerable to the personal attacks and invasions of privacy we have faced since my name became public. In a letter on August 31, 2018, Senator Feinstein wrote that she would not share the letter without my consent. I greatly appreciated this commitment. All sexual assault victims should be able to decide for themselves whether their private experience is made public. As the hearing date got closer, I struggled with a terrible choice: Do I share the facts with the Senate and put myself and my family in the public spotlight? Or do I preserve our privacy and allow the Senate to make its decision on Mr. Kavanaugh's nomination without knowing the full truth about his past behavior? I agonized daily with this decision throughout August and early September 2018. The sense of duty that motivated me to reach out confidentially to The Washington Post, Representative Eshoo's office, and Senator Feinstein's office was always there, but my fears of the consequences of speaking out started to increase. During August 2018, the press reported that Mr. Kavanaugh's confirmation was virtually certain. His allies painted him as a champion of women's rights and empowerment. I believed that if I came forward, my voice would be drowned out by a chorus of powerful supporters. By the time of the confirmation hearings, I had resigned myself to remaining quiet and letting the Committee and the Senate make their decision without knowing what Mr. Kavanaugh had done to me. Once the press started reporting on the existence of the letter I had sent to Senator Feinstein, I faced mounting pressure. Reporters appeared at my home and at my job demanding information about this letter, including in the presence of my graduate students. They called my boss and coworkers and left me many messages, making it clear that my name would inevitably be released to the media. I decided to speak out publicly to a journalist who had responded to the tip I had sent to The Washington Post and who had gained my trust. It was important to me to describe the details of the assault in my own words. Since September 16, the date of The Washington Post story, I have experienced an outpouring of support from people in every state of this country. Thousands of people who have had their lives dramatically altered by sexual violence have reached out to share their own experiences with me and have thanked me for coming forward. We have received tremendous support from friends and our community. At the same time, my greatest fears have been realized – and the reality has been far worse than what I expected. My family and I have been the target of constant harassment and death threats. I have been called the most vile and hateful names imaginable. These messages, while far fewer than the expressions of support, have been terrifying to receive and have rocked me to my core. People have posted my personal information on the internet. This has resulted in additional emails, calls, and threats. My family and I were forced to move out of our home. Since September 16, my family and I have been living in various secure locales, with guards. This past Tuesday evening, my work email account was hacked and messages were sent out supposedly recanting my description of the sexual assault. Apart from the assault itself, these last couple of weeks have been the hardest of my life. I have had to relive my trauma in front of the entire world, and have seen my life picked apart by people on television, in the media, and in this body who have never met me or spoken with me. I have been accused of acting out of partisan political motives. Those who say that do not know me. I am a fiercely independent person and I am no one's pawn. My motivation in coming forward was to provide the facts about how Mr. Kavanaugh's actions have damaged my life, so that you can take that into serious consideration as you make your decision about how to proceed. It is not my responsibility to determine whether Mr. Kavanaugh deserves to sit on the Supreme Court. My responsibility is to tell the truth. I understand that the Majority has hired a professional prosecutor to ask me some questions, and I am committed to doing my very best to answer them. At the same time, because the Committee Members will be judging my credibility, I hope to be able to engage directly with each of you. At this point, I will do my best to answer your questions. READ: Brett Kavanaugh's Opening Statement For Senate Hearing Enlarge this image toggle caption Drew Angerer/Getty Images Drew Angerer/Getty Images On Thursday, Supreme Court nominee Brett Kavanaugh will appear before the Senate Judiciary Committee to testify on a sexual assault allegation by Christine Blasey Ford, who is also testifying. Read Kavanaugh's opening statement below, submitted to the panel on Wednesday. Mr. Chairman, Ranking Member Feinstein, and Members of the Committee: Eleven days ago, Dr. Ford publicly accused me of committing a serious wrong more than 36 years ago when we were both in high school. I denied the allegation immediately, unequivocally, and categorically. The next day, I told this Committee that I wanted to testify as soon as possible, under oath, to clear my name. Over the past few days, other false and uncorroborated accusations have been aired. There has been a frenzy to come up with something—anything, no matter how far-fetched or odious—that will block a vote on my nomination. These are last-minute smears, pure and simple. They debase our public discourse. And the consequences extend beyond any one nomination. Such grotesque and obvious character assassination—if allowed to succeed—will dissuade competent and good people of all political persuasions from serving our country. As I told this Committee the last time I appeared before you, a federal judge must be independent, not swayed by public or political pressure. That is the kind of judge I am and will always be. I will not be intimidated into withdrawing from this process. This effort to destroy my good name will not drive me out. The vile threats of violence against my family will not drive me out. I am here this morning to answer these allegations and to tell the truth. And the truth is that I have never sexually assaulted anyone—not in high school, not in college, not ever. Sexual assault is horrific. It is morally wrong. It is illegal. It is contrary to my religious faith. And it contradicts the core promise of this Nation that all people are created equal and entitled to be treated with dignity and respect. Allegations of sexual assault must be taken seriously. Those who make allegations deserve to be heard. The subject of allegations also deserves to be heard. Due process is a foundation of the American rule of law. Dr. Ford's allegation dates back more than 36 years, to a party that she says occurred during our time in high school. I spent most of my time in high school focused on academics, sports, church, and service. But I was not perfect in those days, just as I am not perfect today. I drank beer with my friends, usually on weekends. Sometimes I had too many. In retrospect, I said and did things in high school that make me cringe now. But that's not why we are here today. What I've been accused of is far more serious than juvenile misbehavior. I never did anything remotely resembling what Dr. Ford describes. The allegation of misconduct is completely inconsistent with the rest of my life. The record of my life, from my days in grade school through the present day, shows that I have always promoted the equality and dignity of women. I categorically and unequivocally deny the allegation against me by Dr. Ford. I never had any sexual or physical encounter of any kind with Dr. Ford. I am not questioning that Dr. Ford may have been sexually assaulted by some person in some place at some time. But I have never done that to her or to anyone. I am innocent of this charge.

She's positive it happened. That's one big theme emerging from Christine Blasey Ford's highly anticipated testimony Thursday before the Senate Judiciary Committee. Ford told senators she has no doubt that it was Supreme Court nominee Brett Kavanaugh who accosted her on a bed at a party in 1982. CNN takes note of an early exchange between Democratic Sen. Dianne Feinstein and Ford. "So what you're telling us is this could not be a case of mistaken identity?" said Feinstein. "Absolutely not," Ford responded. And later, to Democratic Sen. Richard Durbin, Ford said she was "100% certain" it was Kavanaugh. The judge, who testifies later in the day, has adamantly denied the allegation. Other points: Ford's opening statement: "I believed he was going to rape me," Ford said of Kavanaugh in her opening statement. The encounter "drastically altered my life," said Ford, whose emotional testimony seemed to rivet senators, reports the Washington Post. NPR has her full statement, released in advance, here. 'I'm very sorry': Senate Republicans hired a woman-sex-crimes prosecutor Rachel Mitchell from Arizona's Maricopa County-to question Christine Blasey Ford at Thursday's hearing, and NBC News notes that Mitchell's first comments to Ford had a conciliatory tone. Noting that Ford said in her opening remarks that she was "terrified," Mitchell said, "I just wanted to let you know, I'm very sorry. That's not right." Grassley's charge: Panel Chairman Charles Grassley lashed out at how Democrats, particularly Feinstein, have handled the situation. "These allegations could've been investigated in a way that maintained the confidentiality" sought by Ford, he said. "This is a shameful way to treat our witness who insisted on confidentiality." Details: When Democratic Sen. Amy Klobuchar asked Ford what she remembered of the night in question, Ford responded: "The stairwell, the living room, the bedroom, the bed on the right side of the room-as you walk into the room, there was a bed to the right-the bathroom in close proximity, the laughter, the uproarious laughter, and the multiple attempts to escape and the final ability to do so." Laughter: When Democratic Sen. Patrick Leahy asked her what she remembered most about the incident, Ford (a psychologist) responded: "Indelible in the hippocampus is the laughter, the uproarious laughter between the two, and their having fun at my expense," she said, referring to Kavanaugh and friend Mark Judge. Ford alleges both were present in the room with her. Kavanaugh's opening statement: Read it here. "These are last-minute smears, pure and simple," he says of his accusers.

multi_news

Immune synapses formed by T and NK cells both show segregation of the integrin ICAM1 from other proteins such as CD2 (T cell) or KIR (NK cell). However, the mechanism by which these proteins segregate remains unclear; one key hypothesis is a redistribution based on protein size. Simulations of this mechanism qualitatively reproduce observed segregation patterns, but only in certain parameter regimes. Verifying that these parameter constraints in fact hold has not been possible to date, this requiring a quantitative coupling of theory to experimental data. Here, we address this challenge, developing a new methodology for analysing and quantifying image data and its integration with biophysical models. Specifically we fit a binding kinetics model to 2 colour fluorescence data for cytoskeleton independent synapses (2 and 3D) and test whether the observed inverse correlation between fluorophores conforms to size dependent exclusion, and further, whether patterned states are predicted when model parameters are estimated on individual synapses. All synapses analysed satisfy these conditions demonstrating that the mechanisms of protein redistribution have identifiable signatures in their spatial patterns. We conclude that energy processes implicit in protein size based segregation can drive the patternation observed in individual synapses, at least for the specific examples tested, such that no additional processes need to be invoked. This implies that biophysical processes within the membrane interface have a crucial impact on cell∶cell communication and cell signalling, governing protein interactions and protein aggregation. Cell membrane proteins in a number of systems are observed to undergo complex spatial temporal patternation at cell∶cell and cell∶bilayer contact interfaces. Common to these systems is protein segregation according to size, [1], specifically small ligand-receptor pairs (TCR/MHC, KIR/MHC, CD2/CD58, typically 12–15 nm bond length) segregate from proteins with larger extracellular domains (e. g. CD45, ICAM1, LFA1, ranging from 18–50 nm, based on structural arguments [1], [2]), Fig. 1. The contact interface within which patternation is observed is called the immune synapse, a term that encompasses a variety of patterns. The paradigm was established in the 1990s for T cells interacting with protein-rich supported membrane bilayers [3] and at intercellular contacts [4]. This classic synapse comprises the formation of small MHC clusters that then coalesce, repositioning into a mature bulls-eye structure (pattern coarsening) with ICAM1 positioned in a surrounding annulus [3], [4]. However, many other pattern architectures are reported, including multiple foci in thymocytes [5] and NK cells [6]. Fundamental to these systems is the distinction between segregation of proteins - the partitioning of the surface into domains that are enriched in one or other protein - and aggregation (or pattern coarsening) into a single, normally centralised domain. Experimental evidence now suggests that the latter is an active (ATP-dependent) cytoskeleton driven processes [7]; in particular partitioned supported bilayers reveal a clear centrally orientated force in T cells [8]. Further, this active aggregation is absent in systems where cytoskeletal signalling is inactive or disrupted [9]. Protein segregation can be caused by a variety of processes, including differential protein enrichment in lipid raft microdomains [10], ordering by cytoskeletal processes/actin picket fences [11], [12], specific protein-protein interactions such as tetraspanin-mediated microdomains, or segregation driven by different protein exodomain sizes. The latter process has drawn significant attention from modellers given its (dynamic) self organising property, being distinct from the other mechanisms which are dependent on an ancillary structure or process. Three distinct modelling formulations have been used and all confirm the key result that the coupling of receptor-ligand complexes through the elastic cell membrane can order proteins by size. The resulting (stochastic) spatial patterns qualitatively reproduce observed protein patternation [13]–[16]. The common criterion for instability in these models is that the stretching (or compression) energy to bring the receptor and ligand into sufficiently close proximity to form a bond when the inter membrane separation is different than the natural bond length must be sufficiently high, otherwise uniform protein distributions are thermodynamically preferred [14]. These models also indicate a separation of time scales between size driven domain formation (fast) and pattern coarsening (slow), [14], [17], thereby indicating that T-cell synapse maturation to a bulls eye requires an active (cytoskeletal) mechanism as discussed above. Thus, although the bulls-eye is the minimal energy configuration, the energy surface is insufficiently steep for it to be achieved on realistic time scales by size driven segregation alone. To date, the criterion above for patternation has only been indirectly tested using parameter estimates from the literature, these indicating that patternation by this mechanism is a feasible explanation. However, there has been no direct confirmation that this prediction holds in any experimental system, in part because quantitative comparison of models with spatial image data is extremely difficult. We address this challenge here. Given the complexity of immunological synapse dynamics, we selected two minimal systems for our analysis. Specifically, we demanded that active cytoskeletal processes are absent. This means that we cannot examine the classic immunological synapse pattern; however there are two well established systems that display segregation in the absence of cytoskeletal processes, Fig. 1. These are, firstly, T cells interacting with a model protein-rich bilayer system containing two fluorescently labelled proteins: CD58 labelled with the green dye FITC, and ICAM1 labelled with the red dye TRITC [9]. Binding can potentially occur between the T cell and the protein-rich bilayer via the T cell surface proteins CD2, which binds CD58, and LFA1, which binds ICAM1. The absence of cytoskeletal activity was hinted at since there is no central aggregation, and demonstrated by inactivation of signalling to the cytoskeleton [9]. Secondly, we examine segregation in live cell-cell conjugates between a Natural Killer (NK) cell, a YTS cell transfected to express the inhibitory receptor KIR2DL1 which binds class I MHC proteins including HLA-Cw6, and a target cell (721. 221) transfected to express GFP tagged HLA-Cw6 (HLA-Cw6-GFP) [18] and mCherry tagged ICAM1 (ICAM-Cherry). In this case, binding can potentially occur between the receptor ligand pairs KIR/HLA-Cw6, LFA1/ICAM1 as well as many other receptor/ligand pairs at the surface of the two cells. Since the inhibitory ligand HLA-Cw6 is expressed on the target cells, inhibitory synapses form that are independent of the cytoskeleton [6], [18]. Both these systems show strong segregation and patternation with an enrichment of CD58 (HLA-Cw6 respectively) within the contact interface, Fig. 1. Segregation between the labelled ligands CD58 (HLA-Cw6) and ICAM1 is clearly demonstrated in the line intensity profiles along the surface, Fig. 1B/D, whilst correlation coefficients between the fluorophores in the synapse indicate significant levels of mutual exclusion, values range from −0. 39 to −0. 69 per synapse (mean −0. 49, −0. 55 population sd 0. 10,0. 08 for bilayer and NK cells respectively, the latter for pixels on the contour). These characteristics of patternation, specifically the mutual exclusion between different sized fluorophores is typical of immune synapses. However, as we demonstrate here, these two systems also show an even greater simplicity than previously thought. Specifically, there is no enrichment of ICAM1 in the interface, Fig. 1, i. e. the segregation is between the small receptor-ligand complex (CD2/CD58, KIR/HLA-Cw6 respectively) and the larger unbound ICAM1. The theoretical feasibility of segregation by size in such a system has been previously established [19], whilst it probably represents the minimal system capable of exhibiting self organisation through segregation by size. Thus, these two experimental systems are ideal models for establishing a new framework for quantitative analysis and model comparison. In this paper we develop a novel energy model that can be used to analyse protein redistribution. We demonstrate that we can extract previously untapped information from two colour fluorescence images. Applying this analysis to observed synapse patterns we are able to quantify the degree of mutual exclusion and specifically test the hypothesis that differences in protein size are sufficient to drive segregation. Thus, by using an energy model parametrised on each individual synapse, we demonstrate for the first time that observed protein segregation patterns in actual synapses can be explained by differences in protein size alone. In both the bilayer and cell conjugate synapses there is an enrichment of the smaller ligand, CD58 (HLA-Cw6), and a concurrent exclusion of the longer ligand, ICAM1 relative to the free surface in distinct regions of the interface, Fig. 2. The remaining part of the interface has fluorescence levels approaching those of the free surface. This indicates that binding is occurring in the interface between the fluorescently tagged ligand CD58 (HLA-Cw6) and its associated receptor CD2 (KIR2DL1), thereby raising the fluorophore concentration above the free surface levels. We do not observe enrichment of ICAM1 in these synapses, Figs. 1 & 2, indicating that negligible binding with LFA1 is occurring. The affinity and avidity of the primary adhesion receptor LFA-1 are subject to signal-dependent upregulation; thus ICAM1 enrichment and contact stabilisation occurs in activating T (and NK) cell synapses through the activation of this pathway [9]. Inhibitory signalling, through KIR for example prevents this inside-out signal and thus prevents ICAM1 enrichment, abrogating adhesion and conjugate formation [20], [21]. The fluorescence intensity histogram, Fig. 2, demonstrates that there is a wide distribution of intensity levels in the interface (compared to the free surface). In particular, the distributions are not bimodal as may have been expected, indicating that the interface environment is highly heterogeneous with regard to the propensity to form complexes. Thus domains are not idealised demarcated entities and show diffuse domain walls with variable levels of fluorophore intensity within the domains. The small ligand shows high levels of enrichment in most pixels in the cell∶bilayer contact whilst a much more diffuse enrichment in NK synapses, Fig. 2; this is because of dominance of the interface by the enriched CD58 (HLA-Cw6) phase. There is also noticeably higher noise in images of intercellular contacts compared with cells stimulated by a protein-rich bilayer, particularly in the mCherry fluorophore, Fig. 1B/D. The synapse patterns in Fig. 1 are not of the classic T-cell mature synapse variety; there is no centralisation of the small ligand. This is because in both these synapses cytoskeletal processes are not playing a role in reorganisation of the pattern. In the bilayer system, the absence of cytoskeletal transport was confirmed in a truncated CD2 mutant that lacks the cytoskeletal signalling domain [9]. In the inhibitory synapse between YTS: KIR2DL1 and 221: HLA-Cw6-GFP, the organisation of KIR/HLA on a micrometer-scale has been shown to be largely independent of active cytoskeletal rearrangements, at least for these cell types [18]. Mechanisms other than cytoskeletal processes must control protein segregation in these synapses; one possibility is segregation according to protein size [1], [22]. The phenomena works as follows- at the contact interface, bonds form between the small receptor/ligand pair bringing the membranes into close proximity. The intermembrane separation in these regions is likely to be of the order of 12–14 nm, the predicted bond length assuming end on binding [22], [23]. The larger ICAM1, estimated to be 15–20 nm, with dimerisation potentially stiffening the protein, [24], thus experiences an exclusion potential from these regions of close contact. Whether this exclusion is sufficiently strong to give rise to two phases in the interface requires modelling of the system' s dynamics and energetics. Immune synapses have been modelled using a variety of methods. The fundamental division in these approaches is the spatial scale of the modelling. Statistical physics formulations that model individual proteins on a discretised spatial lattice have been simulated (Monte carlo) and analysed, [17], modelling individual receptor-ligand interactions by a rigid square well potential. Thus, binding between facing receptor/ligand pairs occurs only if the membrane separation is within a certain range. These models have previously shown that this system (comprising a long ligand and short receptor-ligand complex) can display patternation [19]. In partial differential equation (PDE) treatments protein concentrations are modelled [13], [14], possibly with noise (stochastic PDEs), and they utilise an effective spring model for receptor/ligand binding in terms of the local membrane separation, [14], (1) where is the natural ‘bond length’ and the spring constant (we absorb into the spring constants for ease of notation). are constants. Here we assume there is no change in the on-rate for simplicity; results are otherwise identical. Since the membrane support is more flexible than the protein, the membrane is essentially the source of this elasticity. A simple model, assuming an infinite elastic sheet gives an effective spring constant of [14], where, are the membrane rigidity and surface tension respectively and is the radius of the protein in the membrane; this analysis requires which is satisfied in practice. Thus, the PDE models work at a different scale than the statistical physics models, using an object comprising a receptor-ligand complex and its local supporting membrane as the fundamental unit. This model is only applicable on scales above, a constraint that is not a problem for light microscopy data of immune synapses. In these models the local membrane separation variable is an average over this length scale. The advantage of these PDE models is their analytically tractability, whilst the fact these two distinctly different modelling formulations give similar predictions indicates that the phenomena is robust to model assumptions. A PDE model can be derived for this system similar to the two receptor/ligand case [13], [14]. There are 5 coupled differential equations; the molecular species are subject to diffusion and binding, (labels and refer to the complex and ICAM1 (long ligand) respectively), (2) where, are the (small) receptor and ligand concentrations, the complex concentration, the long ICAM1 concentration, and, and are diffusion constants. We have parametrised the spring energies of Eqn. (1) as, (3) For ICAM1 this comprises an interplay between compression (pushing against the membranes) and the attractive glycocalyx forces. Here, are the natural bond lengths of the CD2/CD58 (KIR/HLA-Cw6) complex and the extracellular domain length of ICAM1 respectively. These elastic forces act on the complex and large ligand introducing a drift potential in Eqns. (2), dragging the complex, resp. large ligand, towards lower energy regions. Finally, the complex and ligand apply force to the membrane (s) introducing spatial heterogeneities in the local separation against the restoring elasticity forces, [14], (4) where parametrises the response dynamics of the membrane. This model incorporates the fact that complex formation has a degree of flexibility; the supporting membrane can bend to accommodate different sized protein complexes although this incurs an energy penalty in doing so. It is the balance of these energies that is crucial to patternation, patternation in fact only occurring under certain conditions. To derive these conditions we use a stability analysis following [14]. The analysis considers an initial (spatially) uniform steady state, i. e. the inter membrane distance is uniform in the interface and adjusts to establish an equilibrium between bond formation and the cost of exclusion of ICAM1. The protein concentrations determine this balance of energies and thus the equilibrium value (s) of. This homogeneous state is then examined for spatial instability, an instability to spatial fluctuations giving rise to a patterned state since the fluctuations will grow in amplitude. This stability analysis (see Supporting Information file Text S1) gives the following condition for the system to exhibit instability (patternation) in spatial mode with wavenumber (spatial dependence), (5) where concentrations (and) correspond to the uniform steady state. Note that the cell elasticity parameters () only appear with the wave number and thus only distinguish relative stability of the spatial modes; it is the spring constants in the PDE formulation that are the key parameters for stability. Condition (5) applies to the steady states for which there are either 1 or 3; again these are a function of the concentrations and the model parameters. Thus, Eqn. (5) determines, firstly for which values of the model parameters can instability occur under any possible conditions (receptor/ligand concentrations, relative area between free surface and cell interface), and secondly, if instability is possible, then for what initial conditions will patternation be observed. Unfortunately direct fitting of the stochastic analogue of Eqns. (2) to image data is beyond the scope of present methodology. Further, the model implicitly assumes size segregation. The central challenge is thus to model image data using the biophysical principles implicit in the model above in a more general context; i. e. with a model that both incorporates essential biophysical features, can be parametrised from the available data whilst capable of producing testable predictions. Fundamental to an understanding of protein patternation is quantification of the energy demands of protein redistribution and segregation. We thus reparametrise the ICAM1 and complex concentrations in terms of exclusion energies, specifically parametrising in terms of the energy of redistribution relative to a reference (maximum) concentration. For ICAM1 we use the free surface concentration since, in absence of ICAM1 binding, this is the maximum observed concentration in the contact interface, and for CD58 (HLA-Cw6) we define as the optimal (maximal) complex concentration in the interface. By equating chemical potentials this gives, see Fig. 2, (6) where are the local complex and ICAM1 concentration respectively in the contact interface. These relations define the exclusion energies that are dependent on a local environment variable. Under an exclusion by size model the local environment parameter can be identified with the inter membrane separation, achieving a link to the model above. Specifically, this formulation is identical to the PDE model at stationarity, i. e. solving for time invariant solutions to Eqns. (2) under general spring energy functions, or using the special case of a quadratic local energy dependence Eqns. (3). Thus, are the concentrations of the complex and ICAM1 under optimal environmental conditions for each species, i. e. when and respectively. Since there is an asymmetry between the two species, specifically we have a complex with a small bond length that bridges the two membranes and an unbound ligand with a large extracellular domain, there may be a difference in the effective spring elasticities and thus. In general, Eqns. (3) imply that a linear relationship exists between the square roots of the exclusion energies. This is derived by eliminating the unknown (unobserved) intermembrane distance, giving, (7) with regression constants, and. Since there is mutual exclusion between the species, the positive root for is the physical solution. This relation justifies introduction of the square root energies (SQRE) which we use hereafter. The challenge is therefore to use the fluorescence data to estimate the local exclusion energies, Eqns. (6), and determine if there is evidence of this predicted linear relationship. This presents major difficulties since the fluorescence data is noisy and the complex concentration is not immediately measurable because observed fluorescence is the sum of contributions from the complex and free ligand. To deal with these problems we use a statistical model for the fluorescence intensities. A fluorescence measurement is essentially a counting of the number of contributing fluorophores per pixel. Since fluorophore emission events and concentration fluctuations are independent these events are governed by Poisson statistics; we assume that neighbouring pixels are independent and thus that the dependence incurred through the microscope point spread function (PSF) is removed by deconvolution. The fluorescence of channel in a free surface (fs) pixel therefore has distribution, (8) where Po denotes a Poisson distribution. Parameters and represent the combined emission and detection efficiency, and the fluorescence proportionality constant, respectively. In the free surface, pixels are essentially independent samples informing on the model parameter combinations. In the contact interface (ci), individual pixels are modelled with a local environment dependence through the SQRE, as described above in Eqn. (6), giving for pixel, (9) The bilayer model has 5 global parameter (combinations), namely,,,, and local parameters for each pixel in the contact interface. Note that the emission/detection efficiencies are not estimatable separately from the free ligand concentration. The bilayer patterns are imageable directly, but for patternation on cell surfaces the protein distributions need to be reconstructed from a z-stack (see Text S1). We model each image in the z-stack, extracting regions of free surface and the contact interface along the membrane contour (see Text S1). This requires us to deal with the fact that the cell membrane in each slice (even after deconvolution) has a thickness discernible by light microscopy, typically being wider in the free surface than in the contact interface. This is presumably because the interface suppresses ruffling, whilst optical spreading caused by inexact deconvolution may contribute to this width in all regions. Thus, we extended the model to include an apparent thickness of the membrane under a Gaussian model. By modelling each image we reduce processing artifacts, e. g. compared to using a projection which requires distortion of the surface to a plane, whilst also utilising a higher number of pixels in the estimation thereby maximising information extraction. The above model can also be further modified, specifically a Gaussian or Gamma approximation can be used instead of the Poisson distribution above, the latter giving the best fit as it captures the skew in the free surface distribution observed in inter-cell synapses, Fig. 2, cause unknown. In addition we examined a number of model extensions, including inclusion of background autofluorescence and existence of a potential difference for the unbound (small) ligand to diffuse between the contact interface and free surface, this modelling for instance steric or electrostatic effects in the interface. Essentially this discounts the free ligand concentration in the contact interface by factor, i. e. there is a free energy difference of. We fitted the size exclusion model, schematically shown in Fig. 3, to each individual synapse image/z-stack; individual cell fitting allows synapses to be compared and retains key correlations which would otherwise be weakened or lost if synapses are averaged given that synapse patterns are highly variable. We estimate the model parameters for each synapse separately using a Bayesian analysis, (algorithm in Text S1); specifically we estimate concurrently all model parameters by fitting the model to the image data through simulation of the full model posterior distribution. Fluorescence data in the contact interface and a region of the free surface is sufficient to estimate on each channel all model parameters in each synapse (bilayer or cell conjugate); confidence intervals (not shown) for each parameter were reasonable indicating all parameters are estimatable. Our model gives good reproduction of the observed fluorescence histograms, Fig. 2, and further, provides evidence of a strong linear relationship in both the cell∶bilayer and cell∶cell systems, Fig. 4; the first requirement for the segregation by size model. The linear relationship deteriorates at high and high due to degrading signal to noise issues; the fluorescence of the respective species being insufficient to distinguish it from autofluorescence and fluorescence from unbound ligand respectively. This gives rise to the saturation in and the spread of the distribution to the right against the barrier, Fig. 4. The analysis was then repeated with the model for size exclusion assumed, specifically Eqn. (7) was imposed thereby estimating the regression parameters and. The mean line is shown in Figs. 4. There is no evidence that the gradient of Eqn. (7) is equal to 1 in any of the synapses (posterior probability) implying a difference in the energy of compression of ICAM1 between the membranes and the stretching of the bond formed by CD2/CD58 or KIR/HLA-Cw6. This asymmetry suggests that accommodation of the long protein ICAM1 in the interface is energetically cheaper than stretching the small bond (or more likely the supporting surface). This makes sense as ICAM1 has additional degrees of freedom since it is not engaged to ligand, and thus presumably able to tilt or possibly even bend, as illustrated in Fig. 3. We reconstructed the hidden variable for each pixel, the resulting histogram shows a bimodal distribution for the bilayer interfaces with a mode corresponding to the enriched CD58 phase (close to), and one corresponding to the competing phase where the CD2-CD58 complex is excluded (close to), Fig. 5A. For NK cells, the interface is heavily dominated by areas of close contact where HLA-Cw6 is enriched (); thus bimodality is weaker, Fig. 5C. From an energy perspective, ICAM1 experiences exclusion energies up to 1. 5 kT, while in certain parts of the interface the complex experiences an exclusion energy of up to 3 kT, Fig. 5B/D. In the NK synapse, ICAM1 always experiences an exclusion potential relative to the free surface, minimum kT; this explains why the second mode in Fig. 5C is at and not nearer. Our model fit provides estimates of biologically relevant parameters. Typically we obtain an elasticity constant of order 0. 1 (400) which is consistent with a crude model that approximates the membrane as an elastic sheet and gives an order of magnitude of 40, [14]; cytoskeletal pinning of the membrane is ignored in this estimate suggesting that it is an underestimate. Using mass action, the optimum complex enrichment can be interpreted as, the free receptor concentration in the interface and the 2D dissociation constant. Using an order of magnitude estimate of average receptor density on the respective cells (190,100) we obtain 2D affinity constant estimates of order,. We also find significant evidence of an energy barrier for unbound ligands to enter the contact interface in some of the synapses, the density in the contact interface of CD58, HLA-Cw6-GFP being 37%, 14% lower respectively on average than on the free surface and significantly less than 100% in 1 of 3 bilayers, 4 of 8 NK synapses. This compares to a reduction of 30% experimentally measured using CD48, [25] a non binding ligand. The above analysis demonstrates that patternation can be quantitatively parametrised and biologically meaningful parameters determined from experimental images. The next challenge is to address whether extracellular domain size is a primary driver of patternation in these synapses. This requires linking our energy analysis model to the theoretical model of synapse patterning in Eqn. (2). The instability condition, Eqn. (5) for patternation imposes a constraint on the model parameters, a constraint that can be recast in terms of our SQRE coordinates as follows, (10) Here, correspond to the uniform steady state SQREs and we have taken as it is the most unstable mode. The limiting case when the left hand side of Eqn. (10) is equated to zero defines the stability curve. This stability curve is determined by the ratio, which was in fact estimated directly as the gradient of the regression in Eqn. (7); we thus obtain a direct link between the energy profile analysis of an observed synapse pattern and the dynamic instability criterion which must hold in order that patternation is predicted to occur under the exclusion by size model at those estimated parameter values. Whether the condition on the system parameters in Eqn. (10) holds can be tested simply by observing if there are regions in the plane where instability is possible, i. e. if there is an intersection of the stability curve and the observed line of regression, Fig. 6. As the ligand densities are altered, the equilibrium membrane separation shifts and the uniform steady state defined by moves along the line of regression, Eqn. (7); thus this line of regression can also be considered the steady state line. If the small or long molecule dominates, i. e. and respectively, the system moves out of the region where patternation occurs, Fig. 6. This reproduces the intuitive result that patternation requires an appropriate balance between the concentrations of the long and short ligands. To test whether this intersection condition holds for our synapses, we estimate the ratio for each synapse. We find that for every synapse there is an intersection of the stability curve and the line of regression, Fig. 6B/C, and thus there exist ligand concentrations where instability is predicted to be observed under the size exclusion mechanism. This leaves the final issue of whether the receptor/ligand concentrations in these synapses are such that patternation would be realised under the size exclusion model. In practice, in the bilayer experiments tuning of the ligand concentrations is performed to find ligand concentrations where patternation (instability) occurs [26], [27], whilst within a population of cells there is sufficient variation of ligand and receptor densities that a small number of patterned synapses are observed for suitable clones in the cell∶cell system. Thus, our proof that the estimated parameters are such that an instability regime exists is already strong confirmation between theory and experiment. This conclusion is also robust to measurement and system noise, the (posterior) probability of no intersection, and therefore patternation not being predicted in any synapse is. If fluorescence is calibrated in terms of molecule density, some additional progress can be made to assess the likelihood of the patterned states being accessible from the initial configuration in the interface. This is an extremely hard question to answer since the contact interface is dynamic, exhibiting spreading against the adjacent surface and undergoing thermal fluctuations. Further, all the necessary parameters or components governing contact dynamics are not known. However, some suggestive results are possible for the bilayer system. Firstly, we can estimate the location of the initial (uniform) state of the synapse prior to patternation for the cell∶bilayer contacts using a previously measured 2D affinity and average contact area [26]. There are 3 uniform states, Fig. 6B, the two extreme stable states correspond to membrane separations close to the CD2-CD58 bond length and the unbound ICAM1 length respectively. The middle state is a compromise configuration of intermediate membrane separation and is unstable to both homogeneous and spatial perturbations. Thus, as observed experimentally, this suggests cells will initially sit on the bilayer without forming CD2-CD58 bonds corresponding to a steady state with in Fig. 6B, the interface showing no particular enrichment or exclusion of any ligand. In order to patternate, thermal fluctuations are needed to seed a close contact patch where CD2-CD58 bonds can form, thereby leading to exclusion of ICAM1 followed by stabilisation and growth of the patch. Using the analysis and parameters of [28], with an ICAM1 concentration of 500, exodomain size 18 nm, patches with a height separation less than 12 nm have an average size of 30 nm, whilst 7% of the surface will be in this close contact regime corresponding to a patch density of the order of 100. These order of magnitude estimates strongly suggest that seeding of patches is highly likely, and thus the uniform state will eventually patternate. This is the first report of a thermodynamic analysis of molecule patterning in bilayer and cell surface experimental images. Our new method of fluorescence image analysis uses energy principles to extract novel information from either single or multiple fluorescence data. The method integrates image quantification and biophysical modelling, allowing biologically or physically motivated models to be fitted to image data. Applied to immune synapses, we show that through consideration of the local Boltzmann energy of exclusion that a signature for the segregation process can be identified from two colour fluorescence images in 2 and 3D. This is despite the low levels of signal as indicated by the small energies involved in the patternation, typically only of the order of 1–3, Fig. 5B/D, and thus cellular processes can easily reorganise individual protein molecules. Our analysis shows that when a size exclusion model for patternation is fitted to individual synapses, three levels of model consistency can be analysed. Firstly, the predicted linearity between the square root exclusion energies is clearly evident in both cell∶bilayer and cell∶cell systems, Fig. 4, whilst we observe bimodality in the reconstructed distribution, Fig. 5. Further, the parameter estimates for the bond elasticity extracted from this analysis are consistent with the measured flexibility of the cell membrane in similar cells, whilst our estimates of the order of magnitude for the 2D affinities are reasonable compared to previously measured values, [26]. Previous estimates are an order of magnitude lower at 1 per, [29], which may indicate that complex formation is suboptimal in synapses, e. g. because of a difference in the confinement width between patternated and non patternated interfaces, [30]. However, it is known from theoretical considerations that the 2D affinity is environment dependent [17], with a dependence on receptor/ligand concentrations since binding affects the confinement width through a suppression of fluctuations. These theoretical issues remain to be verified experimentally implying that the concept of a 2D affinity estimate is currently poorly defined. Secondly, we were able to show in all the observed synapses, through estimation of synapse specific model parameters that a protein concentration regime exists when patterned states driven by size differences are predicted to be possible. Finally, on those synapses where the fluorescence intensity was calibrated we obtained order of magnitude estimates of close contact patch sizes and patch frequency suggesting that seeding of patterns from an initial (uniform) membrane separation of 18 nm (ICAM1 length) was likely, i. e. the uniform configuration is unstable to thermal fluctuations. We thus conclude that the thermodynamic processes implicit in size exclusion are sufficient to generate the observed patternation and no additional processes need to be invoked. This does not exclude other processes being the cause of, or contributing to segregation; only that as far as has been possible, all predictions of the size exclusion model have been verified. In the NK synapse there are other NK receptor ligands and adhesion molecules that could play a role in the NK synapse patterning; however our results suggest that the main players for synapse organisation are KIR/HLA-Cw6 and ICAM1. Since microscopy required the use of target cells expressing high levels HLA-Cw6 and ICAM1, it is unclear if this also applies to lower expression levels as there are ligand density dependent effects [31]. Our analysis could be improved. Firstly, the PSF also introduces a linear relationship in our plot. We demonstrate that our results our robust to this effect, see Text S1; however the analysis could be improved through using a Bayesian model selection approach. This would entail incorporating the PSF into the model, and thus removing the deconvolution step; clearly advantageous since deconvolution fixes stochastic noise in the images. Secondly, the analysis could be extended to verify additional model predictions. Specifically, the phase boundaries separating receptor/ligand concentrations where patternation occurs [15] could be ascertained and tested. Our analysis also quantitatively describes the effect of ligand length perturbations [27]; length variation shifts the line of regression in Figs. 4, a prediction that could be directly tested. Extending this analysis to more general synapse systems, including the classic synapse pattern, is the next challenge. An extension to 2 receptor/ligand binding pairs is relatively straight forward; however this model has identifiability problems that will need to be dealt with, e. g. through suitable experimental design. The impact of active cytoskeletal processes on our analysis has also not been examined. In summary, our analysis indicates that segregation in the bilayer and (inhibitory) NK synapse can be explained by size exclusion alone; specifically there is strong evidence for the predicted linearity between, and, using model parameters estimated directly from the observed synapse patterns we find that the instability constraints governing patternation are satisfied in each individual synapse. Our results have important implications. At a methodological level we have demonstrated that two colour fluorescence data contains key information on the mechanisms of protein relocation, information that can be extracted through the techniques developed here. Secondly, our application to the immune synapse shows that at a single cell level biophysical interactions between the cell membrane and embedded proteins lead to self organisation, giving rise to protein segregation, control of ligand binding and aggregation. This ultimately has an impact on signalling [32]. Experiments were carried out as detailed in [9]. Images were processed for flat field, illumination gradients and background fluorescence was subtracted. The PSF was measured on 100 nm beads and used to deconvolve the image (Richardson-Lucy algorithm). Chromatic aberration was less than a pixel so not corrected. Pixel size is 167 nm. We present results for 3 separate bilayers with 10 synapses. The HLA-A/B/C negative human EBV-immortalized B-cell line 721. 221 [33], was transfected to express HLA-Cw6-GFP and ICAM-Cherry, and cultured as previously described [18], using hygromycin as an additional selection agent for ICAM-Cherry expression. Cells were sorted for high expression levels of both fluorescent proteins using flow cytometry. The ICAM-Cherry plasmid was generated from an ICAM1 with a C-terminal GFP fusion [34] in a pEGFP N-1 vector. The DNA encoding ICAM1 was ligated using the HindIII/BamHI restriction sites into a pcDNA3. 1 mCherry vector conferring hygromycin resistance (a kind gift from Marco Purbhoo). 221 cells expressing HLA-Cw6-GFP were transfected by electroporation (Amaxa) according to the manufacturer' s instructions and selected with 800 hygromycin (Sigma) for 3 weeks prior to sorting by flow cytometry. The human NK cell line YTS, transfected with the HLA-Cw6 binding inhibitory receptor KIR2DL1 [35], was allowed to form contacts with the target cells for 30 min. A drop of 7 cell suspension in phenol-red-free, HEPES-buffered culture media was mounted between a glass slide and a 22×22 mm coverslip. Imaging was performed at on a confocal laser scanning microscope (TCS SP2, Leica), using a 63× oil immersion objective (1. 32 NA), with voxel sizes of 93×93×360 nm. GFP was excited using a laser wavelength of 488 nm, Cherry using 561 nm, and images obtained by sequential excitation. Deconvolution was performed on the basis of the point-spread function determined by imaging fluorescent beads of sub-resolution size. Chromatic aberration was corrected (typically pixel) by maximising the correlation between the channels on the cell of interest; analysis of two colour beads demonstrated that chromatic aberration was not uniform over the image and varied up to a 2 pixel shift in x and y. Cells appropriate for 3D fluorescence reconstruction and modelling had to satisfy a number of criteria, i) have good surface membrane fluorescence in both channels, ii) have low cytosol fluorescence near the membrane, and iii) possess regions of free cell surface (no cell∶cell contact) that were free of ruffling. We used n = 8 synapses in the presented analysis. We developed Markov chain Monte Carlo algorithms to implement a Bayesian inference method for model parameters for both 2D and 3D data. These algorithms simulate the posterior probability of the model parameters given the data through evaluation of the likelihood (see Text S1), from which we can estimate, for instance, their mean values and correlations. We used uniform priors on all parameters, with the interval for the SQRE. Convergence was ascertained using a multiple chain protocol [36].

A cell interacts with its environment though the thousands of proteins that are expressed on the cell' s surface. A number of these proteins are involved in cell∶cell communication, a complex process where two cells establish a (transient) contact interface forming protein bonds that bridge the interface. In T cells, which are a major component of the immune system, the proteins in this interface are organised into spatial domains with small proteins segregating from large ones, a patternation that is believed to be a crucial step in the recognition of antigens by T cells. The mechanism of this patternation is still unresolved, a mechanism based on maximising the number of bonds by partitioning the interface into regions where short and long proteins can form bonds being the most promising. Here we directly compare image patterns with a mathematical theory of patternation demonstrating that there is a signature of the patternation mechanism in the image data. We developed a framework for quantitative modelling of image data in a biophysical context that can be utilised in a variety of other model validation studies. Using these methods we proved that on these images this theory is sufficient to explain the observed patternation.

lay_plos

Messenger RNA splicing is an essential and complex process for the removal of intron sequences. Whereas the composition of the splicing machinery is mostly known, the kinetics of splicing, the catalytic activity of splicing factors and the interdependency of transcription, splicing and mRNA 3′ end formation are less well understood. We propose a stochastic model of splicing kinetics that explains data obtained from high-resolution kinetic analyses of transcription, splicing and 3′ end formation during induction of an intron-containing reporter gene in budding yeast. Modelling reveals co-transcriptional splicing to be the most probable and most efficient splicing pathway for the reporter transcripts, due in part to a positive feedback mechanism for co-transcriptional second step splicing. Model comparison is used to assess the alternative representations of reactions. Modelling also indicates the functional coupling of transcription and splicing, because both the rate of initiation of transcription and the probability that step one of splicing occurs co-transcriptionally are reduced, when the second step of splicing is abolished in a mutant reporter. The splicing of precursor messenger RNA (pre-mRNA) is an essential process in the expression of most eukaryotic genes. The five small nuclear ribonucleoproteins (snRNPs) and the many non-snRNP-associated proteins that constitute the splicing machinery, assemble anew on each precursor RNA to form the spliceosome complex that catalyses the two chemical reactions of splicing [1]. Both the spliceosome components and the spliceosome assembly process are largely conserved between human and yeast. The complexity of the spliceosome is indicated by the 170 proteins that are associated with it [1]. Adding to the complexity, splicing may occur partly, or entirely, concurrently with transcription. In eukaryotes, the interaction of the spliceosome with the precursor RNA can be considered to be an allosteric cascade in which early recognition steps induce conformational changes required for subsequent steps and for catalytic activation (reviewed by [2]). However, the wealth of knowledge of molecular interactions, obtained mainly through extensive biochemical and genetic analyses, has yet to be formalised as a systems model of transcription and splicing. Spliceosome assembly is thought to occur via a series of events with many points of regulation [3]. In the first step, U1 snRNP binds to the 5′ splice site (5′SS), followed by the U2 snRNP at the branchsite. The U4, U5 and U6 snRNPs join as a tri-snRNP complex and, after the association of other, non-snRNP proteins, the spliceosome complex is activated for the first chemical step of splicing. The 5′ splice site is cleaved and, simultaneously, the 5′ end of the intron becomes covalently attached to the branchsite to form a branched, lariat structure. In the second step, the 3′ splice site (3′SS) is cleaved, which excises the intron, and the exons are joined to produce the mature mRNA. Between the two steps of splicing, a conformational change is required in the catalytic centre of the spliceosome [4], and at several stages during the cycle of spliceosome assembly, splicing and spliceosome dissociation, proofreading mechanisms are thought to operate [5]. Nascent transcripts also have to be matured at their 3′ end, by cleavage and polyadenylation. Figure 1 A illustrates spliceosome assembly and the two steps of splicing for a pre-mRNA with one intron that has already been polyadenylated and released from the DNA template. Splicing can also occur co-transcriptionally, prior to 3′ end maturation (Figure 1 B), and there is considerable experimental evidence for functional coupling of transcription, splicing and 3′ end maturation in vivo [6]–[12]. However, little is known about the impact of coupling on kinetic rates. Splicing has been modelled, but not to the same level of detail as transcription, and models of transcription have yet to fully incorporate the splicing reaction. Quantifying the dynamics of these processes remains a challenge [13], and modelling may have an important role to play in distinguishing functional dependencies from coincidental and contemporaneous effects, and in identifying and characterising the interactions that effect coupling. Existing models of splicing have allowed splicing efficiency to be defined [14], and have shown that transcription by RNA polymerase II (Pol II) greatly increases splicing efficiency in comparison with transcription by T7 polymerase [15]. A correlation between splicing efficiency and the pausing of Pol II on short terminal exons has been reported [11]. Splicing has been represented as a single irreversible reaction that creates the product mRNA from pre-mRNA [11], [14], and as a single irreversible reaction that creates mRNA from the pre-mRNA+spliceosome complex [15]. To-date, steps one and two of splicing have not been modelled as separate reactions, nor have the co- and post-transcriptional splicing pathways been distinguished. Further insights into splicing can be expected by more detailed modelling and analysis. As noted above, splicing can occur during messenger RNA transcription. Transcription begins with the assembly of the pre-initiation complex at the promoter. This complex includes Pol II, which, after initiation, begins the transcript elongation process that transcribes DNA into RNA. Early in elongation, the pre-mRNA is capped at its 5′ end by the capping enzymes. Elongation involves a sequence of many hundreds of individual polymerisation reactions, and hence the time required to complete the elongation of a transcript is predicted to have less variability than a single-step process with an equivalent rate [16], [17]. The mature 3′ end of the RNA is formed by an endonucleolytic cleavage at the so-called poly A site and the newly formed 3′ end is extended by polyadenylation (reviewed by [18]). The elongation process and the 3′ end formation steps can also be accounted for when modelling transcription [16]. The recruitment of Pol II enzymes and spliceosomal proteins are important steps in transcription and splicing, but are not believed to be rate limiting under normal conditions. Kinetic studies of Pol II complexes indicate that a minority of them are actively involved in transcription at any given time. The remainder move by diffusion through the nucleus [19], as do the product mRNA molecules [20]. Three kinetically distinct populations of Pol II have been identified at the site of transcription; those bound to the promoter, those initiating transcription, and those engaged in elongation [21]. The movement of the spliceosomal proteins that catalyse the splicing reactions can be modelled as Brownian diffusion [22]: these RNPs move continuously throughout the nucleus independently of transcription and splicing. We have developed a stochastic model that represents splicing in the context of transcript elongation and RNA 3′ end maturation, as shown diagrammatically in Figure 1 C. (All pathway models are provided as files in Dataset S1.) A stochastic formulation allows the effects of small numbers of molecules to be explored, and simulations of the model can be averaged in order to obtain the population mean over time. Experimental values for the model species (population averages in copies/cell), including fully-spliced mRNA (see Materials and Methods) and two precursor species in both 3′ uncleaved and cleaved/polyadenylated forms, have been obtained by a rapid sampling protocol that is capable of capturing transient species [23]. We first describe the structure of the pathway, then present the data, and subsequently discuss alternative representations of the steps in the RNA pathway in the light of the data. The simplest description that might be adopted for the elongation, 3′ end formation and splicing steps is a single irreversible reaction. However, we find this provides a poor fit to the available data, and consequently a number of alternative representations for these reactions are considered. The extent to which the alternative pathways fit the data is assessed by the Akaike information criterion (AIC) for optimal parameter choices. We propose a multistep model for transcription by dividing the gene into sections to be transcribed. Each section () of the reporter DNA represents approximately 30 nucleotides, corresponding to the footprint of Pol II on the DNA [24]. As the length of the Ribo1 reporter (described below) used in the experimental studies is 1240 bases, we define 40 sections of DNA:. Each section of DNA can be occupied by at most one Pol II, and the progression of Pol II from the 5′ to the 3′ end of the gene is equated with successful extension of the transcript. The number of sections of DNA defines an upper limit on Pol II occupancy, and can limit the effective rate at which a Pol II can complete elongation. Beginning with the initiation of transcription, the reaction (see Figure 1 C) places a Pol II enzyme in the active promoter complex (APC) when the gene is active. Thereafter, this Pol II can progress along the gene at elongation rate (the number of sections of DNA transcribed per unit of time). Letting the rate of polymerisation of nucleotides be (the number of nucleotides incorporated per unit of time):. (Equivalently, the mean time for n polymerisation events:, equals the mean time for one elongation event:). This multistep model of elongation is comparable with the kinetic model of Pol I elongation proposed in [25]. The pathway proposed here does not include a transition between active and inactive states of the promoter, as the rapid rate of mRNA production does not indicate that the promoter switches off during the period immediately after induction. However, such a transition is needed to explain the mRNA distribution in steady state [26] and can easily be included in this model. Kinetic competition between splicing and elongation has been discussed extensively [8], [27], [28], and is modelled here as taking place at the sections of DNA after the branchsite. In these sections, the occurrence of the first step of splicing of an RNA is represented in the model by a change of state of the associated Pol II, which can make a transition to the co-transcriptional splicing path. Sections and represent the same n nucleotides of the DNA and so at most one of these sections can be occupied (by at most one Pol II). The rate for the transition between splicing pathways is 0 prior to the completion of the splicing activation process. The splicing activation process is triggered at rate when the gene switches on. When splicing is active, the transition rate is, where is a constant that determines the ratio of the competing reactions (elongation and splicing) and thereby the probability of co-transcriptional splicing. Activation of co-transcriptional splicing involves co-transcriptional spliceosome assembly as well as the first step splicing reaction (i. e. co-transcriptional spliceosome assembly alone is not sufficient). Each Pol II completes elongation either at, having completed the first step of splicing, or at having failed to do so. Subsequently, on the post-transcriptional path, 3′ end maturation () produces polyadenylated pre-mRNA, step one of splicing () produces polyadenylated lariat-exon2, and step two of splicing () produces mature mRNA and lariat, as indicated in Figure 1 C. On the co-transcriptional pathway, the second step of splicing () produces uncleaved mRNA and lariat, and 3′ end maturation (3′ cleavage, polyadenylation and release;), produces mature mRNA. It is important to note that the species measured experimentally are pre-mRNA, lariat-exon2 (the branched lariat structure) and mRNA, and that the uncleaved and polyadenylated forms can also be distinguished (as illustrated in in Figure 1 C). The assays for these species are described below. Initial estimates for some parameters can be obtained from the literature: the rate of initiation of transcription in yeast has been estimated as [29], [30]. Polymerisation rates in the mammalian nucleus of up to 72 nucleotides/s have been reported for polymerases that do not pause. This is a significant increase on earlier estimates of 18–42 nucleotides/s [13] that may reflect an average or effective rate. A Pol I elongation rate of 90 nucleotides/s has been reported [25]. The time for pre-mRNA 3′ cleavage in HIV-1 has been reported to be 55 s, with release taking 9 s [16]. The probability of co-transcriptional splicing is not known, and this, along with precise values for all other parameters, will be inferred from fitting the pathway to the data. The pathway was developed to explain data from the Ribo1 reporter [23]. Ribo1 is a chimeric yeast gene that contains the single intron from the ACT1 gene and the 3′ end processing signal from PGK1, as shown in Figure 2 A (modified from [31]). The reporter gene is integrated in the genome, transcribed under the control of a doxycycline-responsive promoter in a doxycycline-inducible strain of Saccharomyces cerevisiae. By modelling splicing in this reporter, we aim to define the splicing pathways and to quantify reaction rates. The impact of splice site mutations on the coupling between splicing and transcription can also be explored. Three replicate experiments were performed in which doxycycline was added to a culture to induce reporter gene expression, and transcript levels were measured by reverse transcription and real-time quantitative PCR (RT-qPCR; see Materials and Methods). A time series of values was obtained for accumulation of pre-mRNA, lariat-exon2, and mRNA. The RT-qPCR data were converted to copies per cell (see Materials and Methods; [23]), which allows a quantitative comparison of data obtained for the different RNA species and from different cultures. The merged time series derived from three biological replicates for Ribo1 is shown in Figure 2 B (referred to as Expt 1). In the 120 s interval 420 s–540 s after the addition of doxycycline to the cell culture, the level of Ribo1 mRNA increases from 11 to 45 copies/cell (Figure 2 B). Messenger RNA then reaches 60 copies/cell, on average, 180 s later. The high level of mRNA is notable, as is the rapid rate of transcript synthesis. The delay between the rise in pre-mRNA and the rise in mRNA may indicate a slow, or delayed, splicing reaction. In the substantive phase of transcriptional activity (after 420 s in Figure 2 B), the levels of pre-mRNA and lariat intermediate are only a fraction of the mature mRNA species which shows that the first and second steps of splicing must be rapid. To investigate the effects of blocking the first or second step of splicing, two modified Ribo1 reporters were created with point mutations at the 5′ splice site (5′SSRibo1) or 3′ splice site (3′SSRibo1) respectively [23]. The mutant reporters were induced with doxycycline and the splicing intermediates detected using the primers shown in Figure 2 A. The merged time series for 3′SSRibo1 and 5′SSRibo1 are shown in Figure 2 C and 2 D respectively. As indicated by the error bars in Figure 2 C, the synthesis of lariat-exon2 in the mutant reporter varied between biological replicates, but technical error within each replicate remained at typical levels. The level of pre-mRNA measured in the modified reporters is greater than was observed for Ribo1. This may be attributed to changes in the rates of transcript synthesis, splicing step one or degradation, and modelling can help to resolve this question. For practical reasons, co-transcriptional splicing is defined here as splicing that is completed before the transcript has been released from the transcription complex by 3′ end cleavage. The data shown in Figure 2 were produced using a cDNA primer that hybridises to exon2 (at position C1 in Figure 2 A), which does not distinguish between transcripts that are cleaved and polyadenylated or uncleaved at the 3′ end. Therefore, in order to differentiate between these species and to estimate the rates for 3′ end formation, a modified 3′ cleavage assay used two alternative primers for cDNA synthesis from 3′ end sequences of Ribo1; oligo (dT), anneals to cleaved and polyadenylated transcripts, whereas primer C2 is complementary to a sequence downstream of the mapped 3′ end cleavage sites (Figure 3 A; [23]). By amplifying these cDNAs with specific primers for detection of pre-mRNA, lariat-exon2 and mRNA (Figure 3 A), uncleaved and cleaved/polyadenylated pre-mRNA, lariat-exon2 and mRNA were successfully distinguished in Expt 2 (Figure 3 B and 3 C). The 3′ cleavage assay detected a sharp, transitory peak in uncleaved pre-mRNA at 540 s, followed by a similar peak in polyadenylated pre-mRNA 30 s later (Figure 3 B). This indicates pre-mRNA that is not spliced prior to 3′ end cleavage, i. e. is not spliced co-transcriptionally. However, the rapid accumulation of uncleaved mRNA between 540 and 600 s prior to detection of polyadenylated spliced mRNA at 600 s, clearly shows that co-transcriptional splicing occurs before post-transcriptional splicing. By formally modelling the splicing pathway, we aim to quantify the extent to which mature mRNA is derived from co-transcriptional splicing, and from post-transcriptional splicing respectively. The reactions in the model must be enabled (switched on) progressively in order to explain the data. Following the induction of transcription by doxycycline, a burst of pre-mRNA is postulated to occur. At this time, splicing is not active, and additional transcripts are not initiated. These initial pre-mRNAs are cleaved and polyadenylated, and may then splice or degrade. This process explains the accumulation of pre-mRNA in Figure 2 B, and the peak in uncleaved pre-mRNA in the 3′ cleavage data in Figure 3 B. After a delay (defined by the rate), splicing steps 1 and 2 and the initiation of new transcripts start. This explains the drop in pre-mRNA in Figure 2 B, and the peak in polyadenylated pre-mRNA (Figure 3 B) as the activation of splicing removes these species also. Figure 1 in Text S1 illustrates the sequence of events. The proposition that there are advantages to modelling elongation in detail can be tested. Pathways that include 40 steps of elongation are compared with simpler pathway models where competition between elongation and splicing step one is represented by two reactions and that have APC as the substrate and and as the respective products. The proportion of co-transcriptional splicing can be calculated from these reaction rates and this proportion can be compared with that predicted for the 40 step model (as defined by equation 1 in Materials and Methods). The total time allocated to elongation can also be compared in the alternative models. On completion of elongation, the RNA transcripts undergo 3′ end formation. This involves cleavage, polyadenylation and transcript release, and requires three multi-subunit factors [32]. Polyadenylation adds up to approximately 250 nucleotides to the end of the transcript. Hence, it is uncontroversial to model 3′ end formation as a multi-step process as many steps of maturation are clearly required. When fitting the splicing pathway to the Ribo1 data, a much better qualitative and quantitative fit is obtained when 3′ end maturation is modelled as a five-step process (each of the five steps has rate) in comparison with a single step model. The characteristics of 3′ uncleaved spliced RNA also fit the data better by modelling 3′ end formation in this way. As shown in Figure 3 C, uncleaved mRNA quickly peaks towards its steady state of 10 copies/cell rather than making a slow progression to this level. Replacing the single step model with the 5 step model of 3′ end maturation (reactions and) significantly improves the fit to the data. It is easily shown that a process of five steps, each at the same rate, has a kinetic response that differs significantly from that of a single step. (The distribution of waiting times follows a gamma distribution rather than an exponential distribution.) We do not aim to determine the exact number of steps, rather we aim to test whether a process of multiple steps of maturation or senescence provides a better quantitative and qualitative explanation than a single reaction. Henceforth, we assume that 5 steps constitute an adequate model of a multi-step process for the purpose of testing this hypothesis. Text S2 presents an analysis of Ribo1 degradation kinetics that further illustrates the approach. Genetic studies have identified many splicing factors, but their impact on splicing kinetics in-vivo is difficult to quantify. These factors, and the five snRNPs, are not believed to be rate-limiting and have not been included in the model: We initially consider the kinetics of the splicing intermediates alone. However, we find once more that simple unimolecular models for steps one and two of splicing do not fit the data well. Consequently, we propose two alternative characterisations of the splicing reactions prior to steady state, and quantify the extent to which these models improve the fit of the pathway to the data. The first alternative model of splicing we propose represents these processes as a sequence of several reactions that reflect the many known steps of spliceosome assembly. The precursors and products of multi-step processes show sharp transitions in their kinetics, as observed for pre-mRNA and lariat-exon2 in the experimental data. A multi-step model of this kind has been shown to explain fluorescence recovery after photobleaching (FRAP) data obtained from a splicing reporter in mammalian cells [33]. The second alternative explanation of the rapid processing of pre-mRNA and lariat-exon2 that we propose is based on the proposition that the splicing reactions are catalysed in a manner such that the propensity of the reaction increases on successive splicing events. It is necessary for the initial propensity to be low as otherwise no accumulation of splicing precursor or lariat intermediate would be observed, and for the propensity to increase to remove the accumulation rapidly. The reduction of uncleaved lariat-exon2 over the period of time when mRNA increases rapidly (600 s-700 s in Figure 3 C) may indicate such an increase in reaction propensity: the substrate decreases while the rate of increase of product remains constant. It therefore appears that step two of splicing may not be governed by first-order kinetics when it is co-transcriptional. The observations can be modelled by positive feedback in the splicing reaction. This requires the involvement of additional molecular species in the splicing reaction - the enzyme Y - a role that can be played by factors required for step two of splicing. The following positive feedback mechanism has the property of increasing reaction propensity: Let the enzyme Y have an initial copy number of 1, and increment the copy number on each splicing event to effectively increase the propensity. The enzyme contributes to the reaction propensity according to the formula for bimolecular reactions (). The positive feedback model is proposed for the kinetics of high rates of induction prior to steady state. Due to the uncertainties in pathway structure, and parameter identifiability and estimation, it is of considerable value to explore multiple models of a biological system [34]. The goodness of fit to the experimental data of eight versions of the RNA processing pathway is compared in Table 1. The alternative pathways are distinguished by their representation of elongation, of co-transcriptional splicing step two, and of post-transcriptional splicing steps one and two. Elongation is modelled either as a single step or as 40 steps, as described above. The alternative models considered for the splicing reactions are: a single step, multiple steps (each at the same rate) or positive feedback. It is important to consider each pathway as a whole as the goodness of fit for each observed species is dependent on the reactions that act directly on the observed species, and those that act on its precursors and thereby shape the kinetics of the precursors. Table 1 defines each pathway and lists the AIC scores obtained using the optimal parameters (see Table 1 in Text S1 for the parameter values). Note that pathway slowromancap VIII makes the simplest assumptions about elongation and splicing steps, namely that they are single-step unimolecular reactions, and that the poor fit of this pathway to the data motivates the search for alternative models. Pathway parameters were optimised by a simulated annealing algorithm (see Materials and Methods; [35]) that identified the best fit between each pathway and the nine data series obtained for Ribo1 (those plotted in Figure 2 B, 3 B and 3 C). The total AIC (defined in Materials and Methods) is calculated from the combined residuals from all species/experiments. All data and pathway models are provided as files in Dataset S1. An executable version of the Dizzy simulator [36] is also provided to allow the models to be executed. The AIC scores for pre-mRNA, lariat-exon2 and mRNA are represented separately in the columns of the heat map in Figure 4. It is apparent from the A-pre-mRNA column that all pathways fit well to the pre-mRNA data in Expt 1, and fit to a comparable extent. The majority of pathways also fit the mature mRNA data well (A-mRNA and P-mRNA columns). Pathways I-IV can be optimised to the lariat-exon2 data simultaneously. In contrast, pathways V-VIII have a poor fit to one or more of the lariat-exon2 species. Pathway I has the best overall AIC as a result of fitting the nine data series most consistently. Pathways I-IV incorporate the feedback mechanism for co-transcriptional splicing step two and this feature correlates with a good fit (low AIC) for all lariat-exon2 species. Within these pathways, a multi-step representation of post-transcriptional splicing, combined with a multi-step representation of elongation has the best overall score (pathway I). Feedback in post-transcriptional splicing, combined with a multi-step representation of elongation also explains the data well (III). Pathway VII is ranked in third place, failing to explain the polyadenylated lariat-exon2 data in Expt 2 (as indicated by the white cell in row VII, column P-lariat-exon2 in Figure 4). The predictions of the pathways for each of the nine species measured in Expt 1 and 2 are plotted in Figure 2 in Text S1. Important qualitative differences between the pathways can be seen in these graphs. Pathways with a single elongation step require an initiation rate of 0. 4, and a rate for elongation of 0. 4–0. 54, giving an implausible time of 2–3 s for the elongation of a 1240 nucleotide gene. As a consequence of defining a more realistic elongation time, pathways with a multi-step representation of elongation typically fit the data better, see Table 1. In pathway I, pre-mRNA 3′ end maturation takes 35 s and uncleaved 3′ end mRNA maturation takes 49 s using the measure of the time taken for the sum of intermediate species undergoing the five steps of 3′ end processing to reduce by half (an equivalent to the half life of a single step reaction). The completion of splicing co-transcriptionally in yeast has been a topic of debate. Genome-wide ChIP studies indicated that co-transcriptional spliceosome assembly may not have time to occur if the 3′ exon is short [28]. More recent studies provide evidence for polymerase pausing 3′ of introns, suggesting a mechanism to slow transcription, allowing more time for splicing [10], [11]. With Ribo1 we observe that the initial burst of 3′ uncleaved pre-mRNA is not spliced before it is 3′ end cleaved, as shown by the successive blue and purple peaks in Figure 3 B, and it may undergo post-transcriptional splicing. After this initial burst, the majority of transcripts splice co-transcriptionally, as seen by the accumulation of uncleaved lariat-exon2 and uncleaved mRNA prior to cleaved/polyadenylated mRNA (red, green and black, respectively in Figure 3 C). Optimisation of pathway I computes to be 11. 39, and by substituting this value into equation 1 (see Materials and Methods) it follows that 12% of Ribo1 RNA transcripts splice post-transcriptionally, and 88% of transcripts splice co-transcriptionally. Values for in pathways III, V and VII imply the same proportion of co-transcriptional splicing, as do the values of and in the four remaining pathways where the proportion of co-transcriptional splicing is approximately 85%. Pathways I-IV show a good qualitative fit to the uncleaved lariat-exon2 data (see Figure 2E in Text S1). All four pathways specify a positive feedback mechanism for with estimated rate constants in the range 0. 0061–0. 0068 (see Table 1 in Text S1). In pathway I, the half life of this reaction is 110 s for the first transcript to splice, and, with feedback, the half life reduces to 5. 5 s at 670 s after induction. As, initially, the half life is much greater than the time to transcribe exon 2 (approximately 11 s), the decision to model the second step of splicing as a process that occurs after elongation is justified. The feedback mechanism may be a result of the disassembly and recycling of the snRNPs of the spliceosome for subsequent rounds of splicing [37]. It has been proposed that the branchpoint binding protein (BBP) and Mud2 are recycled between two steps in pre-spliceosome assembly: BBP is released during or after the second step and efficiently recycled to promote the first [38]. The finding that snRNPs do not assemble on a nascent transcript in response to a signal, but move randomly [22], does not preclude them impacting on splicing kinetics in a transcription-dependent manner through an influence on rates of spliceosome assembly, disassembly or recycling. Maintenance of the transient Cajal body (responsible for the maturation of snRNPs) requires continuous recycling of pre-existing snRNPs after each round of spliceosome assembly [22], and may therefore be indirectly dependent on transcription when splicing is co-transcriptional. If recycling mechanisms existed for second step factors, increasing the effective second step reaction rate, this could explain the peak and dip in uncleaved lariat-exon2 in Expt 2. The allosteric effects of second step splicing factors would provide an alternative explanation. Pathways I and III specify a multi-step representation of elongation and feedback in co-transcriptional splicing. They account for 99% of the probability mass available in the Akaike weight analysis (see Table 1). These two pathways differ on the post-transcriptional splicing mechanism: a multi-step representation is more probable (P = 0. 845) but a feedback mechanism cannot be ruled out (P = 0. 152). As pathway I has a better fit to the polyadenylated pre-mRNA and polyadenylated lariat-exon2 data (the precursor and products of post-transcriptional splicing), we tentatively conclude that post-transcriptional splicing has multi-step characteristics. The difficulty in modelling the post-transcriptional splicing process lies in its transient activation. The characteristic features of the feedback mechanism are not clearly revealed. For a multi-step model, the times for the sum of intermediate species undergoing splicing to reduce by half are 34 s for step one and 36 s for step two of splicing. The 3′SSRibo1 data are explained by a variation of the transcription and splicing pathway where step one of splicing can be co-transcriptional or post-transcriptional (as in the full pathway), but where the lariat-exon2 species goes through the five-step cleavage process (at rate) instead of step two of splicing (= 0 and = 0). The pathway used to explain the 5′SSRibo1 data has no co-transcriptional splicing path (cannot be reached), and no post-transcriptional splicing can occur (). The induction of the 3′SSRibo1 reporter (Figure 2 C) shows a greater accumulation of pre-mRNA than observed for Ribo1. The lariat-exon2 product is not spliced, but accumulates and is subject to degradation. The data can be explained by pathway I using the rate inferred for Expt 1 and 2. However, to predict the pre-mRNA response is increased to 30, is reduced to 0. 015, and is reduced to 0. 175. The probability of step 1 occurring co-transcriptionally is therefore reduced to 56% compared with 88% in Ribo1, the time taken for splicing to become active increases two fold, and the rate for the initiation of transcription reduces to 70% of the rate in Ribo1. The prediction for lariat-exon2 is greater than observed, and this may indicate that 3′ end maturation and/or lariat-exon2 degradation pathways differ in the mutant reporter. The induction of 5′SSRibo1 (Figure 2 D) shows that pre-mRNA accumulates and does not splice. The response can be explained by further reduction in to 0. 1, that is, 40% of the rate in Ribo1. The induction of 3′SSRibo1 was repeated using the primers of Expt 2 in order to validate the finding that the probability of co-transcriptional splicing is reduced. The new data are shown in Figure 3A in Text S1. The pathway model predicts only a slow removal of the accumulating uncleaved pre-mRNA (and consequently of polyadenylated pre-mRNA) that is consistent with the new data. In contrast, the large reduction in pre-mRNA that is predicted when the rate for is 11. 39 (as inferred for Ribo1) does not fit the new data, see Figure 3B in Text S1. The overestimation of lariat-exon2 by the model (Figure 2 C) might be explained by a significant underestimation of the degradation rate for this species. This rate has been determined in the 3′SSRibo1 ‘OFF’ strain where transcription is halted by doxycycline, see Text S2. (A second experiment using alternative primers confirms this result [23].) Alternatively, the assumption made when modelling 3SSRibo1 that uncleaved lariat-exon2 would be able to complete 3′ end maturation and contribute to the total population of polyadenylated lariat-exon2 may be incorrect. Modelling shows that polyadenylated lariat-exon2 may be the product of polyadenylated pre-mRNA alone, with no contribution from the co-transcriptional pathway. Despite the biochemical and genetic evidence for multiple steps in the cycle of splicing events, previous in vivo studies of mRNA splicing kinetics have revealed simple first-order monomolecular reactions that exclude the action of a catalyst. The allosteric cascade is yet to be revealed at the systems level, either in terms of the existence of multiple steps, or the impact of enzyme kinetics, and we argue that this is due to the course-graining phenomena associated with stochastic processes [39] and to the lack of experimental quantification of mRNA and its precursors. Using rapid sampling of cultures, combined with RT-qPCR assays that detect the intermediates and products of the splicing reaction in a way that permits quantitative comparisons between different RNA species and between different cultures, we are able to present kinetic data with an unprecedented level of resolution, monitoring pre-mRNA production, the two steps of splicing and 3′ end processing of a reporter transcript in yeast. Our data cannot be explained satisfactorily by single-step unimolecular splicing reactions. We conclude that a systems model of transcription and splicing must distinguish the two steps of splicing, account for their occurrence co- and post-transcriptionally, represent spliceosome assembly, and include the action of an additional partner in the splicing reactions, as we find evidence in the data for each of these processes. While developing the model, we considered including a transition from uncleaved lariat-exon2 to polyadenylated lariat-exon2, which would permit pre-mRNAs that have already undergone the first step of splicing co-transcriptionally to undergo 3′ end maturation. However, when this transition was added to model I, the AIC was found to increase by 1. 4 (after optimisation), meaning model I fits the data better without the additional transition. The proposed transition occurs very slowly, and consequently rarely, does not assist modelling the data, and, therefore, was excluded from the models we analysed further. The model proposed here specifies that pre-mRNAs that have already undergone the first step of splicing co-transcriptionally will be fully spliced co-transcriptionally prior to 3′ end cleavage. This is in contrast with the mammalian model proposed in [40] where splicing is completed after 3′ cleavage (in HeLa nuclear extracts). Both models stipulate that partially-spliced transcripts are not released from the DNA, and both allow for a post-transcriptional splicing pathway. Our model is consistent with the recycling of splicing factors [3]. Recycling of BBP and Mud2 has been proposed for pre-spliceosome formation [38], and similar mechanisms may exist for subsequent spliceosome assembly steps. Alternatively, it has been proposed that an increase in the local concentration of splicing factors is linked to transcription via the C-terminal domain of Pol II [15]. Nuclear speckles may also have a role in keeping spliceosomal components concentrated near nascent transcripts [37]. Cooperativity in the interaction of splicing factors with the spliceosome or with the nascent pre-mRNA may also contribute to the kinetics of co-transcriptional splicing. Addressing the interdependency between RNA processing steps, modelling indicates that mutations at the 3′ and 5′ splice sites reduce the rate of initiation of transcription, and, in the 3′SS mutant, reduce the probability of step one of splicing occurring co-transcriptionally. Quantitative analysis of the mutant data requires establishing a parameterised model for the ‘wild type’ in order to define and test the alternative explanations of the differences observed. A half life for splicing in HeLa cell nuclear extracts of 23 min (splicing rate of 0. 03/min) has been reported [15]. In vivo half-lives of 6–12 min have been reported in mammalian cells [41], as have estimates of 5–10 min for the completion of splicing after intron synthesis [42]. Half lives for splicing in the range 0. 4–7. 5 min have also been reported for the splicing of introns in mammalian cells [43]. The inferred rates for post-transcriptional splicing in Ribo1 equate to half lives of 0. 6 min for each of steps one and two, and are at the faster end of the spectrum reported in [43]. On the co-transcriptional pathway, splicing step one is concurrent with the transcription of the 800 bases from the branchsite until the polyA site (taking approximately 11 s). Co-transcriptional step two occurs with a half life of 110 s for the first transcript, and, with feedback, the half life reduces to 5. 5 s at 670 s after induction. Therefore co-transcriptional splicing is the more efficient pathway under the high induction conditions studied here. This study proposes a mechanistic kinetic model that represents some of the complexity and flexibility of the splicing pathway that is known from biochemical and genetic studies [3]. Co-transcriptional splicing is evident in the data, and modelling shows that this pathway may be activated after a delay. Furthermore, the second step of splicing benefits from positive feedback when co-transcriptional. These could be explained by the coordination of splicing factor recruitment/recycling with transcription, possibly facilitated by polymerase pausing [10], [11] and/or dynamic chromatin modification [9], [44], [45]. To analyse the transcription, splicing, degradation and 3′ end formation of yeast pre-mRNA, the Ribo1 reporter was integrated into the yeast genome at the his3 locus. The reporter is based on a hybrid ACT1/PGK1 gene [31], modified as described in [23] by inserting two copies of the boxB sequence (57 bp each) in the ACT1 intron, enabling it to be readily distinguished by RT-qPCR from the endogenous ACT1 intron without affecting splicing. Primer pairs were created to measure the unspliced pre-mRNA (5′ primer upstream of ATG, 3′ primer over the exon 1 - intron junction), the lariat-exon2 intermediate (5′ primer upstream of 3′end of intron, 3′ primer over exon 2; the pre-mRNA level was subtracted from this measurement) and the spliced mRNA (5′ primer upstream of ATG, 3′ primer over exon 2). Measurements of mRNA in copies per cell, averaged over a population, were obtained by carefully quantifying the efficiency of cell lysis, recovery of RNA, reverse transcription and qPCR. For full details see [23]. The first step of post-transcriptional splicing, and all transitions to the path, decrease pre-mRNA and increase lariat-exon2. The second step of splicing decreases lariat-exon2 and increases spliced mRNA, according to the pathway. All species in the pathway, with the exception of the excised intron product of step two, are measurable by RT-qPCR, provided that they extend beyond the position of the cDNA primer. Splicing events on transcripts that have not been elongated to the cDNA point are not detected until this sequence is transcribed, and the calculation of RT-qPCR signal intensity from the species in the pathway reflects this. For example, the (simulated) pre-mRNA signal is not incremented until the species is incremented, despite the PCR primers for pre-mRNA being located several hundred bases upstream. Considering a single Pol II complex (and ignoring the effect of other Pol IIs on its movement), the probability of transitioning from states to the co-transcriptional path is simply calculated from the elongation rate and the transition rate. This choice can be made 25 times, allowing the probability of the Pol II exiting on the post-transcriptional pathway to be estimated independently of by: (1) Unless otherwise stated, reaction rates are expressed as the probability density per unit time, per distinct combination of reactant molecules. Where there is a single reactant species, the number of distinct combinations is just the population of reactants. The half life is the time a molecular species takes to reduce by half, and is computed for unimolecular reactions by in units of seconds. Pathway models were optimised by the simulated annealing algorithm specified in [35] (see Figure 1). Following [35], the error E is defined by equation 2 where S is a time series simulated from a pathway model, D is the observed data, n is the number of time points and d the number of dependent variables (the dimension of and is d). (2) On each iteration of the algorithm, each parameter is assigned a new value () and the error for the new set of parameters () is calculated from a simulation of the model using the updated parameter set. The new parameter value is always accepted if, otherwise it is accepted with probability, where T is the current temperature and E is the error of the current parameter set. The new parameter value is generated from the current value by adding a normally-distributed random value. We define the scale constant k in equation 3 using the error of a set of parameter values that are given as input at the start of optimisation (these must provide an approximate fit to the data), and then update each parameter value according to equation 4, where N (0,1) is a normally-distributed random value (mean 0, standard deviation 1) and and are the maximum and minimum values respectively that is allowed to take. See [35] for further details. (3) (4) The Akaike information criterion (AIC; eqn. 5) was used to assess the fit between a time series S simulated from a pathway model of k optimised parameters, a data set D of n values [46]. Assuming normally distributed errors, AIC can be computed from the model residuals (eqn. 6) [47]. The values for total AIC incorporate the 2 k penalty for the number of parameters optimised. (5) (6) When comparing m pathway models, the Akaike weight w of model i can be defined in terms of the relative likelihood, where is the difference between the AIC for model i and the AIC of the best model [47]. Akaike weights computed by equations 7 and 8 are listed in Table 1. (7) (8)

The coding information for the synthesis of proteins in mammalian cells is first transcribed from DNA to messenger RNA (mRNA), before being translated from mRNA to protein. Each step is complex, and subject to regulation. Certain sequences of DNA must be skipped in order to generate a functional protein, and these sequences, known as introns, are removed from the mRNA by the process of splicing. Splicing is well understood in terms of the proteins and complexes that are involved, but the rates of reactions, and models for the splicing pathways, have not yet been established. We present a model of splicing in yeast that accounts for the possibilities that splicing may take place while the mRNA is in the process of being created, as well as the possibility that splicing takes place once mRNA transcription is complete. We assign rates to the reactions in the pathway, and show that co-transcriptional splicing is the preferred pathway. In order to reach these conclusions, we compare a number of alternative models by a quantitative computational method. Our analysis relies on the quantitative measurement of messenger RNA in live cells - this is a major challenge in itself that has only recently been addressed.

lay_plos

Medulloblastoma is the most common malignant brain tumor in children. A subset of medulloblastoma originates from granule cell precursors (GCPs) of the developing cerebellum and demonstrates aberrant hedgehog signaling, typically due to inactivating mutations in the receptor PTCH1, a pathomechanism recapitulated in Ptch1+/− mice. As nitric oxide may regulate GCP proliferation and differentiation, we crossed Ptch1+/− mice with mice lacking inducible nitric oxide synthase (Nos2) to investigate a possible influence on tumorigenesis. We observed a two-fold higher medulloblastoma rate in Ptch1+/− Nos2−/− mice compared to Ptch1+/− Nos2+/+ mice. To identify the molecular mechanisms underlying this finding, we performed gene expression profiling of medulloblastomas from both genotypes, as well as normal cerebellar tissue samples of different developmental stages and genotypes. Downregulation of hedgehog target genes was observed in postnatal cerebellum from Ptch1+/+ Nos2−/− mice but not from Ptch1+/− Nos2−/− mice. The most consistent effect of Nos2 deficiency was downregulation of growth-associated protein 43 (Gap43). Functional studies in neuronal progenitor cells demonstrated nitric oxide dependence of Gap43 expression and impaired migration upon Gap43 knock-down. Both effects were confirmed in situ by immunofluorescence analyses on tissue sections of the developing cerebellum. Finally, the number of proliferating GCPs at the cerebellar periphery was decreased in Ptch1+/+ Nos2−/− mice but increased in Ptch1+/− Nos2−/− mice relative to Ptch1+/− Nos2+/+ mice. Taken together, these results indicate that Nos2 deficiency promotes medulloblastoma development in Ptch1+/− mice through retention of proliferating GCPs in the external granular layer due to reduced Gap43 expression. This study illustrates a new role of nitric oxide signaling in cerebellar development and demonstrates that the localization of pre-neoplastic cells during morphogenesis is crucial for their malignant progression. Medulloblastoma (MB) is a highly malignant tumor of the cerebellum that preferentially develops in children and adolescents. Although the survival rate for standard risk MB is around 70% [1] surviving patients often suffer from neurodevelopmental and cognitive side effects of the aggressive therapy [2]. Therefore, improved understanding of the molecular pathomechanisms driving MB growth is necessary to develop less toxic and more effective treatments. Recent molecular profiling studies suggested at least four MB subtypes that are associated with distinct expression profiles, genomic aberrations and clinical features [3], [4]. One of these MB subtypes is characterized by aberrant activation of the hedgehog (Hh) pathway and typically corresponds to the desmoplastic (nodular) MB variant. This subtype is supposed to develop from granule cell precursors (GCPs) of the external granular layer (EGL) [5]. The EGL is a transient germinal zone at the subpial cerebellar surface consisting of rhombic lip-derived progenitor cells that have migrated tangentially to the emerging cerebellar cortex at late stages of embryonal brain development [6]. During the early postnatal period in mouse, the morphogenic factor sonic hedgehog (Shh) is secreted by subjacent Purkinje cells and binds to patched receptors (Ptch1 and Ptch2) expressed on the GCP surface [7]. Ligand binding to Ptch1 then leads to functional de-repression of Smoh (Drosophila smoothened homolog) and subsequent activation of Gli (Glioma-associated oncogene family zinc finger) transcription factors [8]. This launches a temporally concerted gene expression pattern causing a proliferation burst and massive expansion of the GCP population during the first two postnatal weeks [7]. In particular, the direct Gli-target N-myc [9], [10] and D type cyclins [11] were shown to be crucial for the growth and neoplastic transformation of GCPs [12]. In addition, the set of genes targeted by activated Gli transcription factors also include components of the canonical Hh pathway for feedback-loop regulation, such as the receptors Ptch1 and Ptch2 as well as the hedgehog-interacting protein (Hip) [10], [13]. After several rounds of cell division, GCPs normally exit cell cycle and accumulate at the inner site of the EGL [14], where they start to migrate through the molecular layer (ML) and the Purkinje cell layer to form the internal granular layer (IGL) [15]. The mechanisms underlying the attenuation of the mitotic response and eventually the stop of GCP proliferation are not well understood. The most evident concepts describe extrinsic cues in gradient-based models to trigger GCP differentiation with increasing distance to the region of the outer EGL [16]. Finally, the EGL disappears at about three weeks after birth in mice. PTCH1 was identified as a frequent target of inactivating mutations or genomic loss in sporadic MBs [17]–[19] that belong to the molecular subtype hallmarked by an aberrant activity of hedgehog signaling. The monoallelic inactivation of the Ptch1 gene in mice and thus downstream activation of the Hh pathway leads to MB development at a frequency of about 10–15% [20]. This mouse model has provided substantial insights into the pathogenesis of Hh-dependent MBs and has been used in different cross-breeding experiments to investigate tumor suppressor gene functions in this particular context [21], [22]. Nitric oxide (NO) is a highly reactive gaseous molecule involved in various physiological processes ranging from vasculature modulation to neurotransmission [23], [24]. NO is produced by three distinct enzyme isoforms: i) neuronal nitric oxide synthase (nNos/Nos1), ii) inducible nitric oxide synthase (iNos/Nos2), and iii) endothelial nitric oxide synthase (eNos/Nos3). Though being constitutively expressed in their respective tissue, nNos and eNos activity strongly depends on calcium [25], whereas calcium-independent iNos is primarily regulated by transcriptional induction, e. g. by inflammatory cytokines and endotoxins [26], which permits higher quantities of NO generation. The role of NO in cancer initiation and progression is heterogeneous with opposing effects in different malignancies [27]. Considering effects of tumor stroma, increased angiogenesis was reported to be associated with elevated Nos activity [28] and some immune-related processes were found to be mediated by NO [29], including cytotoxicity of activated microglia [30]. Finally, NO released by vascular endothelial cells was reported to build a niche-like microenvironment for maintenance of glioma stem cells [31]. In the context of cerebellar development, Nos2 (inducible Nos) is initially expressed in early GCPs, whereas Nos1 (neuronal Nos) is hardly present before postnatal day 7 (Cerebellar Development Transcriptome Database [32]). Successively, Nos1 expression increases along with granule cell differentiation [33] and predominantly contributes to the common NO signaling that becomes apparent in the IGL as development proceeds [34]. Evidence has been provided that NO negatively acts on proliferation of neuronal precursors during adult neurogenesis [35]. Similarly, Ciani and colleagues demonstrated enhanced proliferation of cerebellar precursor cells upon inhibition of NO synthases [36]. Here, we report on the generation of Ptch1+/− Nos2−/− mice to investigate the impact of Nos2 on tumor development in Ptch1 hemizygous mutant mice. Interestingly, we observed an approximately two-fold increase in the incidence of spontaneous MB in Ptch1+/− Nos2−/− mice in comparison to Ptch1+/− Nos2+/+ mice. To characterize the molecular pathomechanism underlying the tumor-promoting effect of Nos2 deficiency in Ptch1+/− mice, we performed comprehensive expression and DNA copy number profiling of MB tumors (Ptch1+/− Nos2+/+ versus Ptch1+/− Nos2−/−) as well as expression profiling of normal cerebellar tissue samples from different developmental stages and various genotypes (Ptch1+/− Nos2+/+, Ptch1+/− Nos2−/−, Ptch1+/+ Nos2−/− and wild-type mice). Downregulation of the growth-associated protein 43 (Gap43) was the most striking feature in the cerebellum of Nos2-deficient mice when compared to Ptch1+/− Nos2+/+ and wild-type mice. Subsequent functional analyses and results from in situ studies of GCPs in postnatal cerebellum allowed us to formulate a model for the tumor promoting role of Nos2 deficiency in Ptch1 mutant mice via deregulation of Gap43-dependent migration of GCPs. Survival analyses of 315 wild-type mice, 412 Ptch1+/+ Nos2−/− mice, 215 Ptch1+/− Nos2+/+ mice and 221 Ptch1+/− Nos2−/− mice demonstrated a significantly higher MB incidence in the group of Ptch1+/− Nos2−/− mice relative to the group of Ptch1+/− Nos2+/+ mice (p = 0. 0007, Logrank test, Figure 1A). In total, 11% of the Ptch1+/− Nos2+/+ mice (24/215) and 21% of the Ptch1+/− Nos2−/− mice (47/221) were sacrificed due to the development of cerebellar MB. None of the 315 wild-type and the 412 Ptch1+/+ Nos2−/− mice developed MBs. These observations indicate a MB-promoting role of Nos2 deficiency in Ptch1+/− mice. In humans, Hh-dependent MBs typically correspond to the desmoplastic subtype. MBs in Ptch1+/− mice, however, microscopically resemble the classic MB subtype [20]. Histological analysis of MBs in Ptch1+/− Nos2+/+ and Ptch1+/− Nos2−/− mice demonstrated similar morphological features (Figure 1B–1E). The tumors were composed of densely packed sheets of cells with hyperchromatic carrot-shaped nuclei and scant cytoplasm. There were no obvious histopathological differences between MBs of the two genotypes. For an initial assessment of the molecular tumor characteristics, gene expression of hedgehog signaling pathway components were measured in 21 MBs and 24 normal (adult) cerebellar tissue samples from both Ptch1+/− Nos2+/+ and Ptch1+/− Nos2−/− mice. Using quantitative real-time PCR (qRT-PCR), significant downregulation of the wild-type Ptch1 transcript and upregulation of the Shh target genes Gli1 and N-myc were generally observed in the tumor samples (Figure S1), indicating all examined MBs to be of the same Hh-dependent molecular subtype. However, there were no significant differences for these genes between MBs of the two genotypes. Furthermore, targeted genetic analyses showed a loss of the wild-type Ptch1 allele in 10 of the 21 MBs investigated, while none of the tumors demonstrated a Tp53 mutation or N-myc amplification. The Cdkn2a/p16INK4a locus was retained in all tumors while a single MB demonstrated a homozygous p19ARF deletion (see Table S1 and Text S1 for details). In order to identify the molecular pathomechanism contributing to the increased MB rate in Nos2-deficient Ptch1 mutant mice, we performed array-based gene expression profiling of three Ptch1+/− Nos2+/+ versus six Ptch1+/− Nos2−/− and comparative genomic hybridization (array-CGH) analyses of five Ptch1+/− Nos2+/+ versus seven Ptch1+/− Nos2−/− MB tissue samples. All specimens investigated had tumor cell contents between 70% and 90% as determined on corresponding formalin-fixed and paraffin-embedded (FFPE) reference sections. Differential expression of selected candidate genes was validated by qRT-PCR on an expanded, partially overlapping tumor set of seven Ptch1+/− Nos2+/+ versus seven Ptch1+/− Nos2−/− MB samples. The expression profiling analysis revealed a total of 87 differentially regulated genes between tumors of the two genotypes (Table S2) with the vast majority (87%) showing lower transcript levels in Ptch1+/− Nos2−/− when compared to Ptch1+/− Nos2+/+ mice. As expected from the initial targeted qRT-PCR measurements, there was no difference detectable concerning the activation of Hh pathway genes. Due to the important role of Nos2 during angiogenesis and cancer-associated immune response, including microglia, stromal effects need to be particularly considered in a systemic Nos2 knockout model. However, neither the set of significantly deregulated genes nor selective determination of marker expression for pericytes, vascular endothelial cells or microglia suggested any differences in the tumor stroma between the two genotypes (see Table S3 and Text S1 for details). According to the findings of Ciani and co-workers [37], reduction of NO enhances GCP proliferation through an increased expression of the proto-oncogene N-myc. Therefore, protein levels were particularly examined for differences between tumor samples from Ptch1+/− Nos2+/+ and Ptch1+/− Nos2−/− mice. The results shown in Figure S2, however, revealed similar expression of N-myc in all MBs. Analyses of genomic copy number alterations revealed a trisomy of chromosome 6 in the majority of MBs from both groups (11/12, Figure 2A and 2B). Moreover, a small region on chromosome 13, approximately 1. 5 Mb upstream of the Ptch1 gene, showed a hemizygous deletion in healthy cerebella of Ptch1-mutant mice (data not shown) but a homozygous deletion in most tumors (10/12). Similarly, a second small region 3. 8 Mb downstream of the last Ptch1 exon exhibited a loss in 9 of 12 MBs. This suggests structural changes flanking the Ptch1 locus that likely contribute to inactivation of the wild-type allele. The frequencies of genomic aberrations showed a more heterogeneous karyotype with gross structural changes in Ptch1+/− Nos2+/+ MBs when compared to Ptch1+/− Nos2−/− MBs (see Figure 2A, 2B and Text S1 for details). However, a general difference in chromosomal instability was not obvious between both genotypes. Only a small region (205. 6 kb) on chromosome 14 containing the Entpd4 (ectonucleoside triphosphate diphosphohydrolase 4) gene was more frequently gained in Ptch1+/− Nos2−/− MBs (7/7) than in Ptch1+/− Nos2+/+ MBs (1/5, Figure 2A). Accordingly, Entpd4 expression appeared to be specifically upregulated in expression profiles of Ptch1+/− Nos2−/− tumors. QRT-PCR validation confirmed an elevated mean expression in Ptch1+/− Nos2−/− compared to Ptch1+/− Nos2+/+ MBs in those samples that overlapped with the array-CGH analysis but revealed no significant difference across the expanded tumor set (Figure 2C). As GCPs are considered the cells of origin for the Hh-dependent MB subtype, we examined the effect of Nos2 ablation in the context of cerebellar development. Therefore, gene expression profiles of normal cerebellar tissue samples from postnatal day 9 (P9), 6 weeks after birth (6W), and 1 year of age (1Y) were generated from wild-type, Ptch1+/− Nos2+/+, Ptch1+/− Nos2−/−, and Ptch1+/+ Nos2−/− animals. While specimens of mature cerebellum (6W and 1Y) were investigated separately in 3 biological replicates per genotype and developmental stage, samples of postnatal cerebellum consisted of pooled RNA from 4–5 individuals processed in technical replicates due to limited tissue amounts. An unsupervised hierarchical cluster analysis of transcriptome data clearly separated developing cerebellum (P9) of wild-type mice and the two Ptch1-mutated genotypes from mature cerebellum. Interestingly, P9 cerebellum of Ptch1+/+ Nos2−/− mice displayed different properties highly similar to mature cerebellum (Figure 3A). Expression profiles of MBs formed a distinct cluster clearly separated from all healthy tissue samples. A direct comparison between gene expression profiles from Ptch1+/+ Nos2−/− and wild-type P9 cerebellar tissue samples resulted in a total of 984 deregulated genes with 755 genes (76. 7%) showing a decreased expression in Ptch1+/+ Nos2−/− mice (Table S4). P9 cerebellum from Ptch1+/− Nos2+/+ and Ptch1+/− Nos2−/− mice revealed only 5 and 32 deregulated genes relative to wild-type, respectively (Table S5 and Table S6). This large deviation of postnatal gene expression in the Ptch1+/+ Nos2−/− genotype included a set of downregulated genes that are essential for proliferation of GCPs (e. g. cyclin D1, cyclin D2 and N-myc, Figure 3B). As hedgehog signaling constitutes the main regulatory pathway for neonatal cell proliferation in GCPs of the EGL, the 984 deregulated genes were analyzed for enrichment of Gli transcription factor targets. Matching this list to a set of recently identified Gli-targets in GCPs [10] yielded a significant overrepresentation of Gli1-regulated genes (p = 0. 005, chi-square test). Hence, the reduced transcript levels of these target genes suggests an attenuated hedgehog signaling in postnatal Ptch1+/+ Nos2−/− cerebellum compared to wild-type (or any other genotype). Notably, the decreased expression of Gli1-targets and proliferation-associated genes observed in Nos2-deficient cerebellar tissue was abolished upon additional inactivation of the hedgehog receptor Ptch1 (in Ptch1+/− Nos2−/− mice). Therefore, we examined the transcript levels of patched receptors themselves in more detail. While neither Ptch1 nor Ptch2 expression was changed between wild-type and Ptch1+/− Nos2+/+ P9 cerebellum, a significant increase of Ptch1 and a minor increase of Ptch2 expression were observed in Ptch1+/+ Nos2−/− mice relative to wild-type mice (Figure 3C). Notably, in Ptch1+/− Nos2−/− cerebellar tissue samples, Ptch2 expression was more elevated than Ptch1. However, since Ptch2 is not capable of inhibiting smoothened (Smoh), it probably failed to take over the attenuating effect on Gli activity [38]. MB specimens from Ptch1+/− Nos2+/+ versus Ptch1+/− Nos2−/− mice showed no significant difference in expression levels of either patched receptor, with Ptch2 being substantially increased over Ptch1 in both groups (Figure 3C). These findings indicate that Nos2 deficiency leads to an upregulation of Ptch1 in GCPs, which results in a downregulation of mitotic genes and Gli-targets only in a Ptch1-wild-type background. So far, Nos2 inactivation was shown to counteract proliferation and antagonize hedgehog signaling in developing cerebella. To identify those Nos2-dependent effects promoting MB induction, we determined the features that were common to Ptch1+/+ Nos2−/− and Ptch1+/− Nos2−/− genotypes and persisted in the tumor tissues. Accordingly, the overlap of differential gene expression from three comparisons was built: i) Ptch1+/+ Nos2−/− versus wild-type P9 cerebellum, ii) Ptch1+/− Nos2−/− versus Ptch1+/− Nos2+/+ P9 cerebellum; and iii) Ptch1+/− Nos2−/− versus Ptch1+/− Nos2+/+ MB. As a result, only 2 genes were observed to be deregulated in a Nos2-dependent manner during cerebellar development and in MBs (Figure 4A). Although Stmn1 (stathmin 1) appeared to be upregulated in Ptch1+/− Nos2−/− MBs relative to Ptch1+/− Nos2+/+ MBs, this could not be confirmed by qRT-PCR (Figure S3). Gene expression of Gap43 was consistently reduced in Nos2-deficient cerebellar tissue samples and downregulation in Ptch1+/− Nos2−/− tumors relative to Ptch1+/− Nos2+/+ tumors was also significant in the expanded validation set (Figure 4C and 4D). To further assess the immediacy of Nos2 inactivation and Gap43 deregulation, Gap43 transcript levels were determined in expression profiles of healthy cerebella from all developmental stages (P9,6W and 1Y). Groups for comparison were built according to presence or absence of Nos2, irrespective of the Ptch1 status. The results clearly demonstrated a close association of altered Gap43 transcript levels and Nos2 status (Figure 4B), and indicated downregulation of Gap43 to be the most common effect of Nos2 deficiency in the cerebellum. To investigate differences in Gap43 expression on protein level in situ we performed immunofluorescent double stainings of Gap43 and the proliferation marker Ki-67 on FFPE sections of P9 cerebella from wild-type, Ptch1+/− Nos2+/+, Ptch+/+ Nos2−/− and Ptch1+/− Nos2−/− mice. As illustrated in Figure 4E, Gap43 immunofluorescence was particularly prominent in the outer region of the molecular layer (ML) that is connected to and partially comprised of radial GCP process extensions. Image quantification further indicate a quantitative difference of Gap43 expression in this region with sections from wild-type and Ptch1+/− Nos2+/+ mice showing a more intense staining than sections from Ptch1+/− Nos2−/− and Ptch1+/+ Nos2−/− mice (Figure 4F). The association of Nos2 inactivation and decreased Gap43 expression suggests a gene-regulatory function of NO signaling. In order to investigate this possible link in vitro, we used the murine cerebellar precursor cell line c17. 2 and the human MB cell line D458 (see Text S2 for details). Both cell lines were treated either with the Nos inhibitor L-NAME (Nω-nitro-L-arginine methyl ester) to reduce NO levels or solvent control (Figure S4). Relative expression of Gap43 was assessed every 24 hours by qRT-PCR. In c17. 2 cells, Gap43 transcript abundance was generally low and increased with culture duration. We observed a slightly decreased expression of Gap43 upon L-NAME treatment that reached significance (p = 0. 023) after 120 hours (Figure 5A). NOS inhibition in D458 human MB cells resulted in a significant reduction of Gap43 transcript levels starting already after 72 hours with further decrease after 96 hours and 120 hours (Figure 5B). FACS analyses of apoptosis and cell cycle excluded these observations to be attributed to secondary effects of changing cell conditions (Figure S5). These results suggest Gap43 downregulation as a direct consequence of reduced NO levels in murine neuronal precursors and human MB cells. The dependency of Gap43 expression on NO signaling suggests this gene as key mediator of the effects observed in Nos2-deficient P9 cerebellum and Ptch1+/− Nos2−/− MB, in particular, the upregulation of functional Ptch1 in Ptch1+/+ Nos2−/− mice. Mishra et al. recently reported a central role of Gap43 in the polarization of developing GCPs by regulating centrosome positioning and thus defining correct orientation towards the IGL [39]. Since this is a prerequisite for directed migration, reduced levels of Gap43 in P9 cerebellar tissue may lead to retention of GCPs in the EGL. To test these hypotheses, shRNA-mediated knockdown of Gap43 was performed in c17. 2 cells (see Text S1 for details). Upon knockdown of Gap43 we observed a strong inverse behavior of Ptch1 and Gap43 transcript levels (Figure 5C). Changes in migration characteristics were assayed in a Boyden chamber using recombinant SDF-1α (CXCL12) as chemoattractant, which was reported to participate in guiding migration of embryonal GCPs in vivo [40]. Downregulation of Gap43 yielded a significant decrease in cell migration between 14% (p = 0. 013) and 20% (p = 0. 007) (Figure 5D; Figure S6C). A pseudo-effect of the knockdown due to altered proliferation of the v-myc-immortalized c17. 2 cells was excluded by FACS-based cell cycle analysis (Figure S7). Transcriptome and functional analyses suggest that a decreased Gap43 expression accounts for Ptch1 upregulation and impairment of directed neuronal precursor migration in vitro. Accordingly, Ptch1+/+ Nos2−/− P9 cerebella are supposed to increasingly retain GCPs with reduced mitotic activity in the EGL compared to wild-type and Ptch1+/− Nos2+/+ mice. Moreover, the Ptch1+/− Nos2−/− genotype is also expected to exhibit retention of GCPs, but not to show any cell cycle arrest. To further verify this hypothesis in situ we performed immunofluorescent double staining of proliferating (Ki-67+) and post-mitotic GCPs on FFPE sections of postnatal cerebellum (Figure 6A). Here, post-mitotic cells were delineated by the neuronal marker NeuN (neuronal nuclear antigen A60) [41]. At least three different regions of each mouse cerebellum were analyzed in three to four animals per genotype using confocal laser scanning microscopy. In accordance with the microarray data, averaged cell counts of wild-type and Ptch1+/− Nos2+/+ mice did not show significant difference. In contrast, an increase of post-mitotic GCPs (NeuN+, Ki-67−) was detectable in the EGL of Ptch1+/− Nos2−/− and Ptch1+/+ Nos2−/− mice (Figure 6C). Concurrently, the ratio of dividing to non-dividing GCPs was similar in Ptch1+/− Nos2−/−, wild-type and Ptch1+/− Nos2+/+ P9 cerebella but markedly decreased in Ptch1+/+ Nos2−/− mice. This recapitulated the downregulation of mitotic genes observed in the expression profiles. However, the total amount of proliferating GCPs per EGL section was significantly higher in Ptch1+/− Nos2−/− mice compared to any other genotype (Figure 6C). These results demonstrate a tissue phenotype that corresponds to the effects of reduced Gap43 in developing cerebellar neuronal precursors (in vitro). The increased accumulation of proliferating GCPs in the EGL observed in the Ptch1+/− Nos2−/− genotype supposedly leads to a larger pool of cells susceptible to neoplastic transformation and is therefore likely to promote medulloblastoma development. The Ptch1+/− MB mouse model has been intensively studied and has greatly contributed to our understanding of Hh-dependent MB tumorigenesis in the context of cerebellar development. The data presented here indicate a role of Nos2 and hence NO signaling in Hh-dependent MB by demonstrating a significantly increased MB rate in Ptch1+/− Nos2−/− mice compared to Ptch1+/− Nos2+/+ mice. The global genome-wide screens performed in the present study did not reveal obvious molecular differences between MBs in Ptch1+/− Nos2+/+ versus Ptch1+/− Nos2−/− animals. Assessment of genomic alterations using array-CGH identified trisomy of chromosome 6 as a recurrent feature in tumors of both genotypes. This corresponds to a recent report on MBs of the same molecular subtype with inactivated double-strand break repair proteins targeted to neuronal progenitors of p53−/− mice [42]. The most common loss identified in our analyses affected two small regions on chromosome 13 encompassing the Ptch1 gene and possibly indicate acquired homozygosity for the mutant allele or somatic rearrangements rather than a broad deletion of the locus. Targeted duplex PCR further confirmed loss of the functional wild-type allele to be a frequent event in these MBs. Notably, tumors of the Ptch1+/− Nos2−/− genotype showed a higher frequency of a small gain on chromosome 14. The affected Entpd4 gene encodes for an apyrase located at the internal membrane of lysosomal vacuoles and the Golgi apparatus. It preferentially catalyzes the hydrolysis of UDP to UMP [43] and thereby facilitates the inverse directed import of UDP-GlcNAc [44]. This in turn was reported to increase glycosylation of surface receptors (e. g. EGFR and PDGFR) and foster cell growth [45]. According to the microarray and qRT-PCR expression data, Entpd4 transcript levels were indeed increased in tumors with this chromosomal gain. However, this effect did not turn out to be Nos2-dependent in an expanded sample set. Consequently, Entpd4 likely plays a role in MB pathogenesis but is not directly linked to loss of Nos2. The examination of tumor-relevant changes in developing cerebellum as a consequence of impaired Nos2 activity and hence NO signaling surprisingly revealed a decreased proliferation of GCPs in the cerebellum of Ptch1+/+ Nos2−/− mice. The concurrent upregulation of Ptch1 and the significant enrichment of downregulated Gli1-target genes strongly suggest that this effect is a consequence of reduced hedgehog signaling. Moreover, this phenotype was completely abrogated by a concomitant Ptch1 mutation. The slight increase of Ptch2 in Ptch1+/− Nos2−/− cells points to a compensatory effect and further supports the notion of an inhibitory function of Nos2 loss on the hedgehog pathway in postnatal cerebellum. Since neither a Smoh-regulating domain [38] nor a function for cell cycle arrest through seizing cyclin B1 [46] were reported for Ptch2, its upregulation may be insufficient for preventing MB induction. In contrast to these observations, Ciani et al. demonstrated that proliferation of cultured GCPs increased upon withdrawal of NO and that this effect was mediated by augmented N-myc levels [37]. However, N-myc was not differentially expressed between Ptch1+/− Nos2−/− and Ptch1+/− Nos2+/+ MBs of our series. A possible explanation for this discrepancy might be an unrecognized heterogeneity in the isolated cerebellar cell population used in the Ciani study. Since eNos and nNos are known to attenuate the mitotic activity of subventricular neuronal stem cells [47], [48] Nos inhibitor treatment possibly resulted in a selective growth advantage over GCPs. Downregulation of Gap43 was the only feature observed in Nos2-deficient versus Nos2-proficient postnatal cerebella irrespective of the Ptch1 status. This difference was also conserved between Ptch1+/− Nos2−/− and Ptch1+/− Nos2+/+ MBs, and particularly visible in outer regions of the molecular layer, where maturating GCPs of the EGL develop contact forming projections prior to radial migration. Other studies already suggested a link between Gap43 mRNA levels and NO signaling due to co-induction of nNos and Gap43 during axon regeneration and reactive synaptogenesis following injury of spinal motoneurons [49] and sensory neurons [50]. Furthermore, a downregulation of Gap43 was reported after silencing of soluble guanylate cyclase subunits, the central elements of cGMP-mediated NO signaling [51]. Finally, the present study demonstrates Gap43 downregulation to be a consequence of NO withdrawal in neuronal progenitors and MB cells. A possible mechanism for this regulation refers to decreased protein levels of the poly (U) -binding and degradation factor AUF1 upon NO-dependent cGMP production [52]. AUF-proteins generally bind to AU-rich elements of the 3′UTR (untranslated region) of coding transcripts and associate with proteins of the ELAV-like family to control gene expression via mRNA decay [53]. Tsai et al. demonstrated that Gap43 mRNA levels are post-transcriptionally regulated during neuronal differentiation and that elements of the 3′UTR confer transcript instability, which is abolished upon TPA treatment (inter alia inducing NOS2) [54]. At the same time, Chung et al. demonstrated that indeed ELAV-like family member HuD was binding to 3′UTR regions of GAP43 [55]. Taken together, NO accumulation possibly decreases cellular levels of mRNA-destabilizing AUF1 protein and thus might contribute to a high transcript abundance of Gap43. Gap43 is a membrane-anchored protein at the cytoplasmic side of neuronal cell projections and found to be highly expressed during development of the CNS [56]. It is particularly localized in axonal growth cones and participates in the coordination of extrinsic stimuli and intrinsic cell remodeling [57] by regulating cytoskeleton dynamics [58]. Granule cell (GC) migration follows a sequence of tangential and radial movements controlled by successive formation of leading projections [59]. As maturating GCPs exit cell cycle, positioning of the centrosome determines the site of axon growth cone emergence and thus neuronal polarity including localization of such projections [60]. This defines the structural orientation of GCPs in terms of directing its dendrite to descend across the molecular and Purkinje cell layers to populate the IGL. However, centrosome positioning and therefore accurate polarization of GCPs require phosphorylated Gap43 to bind to the centrosome-associated microtubule-organizing center [61]. Hence, inaccurate GCP migration was observed in Gap43−/− animals [39], a finding that is in full agreement with our data from the functional Gap43 knockdown assays. Downregulation of Gap43 in Nos2-deficient P9 cerebellum therefore likely mediates the retention of GCPs observed in FFPE sections. Accordingly, NO/cGMP signaling was demonstrated to be crucial for accurate migration of the neuronal precursor cell line NT2 [62]. Furthermore, slice culture experiments of neonatal cerebella (P9) exhibited a substantial reduction of proliferation and migration of maturating granule cells to the IGL upon application of NO synthases inhibitors [63]. The elevation of Ptch1 levels upon Gap43 reduction in vitro fits to the data by Shen et al. who reported an upregulation of Ptch1 gene expression in inner EGL regions of Gap43−/− mice compared to wild-type animals. Moreover, cultured Gap43-deficient GCPs show decreased proliferation in response to administered recombinant Shh protein [64]. A possible regulatory link was recently provided as the activation of the hedgehog signaling component Smoh was found to depend on PI4P (phosphatidylinositol 4-phosphate) levels that immediately increase when Shh binds to Ptch1 or when functional Ptch1 is absent [65]. The authors further showed that imbalanced conversion of the precursor molecule PI into PI4P influences hedgehog pathway activity. Alternatively, the production of PI4P can also result from a specific dephosphorylation of PI (4,5) P2 [66]. In this context, Gap43 protein was recently demonstrated to build oligomeric structures in the plasma membrane which sequester specifically PI (4,5) P2 [67]. A similar finding has been reported earlier showing that GAP43 participates in the accumulation of plasmalemma rafts, which promoted retention of PI (4,5) P2 [68]. The amount of Gap43 associated with the plasma membrane therefore possibly modulates the utilization of PI (4,5) P2, including its conversion into PI4P, which in turn directly affects hedgehog signaling through Smoh activation. However, the effective impact on downstream Gli-targets would still be difficult to conclude regarding the multitude of responses to Shh, including negative feedback regulation [13]. Further studies applying depletion and enrichment of specific phosphatidyl derivatives and selective silencing of hedgehog pathway elements will be necessary to elucidate the molecular nature of this proposed signaling axis. The increased accumulation of mitotic granule cells at the EGL seen in the combined Ptch1+/− Nos2−/− genotype supposedly gives a special clue to MB induction. In contrast to the classical view of neonatal EGL organization, which describes radial migration of granule cells to follow a proliferation stop, more and more evidence arises showing that cell cycle arrest is not a prerequisite for migration but rather occurs during a temporally coordinated interplay of gene expression patterns [69]. This corroborates our data shown in Figure 6 (white arrows), where proliferation is still maintained in migrating granule cells and even in cells of the IGL of wild-type cerebellum. Regulation of such expression patterns is largely dependent on Shh stimuli being most intensive in the EGL [70], as well as gradients of other soluble factors such as Bmps, which were reported to account for a regulatory environment along the transition through the cerebellar layers [71]. Further evidence for a niche-like-concept was provided by Choi et al. in Bdnf−/− mice that displayed a severe retardation of GCP migration [16]. The authors could demonstrate that mitotic activity of maturating GCPs was significantly enhanced when cells were retained in the EGL and declined with increasing distance from outer EGL regions. Therefore, the accumulation of GCPs in the EGL in combination with the insensitivity to Ptch1-mediated cell cycle arrest in Ptch1+/− Nos2−/− mice provide a growth advantage and increase the number of putative transformation targets over Ptch1+/− Nos2+/+ mice (Figure 7C). In conclusion, the following picture emerged from our data: Homozygous deletion of Nos2 leads to a reduction of basic NO levels in immature GCPs of the EGL during postnatal development of the cerebellum. This reduction causes a downregulation of Gap43 expression, which results in an increased expression of Ptch1 and impaired directed migration of maturating GCPs. As a consequence, undifferentiated granule cell precursors exit cell cycle and are retained at the EGL (Figure 7B). In case of an additional heterozygous Ptch1 mutation, upregulation of this receptor does not suffice to exert the anti-proliferative stimulus following Gap43 decrease, which results in an increased fraction of continuously dividing cells in the EGL (Figure 7C). As reduced migration towards the IGL further leads to a withdrawal of growth-limiting signals, expansion of the GCP population is additionally supported. Finally, this advances medulloblastoma development in Ptch1+/− Nos2−/− mice compared to Ptch1+/− Nos2+/+ mice. The mechanism described here illustrates a new tumor-promoting concept in MB showing that the localization of pre-neoplastic cells within the developing cerebellum is important for pathogenesis. Ptch1+/− mice (B6; 129P2-Ptch1tm1Mps/Ptch+; [20]) and Nos2−/− mice (B6; 129P2-Nos2tm1Lau; [72]) were obtained from the Jackson Laboratory (Bar Harbor, Maine, USA) and crossbred to generate double heterozygous mice (Ptch1+/− Nos2+/−). The F1 hybrids were backcrossed with Ptch1+/+ Nos2−/− mice to generate Ptch1+/− Nos2−/− mice. Later on, Ptch1+/− Nos2−/− mice were directly mated. For details on housing and genotyping see Text S2 and Table S9. All animal experiments were approved by the responsible federal authorities (Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen, Recklinghausen, Germany, Az. 50. 05-230-17/06). Total RNA of tumor specimens and normal cerebellar tissue samples was isolated via CsCl density gradient centrifugation [73] and assessed for integrity using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA). For gene expression microarrays, linear amplification of mRNA and labeling of cDNA were conducted on samples and Mouse Universal Reference RNA (Stratagene, La Jolla, USA) according to the TAcKLE protocol [74]. Both were combined for two-color hybridizations with each sample being performed as two replicates of inverse dye orientation. Global gene expression profiling was performed on self-printed oligonucleotide microarrays. Further details of microarray production and hybridization are described in Text S2. Genomic DNA of tumor specimens was isolated from the interphase of the CsCl gradient by ethanol precipitation, proteinase K digest, and phenol/chloroform extraction. DNA samples were monitored for purity and adequate fragment size using spectrophotometric measurements and gel electrophoresis. Array-based comparative genomic hybridization (array-CGH, matrix-CGH, [75]) was performed on Mouse Genome CGH 244 k Microarrays (Agilent). Cy5-labeled tumor DNA was combined with corresponding Cy3-labeled reference (wild-type genomic DNA) to receive either sex-matched sample pairs or pairs of different gender for internal negative or positive control. Sample preparation, microarray hybridization, and washing procedures were carried out as described in the manufacturer' s protocol. Microarray data are available in GEO (http: //www-ncbi. nlm. nih. gov/geo), under accession number GSE29201. Total RNA isolated from cell culture samples using the RNeasy Mini Kit (Qiagen, Hilden, Germany) or RNA from tissue specimens was subjected to oligo (dT) -primed reverse transcription. QRT-PCR measurements were conducted in an ABI PRISM 7900HT thermal cycler (Applied Biosystems, Foster City, USA) using the SYBR green reaction and detection system (ABgene, Epsom, UK). For relative quantification mean ratios were calculated between genes of interest and a set of five housekeeping genes (Table S7) according to the Pfaffl method [76]. The expression levels of Ptch1, Gli1, N-myc, and Nos2 were determined by real-time reverse transcription PCR analysis using the ABI PRISM 5700 system (Applied Biosystems) as reported before [73]. For these experiments, the mRNA expression level of mitochondrial ribosomal protein L32 (Mrpl32) served as housekeeping reference. All primer sequences are depicted in Table S8. Inhibition of NO synthases was performed in c17. 2 and D458 cells which were seeded at densities of 2×105 and 4×105 cells per well in 12-well plates, respectively. Cells were daily treated with either 1 mM of the inhibitor L-NAME or 1× PBS as solvent control. For knockdown experiments of Gap43, c17. 2 cells were grown in a 12-well plate to 80% confluency and transfected with 2 µg of pLKO. 1-puro vector that contained either shRNA constructs targeting Gap43, shRNA against GFP, or non-target shRNA as a control (Sigma-Aldrich, St. Louis, USA) using 9 µl FuGene HD reagent (Roche, Basel, Switzerland). Transfection was repeated 2 times each after 8 hours and subjected to selection conditions (1 µg/ml puromycin) for 24 hours. Subsequently, cells were trypsinized, adjusted to 4×105 cells/ml and seeded into the inserts of a Costar Polycarbonate Membrane Transwell plate (8 µm pores, Corning, USA). After 24 hours cells were either harvested for gene expression and protein analyses or 0. 1 µg/µl recombinant SDF-1α was applied to the lower compartment for migration assays. Following 12 hours of incubation, cells at the bottom of the insert membrane were methanol-fixed, hematoxylin-stained, and counted. FFPE sections of postnatal cerebella were pre-processed as described in Text S2. For immunofluorescence co-staining, Gap43 (Sigma-Aldrich, clone GAP-7b10) or NeuN (Millipore, clone A60) first primary antibodies were diluted 1∶1000 or 1∶200, respectively and applied using the Dako REAL Detection System (Dako, Glostrup, Denmark). Following over night incubation at 4°C, washing in TBS, and blocking of residual biotin/streptavidin, sections were subsequently incubated with biotinylated anti-mouse secondary antibody (Dako) and stained with 20 ng/µl FITC-conjugated streptavidin (Invitrogen, Carlsbad, USA). The second primary antibody against Ki-67 (Novocastra, Wetzlar, Germany) was diluted 1∶1000 and accordingly applied using biotinylated anti-rabbit secondary antibody (Dako) and 20 ng/µl Cy5-conjugated streptavidin (Invitrogen). Co-stained sections were then covered with DAPI-containing VECTASHIELD Mounting Medium (Vector, Burlingame, USA) and subjected to confocal laser scanning microscopy. Quantification of Gap43 staining was performed for areas of interest using Image J software (NIH). Numbers of dividing and non-dividing cells in the EGL of postnatal cerebellar tissue sections were counted manually and normalized to the corresponding length of the EGL edge. Cell counts for each region were averaged across three sections, each with 10–20 µm distance in z-axis, and per individual. Kaplan-Meier survival plots were calculated for a total of 1167 mice, including 315 wild-type mice, 412 Ptch1+/+ Nos2−/− mice, 215 Ptch1+/− Nos2+/+ mice and 221 Ptch1+/− Nos2−/− mice. MB-free survival was plotted using the GraphPad Prism 5 software (GraphPad, La Jolla, USA). The logrank test was applied to compare survival (tumor occurrence) of the different genotypes. For comparisons of differences of means between two groups of replicates, p-value calculations were performed using an unpaired, two-tailed t-test, unless indicated otherwise. Calculated error bars represent the standard error of the mean (SEM). For details on microarray statistics please refer to Text S2.

Medulloblastoma is a common pediatric brain tumor, a subtype of which is driven by aberrant hedgehog pathway activation in cerebellar granule cell precursors. Although this tumor etiology has been intensively investigated in the well-established Ptch1+/− mouse model, knowledge is still lacking about the molecular interactions between neoplastic transformation and other developmental processes. Nitric oxide (NO) has been reported to be involved in controlling proliferation and differentiation of these cells. Therefore, inactivation of the NO-producing enzyme Nos2 in combination with the mutated Ptch1 gene should provide insight into how developmental regulation influences pathogenesis. Here, we describe a new role for NO in developing neuronal precursors of the cerebellum facilitating physiologically accurate migration via regulation of Gap43. We further demonstrate that disturbance of these processes leads to retention of granule precursor cells to the cerebellar periphery. Together with the sustained proliferation of these cells in combined Ptch1+/− Nos2−/− mice, this effect results in an increased medulloblastoma incidence relative to Ptch1+/− mice and demonstrates a new disease-promoting mechanism in this tumor entity.

lay_plos

Chikungunya virus (CHIKV) is the most common alphavirus infecting humans worldwide, causing acute and chronically debilitating arthralgia at a great economic expense. To facilitate our study of CHIKV, we generated a mCherry tagged replication-competent chimeric virus, CHIKV 37997-mCherry. Single particle cryoEM demonstrated icosahedral organization of the chimeric virus and the display of mCherry proteins on virus surface. CHIKV 37997-mCherry is attenuated in both IFNαR knockout and wild-type mice. Strong anti-CHIKV and anti-mCherry antibody responses were induced in CHIKV 37997-mCherry infected mice. Our work suggests that chimeric alphaviruses displaying foreign antigen can serve as vaccines against both aphaviruses and other pathogens and diseases. Chikungunya virus (CHIKV) is a mosquito-transmitted, enveloped positive stranded RNA virus that belongs to the Alphavirus genus of the Togaviridae family. CHIKV infection causes an acute febrile illness typically accompanied by severe arthralgia, with relapses for weeks to months [1]. In the past decade, CHIKV has spread from endemic areas of Africa and Asia to new populations in Europe and the Americas, making CHIKV a global threat and the most common alphavirus infecting humans. Millions of individuals were infected during the 2000s, resulting in thousands of deaths [2]. The CHIKV RNA genome encodes four nonstructural (nsP1 to nsP4) and five structural (C, E3, E2,6K and E1) proteins. All five structural proteins are translated as a single polyprotein from which the capsid (C) protein is released via self-cleavage. The envelope polyprotein precursor (E3-E2-6K-E1) is translocated into the endoplasmic reticulum (ER) and processed by host signalase into E1,6K and E3E2 polyprotein (p62). In the ER, E1 and p62 assemble into heterodimers and subsequently trimerize to form viral spikes. p62 is cleaved by host furin or furin-like proteases into E3 and E2 during trafficking through the Golgi and trans-Golgi network (TGN). The mature CHIKV particles bud at the plasma membrane and have T = 4 quasi-icosahedral symmetry with 240 copies of the E1E2 heterodimer, assembled into 80 spikes on the viral surface; 240 copies of C form an icosahedral nucleocapsid core enclosing viral genomic RNA within the lipid membrane [3]. E1 is a type II membrane fusion protein and sits at the base of the trimeric spike with E2 positioned on top of E1. The ectodomain of E1 consists of three domains [4]. Domain I links distal domain II and membrane proximal domain III. A fusion loop is situated at the distal end of E1 domain II, and is protected by domain B of E2, located at the distal end of E2 [4,5]. After viral entry into target cells, the acidic environment of endosomes triggers conformational rearrangements within E2, leading to domain B dissociating from the fusion loop [6]. E1 then forms a homotrimer, further exposing the fusion loops of each monomer at the end of the trimeric complex for insertion into the host membrane [7]. E3 limits the movement of E2 domain B until it is cleaved from E2 by furin or furin-like proteases to prevent accidental activation of the fusion peptide before the assembly and budding of mature virions [5,8]. Studies show alphaviruses can tolerate insertion of foreign antigens to N-terminal of E2 generating chimeric virus presenting foreign antigens [9–11]. There are currently no licensed vaccines or treatments for CHIKV infection. We hypothesize that an attenuated CHIKV carrying foreign antigens may induce immune responses against both CHIKV and the foreign antigen. In the current study, we report a replication-competent CHIKV with mCherry fused to the N-terminus of E2. The resulting chimeric virus presents mCherry on the virion surface in a repetitive pattern and induces strong antibody responses against both CHIKV and mCherry. The virus is attenuated in vivo and provides a safe research tool to study CHIKV virology, as well as demonstrating a useful vaccine platform. Vero and BHK21 cells (ATCC CRL-1586 and CCL-10) were maintained at 37°C in a fully humidified atmosphere with 5% CO2 in DMEM (Invitrogen) medium supplemented with penicillin and streptomycin, 10 mM HEPES, non-essential amino acids, and 10% FBS (Hyclone). The CHIKV 37997 and La Reunion (LR) strain virus clone was gift of S. Higgs (Kansas State University) and the CHIKV vaccine strain 181/clone 25 was gift of S. Weaver (University of Texas Medical Branch). Viruses were produced from infectious cDNA clones as previously described [12,13]. The mCherry gene was inserted between E3 and E2 after the furin cleavage site in CHIKV 37997 genome to make CHIKV 37993-mCherry cDNA clone [14]. mCherry protein fused with 6xHis-tag at the N-terminus was expressed in bacterial cells and affinity purified through Cobalt resins (ThermoFisher Scientific). Vero cells were infected with CHIKV 37993-mCherry at a multiplicity of infection (MOI) of 5. At 8 h post infection, the cells were fixed and imaged with a Nikon Ti inverted fluorescence microscope equipped with a Yokogawa CSU-22 spinning disk confocal with a 561 nm laser. Purified viruses were subjected to electrophoresis with 4–12% sodium dodecyl sulfate-polyacrylamide gel (ThermoFisher Scientific) followed by Coomassie staining with SimpleBlue SafeStain (ThermoFisher Scientific) or Western blot analysis with rabbit polyclonal anti-CHIKV 181/25 (IBT Bioservices) and mouse monoclonal anti-mCherry (Sigma). C57BL/6J mice were purchased from The Jackson Laboratory (Sacramento, CA). Ifnar-/- mice [15], on a C57BL/6J background, were a gift from Dr. Michael Diamond (Washington University) and bred in house under specific-pathogen-free conditions in microisolator cages (Innovive Inc., San Diego, CA). To study viral pathogenesis in wild type mice, three week-old C57BL/6J mice were inoculated with CHIKV subcutaneously in the left footpad with 103 or 106 PFU of CHIKV in PBS supplemented with 1% heat inactivated FBS. Joint swelling was monitored via left and right foot measurements at the peritarsal region (width x height) using digital calipers. Sera were collected at day 3 after infection and rear ankles were collected at day 7 after infection. To study viral pathogenesis in a lethal mouse model, 6–8 week-old Ifnar-/- mice were inoculated with CHIKV subcutaneously in the left footpad with 10,100 or 1,000 PFU of CHIKV in PBS supplemented with 1% heat inactivated FBS. Joint swelling was monitored via left and right foot measurements at the peritarsal region (width x height) using digital calipers, and mouse survival was monitored for at least 3 weeks. Immunization and challenge with CHIKV were performed by injection of virus subcutaneously in the left footpad. To immunize mice with mCherry, 8 week-old C57BL/6J mice were administered with purified mCherry protein via intraperitoneal injection. Mice were prime immunized with 100 μg of mCherry protein mixed with complete Freund’s adjuvant (Sigma-Aldrich) followed by boosting with 50 μg of mCherry proteins mixed with incomplete Freund’s adjuvant (Sigma-Aldrich) 2 weeks later. All animal experiments were performed with the approval of the Institutional Animal Care and Use Committee at PMI Preclinical, LLC (San Carlos, CA), protocol number IAC 1705. Mice received humane care according to the criteria outlined by the National Research Council’s Institute of Laboratory Animal Resources in the “Guide for the Care and Use of Laboratory Animals”. All injections of mice with virus were performed under anesthesia with isoflurane or ketamine and xyalzine. Viral RNA in culture supernatant or mouse blood was extracted by the QIAamp Viral RNA Mini kit (Qiagen) following the manufacturer’s protocol. To extract viral RNA in mouse tissues, tissues were first lysed in TRIzol (ThermoFisher Scientific) using Qiagen Tissue Lyser II (Qiagen). RNA was extracted following the manufacturer’s protocol. Isolated RNA was analyzed by qRT-PCR and compared to a standard curve generated from CHIKV-GLuc plasmid [16] with the following primers: CKV_For, 5’-TGGCCACCTTTGCAAGCTC-3’; CKV_Rev, 5’-GGGATGAACTCCATTGTAGC-3’; and CKV_Probe, 5’/56-FAM/AGGTACGCACTACAGCTACC/36-TAMSp/3’. Anti-CHIKV and anti-mCherry antibodies in mouse sera were quantified using virion-based ELISA. To detect anti-CHIKV antibodies, Immulon 4HBX plates (96-well, Thermo Scientific) were coated with CHIKV 181/25 virus purified through sucrose cushion (2. 5 x 108 genome copies/well). To detect anti-mCherry antibodies, Immulon 4HBX plates were coated with purified 6xHis-mCherry protein (1 μg/well). Serial dilutions of mouse serum were added to the plates. Serum of naïve mice was used as the background control. Next, plate-bound antibodies were detected with biotin-conjugated goat anti-mouse IgG antibodies (Southern Biotech), followed with streptavidin-conjugated horseradish peroxidase (Southern Biotech). Binding was detected with 3,3’5,5’-tetramethylbenzidine substrate (Neogen). Endpoint titers were defined as the reciprocal of the last dilution to have an absorbance two times greater than the background control. Neutralizing activity of mouse serum was quantified using a plaque reduction neutralization test (PRNT). Serial dilutions of mouse serum were incubated with 50 PFUs of challenge virus for 1 hour at 37°C, followed by infection of Vero cells for 1 hour at 37°C. To quantify virus by plaque assay, Vero cells were incubated with serial dilutions of virus containing supernatant for 1 hour at 37°C. Next, cells were overlaid with medium containing 2% FBS and 0. 8% agarose, followed by culture at 37°C. Plaques were counted 2 days later. To coat ELISA plates, CHIKV 181/25 was produced in BHK21 cells virus and was pelleted through a 20% sucrose cushion. To make gradient-purified viruses for single particle cryoEM study, we adapted a published protocol [17]. CHIKV 37997 and CHIKV 37997-mCherry viruses were produced in BHK21 cells and first concentrated by pelleting the culture supernatant through a 20% sucrose cushion. Then, the resuspended virus was layered onto 2 ml of 60% OptiPrep (Sigma-Aldrich) and spun at 50,000 x g for 1. 5 hour using a SW28 rotor. After ultracentrifugation, the supernatants were removed to leave 4 ml above the virus band. The remaining 4 ml supernatant, the virus band and the underlay of 2 ml of 60% OptiPrep were mixed to reach a final concentration of 20% OptiPrep. The mixture was spun at 360,000 x g for 3. 5 hours with a NVT65. 2 rotor. The virus band was extracted and buffer exchanged to NTE buffer (20 mM Tris, pH 8. 0,120 mM NaCl, 1 mM EDTA) using an Amicon Ultra-2 Centrifugal Filter Unit with Ultracel-100 membrane (Millipore). Purified CHIKV 37997-mCherry virus was flash-frozen on C-Flat copper grids (R2/2,200 mesh) in liquid ethane. The movie-mode data of CHIKV 37997-mCherry was taken on a DE-20 direct electron detector (Direct Electron, LP, San Diego, CA) on a 200-kV JEOL 2200FS microscope under low-dose conditions. We used a frame rate of 25 frames/s and 1. 52 s exposure that corresponded to ∼38 e/Å2 dose/image. Individual frames in each image were aligned using DE_process_frames. py script provided by Direct Electron (Direct Electron, LP, San Diego, CA) using radiation damage compensation to increase low-frequency content in the final images, thus facilitating subsequent particle alignment. Particle images were automatically boxed out using the E2BOXER program from the EMAN2 suite [18]. EMAN2 was used for CTF determination and correction; the subsequent processing (particle alignment, orientation search and refinement, and 3D reconstruction) was done in IMAGIC-5 [19]. All data were analyzed using Prism software (La Jolla, CA) and statistical significance was assigned when P values were < 0. 05. Neutralization curves were calculated using non-linear regression. Mouse survival curves were compared with Gehan-Breslow-Wilcoxon test. Viral titers and mouse joint swelling were analyzed using a one-way or two-way ANOVA test. To make a fluorescently tagged CHIKV, we inserted DNA encoding mCherry into a cDNA clone immediately downstream of the furin cleavage site between E3 and E2 in CHIKV 37997 (Fig 1A). The resulting chimeric CHIKV 37997-mCherry virus was then produced as previously described [16]. In CHIKV 37997-mCherry infected cells, mCherry-E2 displayed a typical glycoprotein expression pattern: from Golgi apparatus to endosomes to the plasma membrane, and alphavirus-induced Env positive membrane extensions were observed (Fig 1B). Next, we compared viral growth curves of CHIKV 37997-mCherry with the parental CHIKV 37997 virus in vitro. Viruses released in the culture supernatants were quantified by viral RNA level measured by qRT-PCR and infectious particle number measured by plaque assay. In BHK21 cells, CHIKV 37997-mCherry demonstrated slightly slower growth kinetics compared to parental wild-type virus at early time points (before 12 hours post-infection), however no differences were seen at later time points (after 18 hours post-infection) (Fig 1C). CHIKV 37997-mCherry formed plaques similar to parental viral plaques (Fig 1D) and had a similar viral genome copy to PFU ratio (2. 49 x 103) as the parental CHIKV 37997 (2. 75 x 103). In gradient purified CHIKV 37997-mCherry virus E1 and capsid proteins were present at the expected size (Fig 1E). The double bands that react with both anti-mCherry monoclonal antibody and anti-CHIKV polyclonal antibody are probably E3-mCherry-E2 precursor and mCherry-E2 fusion proteins. Insertion of mCherry between E3 and E2 might slow down the furin cleavage of E3 because the parental CHIKV 37997 virus contains very little uncleaved E3-E2 (p62) precursor. The smaller sizes of E3-mCherry-E2 and mCherry-E2 than expected may due to some glycosylation difference with mCherry insertion rather than truncation within the fusion protein as the viral RNA genome was intact in gradient purified CHIKV 37997-mCherry (S1 Fig). To understand the presentation of tagged mCherry proteins on chimeric CHIKV 37997-mCherry virus, we performed single-particle cryoEM analysis of purified CHIKV 37997-mCherry virus particles (Fig 2). CHIKV 37997-mCherry virions display a typical T = 4 icosahedral structure similar to wild-type CHIKV and other alphaviruses (Fig 2A and 2B). Compared with the cryoEM map of CHIK virus-like particles that we previously obtained (Fig 2C) [16], CHIKV 37997-mCherry displayed extra density on the particle surface representing mCherry proteins fused at the N-terminal of E2 (Fig 2D). The mCherry proteins were well exposed, occupying the space between viral spikes on the particle surface (Fig 2D). After we confirmed that CHIKV 37997-mCherry replicated to a similar level as wild-type CHIKV 37997 in vitro, we next compared their growth and pathogenesis in vivo. In an IFNαR-/- lethal infection model, injection of 10 PFU of CHIKV 37997 into the mouse footpad resulted in death in 100% of the mice in 4 days. In contrast, all the mice infected with 10 PFU of CHIKV 37997-mCherry survived through the period of observation (23 days) (Fig 3A). Surprisingly, infection with the CHIKV vaccine strain 181/clone 25 (CHIKV 181/25) also resulted in death in 40% of the mice within 9 days (Fig 3A). Infection of all three strains caused footpad swelling on the ipsilateral side, but with a delay observed with CHIKV 37997-mCherry and CHIKV 181/25 infections compared to CHIKV 37997 (Fig 3B). Because CHIKV 37997-mCherry was found to be attenuated compared with the parental virus at a 10 PFU dose, we next compared its pathogenesis with the vaccine strain CHIKV 181/25 in IFNαR-/- mice over a range of different inoculation doses. Survival curves of mice infected with 10 PFU to 104 PFU of CHIKV 37997-mCherry vs CHIKV 181/25 showed no significant difference (Fig 4), indicating CHIKV 37997-mCherry is similarly attenuated as CHIKV 181/25 in IFNαR-/- mice. Because CHIKV 37997-mCherry is attenuated like the vaccine strain, we next tested if infection with CHIKV 37997-mCherry provides a protection similar to the vaccine strain (Fig 5). The IFNαR-/- mice that survived infection with 10 PFU to 103 PFU of CHIKV 37997-mCherry or CHIKV 181/25 were challenged with 10 PFU of pathogenic CHIKV LR strain. All the mice pre-exposed to either CHIKV 37997-mCherry or CHIKV 181/25 survived (Fig 5A), although it was apparent sterilizing immunity was not induced in some animals. However, viral loads of challenge virus were limited compared to CHIKV LR replication in the control mice (Fig 5B), which demonstrated death in 100% of the mice within 4 days (Fig 5A). We next compared antibody responses in CHIKV 37997-mCherry vs CHIKV 181/25 infected IFNαR-/- mice before (Fig 6A) and after (Fig 6B) re-challenge with CHIKV LR. Infection with 10,100 or 1,000 PFU of CHIKV 37997-mCherry and CHIKV 181/25 induced similar levels of antibodies against CHIKV in mice at 3 weeks post-infection and 11 days post-challenge with CHIKV LR. As expected, antibodies against mCherry were induced in mice infected with 37997-mCherry. Neutralizing activities of the antiserum in CHIKV 37997-mCherry and CHIKV 181/25 infected mice were next tested using a plaque reduction neutralization assay (Fig 7). Serum from CHIKV 37997-mCherry and CHIKV 181/25 vaccinated mice at both 3 weeks post-infection (Fig 7A) and 11 days post-re-challenge (Fig 7B) neutralized virus entry of CHIKV 181/25 at similar efficiency. Interestingly, serum from CHIKV 37997-mCherry vaccinated mice neutralized virus entry of CHIKV 37997-mCherry more efficiently than serum from CHIKV 181/25 vaccinated mice, with IC50s for the former ~30-fold higher than those for the latter (Fig 7C and 7D). Serum from CHIKV 181/25 infected mice neutralized CHIKV 181/25 and CHIKV 37997-mCherry at similar efficiency (Fig 7), excluding the possibility that serum from CHIKV 181/25 infected mice only weakly neutralized CHIKV 37997-mCherry. After we found CHIKV 37997-mCherry was attenuated in the lethal mouse model of immunocompromised IFNαR-/- mice, we next compared its pathogenesis with the parental and vaccine strains in a chronic infection mouse model (Fig 8). Three weeks old wild-type mice were infected with 103 PFU of CHIKV 37997, CHIKV 37997-mCherry or CHIKV 181/25 by injection in the rear footpad. As expected, CHIKV 37997 infection caused significant swelling of the footpads on the ipsilateral side at day 3 and day 7 post-infection (Fig 8A). In contrast, infection with CHIKV 37997-mCherry did not cause significant footpad swelling on day 3 and significantly less swelling than CHIKV 37997 on day 7 (Fig 8A). Tissue inflammation induced by infection of the vaccine strain CHIKV 181/25 in this chronic infection model was also attenuated to a similar level as CHIKV 37997-mCherry (Fig 8A). Consistently, plasma RNA viral loads at 3 days post-infection of CHIKV 37997-mCherry or CHIKV 181/25 were significantly lower than those in CHIKV 37997 infected mice (Fig 8B). At 28 days post-infection, in the chronic phase of CHIKV infection, persistent viral RNA in ankle tissues was compared (Fig 8C). As we, and other researchers, have reported before [16,20], CHIKV persists in both the ipsilateral and contralateral ankles after CHIKV 37997 infection. In contrast, viral RNA in the ankles on the contralateral side of CHIKV 37997-mCherry infected mice were not significantly higher than the assay limit of detection, and viral loads in ankles on the ipsilateral side were significantly lower than those in parental CHIKV 37997 infected mice (Fig 8C). In vaccine strain CHIKV 181/25 infected mice viral loads varied extensively between individual mice in ankles of both sides (Fig 8C), consistent with the large variation of plasma viral load in CHIKV 181/25 infected mice (Fig 8B). In some CHIKV 181/25 infected mice, virus replicated and persisted at high loads relative to those in CHIKV 37997-mCherry infected mice, although no statistical significant difference could be demonstrated between the two groups. Similar attenuation of CHIKV 37997-mCherry at a dose as high as 106 PFU was observed in 3-weeks old mice (Fig 9). We observed presentation of mCherry on the surface of chimeric CHIKV 37997-mCherry in a repeating pattern with 240 copies of mCherry on each virion. Strong anti-CHIKV responses were induced by infection of CHIKV 37997-mCherry, so we next tested if strong antibody responses against mCherry could be induced by CHIKV 37997-mCherry. In adult mice, infection with 104 or 106 PFU of CHIKV 37997-mCherry induced high antibody responses against mCherry when assessed at day 14 post-infection and the high levels of antibody lasted for at least 34 days (Fig 10A). Immunization with 100 μg of purified mCherry protein in complete Freud’s adjuvant induced antibodies against mCherry at more than 20 fold lower level than induced by CHIKV 37997-mCherry infection at day 14 (Fig 10A). Boosting with 50 μg of mCherry enhanced antibody responses to the level induced by CHIKV 37997-mCherry infection in only 2 of 7 mice (Fig 10A). Interestingly, serum from mCherry immunized mice could neutralize CHIKV 37997-mCherry virus entry, although at a lower efficiency than the serum from CHIKV 37997-mCherry infected mice (Fig 10B). This is consistent with the results that the serum from CHIKV 37997-mCherry infected IFNαR-/- mice neutralized CHIKV 37997-mCherry at about a ~30-fold higher efficiency than the serum from CHIKV 181/25 infected IFNαR-/- mice (Fig 7C & 7D). CHIKV is the most common alphavirus infecting humans–with millions of individuals infected during the 2000s, resulting in thousands of deaths [2]. Currently there are no licensed vaccines or treatments for CHIKV infection. An attenuated live vaccine strain (CHIKV 181/25) was developed and reported to be safe, highly immunogenic and produced well-tolerated side effects in a phase II clinical trial [21–23]. Two point mutations at I12 and R82 in the E2 glycoprotein of CHIKV 181/25 are responsible for its attenuation of acute disease in mice [24,25]. Acquisition of R82 in E2 enhances the affinity of CHIKV 181/25 particles for glycosaminoglycans (GAGs) in vitro [26], which may limit the capacity of CHIKV to disseminate from early sites of primary replication in vivo [24,27]. A recent study reported that R82 in E2 also renders CHIKV 181/25 vulnerable to neutralization by antibodies targeting E2 domain B and therefore more likely to be cleared [28]. In our study, CHIKV 37997-mCherry was more efficiently neutralized by serum from CHIKV 37997-mCherry infected mice than from CHIKV 181/25 infected mice (Fig 7). This may explain the quick clearance of CHIKV 37997-mCherry in blood and ankle tissues (Figs 8 & 9) and therefore the attenuation of CHIKV 37997-mCherry in vivo. Interestingly, we found antiserum against mCherry neutralized virus entry of CHIKV 37997-mCherry although at a lower efficiency than the antiserum raised in CHIKV 37997-mCherry infected mice (Fig 10). mCherry proteins displayed on the surface of CHIKV 37997-mCherry particle was not expected to mediate either the virus-receptor interaction or membrane fusion during virus entry. How anti-mCherry antibodies neutralize CHIKV 37997-mCherry warrants further studies. We can speculate that binding of antibodies to mCherry may limit the flexibility of E2 domain B and therefore prevent the activation of the fusion loop for virus entry. This additional neutralizing epitope and the presence of anti-mCherry antibodies provides the likely explanation for the ~30-fold more efficient neutralization of CHIKV 37997-mCherry by the antiserum raised in CHIKV 37997-mCherry infected mice compared to antiserum raised in CHIKV 181/25 infected mice, and therefore the attenuation of CHIKV 37997-mCherry (Fig 7). Whether or not chimeric CHIKV virus carrying other foreign antigens is attenuated depends on whether the foreign antigens are presented as neutralizing epitopes. Reversion of attenuating mutations was reported in CHIKV 181/25 infected wild-type mice [24,25]. The insertion of mCherry between E3 and E2 in CHIKV 37997 genome may not be so easy to revert compared to reversion of single amino acid mutation. This could explain the large variation of virus titers in blood and ankle tissues in mice infected with CHIKV 181/25 compared to consistent low virus titers in mice infected with CHIKV 37997-mCherry (Figs 8 & 9). Future studies on virus stability in vitro and in vivo as well as in vivo tropism of CHIKV 37997-mCherry are needed to understand the dynamics of the virus populations. On each chimeric CHIKV 37997-mCherry virion, 240 copies of mCherry are presented in a repetitive pattern between viral spikes (Fig 2). The high density of mCherry can be easily recognized by B cells and, therefore, activate B cells efficiently. Consistently, a single infection of CHIKV 37997-mCherry induced long-lasting and high titer antibody responses against mCherry that can only be reached after boosting in mice conventionally immunized with mCherry proteins (Fig 10). Our results suggest that fusions to E2 may be a promising vaccine platform. Fusion of foreign antigens to E2 generates replication-competent chimeric virus able to present these antigens in a repetitive array on virion surface [9,10]. Live attenuated chimeric aphaviruses are able to induce strong antibody responses against the foreign antigen as well as anti-alphavirus response. Recently, a study was reported using CHIKV virus-like particles as a vaccine platform to present NANP repeats from the circumsporozoite protein (CSP) of the Plasmodium falciparum malaria parasite [11]. Similarly the dense array of NANP repeats on VLPs induced strong antibody responses that protected mice from malaria infection. In that study NANP repeats of 58 amino acid were inserted on E3 and E2 separately. In our study, a much larger antigen, mCherry (236 amino acid) was fused to E2 with little effect on E3 cleavage efficiency, which permits infectivity of the chimeric virus. Virus replication generates viral RNA replication intermediates and viral RNA that strongly activate host innate immune responses, which may explain the strong immunogenicity of our replication-competent chimeric virus[29]. In summary, we report an attenuated mCherry tagged replication-competent CHIKV that may suggest an additional risk mitigation should an individual be accidentally infected. In addition our work suggests similar chimeric replication-competent alphaviruses can serve as vaccines against other pathogens and diseases, in addition to the chimeric VLP-based vaccines.

Chikungunya virus (CHIKV) is an alphavirus capable of causing long term debilitating joint and muscle pain at a great economic expense. Currently there are no licensed vaccines or treatment for CHIKV infection. We generated a modified version of the virus, termed CHIKV 37997-mCherry, stably expressing a fluorescent tag on the surface of the virus. To achieve this, a red fluorescent protein, mCherry, was fused to the virus envelope protein E2. Structural studies demonstrated the presence of mCherry on the virus surface. Infection of mice with CHIKV 37997-mCherry caused less severe disease in the animals compared to wild-type virus. Infection with CHIKV 37997-mCherry induced immune responses against both the mCherry protein and the virus. Furthermore, CHIKV 37997-mCherry is as attenuated as the vaccine strain CHIKV 181/clone 25 in different mouse models, causing less joint swelling and reduced persistence of viral genomes in tissue. Our work suggests that chimeric alphaviruses carrying foreign antigen on virus particles may serve as vaccines against both aphaviruses and other pathogens and diseases.

lay_plos

Cyrano, Ragueneau, poets, Carbon de Castel-Jaloux, the cadets, a crowd, then De Guiche. RAGUENEAU: Can we come in? CYRANO (without stirring): Yes... (Ragueneau signs to his friends, and they come in. At the same time, by door at back, enters Carbon de Castel-Jaloux, in Captain's uniform. He makes gestures of surprise on seeing Cyrano.) CARBON: Here he is! CYRANO (raising his head): Captain!... CARBON (delightedly): Our hero! We heard all! Thirty or more Of my cadets are there!... CYRANO (shrinking back): But... CARBON (trying to draw him away): Come with me! They will not rest until they see you! CYRANO: No! CARBON: They're drinking opposite, at The Bear's Head. CYRANO: I... CARBON (going to the door and calling across the street in a voice of thunder): He won't come! The hero's in the sulks! A VOICE (outside): Ah! Sandious! (Tumult outside. Noise of boots and swords is heard approaching.) CARBON (rubbing his hands): They are running 'cross the street! CADETS (entering): Mille dious! Capdedious! Pocapdedious! RAGUENEAU (drawing back startled): Gentlemen, are you all from Gascony? THE CADETS: All! A CADET (to Cyrano): Bravo! CYRANO: Baron! ANOTHER (shaking his hands): Vivat! CYRANO: Baron! THIRD CADET: Come! I must embrace you! CYRANO: Baron! SEVERAL GASCONS: We'll embrace Him, all in turn! CYRANO (not knowing whom to reply to): Baron!...Baron!...I beg... RAGUENEAU: Are you all Barons, Sirs? THE CADETS: Ay, every one! RAGUENEAU: Is it true?... FIRST CADET: Ay--why, you could build a tower With nothing but our coronets, my friend! LE BRET (entering, and running up to Cyrano): They're looking for you! Here's a crazy mob Led by the men who followed you last night... CYRANO (alarmed): What! Have you told them where to find me? LE BRET (rubbing his hands): Yes! A BURGHER (entering, followed by a group of men): Sir, all the Marais is a-coming here! (Outside the street has filled with people. Chaises a porteurs and carriages have drawn up.) LE BRET (in a low voice, smiling, to Cyrano): And Roxane? CYRANO (quickly): Hush! THE CROWD (calling outside): Cyrano!... (A crowd rush into the shop, pushing one another. Acclamations.) RAGUENEAU (standing on a table): Lo! my shop Invaded! They break all! Magnificent! PEOPLE (crowding round Cyrano): My friend!...my friend... Cyrano: Meseems that yesterday I had not all these friends! LE BRET (delighted): Success! A YOUNG MARQUIS (hurrying up with his hands held out): My friend, Didst thou but know... CYRANO: Thou!...Marry!...thou!...Pray when Did we herd swine together, you and I! ANOTHER: I would present you, Sir, to some fair dames Who in my carriage yonder... CYRANO (coldly): Ah! and who Will first present you, Sir, to me? LE BRET (astonished): What's wrong? CYRANO: Hush! A MAN OF LETTERS (with writing-board): A few details?... CYRANO: No. LE BRET (nudging his elbow): 'Tis Theophrast, Renaudet,...of the 'Court Gazette'! CYRANO: Who cares? LE BRET: This paper--but it is of great importance!... They say it will be an immense success! A POET (advancing): Sir... CYRANO: What, another! THE POET:...Pray permit I make A pentacrostic on your name... SOME ONE (also advancing): Pray, Sir... CYRANO: Enough! Enough! (A movement in the crowd. De Guiche appears, escorted by officers. Cuigy, Brissaille, the officers who went with Cyrano the night before. Cuigy comes rapidly up to Cyrano.) CUIGY (to Cyrano): Here is Monsieur de Guiche? (A murmur--every one makes way): He comes from the Marshal of Gassion! DE GUICHE (bowing to Cyrano):...Who would express his admiration, Sir, For your new exploit noised so loud abroad. THE CROWD: Bravo! CYRANO (bowing): The Marshal is a judge of valor. DE GUICHE: He could not have believed the thing, unless These gentlemen had sworn they witnessed it. CUIGY: With our own eyes! LE BRET (aside to Cyrano, who has an absent air): But...you... CYRANO: Hush! LE BRET: But...You suffer? CYRANO (starting): Before this rabble?--I?... (He draws himself up, twirls his mustache, and throws back his shoulders): Wait!...You shall see! DE GUICHE (to whom Cuigy has spoken in a low voice): In feats of arms, already your career Abounded.--You serve with those crazy pates Of Gascons? CYRANO: Ay, with the Cadets. A CADET (in a terrible voice): With us! DE GUICHE (looking at the cadets, ranged behind Cyrano): Ah!...All these gentlemen of haughty mien, Are they the famous?... CARBON: Cyrano! CYRANO: Ay, Captain! CARBON: Since all my company's assembled here, Pray favor me,--present them to my lord! CYRANO (making two steps toward De Guiche): My Lord de Guiche, permit that I present-- (pointing to the cadets): The bold Cadets of Gascony, Of Carbon of Castel-Jaloux! Brawling and swaggering boastfully, The bold Cadets of Gascony! Spouting of Armory, Heraldry, Their veins a-brimming with blood so blue, The bold Cadets of Gascony, Of Carbon of Castel-Jaloux: Eagle-eye, and spindle-shanks, Fierce mustache, and wolfish tooth! Slash-the-rabble and scatter-their-ranks; Eagle-eye and spindle-shanks, With a flaming feather that gayly pranks, Hiding the holes in their hats, forsooth! Eagle-eye and spindle-shanks, Fierce mustache, and wolfish tooth! 'Pink-your-Doublet' and 'Slit-your-Trunk' Are their gentlest sobriquets; With Fame and Glory their soul is drunk! 'Pink-your-Doublet' and 'Slit-your-Trunk,' In brawl and skirmish they show their spunk, Give rendezvous in broil and fray; 'Pink-your-Doublet' and 'Slit-your-Trunk' Are their gentlest sobriquets! What, ho! Cadets of Gascony! All jealous lovers are sport for you! O Woman! dear divinity! What, ho! Cadets of Gascony! Whom scowling husbands quake to see. Blow, 'taratara,' and cry 'Cuckoo.' What, ho! Cadets of Gascony! Husbands and lovers are game for you! DE GUICHE (seated with haughty carelessness in an armchair brought quickly by Ragueneau): A poet! 'Tis the fashion of the hour! --Will you be mine? CYRANO: No, Sir,--no man's! DE GUICHE: Last night Your fancy pleased my uncle Richelieu. I'll gladly say a word to him for you. LE BRET (overjoyed): Great Heavens! DE GUICHE: I imagine you have rhymed Five acts, or so? LE BRET (in Cyrano's ear): Your play!--your 'Agrippine!' You'll see it staged at last! DE GUICHE: Take them to him. CYRANO (beginning to be tempted and attracted): In sooth,--I would... DE GUICHE: He is a critic skilled: He may correct a line or two, at most. CYRANO (whose face stiffens at once): Impossible! My blood congeals to think That other hand should change a comma's dot. DE GUICHE: But when a verse approves itself to him He pays it dear, good friend. CYRANO: He pays less dear Than I myself; when a verse pleases me I pay myself, and sing it to myself! DE GUICHE: You are proud. CYRANO: Really? You have noticed that? A CADET (entering, with a string of old battered plumed beaver hats, full of holes, slung on his sword): See, Cyrano,--this morning, on the quay What strange bright-feathered game we caught! The hats O' the fugitives... CARBON: 'Spolia opima!' ALL (laughing): Ah! ah! ah! CUIGY: He who laid that ambush, 'faith! Must curse and swear! BRISSAILLE: Who was it? DE GUICHE: I myself. (The laughter stops): I charged them--work too dirty for my sword, To punish and chastise a rhymster sot. (Constrained silence.) The CADET (in a low voice, to Cyrano, showing him the beavers): What do with them? They're full of grease!--a stew? CYRANO (taking the sword and, with a salute, dropping the hats at De Guiche's feet): Sir, pray be good enough to render them Back to your friends. DE GUICHE (rising, sharply): My chair there--quick!--I go! (To Cyrano passionately): As to you, sirrah!... VOICE (in the street): Porters for my lord De Guiche! DE GUICHE (who has controlled himself--smiling): Have you read 'Don Quixote'? CYRANO: I have! And doff my hat at th' mad knight-errant's name. DE GUICHE: I counsel you to study... A PORTER (appearing at back): My lord's chair! DE GUICHE:...The windmill chapter! CYRANO (bowing): Chapter the Thirteenth. DE GUICHE: For when one tilts 'gainst windmills--it may chance... CYRANO: Tilt I 'gainst those who change with every breeze? DE GUICHE:...That windmill sails may sweep you with their arm Down--in the mire!... CYRANO: Or upward--to the stars! (De Guiche goes out, and mounts into his chair. The other lords go away whispering together. Le Bret goes to the door with them. The crowd disperses.) Cyrano, Le Bret, the cadets, who are eating and drinking at the tables right and left. CYRANO (bowing mockingly to those who go out without daring to salute him): Gentlemen...Gentlemen... LE BRET (coming back, despairingly): Here's a fine coil! CYRANO: Oh! scold away! LE BRET: At least, you will agree That to annihilate each chance of Fate Exaggerates... CYRANO: Yes!--I exaggerate! LE BRET (triumphantly): Ah! CYRANO: But for principle--example too,-- I think 'tis well thus to exaggerate. LE BRET: Oh! lay aside that pride of musketeer, Fortune and glory wait you!... CYRANO: Ay, and then?... Seek a protector, choose a patron out, And like the crawling ivy round a tree That licks the bark to gain the trunk's support, Climb high by creeping ruse instead of force? No, grammercy! What! I, like all the rest Dedicate verse to bankers?--play buffoon In cringing hope to see, at last, a smile Not disapproving, on a patron's lips? Grammercy, no! What! learn to swallow toads? --With frame aweary climbing stairs?--a skin Grown grimed and horny,--here, about the knees? And, acrobat-like, teach my back to bend?-- No, grammercy! Or,--double-faced and sly-- Run with the hare, while hunting with the hounds; And, oily-tongued, to win the oil of praise, Flatter the great man to his very nose? No, grammercy! Steal soft from lap to lap, --A little great man in a circle small, Or navigate, with madrigals for sails, Blown gently windward by old ladies' sighs? No, grammercy! Bribe kindly editors To spread abroad my verses? Grammercy! Or try to be elected as the pope Of tavern-councils held by imbeciles? No, grammercy! Toil to gain reputation By one small sonnet,'stead of making many? No, grammercy! Or flatter sorry bunglers? Be terrorized by every prating paper? Say ceaselessly, 'Oh, had I but the chance Of a fair notice in the "Mercury"!' Grammercy, no! Grow pale, fear, calculate? Prefer to make a visit to a rhyme? Seek introductions, draw petitions up? No, grammercy! and no! and no again! But--sing? Dream, laugh, go lightly, solitary, free, With eyes that look straight forward--fearless voice! To cock your beaver just the way you choose,-- For 'yes' or 'no' show fight, or turn a rhyme! --To work without one thought of gain or fame, To realize that journey to the moon! Never to pen a line that has not sprung Straight from the heart within. Embracing then Modesty, say to oneself, 'Good my friend, Be thou content with flowers,--fruit,--nay, leaves, But pluck them from no garden but thine own!' And then, if glory come by chance your way, To pay no tribute unto Caesar, none, But keep the merit all your own! In short, Disdaining tendrils of the parasite, To be content, if neither oak nor elm-- Not to mount high, perchance, but mount alone! LE BRET: Alone, an if you will! But not with hand 'Gainst every man! How in the devil's name Have you conceived this lunatic idea, To make foes for yourself at every turn? CYRANO: By dint of seeing you at every turn Make friends,--and fawn upon your frequent friends With mouth wide smiling, slit from ear to ear! I pass, still unsaluted, joyfully, And cry,--What, ho! another enemy? LE BRET: Lunacy! CYRANO: Well, what if it be my vice, My pleasure to displease--to love men hate me! Ah, friend of mine, believe me, I march better 'Neath the cross-fire of glances inimical! How droll the stains one sees on fine-laced doublets, From gall of envy, or the poltroon's drivel! --The enervating friendship which enfolds you Is like an open-laced Italian collar, Floating around your neck in woman's fashion; One is at ease thus,--but less proud the carriage! The forehead, free from mainstay or coercion, Bends here, there, everywhere. But I, embracing Hatred, she lends,--forbidding, stiffly fluted, The ruff's starched folds that hold the head so rigid; Each enemy--another fold--a gopher, Who adds constraint, and adds a ray of glory; For Hatred, like the ruff worn by the Spanish, Grips like a vice, but frames you like a halo! LE BRET (after a silence, taking his arm): Speak proud aloud, and bitter!--In my ear Whisper me simply this,--She loves thee not! CYRANO (vehemently): Hush! (Christian has just entered, and mingled with the cadets, who do not speak to him; he has seated himself at a table, where Lise serves him.)

The Gascony Guards enter, proud of Cyrano. There is also a poet who wants to immortalize the exploit, and a newspaper editor who wants to interview Cyrano. The little pastry shop is suddenly full and noisy. Cyrano, of course, cares nothing for poets and reporters. When Le Bret asks about his interview with Roxane, Cyrano simply tells him to be quiet. De Guiche, Richelieu's powerful nephew who wants Roxane for his mistress, offers the services of himself and his uncle. Cyrano refuses, though he has written a play that he would like to see produced. As De Guiche leaves, he asks Cyrano if he knows of Don Quixote. Cyrano acknowledges that he recognizes himself. De Guiche tells him that the arm of the windmill could cause his downfall, but Cyrano refuses to be intimidated. Le Bret chides Cyrano for throwing away such a brilliant opportunity. Cyrano describes the life of a protege in disparaging terms. He wants to be free, to sing, to dream. He still refuses to discuss Roxane.

booksum

Jomati Consultants LLP is the leading UK-based international management consultancy specialising in the legal profession. Its consultants have held worldwide level senior roles in law firm management, as general counsel and as providers of financial analysis for law firms. Our advice is exclusive, tailored and discrete. (read more about our services) Jomati publishes a series of in-depth research reports on aspects of the legal industry. (read more about our reports) If you wish to receive a copy of any of our reports, please contact tony.williams@jomati.com A new report is predicting that robots and artificial intelligence will dominate most legal practices within 15 years, leading to the "structural collapse" of law firms. Expert systems fuelled by sophisticated algorithms, natural language processing capabilities, and unhindered access to stores of data are poised to uproot many well established industries and institutions. And as a new report compiled by Jomati Consultants points out, lawyers — like many other white collar workers — are in danger of being supplanted. Legal Futures reports: The report's focus on the future of work contained the most disturbing findings for lawyers. Its main proposition is that AI is already close in 2014. "It is no longer unrealistic to consider that workplace robots and their AI processing systems could reach the point of general production by 2030… after long incubation and experimentation, technology can suddenly race ahead at astonishing speed." By this time, 'bots' could be doing "low-level knowledge economy work" and soon much more. "Eventually each bot would be able to do the work of a dozen low-level associates. They would not get tired. They would not seek advancement. They would not ask for pay rises. Process legal work would rapidly descend in cost." The human part of lawyering would shrink. "To sustain margins a law firm would have to show added value elsewhere, such as in high-level advisory work, effectively using the AI as a production tool that enabled them to retain the loyalty and major work of clients… "Clients would instead greatly value the human input of the firm's top partners, especially those that could empathise with the client's needs and show real understanding and human insight into their problems." This will be a disaster for associate lawyers owing to the dramatic reduction in positions available. Advertisement Related: How Universal Basic Income Will Save Us From the Robot Uprising As for the impact on law firms, and in the words of the Jomati report: "The economic model of law firms is heading for a structural revolution, some might say a structural collapse. We may have heard a lot about 'New Law' and alternative business structures [ABI], but the impact of AI will make such developments pale in comparison." Sponsored The report is titled "Civilisation 2030: The near future for law firms." More at Legal Futures. Image: Hiro-Hideki/Shutterstock Robots and artificial intelligence (AI) will dominate legal practice within 15 years, perhaps leading to the “structural collapse” of law firms, a report predicting the shape of the legal market has envisaged. Civilisation 2030: The near future for law firms, by Jomati Consultants, foresees a world in which population growth is actually slowing, with “peak humanity” occurring as early as 2055, and ageing populations bringing a growth in demand for legal work on issues affecting older people. This could mean more advice needed by healthcare and specialist construction companies on the building and financing of hospitals, and on pension investment businesses, as well as financial and regulatory work around the demographic changes to come; more age-related litigation, IP battles between pharmaceutical companies, and around so-called “geriatric-tech” related IP. The report’s focus on the future of work contained the most disturbing findings for lawyers. Its main proposition is that AI is already close in 2014. “It is no longer unrealistic to consider that workplace robots and their AI processing systems could reach the point of general production by 2030… after long incubation and experimentation, technology can suddenly race ahead at astonishing speed.” By this time, ‘bots’ could be doing “low-level knowledge economy work” and soon much more. “Eventually each bot would be able to do the work of a dozen low-level associates. They would not get tired. They would not seek advancement. They would not ask for pay rises. Process legal work would rapidly descend in cost.” The human part of lawyering would shrink. “To sustain margins a law firm would have to show added value elsewhere, such as in high-level advisory work, effectively using the AI as a production tool that enabled them to retain the loyalty and major work of clients… “Clients would instead greatly value the human input of the firm’s top partners, especially those that could empathise with the client’s needs and show real understanding and human insight into their problems.” Jomati pointed out that the managing partners of 2030 are in their 30s today and will embrace the advantages of AI. Alternative business structures (ABSs) in particular will be receptive, it predicted, “as it will greatly suit the type of matters they handle”. It continued: “With their external investors able to provide significant capital, they will invest in the latest AI when it becomes available and use it to rapidly increase the volume of matters. This increased efficiency will not harm their model, but rather make the shareholders in their narrow equity model extremely wealthy.” For associate lawyers, the rise of AI will be a disaster: “The number of associates that firms need to hire will be greatly reduced, at least if the intention is to use junior lawyers for billable work rather than primarily to educate and train them ready to become business winners. “Firms will struggle to overcome this gap in the usual career paths of their lawyers, i.e. firms need to hire young lawyers to become the next client winners, but they will be far less profitable at the start of their careers when knowledge bots take over most work up to [three years’] PQE.” On the impact of AI on law firms, Jomati concluded: “The economic model of law firms is heading for a structural revolution, also some legal wedding dress company some might say a structural collapse. We may have heard a lot about ‘New Law’ and [ABS], but the impact of AI will make such developments pale in comparison.” Small, specialist advisory firms and those focused on process matters might not be affected by AI, the report predicted, adding: “The firms that will be most affected would be the very large, high-value commercial firms whose associates expect to be given interesting work and many of whom aspire to the status and wealth that equity partnership affords. These fee-earners are also incredibly profitable as they clock up the hours on matters the partners have brought in.” The report forecast a big rise in the number of cities of over 10m people. Jomati’s top five cities in 2030 for their legal markets were New York, London, Paris, Frankfurt, and Singapore. It anticipated: “Far greater global balance in the largest law firms as they seek to follow clients into developing markets and key megacities and global cities around the world. While some global firms already have more than 50% of their revenues and staff based outside the ‘home nation’, by 2030 this will become standard for nearly all major commercial firms.” Software is also making its way into tasks that were the exclusive province of human decision makers, like loan and mortgage officers and tax accountants. These new forms of automation have renewed the debate over the economic consequences of technological progress. David H. Autor, an economics professor at the Massachusetts Institute of Technology, says the United States economy is being “hollowed out.” New jobs, he says, are coming at the bottom of the economic pyramid, jobs in the middle are being lost to automation and outsourcing, and now job growth at the top is slowing because of automation. “There is no reason to think that technology creates unemployment,” Professor Autor said. “Over the long run we find things for people to do. The harder question is, does changing technology always lead to better jobs? The answer is no.” Automation of higher-level jobs is accelerating because of progress in computer science and linguistics. Only recently have researchers been able to test and refine algorithms on vast data samples, including a huge trove of e-mail from the Enron Corporation. “The economic impact will be huge,” said Tom Mitchell, chairman of the machine learning department at Carnegie Mellon University in Pittsburgh. “We’re at the beginning of a 10-year period where we’re going to transition from computers that can’t understand language to a point where computers can understand quite a bit about language.” Nowhere are these advances clearer than in the legal world. E-discovery technologies generally fall into two broad categories that can be described as “linguistic” and “sociological.” Advertisement Continue reading the main story The most basic linguistic approach uses specific search words to find and sort relevant documents. More advanced programs filter documents through a large web of word and phrase definitions. A user who types “dog” will also find documents that mention “man’s best friend” and even the notion of a “walk.” The sociological approach adds an inferential layer of analysis, mimicking the deductive powers of a human Sherlock Holmes. Engineers and linguists at Cataphora, an information-sifting company based in Silicon Valley, have their software mine documents for the activities and interactions of people — who did what when, and who talks to whom. The software seeks to visualize chains of events. It identifies discussions that might have taken place across e-mail, instant messages and telephone calls. Then the computer pounces, so to speak, capturing “digital anomalies” that white-collar criminals often create in trying to hide their activities. For example, it finds “call me” moments — those incidents when an employee decides to hide a particular action by having a private conversation. This usually involves switching media, perhaps from an e-mail conversation to instant messaging, telephone or even a face-to-face encounter. “It doesn’t use keywords at all,” said Elizabeth Charnock, Cataphora’s founder. “But it’s a means of showing who leaked information, who’s influential in the organization or when a sensitive document like an S.E.C. filing is being edited an unusual number of times, or an unusual number of ways, by an unusual type or number of people.” The Cataphora software can also recognize the sentiment in an e-mail message — whether a person is positive or negative, or what the company calls “loud talking” — unusual emphasis that might give hints that a document is about a stressful situation. The software can also detect subtle changes in the style of an e-mail communication. Photo A shift in an author’s e-mail style, from breezy to unusually formal, can raise a red flag about illegal activity. “You tend to split a lot fewer infinitives when you think the F.B.I. might be reading your mail,” said Steve Roberts, Cataphora’s chief technology officer. Advertisement Continue reading the main story Another e-discovery company in Silicon Valley, Clearwell, has developed software that analyzes documents to find concepts rather than specific keywords, shortening the time required to locate relevant material in litigation. Newsletter Sign Up Continue reading the main story Please verify you're not a robot by clicking the box. Invalid email address. Please re-enter. You must select a newsletter to subscribe to. Sign Up You will receive emails containing news content, updates and promotions from The New York Times. You may opt-out at any time. You agree to receive occasional updates and special offers for The New York Times's products and services. Thank you for subscribing. An error has occurred. Please try again later. View all New York Times newsletters. Last year, Clearwell software was used by the law firm DLA Piper to search through a half-million documents under a court-imposed deadline of one week. Clearwell’s software analyzed and sorted 570,000 documents (each document can be many pages) in two days. The law firm used just one more day to identify 3,070 documents that were relevant to the court-ordered discovery motion. Clearwell’s software uses language analysis and a visual way of representing general concepts found in documents to make it possible for a single lawyer to do work that might have once required hundreds. “The catch here is information overload,” said Aaref A. Hilaly, Clearwell’s chief executive. “How do you zoom in to just the specific set of documents or facts that are relevant to the specific question? It’s not about search; it’s about sifting, and that’s what e-discovery software enables.” For Neil Fraser, a lawyer at Milberg, a law firm based in New York, the Cataphora software provides a way to better understand the internal workings of corporations he sues, particularly when the real decision makers may be hidden from view. He says the software allows him to find the ex-Pfc. Wintergreens in an organization — a reference to a lowly character in the novel “ Catch-22 ” who wielded great power because he distributed mail to generals and was able to withhold it or dispatch it as he saw fit. Such tools owe a debt to an unlikely, though appropriate, source: the electronic mail database known as the Enron Corpus. In October 2003, Andrew McCallum, a computer scientist at the University of Massachusetts, Amherst, read that the federal government had a collection of more than five million messages from the prosecution of Enron. He bought a copy of the database for $10,000 and made it freely available to academic and corporate researchers. Since then, it has become the foundation of a wealth of new science — and its value has endured, since privacy constraints usually keep large collections of e-mail out of reach. “It’s made a massive difference in the research community,” Dr. McCallum said. Advertisement Continue reading the main story The Enron Corpus has led to a better understanding of how language is used and how social networks function, and it has improved efforts to uncover social groups based on e-mail communication. Now artificial intelligence software has taken a seat at the negotiating table. Two months ago, Autonomy, an e-discovery company based in Britain, worked with defense lawyers in a lawsuit brought against a large oil and gas company. The plaintiffs showed up during a pretrial negotiation with a list of words intended to be used to help select documents for use in the lawsuit. “The plaintiffs asked for 500 keywords to search on,” said Mike Sullivan, chief executive of Autonomy Protect, the company’s e-discovery division. In response, he said, the defense lawyers used those words to analyze their own documents during the negotiations, and those results helped them bargain more effectively, Mr. Sullivan said. Some specialists acknowledge that the technology has limits. “The documents that the process kicks out still have to be read by someone,” said Herbert L. Roitblat of OrcaTec, a consulting firm in Altanta. Quantifying the employment impact of these new technologies is difficult. Mike Lynch, the founder of Autonomy, is convinced that “legal is a sector that will likely employ fewer, not more, people in the U.S. in the future.” He estimated that the shift from manual document discovery to e-discovery would lead to a manpower reduction in which one lawyer would suffice for work that once required 500 and that the newest generation of software, which can detect duplicates and find clusters of important documents on a particular topic, could cut the head count by another 50 percent. The computers seem to be good at their new jobs. Mr. Herr, the former chemical company lawyer, used e-discovery software to reanalyze work his company’s lawyers did in the 1980s and ’90s. His human colleagues had been only 60 percent accurate, he found. “Think about how much money had been spent to be slightly better than a coin toss,” he said.

A new report looks at the state of the legal profession in 2030, and it doesn't look too pretty as far as employment is concerned, io9 reports. "It is no longer unrealistic to consider that workplace robots and their AI processing systems could reach the point of general production by 2030," the report, by Jomati Consultants, says. And those robots could eventually "do the work of a dozen low-level associates. They would not get tired. They would not seek advancement (or) pay rises." A firm's upper echelons would still be populated by actual human lawyers, but the need for associates would shrink dramatically. As such, legal firms could see a "structural collapse," Legal Futures reports. Those in the top rungs of firms would offer "real understanding and human insight" to clients, the report says, and it suggests that because the top partners of 2030 are currently in their 30s, they're likely to push the AI business model. All these changes will apply most to "very large, high-value commercial firms," whereas smaller, specialist firms might see less of an impact, the report finds. Artificial intelligence has already had a big effect on law firms, as the New York Times reported in 2011. It juxtaposed two cases: a 1978 case that required $2.2 million worth of work by lawyers and paralegals who sorted through 6 million documents, compared to a 2011 case in which software was able to pore over 1.5 million documents for less than $100,000.

multi_news

37 2311 S 35th St Fort Pierce, FL 34981 (772) 466-8303 I used to frequent the Big Apple pizza in North Palm Beach, so when we were looking for a midpoint place to meet family, we chose this Big Apple Pizza location. When we arrived, it was empty, but there is ample seating available. Their tv's are tuned to sports, and many pictures are on the walls, including one of President Obama visiting there. We got garlic knots which are good, but there are the harder ones, I prefer soft, but they were still good. The salad and dressing was also good. We got a loaded pizza and a plain cheese pizza, both were huge and very good. While this location is off the beaten path, it is worth the drive. Great service and a great lunch for a good price. For $9 I got two slices of pizza, a drink, and a large salad. It's about five to ten minutes off the turnpike. They've also got a nice draft beer selection if that's something you're interested in. I got to this one because it's close to home. I like the owners and the employees there are very hospitable. No complaints here! We ordered takeout from here last night When I placed my order we were disappointed because we were told they did not have any soups at that location nor did they have the wedge salad Both were on their internet menu... We got the sampler dinner... It was very good Spaghetti and sausage.... Not much sauce kind of plain tasting however the sausage was good but nothing exceptional Two slices of pizza... Was ok again not much sauce and the crust was very tough and chewy Their garlic knots were tasty but again very tough and chewy... Fed them to the dogs Side salads were good Canoili was good and the cheesecake was average Maybe it would have been much better at the restaurant but not so much delivery If in the area again I know we would not order takeout again but might give it a second chance and eat dine in Was disappointed but will give a second try My husband and I were new to the area and were looking for a good sandwich and pizza restaurant. And we found Big Apple..... great subs and great pizza. Fresh ingredients. Clean. Definitely will go again!!! Yes! It is worth it! So a funny thing happened while on another Ocala to Miami drive - Yup, I got hungry!! Was in a rush but just not in the mood for crappy food. -- Yelp-- Benchmarks -- I honestly did not remember why I tagged this pizza shop. I'm not a aficionado, heck, I don't even eat pizza that often. But trusting there had to be a good reason I took a sizable detour from the Turnpike. And IT WAS WORTH IT! Yep. I was very happy with my 2 slices mushrooms, roasted peppers and sausage. Plus a draft of 312 (don't judge me) So while waiting for the order, I went back through the reviews and quickly remember why I wanted to make a visit here. The social brouhaha after the POTUS hugged the owner, the subsequent dirty responses from (XXXXXX) people who have issue with the POTUS, and the noble response from the owner really demonstrated the depth of character of this man. As I pondered the reason why POTUS would "randomly" select here (did the secret service rely on Yelp also?), I also started to notice many of the pictures, T-shirts and letters of Thank you. Very near and dear to my heart, was the letter of from the Local Fire Chief asking patrons to consider supporting MDA and the "Fill the Boot" drive. What is clear, is the person who owns this restaurant has deep relationship with his community from what appears to be a generous heart. The pizza arrived, with these thoughts contemplating in my head. The first bite in and I was rewarded. Of special note, the crust has a special springy lightness, that I have found in only one other pizza shop, who owner comes from Philly. Coincidence? Not sure, but I suspect that when I finally get to Philly, I will be seeking out this special type of crust. In short - just go and enjoy!! Wonderful pizza tastes like home! Meatballs were awesome too. Little pricy but wonderful and worth the stop. Came in and sat down and waited. Never been here before and I was the only person in here. It took some type before someone asked if I needed something. Wasn't the best pizza just ok. This is a great place to go for a quick bite while at the school or in the area. Awesome decor, the employees are very friendly. Food is great and easy. Highly recommend this place It's pizza - no pizazz - pizza maker was yelling and using profanity to another person in the kitchen about another worker. Not very professional. Garlic knots gave the worst indigestion that I have had in years. We stopped by yesterday, on Labor Day, and were pleased to learn that the Monday-Tuesday special (a large pie for $8.49) was in effect even on a holiday. We added artichokes as a topping for $1.75, and between the two of us we very nearly finished the 16-inch pie. That's especially impressive considering that my wife doesn't usually like pizza. This one she liked. She remembered the place from the early 2000s, back when the restaurant's most famous guest was still in the Illinois State Senate, when she was going to IRCC. (Kids, that's what your mother called IRSC back in the day. She pronounced it 'irk'.) Since then, the Big Apple has gotten bigger, and has completely redecorated. We weren't quite sure whether to expect table service or counter service, since there was an 'order here' sign above the register but we were invited to choose a table and our server brought menus and took our order. Later, I refilled my own diet Pepsi from the fountain, but we also had refills brought to us. Maybe that's because it was dinnertime; maybe it's because my wife was using her crutches. When the pizza arrived, we dug in. The crust was thin and crackled a little bit, like you'd expect from a New York style pie. Artichokes were scattered lightly around, but as my wife pointed out, the amount of artichokes you'd hope for is charged in most other places as a double topping -- so we got a reasonable value for the money. If you want lots of artichoke, you can always order your 16" pizza with double artichokes for an extra $3.50 instead of $1.75. Easy enough. We were saddened that there was no lemon for the freshly brewed iced tea, not even packets of lemon juice. And when it was time to pile back into our car (which we just bought this weekend, and have not yet ruined), we hoped to have a plastic grocery sack to put our clamshell of leftovers into. A place that serves garlic knots really should have a leakproof takeout solution. But even missing those two things, we enjoyed ourselves and heard a great retelling of Big Apple's big day. Our server told the story well for someone who had just barely missed coming in on the day the Secret Service arrived to secure the place. She says the owner himself had even scolded the employee for interrupting his golf game, until he realized he really *did* need to get back to the store within an hour for the photo op of his life. I might not come in off the links just for Big Apple Pizza, especially if I was only shooting 10 above par (per hole -- hey, everyone starts somewhere). But I sure would return the next time I'm hungry in Fort Pierce. I have been traveling a lot, so I have been eating out a lot. Usually I am looking for something simple, quick and tasty. This place was recommended by a person at the place I was auditing and they were not wrong. Coming from Chicago, my expectations are high and I was not disappointed. Lovely, lovely food. Got the Big Apple White Pizza...very nice balance of cheeses and tomatoes. Also ordered some Sicilian slices with sausage that was amazing. I love a good sauce and this place does it right. My co-worker got the Big Apple Sampler Meatball Sliders. Sliders and wings were nice but the garlic knots were too much for me but I am not big on mega garlic. Co-worker thought they were great. What I will say though they were tasty with a nice buttery flavor. Will be my go to place when I return in 4 months for a follow up audit. Coming back from a weekend road trip Fort Pierce was the right distance in which to stop to get some grub. Kinda of in the mood for some tasty pizza, we launched Yelp and it pointed us to Big Apple Pizza. It was just ok. Here's the run down... We ordered a large pizza, 1/2 mushroom and 1/2 sausage. We ordered a small Antipasto salad. 1) 2 garlic rolls came with the salad. They weren't fully baked and in fact almost cold. 2) Salad was decent, and we ordered the house dressing (which was good). What threw me for a bit was that it was a pre packaged (ie commercial type) dressing, but it had big apple pizza's name on it. This made sense now that I remember seeing on the menu that you could take a bottle of dressing home. The impression it left me however was that stuff was made a long time ago, not fresh in the kitchen today or yesterday. Just my perception, but not a good one however. 3) They used canned mushrooms. We are a bit of stickler on this. Mushrooms shouldn't be green'ish. 4) I liked the sauce on the pizza. Sauce to me is the most important factor. 5) The cleanliness and upkeep of the restaurant was so so. Bathroom was decently clean, yet no soap. The soda machine was a bit broken. The buffet serving center looked a little run down. Good enough for the pass through. The wings here are amazing. Pizza is fantastic. Screw these negative reviewers. Yelp is not the place for political BS. Really liked this spot...the server was the owners daughter, not sure if she is always there, but I like when family works together at a restaurant. We had just got done moving to PSL, and after a week of hell, we were hungry and stumbled into this pizza joint... Very friendly server, and after ordering, the owner came and introduced himself, asking if we would like to try the vodka sauce over some penne...umm, cha...and so happy we did...best I have ever had. Then, the food came...wedding soup was top notch...garlic knots were right on...spag and meatball were very good, might have been a box pasta but very good sauce and meatball...and the cheese pizza was tasty as hell, very thin and foldable, just like a New York pie should be... Then, the owner came over and invited us to meet up and ride sometimes, I think because he knew we were new in town, and it was a very genuine offer...which we are going to take him up on... Our one regret, is that we did not save room for the cannolli...this time...I mean, since we will be back! Pizza was absolutely delicious. Just like I get in NY. Great antipasto salad and calzone was delicious. Highly recommend. I have been here several times. The canoles and strombolis are very good. The pizza is okay. One of the young ladies (I am not the best at descriptions) that works there has a bad attitude and seems indifferent, like she could care less whether you were there or not. When I go there, I always hope she isn't there so I will have a better dining experience. The owner seems to have the opposite attitude, I wish he could see she is not good for business. I stopped by on my summer family vacation this year. I was surprised to find a pizza joint that actually has the new york flavor and being from the Bronx, I know what am taking about... Pizza was good, staff equally and prices were affordable. A family of 4 bill came to $25 and we were all stuff... Will definitely recommend it and return next time am in town.. Decent food with a typical pizza shoppe look. Maybe it's because of the time we came to eat or the dishes that we ordered, but it wasn't exactly what we expected when going by the yelp reviews. The egg plant was definitely fresh but it was not cooked all the way through and my girlfriends chicken was somewhat chewy. Aside from the food, I respect what the owner is doing for his local community! I give him two thumbs up for that! A pizza shop owner lifted President Obama about a foot off the ground in a giant bearhug. Obama arrived at the Big Apple Pizza & Pasta Restaurant in Fort Pierce, Fla., at 2:55 p.m. There were only 10 customers at the time, but the owner, Scott Van Duzer, made up for the relatively empty shop. "Scott, what's going on man?" Obama shouted as he came through the door. "Scott, let me tell you, you are like the biggest pizza shop owner I've ever seen." He approached Van Duzer, 46, of Port St. Lucie, who stands 6-foot-3 and weighs 260 pounds. "Everybody look at these guns," Obama said, pointing to Van Duzer's monstrous pectoral muscles. "If I eat your pizza, will I look like that?" The two men laughed. They embraced. Van Duzer lifted the president a good foot off the ground. "Look at that!" Obama exclaimed once back on firm ground. "Man, are you a powerlifter or what?" Indeed, Van Duzer said later that he can bench-press 350 pounds. Van Duzer then gave Obama a tour of the restaurant. After their brief tour, Obama approached reporters and told why we stopped at Van Duzer's shop. "One of the reasons that we wanted to stop by is that Scott has been doing unbelievable work out of this pizza shop in promoting the importance of donating blood," Potus said. "And so he has set some records here in Florida. He has received commendations from the White House, the surgeon general, he has galvanized and mobilized the local community and he's educating kids and folks all across the country on this issue. "So here's an example of somebody who is doing well but he's also giving back. So, we just want to say how proud we are of him. I still wonder how he got these biceps, but what we know is that the guy's just got a big heart along with big pecs. So, we're very proud of him and we just want to say thank you for all the great work." Van Duzer, who said he played basketball, football and baseball in high school, said he is a registered Republican, yet voted for Obama in 2008 and plans to do so again in November. "I don't vote party line, I vote who I feel comfortable with, and I do feel extremely comfortable with him," Van Duzer said. Of the Secret Service agents possibly flipping out over Van Duzer lifting the president of the United States, he said: "He said I was all right as long as I didn’t take him away." Read more about: Barack Obama, Florida 2012, Scott Van Duzer Vice President Joe Biden was looking to cozy up with voters as he toured Ohio this weekend, but he did not imagine that an Ohio woman would nearly end up in his lap. Vice President Joe Biden's talks to customers during a stop at Cruisers Diner, Sunday, Sept. 9, 2012, in Seaman, Ohio. (AP Photo/Carolyn Kaster) (Associated Press) Vice President Joe Biden visits with patrons over lunch at Cruisers Diner, Sunday, Sept. 9, 2012, in Seaman, Ohio. (AP Photo/Carolyn Kaster) (Associated Press) Biden was chatting up customers in the Cruisers Diner in southern Ohio Sunday when he met a group of motorcycle riders in black leather vests and bandanas. A female group member was watching, and Biden waved her over, telling her, "I know who runs the show." The woman had no place to sit, so Biden pulled a chair in front of himself and pulled her nearly into his lap. He put his hands on her shoulders and leaned in for a conversation as photographers snapped away.

In a weird day on the trail, President Obama was picked up and Joe Biden nearly picked up a biker gal-at least judging by the photographs. Obama visited a pizza joint in Florida yesterday where owner Scott Van Duzer hugged him so hard that the president was lifted right off his feet, Politico reports. Even weirder: Van Duzer is a registered Republican who voted for Obama in 2008 and plans to vote for him again. Sadly, Obama's airlift triggered a war of comments on the restaurant's Yelp page, with reviewers trading politically charged one-star and five-star reviews, CBS News reports. As for Biden, he was pressing the flesh at a diner in southern Ohio when he encountered a group of motorbike riders. Saying "I know who runs the show," he called over a female biker and pulled up a chair so close that the two practically snuggled, reports the AP. Note how nervous her biker buddies are watching her have fun with the VP.

multi_news

Developing HIV-1 vaccines that trigger broadly neutralizing antibodies (bnAbs) is a priority as bnAbs are considered key to elicitation of a protective immune response. To investigate whether the breadth of a neutralizing antibody (nAb) depended on the conservation of its epitope among circulating viruses, we examined Antibody: Envelope (Ab: Env) interactions and worldwide Env diversity. We found that sites corresponding to bnAb epitopes were as variable as other accessible, non-hypervariable Env sites (p = 0. 50, Mann-Whitney U-test) with no significant relationship between epitope conservation and neutralization breadth (Spearman’s ρ = -0. 44, adjusted p = 0. 079). However, when accounting for key sites in the Ab: Env interaction, we showed that the broadest bnAbs targeted more conserved epitopes (Spearman’s ρ = -0. 70, adjusted p = 5. 0e-5). Neutralization breadth did not stem from the overall conservation of Ab epitopes but depended instead on the conservation of key sites of the Ab: Env interaction, revealing a mechanistic basis for neutralization breadth that could be exploited for vaccine design. There is an urgent need for a vaccine against HIV-1. Since HIV-1 shows remarkable diversity, it is assumed that a vaccine should elicit bnAbs to block the most extensive array of HIV-1 strains[1–4]. Neutralizing Abs develop over the course of HIV-1 infection and there is a continuum in the extent of neutralization breadth developed across individuals, with typically half of a cohort being able to neutralize about half of a virus panel [5]. A number of studies have focused on the fraction of individuals who can develop bnAbs that can neutralize a majority of the viruses in a panel[6–9]. These bnAbs arise after Ab lineages have matured typically over multiple years[5,10–13]. Highlighting the many paths that can lead to bnAb development, bnAbs have been isolated from individuals with different HIV-1 subtypes and presenting different clinical disease progression profiles. For example, VRC01 was isolated from patient 45, an African-American male who had been infected with HIV-1 subtype B for 11 years at the time of Ab isolation; he was considered a long term non-progressor as his viremia was maintained around 10,000 copies/ml [14]. In contrast, the bnAb CH103 was isolated from patient CH505, a male from Malawi who had been infected with HIV-1 subtype C for 2. 5 years when Ab were isolated; patient CH505 was followed for six years and maintained a high median viral load of 173,667 over that time [15]. BnAbs recognize exposed regions of the Env trimer and tend to target five sites: the V1V2-glycan site (e. g. PG9), the V3-glycan site (e. g. PGT128), the CD4 binding site (e. g. VRC01), the gp120-gp41 interface (e. g. 8ANC195,35O22) and the membrane proximal region of Env-gp41 (e. g. 10E8) [16]. It is generally believed that bnAbs target conserved epitopes on HIV-1 Env trimers [17–20]. Yet, no study has systematically quantified the relationship between the neutralization breadth of bnAbs and the conservation of their respective epitopes on Env. Here we analyzed publicly-available Ab: Env complex structures and characterized how the neutralization breadth of an Ab was influenced by the conservation of its epitope. We describe how neutralization breadth was positively associated with Env epitope conservation only when the epitope conservation was defined by taking into account the strength of the Ab: epitope interaction, i. e. specifically weighting structurally important sites, and not simply sequence conservation. To characterize the diversity of HIV-1 circulating strains, we created an HIV-1 group M alignment of 239 Env sequences that reflected the global representation of HIV-1 subtypes (gp M), as well as specific datasets for subtype A1 (n = 203), B (n = 1035), C (n = 1184), D (n = 116) and CRF01_AE (n = 577). We analyzed 34 Abs for which neutralization breadth had been measured using a panel of 136 viruses and ranged between 31 and 97% [21] and for which Ab: Env complex structures were available (S1 Table, S1 Fig). For these 34 Abs, the epitope consisted of 8 to 36 sites. We looked at the diversity among group M sequences at each accessible, non-hypervariable site on the surface of Env and found no difference between sites that belonged to Ab epitopes (n = 31 Abs, epitopes of 3 MPER antibodies were excluded as they are partially/totally missing from the Env structure 5FYJ, which was used to define surface sites) and sites that were outside of epitopes: median Shannon entropy = 0. 47 vs. 0. 32 bits, respectively (p-value = 0. 50, Mann-Whitney U-test) (Fig 1). This result was confirmed when the analysis was restricted to the 15 antibodies that showed over 70% breadth: median Shannon entropy = 0. 44 vs. 0. 32 bits, respectively (p-value = 0. 47, Mann-Whitney U-test) (Fig 1B). Thus, bnAbs targeted Env sites that were as variable as other accessible Env sites. To define the epitope diversity, we summed the Shannon entropy of all the epitope sites and adjusted with the mutual entropy of neighbor pairs of sites. This corresponds to the diversity of the whole epitope patch, which we then normalized based on the size of the epitope. There was no relationship between the epitope diversity (or conservation) and the breadth of neutralization of HIV-1 strains by nAbs if we considered the epitope diversity of the whole epitope (Spearman’s ρ = -0. 20, adjusted p-value = 0. 70) or when the epitope diversity was normalized based on the size of the epitope (Spearman’s ρ = -0. 44, adjusted p-value = 0. 079; Fig 2A) when using the set of sequences representative of the global HIV-1 distribution. Results were similar when we tested the sequence sets corresponding to different subtypes/CRF: Spearman correlation coefficient ρ ranged between -0. 31 and -0. 43 with adjusted p-values > 0. 078, showing that the finding was not dependent on the dataset tested (group M versus subtype-specific alignments) (Fig 3). We modified our definition of epitope diversity to integrate structural factors beyond sequence conservation that may be critical to efficient Ab neutralization. These epitope diversity measures weighted each site in the Env epitope by i) the number of Ab atom contacts for each Env epitope residue, ii) the number of atom pairs in contact between the epitope and the Ab, iii) the number of neighboring Ab residues for a given epitope residue in the Ab: Env complex and iv) the reduced accessible surface area after Ab binding. These definitions were also normalized for the size of the epitope (8–36 sites). The results reported below correspond to the nine epitope sites that had the highest number of neighboring Ab residues (we focused on nine sites as it showed the best correlation when comparing between seven and eleven key sites, S2 Fig). Fig 3 shows the effect of weighting by the features described above, we found a negative relationship between epitope diversity and neutralization breadth: the more conserved the structurally weighted Env epitope, the greater the neutralization breadth. If we consider the alignment of 239 representative group M sequences, the Spearman correlation coefficient ρ was -0. 70 (adjusted p-value = 5. 0e-5) (Fig 2B, S2 Table). The negative relationship between the Env epitope diversity and neutralization breadth was replicated when restricting the analysis to specific subtypes/CRF: considering the top nine target sites, the Spearman correlation coefficient ρ ranged between -0. 57 and -0. 73 with adjusted p-values ≤ 6. 1e-3. We note that the relationship was similar when we considered only the dataset corresponding to the subtype matching the subtype of the infected individual from whom the Ab was obtained (Spearman’s ρ = -0. 70, adjusted p-value = 1. 7e-3; S3 Fig and S4 Fig). Since most of the 34 Abs targeted the CD4 binding site (CD4bs) (n = 21), we analyzed data separately for these Abs. We found that the relationship between epitope diversity and neutralization breadth was largely driven by CD4bs Abs (Spearman’s ρ ranged between -0. 61 and -0. 73 with adjusted p-values ≤ 3. 1e-3 when analyzing the different sequence sets, S3 Table). However, for the 13 other Abs, the relationship was not improved by the structural-weighting (Spearman’s ρ ranged between -0. 39 and -0. 66,0. 18 ≤ adjusted p-values ≤ 1. 00, S3 Table; the lack of significance may be due to the small sample size). The fact that epitope conservation failed to strictly derive from sequence conservation but corresponded to a structurally-weighted conservation measure is illustrated by the comparison of the Env epitopes of 3BNC117 and VRC03, two Abs that target the CD4 binding site. The bnAb 3BNC117 neutralizes 82% of HIV-1 strains while VRC03 neutralizes 48% of HIV-1 strains. Because the Env epitopes of 3BNC117 and VRC03 are very similar, the unweighted measure of diversity gave similar diversity values. However, when we considered only the structurally key epitope sites for Ab binding, 3BNC117 engaged conserved sites while VRC03 had many atom contacts with more variable sites such as amino acids 460 and 461 in Env-V5. Hence, the VRC03 epitope had a higher diversity value for its top nine sites than the 3BNC117 epitope and was associated with a more limited breadth of neutralization, highlighting that the mode of interaction between the Ab and epitope and not just the location of the epitope was associated with increased neutralization coverage (Fig 2). We obtained similar results when we used as a measure of epitope conservation the similarity of a given epitope to its counterpart in the three strains experimentally-defined as the most susceptible to neutralization. The three most susceptible strains were selected using the data from the panel of 136 viruses assayed by Doria-Rose and colleagues [21] (up to five strains were tested before choosing a combination of three strains, S5 Fig). For each Ab, we calculated the fraction of viruses with epitopes matching the epitope in the three most susceptible strains. When we considered whole epitopes, the nAbs that showed a higher fraction of epitopes similar to the three most susceptible strains were associated with increased neutralization breadth (Spearman’s ρ = 0. 60, adjusted p = 1. 9e-3). As seen above, this relationship was stronger when we focused on the top nine epitope sites (ranked by the number of neighboring Ab residues) in the Ab: Env interaction: Spearman’s ρ = 0. 80, adjusted p = 2. 7e-7 (Fig 4). Similar to what we noted above, the Spearman’s correlation coefficient was stronger for Abs that targeted the CD4bs (Spearman’s ρ = 0. 78, adjusted p = 5. 2e-4, S4 Table) than for Abs that targeted other epitopes (Spearman’s ρ = 0. 73, adjusted p = 0. 070, S4 Table). One shortcoming of this study is that Env accessibility was measured using Env structures with glycans removed, while we know that HIV-1 Env trimers are covered by a glycan shield of ∼90 N-linked oligosaccharides constituting about half of the Env mass—a key factor in HIV-1 evading humoral immunity[22–24]. We weighted the epitope diversity calculations (described above) for the presence of glycans at specific epitope sites, yet this modification did not reveal any significant difference in the relationship between epitope diversity and neutralization (Fig 4). We compared the 9-, 7- and 5-mannose models sampled in molecular dynamics simulations [25] and found that the median Ab accessibility was diminished by about three-fold when glycans were integrated: 13. 6 Å in the 9-mannose model vs. 4. 6 Å in the absence of glycans. The 7- and 5- mannose models provided better Ab accessibility than the 9-mannose model (median depth of 11. 1 Å, 12. 2 Å and 13. 6 Å for 5-, 7 and 9-mannose Env models, respectively), indicating that engineering the glycan shield to have only 5 or 7 mannose residues at each glycan site may improve Ab accessibility. This is consistent with the finding that restricting the glycan site to be 5 mannose greatly increased its susceptibility to an array of bnAbs[26]. In summary, we systematically analyzed the interaction between nAbs and their corresponding Env epitopes to identify the mechanistic basis of HIV-1 neutralization breadth. Surprisingly, although it is widely accepted that HIV-1 bnAbs target conserved segment of HIV-1 Env, we demonstrated that bnAbs targeted Env sites that were no more conserved than other accessible, non-hypervariable Env sites and that the breadth of a nAb was not significantly related to the conservation of its epitope among circulating viruses if we used a standard measure of epitope conservation. It is only when HIV-1 conservation was measured by accounting for the structural strength of the Ab: Env interaction that we found a positive relationship between sequence conservation and neutralization breadth. We note, however, that certain factors complicated our analysis. For example, it is difficult to account for the influence of the glycan shield or for the missingness of some structural information such as for MPER antibodies where only a fragment of Env is complexed with the antibody. Future studies will also be needed to evaluate whether non-neutralizing antibodies differ from bnAbs in their mode of interaction with Env. Our finding has implications for vaccine development. We showed that epitopes were not more conserved than any other non-hypervariable sites at the surface of the prefusion-closed Env trimer, yet the broadest bnAbs showed key interactions at very conserved sites. Our study indicates that targeting conserved epitopes is necessary but not sufficient to promote breadth and that an antibody’s interactions with key conserved sites are primordial to achieve neutralization breadth. This would suggest that targeting conserved epitopes may not be sufficient if there is no further Ab maturation to focus on key epitope sites. Hence, this work suggests Ab breadth could be improved by designing epitope patches on immunogens to favor breadth-promoting Ab interactions. In addition, this knowledge can directly be used to characterize breakthrough and rebound viruses following bnAb-based interventions, providing a novel blueprint to prospectively interpret results of clinical trials that use bnAbs as a therapeutic agent. An Ab epitope was defined based on the Ab: Env complex structure as the Env sites with heavy atoms (called atoms below and in the main text) that were located within 4 Å of the Ab. Weights were assigned to specific Env sites in different ways, using: i) the number of Ab atoms contacted (w. natom), ii) the number of atom pairs in contact between the epitope and the Ab (w. npairs), iii) the number of neighboring Ab residues in the Delaunay tetrahedralization of Cβ (Cα of Gly) atoms of the Ab: Env complex (w. nnbs), and iv) the reduced accessible surface area after Ab binding (w. asa) [31]. The Delaunay tetrahedralization was obtained using Quickhull [32] and edges longer than 8. 5 Å were removed. If multiple complex structures were available for an Ab, the mean weight from the different complex structures was used as the final weight of the epitope. To avoid overweighting, the weight was capped at the 98th percentile of all sites; any site with a weight above the 98th percentile was set to the 98th percentile. The weight of N-linked glycans was scaled such that its 98th percentile was equal to the 98th percentile of the amino acids before adding to the corresponding asparagine. We defined the epitope diversity as the Shannon entropy [33] of the epitope: H=∑iHi−∑i, jIi, j (1) Hi=−∑kp (k) log2p (k) (2) Ii, j=∑m, npi, j (m, n) log2pi, j (m, n) pi (m) ∙pj (n) (3) where Hi is the Shannon entropy of epitope site i; Ii, j is the mutual entropy between a pair of neighbor sites (i, j); p (k) is the fraction of amino acid k on a site; pi, j (m, n) is the fraction of amino acid combinations (m, n) on a pair of neighbor sites (i, j) (m on site i and n on site j). The summation is over all epitope sites in the first term and over all neighbor sites in the second term of Eq 1. In Eq 2, the summation is over all amino acids. In Eq 3, the summation is over all amino acid combinations on neighbor sites i and j. All neighbor pairs were identified by Delaunay tetrahedralization of Cβ (Cα of Gly) atoms in the ab: Env complex structure. An example based on a toy epitope is provided in the supplementary material to illustrate how the diversity is estimated. To account for the contribution of specific sites in the Ab: Env complex, we used: H=∑iwiHi−∑i, jwi, jIi, j (4) wi, j= (wiHi+wjHj) / (Hi+Hj) (5) where wi, wj and wi, j are the weights assigned to epitope sites i, epitope site j, and a pair of neighbor sites (i, j), respectively. The summation is over all epitope sites in the first term and over all neighbor pairs in the second term of Eq 4. When the epitope diversity is normalized, the weight of each site is adjusted as wi′=wi/∑iwi, in which the summation is over all epitope sites. Based on the chemical similarity between certain amino acids, we grouped amino acids D and E as ‘a’, R and K as ‘b’, N and Q as ‘n’, L and M as ‘l’, V and I as ‘i', and F and Y as ‘f’ before estimating the Shannon entropy. If N was a potential N-linked glycosylation site, it was flagged as ‘g’. The epitope similarity between a sequence X and a reference sequence R was defined as: S (R, X) =−[M (R, R) −M (R, X) ] (6) M (R, X) =[∑iwi∙Sim (Ri, Xi) ]/∑iwi (7) where M (R, X) is the match score between R and X, wi is the weight assigned to epitope site i, and Sim (Ri, Xi) is from either the BLOSUM62 [34] or the VTML200 [35] matrix, which describes the similarity between Ri (amino acid on site i of R) and Xi (amino acid on site j of X). The minus sign on the right side of Eq (6) converted a distance to the similarity. To avoid relying on a single strain as the reference, we selected three references strains (n one to five strains with lowest IC50s from 136 strains were tested). The highest epitope similarity to the three references were used as the epitope similarities to the susceptible strains (S (R, X) = max (S (R1, X), S (R2, X), S (R3, X) ) ). Then, we predicted the breadth of an antibody as the fraction of strains with an epitope similarity below a similarity threshold (TH). The number of resistant viruses predicted by the epitope similarity should be same as the number of resistant viruses determined in the neutralization assays. Thus, we set the threshold such that ∑NIC50≥25μg/ml = ∑NSim<TH, in which the summation is over all 34 antibodies. Specifically, given the 136 viruses tested against 34 antibodies, there were 34×136 = 4,624 epitope similarities. The neutralization assays identified 1,561 = 33. 8% of virus-Ab combinations as resistant (with IC50 ≥ 25 μg/ml). Thus, we set the similarity threshold as the 1561th element after all the 4,624 epitope similarities were sorted ascendingly. Data analysis, visualization and statistical testing were performed in the Python environment[41–47]. Statistical details of analyses can be found in the main text and figure captions where applicable; significance was established at p < 0. 05. A link to the data archive and code to reproduce the analysis is provided below.

So far, no HIV-1 vaccine has elicited broadly neutralizing antibodies (bnAbs) in humans. HIV-1, one of the most rapidly evolving pathogens, is remarkable for its high variability across individuals and adaptability within hosts. We tested the relationship between HIV-1 diversity and neutralization breadth. While bnAbs did not specifically target more conserved regions of HIV-1 Env, we found that the broadest bnAbs relied forcibly more on structural interactions at key sites of the Ab: Env interaction than other Abs. Understanding mechanisms underlying neutralization breadth provides guidelines to design more efficacious vaccines and antibody-based therapeutics.

lay_plos

To form a percept of the environment, the brain needs to solve the binding problem—inferring whether signals come from a common cause and are integrated or come from independent causes and are segregated. Behaviourally, humans solve this problem near-optimally as predicted by Bayesian causal inference; but the neural mechanisms remain unclear. Combining Bayesian modelling, electroencephalography (EEG), and multivariate decoding in an audiovisual spatial localisation task, we show that the brain accomplishes Bayesian causal inference by dynamically encoding multiple spatial estimates. Initially, auditory and visual signal locations are estimated independently; next, an estimate is formed that combines information from vision and audition. Yet, it is only from 200 ms onwards that the brain integrates audiovisual signals weighted by their bottom-up sensory reliabilities and top-down task relevance into spatial priority maps that guide behavioural responses. As predicted by Bayesian causal inference, these spatial priority maps take into account the brain’s uncertainty about the world’s causal structure and flexibly arbitrate between sensory integration and segregation. The dynamic evolution of perceptual estimates thus reflects the hierarchical nature of Bayesian causal inference, a statistical computation, which is crucial for effective interactions with the environment. In our natural environment, our senses are exposed to a barrage of sensory signals: the sight of a rapidly approaching truck, its looming motor noise, the smell of traffic fumes. How the brain effortlessly merges these signals into a seamless percept of the environment remains unclear. The brain faces two fundamental computational challenges: First, we need to solve the ‘binding’ or ‘causal inference’ problem—deciding whether signals come from a common cause and thus should be integrated or instead be treated independently [1,2]. Second, when there is a common cause, the brain should integrate signals taking into account their uncertainties [3,4]. Hierarchical Bayesian causal inference provides a rational strategy to arbitrate between sensory integration and segregation in perception [2]. Bayesian causal inference explicitly models the potential causal structures that could have generated the sensory signals—i. e., whether signals come from common or independent sources. In line with Helmholtz’s notion of ‘unconscious inference’, the brain is then thought to invert this generative model during perception [5]. In case of a common signal source, signals are integrated weighted in proportion to their relative sensory reliabilities (i. e., forced fusion [3,4, 6–10]). In case of independent sources, they are processed independently (i. e., full segregation [11,12]). Iin a particular instance, the brain does not know the world’s causal structure that gave rise to the sensory signals. To account for this causal uncertainty, a final estimate (e. g., object’s location) is obtained by averaging the estimates under the two causal structures (i. e., common versus independent source models) weighted by each causal structure’s posterior probability—a strategy referred to as model averaging (for other decisional strategies, see [13]). A large body of psychophysics research has demonstrated that human observers combine sensory signals near-optimally as predicted by Bayesian causal inference [2,13–16]. Most prominently, when locating events in the environment, observers gracefully transition between sensory integration and segregation as a function of audiovisual spatial disparity [12]. For small spatial disparities, they integrate signals weighted by their reliabilities, leading to cross-modal spatial biases [17]; for larger spatial disparities, audiovisual interactions are attenuated. A recent functional MRI (fMRI) study showed how Bayesian causal inference is accomplished within the cortical hierarchy [14,16]: While early auditory and visual areas represented the signals on the basis that they were generated by independent sources (i. e., full segregation), the posterior parietal cortex integrated sensory signals into one unified percept (i. e., forced fusion). Only at the top of the cortical hierarchy, in anterior parietal cortex, the uncertainty about the world’s causal structure was taken into account and signals were integrated into a spatial estimate consistent with Bayesian causal inference. The organisation of Bayesian causal inference across the cortical hierarchy raises the critical question of how these neural computations unfold dynamically over time within a trial. How does the brain merge spatial information that is initially coded in different reference frames and representational formats? Whereas the brain is likely to recurrently update all spatial estimates by passing messages forwards and backwards across the cortical hierarchy [18–20], the unisensory estimates may to some extent precede the computation of the Bayesian causal inference estimate. To characterise the neural dynamics of Bayesian causal inference, we presented human observers with auditory, visual, and audiovisual signals that varied in their spatial disparity in an auditory and visual spatial localisation task while recording their neural activity with electroencephalography (EEG). First, we employed cross-sensory decoding and temporal generalisation matrices [21] of the unisensory auditory and visual signal trials to characterise the emergence and the temporal stability of spatial representations across the senses. Second, combining psychophysics, EEG, and Bayesian modelling, we temporally resolved the evolution of unisensory segregation, forced fusion, and Bayesian causal inference in multisensory perception. Combining psychophysics, multivariate EEG pattern decoding, and computational modelling, we next investigated the computational principles and neural dynamics underlying audiovisual integration of spatial representations using a general linear model (GLM) -based wAV and a Bayesian modelling analysis. As shown in Fig 3, both analyses were applied to the spatial estimates that were either reported by participants (i. e., behaviour, Fig 3B left) or decoded from EEG activity patterns independently for each poststimulus time point (i. e., neural, Fig 3B right, for further details, see the Methods section and the Fig 3 legend). The GLM-based wAV analysis quantifies the influence of the true auditory and true visual location on (1) the reported or (2) EEG decoded auditory and visual spatial estimates in terms of an audiovisual weight index wAV. The Bayesian modelling analysis formally assessed the extent to which (2) the full-segregation model (s) (Fig 3C, encircled in light blue, red or green), (2) the forced-fusion model (Fig 3C, yellow), and (3) the Bayesian causal inference model (i. e., using model averaging as decision function, encircled in dark blue; see supporting material S1 Table for other decision functions) can account for the spatial estimates reported by observers (i. e., behaviour) or decoded from EEG activity pattern (i. e., neural). Integrating information from vision and audition into a coherent representation of the space around us is critical for effective interactions with the environment. This EEG study temporally resolved the neural dynamics that enable the brain to flexibly integrate auditory and visual signals into spatial representations in line with the predictions of Bayesian causal inference. Auditory and visual senses code spatial location in different reference frames and representational formats [26]. Vision provides spatial information in eye-centred and audition in head-centred reference frames [27,28]. Furthermore, spatial location is directly coded in the retinotopic organisation in primary visual cortex [29], whereas spatial location in audition is computed from sound latency and amplitude differences between the ears, starting in the brainstem [27]. In auditory cortices of primates, spatial location is thought to be represented by neuronal populations with broad tuning functions [30,31]. In order to merge spatial information from vision and audition, the brain thus needs to establish coordinate mappings and/or transform spatial information into partially shared ‘hybrid’ reference frames, as previously suggested by neurophysiological recordings in nonhuman primates [30,32]. In the first step, we therefore investigated the neural dynamics of spatial representations encoded in EEG activity patterns separately for unisensory auditory and visual signals using the method of temporal generalisation matrices [21]. In vision, spatial location was encoded initially at 60 ms in transient neural activity associated with the early P1 and N1 components and then turned into temporally more stable representations from 200 ms and particularly from 350 ms (Fig 2, upper right quadrant, S2 Fig). In audition, spatial location was encoded by relatively stable EEG activity from 95 ms and particularly from 250 ms, which is associated with the auditory long latency P2 component [22–24] (S3 Fig). Activity patterns encoding spatial location generalised not only across time but also across sensory modalities between 160 and 360 ms. As indicated in Fig 2, SVR models trained on visual-evoked responses generalised to auditory-evoked responses and vice versa (upper left and lower right quadrant, significant cross-sensory generalisation encircled by thick grey line). These results suggest that unisensory auditory and visual spatial locations are initially represented by transient and modality-specific activity patterns. Later, at about 200 ms, they are transformed into temporally more stable representations that may rely on neural sources in frontoparietal cortices that are at least to some extent shared between auditory and visual modalities [22,33,34]. Next, we asked when and how the human brain combines spatial information from vision and audition into a coherent representation of space. The brain should integrate sensory signals only when they come from a common event but should segregate signals from independent events [1,2, 12]. To investigate how the brain arbitrates between sensory integration and segregation, we presented observers with synchronous audiovisual signals that varied in their spatial disparity across trials. On each trial, observers reported either the auditory or the visual location. Our results show that a concurrent yet spatially disparate visual signal biased observers’ perceived sound location towards the visual location—a phenomenon coined spatial ventriloquist illusion [17,35]. Consistent with reliability-weighted integration, this audiovisual spatial bias was significantly stronger when the visual signal was more reliable (Fig 1C left, grey solid versus dashed lines). Furthermore, observers reported different locations for auditory and visual signals, and this difference was even greater for large- relative to small-spatial-disparity trials. This significant interaction between spatial disparity and task relevance indicates that human observers arbitrate between sensory integration and segregation depending on the probabilities of different causal structures of the world that can be inferred from audiovisual spatial disparity. Using EEG, we then investigated how the brain forms neural spatial representations dynamically post stimulus. Our analysis of the neural audiovisual weight index wAV shows that the spatial estimates decoded from EEG activity patterns are initially dominated by visual inputs (i. e., wAV close to 90°). This visual dominance is most likely explained by the retinotopic representation of visual space that facilitates EEG decoding of space leading to visual predominance (for further discussion, see the Methods section). From about 65 ms onwards, visual reliability significantly influenced wAV (Fig 4A): as expected, the location of the visual signal exerted a stronger influence on the spatial estimate decoded from EEG activity patterns when the visual signal was more reliable than unreliable. By contrast, the signal’s task relevance influenced the audiovisual weight index only later, from about 190 ms (Fig 4B). Thus, visual reliability as a bottom-up stimulus-bound factor impacted the sensory weighting in audiovisual integration prior to top-down effects of task relevance. We observed a significant interaction between task relevance and spatial disparity as the characteristic profile for Bayesian causal inference from about 310 ms: the difference in wAV between auditory and visual report was significantly greater for large- than for small-disparity trials (Fig 4D, Table 2). Thus, spatial disparity determined the influence of task-irrelevant signals on the spatial representations encoded in EEG activity from about 310 ms onwards. A task-irrelevant signal influenced the spatial representations mainly when auditory and visual signals were close in space and hence likely to come from a common event, but it had minimal influence when they were far apart in space. Collectively, our statistical analysis of the audiovisual weight index revealed a sequential emergence of visual dominance, reliability weighting (from about 100 ms), effects of task relevance (from about 200 ms), and finally the interaction between task relevance and spatial disparity (from about 310 ms, Fig 4A–4D). This multistage process was also mirrored in the time course of exceedance probabilities furnished by our formal Bayesian model comparison: The unisensory visual segregation (SegV) model was the winning model for the first 100 ms, thereby modelling the early visual dominance. The audiovisual forced-fusion model embodying reliability-weighted integration dominated the time interval of 100–250 ms. Finally, the Bayesian causal inference model that enables the arbitration between sensory integration and segregation depending on spatial disparity outperformed all other models from 350 ms onwards. Hence, both our Bayesian modelling analysis and our wAV analysis showed that the hierarchical structure of Bayesian causal inference is reflected in the neural dynamics of spatial representations decoded from EEG. The Bayesian causal inference model also outperformed the audiovisual full-segregation (SegV, A) model that enables the representation of the location of the task-relevant stimulus unaffected by the location of the task-irrelevant stimulus. Instead, our Bayesian modelling analysis confirmed that from 350 ms onwards, the brain integrates audiovisual signals weighted by their bottom-up reliability and top-down task relevance into spatial priority maps [36,37] that take into account the probabilities of the different causal structures consistent with Bayesian causal inference. The spatial priority maps were behaviourally relevant for guiding spatial orienting and actions, as indicated by the correlation between the neural and behavioural audiovisual weight indices, which progressively increased from 100 ms and culminated at about 300–400 ms. Two recent studies have also demonstrated such a temporal evolution of Bayesian causal inference in an audiovisual temporal numerosity judgement task [38] and an audiovisual rate categorisation task [39]. The timing and the parietal-dominant topographies of the AV potentials (see S2 and S3 Figs) that form the basis for our spatial decoding (and hence for wAV and Bayesian modelling analyses) closely match the P3b component (i. e., a subcomponent of the classical P300). Although it is thought that the P3b relies on neural generators located mainly in parietal cortices [40,41], its specific functional role remains controversial [42]. Given its sensitivity to stimulus probability [43–45] and discriminability [46] as well as task context [42,47,48], it was proposed to reflect neural processes involved in transforming sensory evidence into decisions and actions [49]. Most recent research has suggested that the P3b may sustain processes of evidence accumulation [50] that are influenced by observers’ prior [51], incoming evidence (i. e., likelihood [52]), and observers’ belief updating [53]. Likewise, our supplementary time-frequency analyses revealed that alpha/beta power, which has previously been associated with the generation of the P3b component [54], depended on bottom-up visual reliability between 200 and 400 ms and top-down task relevance between 350 and 550 ms post stimulus (see S5 Fig, S2 Table and S1 Text), thereby mimicking the temporal evolution of bottom-up and top-down influences observed in our main wAV and Bayesian modelling analysis. Yet, our main analysis took a different approach. Rather than focusing on the effects of visual reliability, task relevance/attention, and spatial disparity directly on event-related potentials (ERPs) or time-frequency power, the wAV analysis investigated how these manipulations affect the spatial representations encoded in EEG activity patterns, and the Bayesian modelling analysis accommodated those effects directly in the computations of Bayesian causal inference. Along similar lines, two recent fMRI studies characterised the computations involved in integrating audiovisual spatial inputs across the cortical hierarchy [14,16]: whereas low level auditory and visual areas predominantly encoded the unisensory auditory or visual locations (i. e., full-segregation model) [55–64], higher-order visual areas and posterior parietal cortices combined audiovisual signals weighted by their sensory reliabilities (i. e., forced-fusion model) [65–68]. Only at the top of the hierarchy, in anterior parietal cortices, did the brain integrate sensory signals consistent with Bayesian causal inference. Thus, the temporal evolution of Bayesian causal inference observed in our current EEG study mirrored its organisation across the cortical hierarchy observed in fMRI. Fusing the results from EEG and fMRI studies (see caveats in the Methods section) thus suggests that Bayesian causal inference in multisensory perception relies on dynamic encoding of multiple spatial estimates across the cortical hierarchy. During early processing, multisensory perception is dominated by full-segregation models associated with activity in low-level sensory areas. Later audiovisual interactions that are governed by forced-fusion principles rely on posterior parietal areas. Finally, Bayesian causal inference estimates are formed in anterior parietal areas. Yet, although our results suggest that full segregation, forced fusion, and Bayesian causal inference dominate EEG activity patterns at different latencies, they do not imply a strictly feed-forward architecture. Instead, we propose that the brain concurrently accumulates evidence about the different spatial estimates and the underlying causal structure (i. e., common versus independent sources) most likely via multiple feedback loops across the cortical hierarchy [18,19]. Only after 350 ms is a final perceptual estimate formed in anterior parietal cortices that takes into account the uncertainty about the world’s causal structure and combines audiovisual signals into spatial priority maps as predicted by Bayesian causal inference. Sixteen right-handed participants participated in the experiment; three of those participants did not complete the entire experiment: two participants were excluded based on eye tracking results from the first day (the inclusion criterion was less than 10% of trials rejected because of eye blinks or saccades; see the Eye movement recording and analysis section for details), and one participant withdrew from the experiment. The remaining 13 participants (7 females, mean age = 22. 1 years; SD = 3. 0) completed the 3-day experiment and are thus included in the analysis. All participants had no history of neurological or psychiatric illnesses, had normal or corrected-to-normal vision, and had normal hearing. All participants gave informed written consent to participate in the experiment. The study was approved by the research ethics committee of the University of Birmingham (approval number: ERN_11_0470AP4) and was conducted in accordance with the principles outlined in the Declaration of Helsinki. The visual (‘V’) stimulus was a cloud of 20 white dots (diameter = 0. 43° visual angle, stimulus duration: 50 ms) sampled from a bivariate Gaussian distribution with vertical standard deviation of 2° and horizontal standard deviation of 2° or 12° visual angle presented on a dark grey background (67% contrast). Participants were told that the 20 dots were generated by one underlying source in the centre of the cloud. The visual cloud of dots was presented at one of four possible locations along the azimuth (i. e., −10°, −3. 3°, 3. 3°, or 10°). The auditory (‘A’) stimulus was a 50-ms-long burst of white noise with a 5-ms on/off ramp. Each auditory stimulus was delivered at a 75-dB sound pressure level through one of four pairs of two vertically aligned loudspeakers placed above and below the monitor at four positions along the azimuth (i. e., −10°, −3. 3°, 3. 3°, or 10°). The volumes of the 2 × 4 speakers were carefully calibrated across and within each pair to ensure that participants perceived the sounds as emanating from the horizontal midline of the monitor. In a spatial ventriloquist paradigm, participants were presented with synchronous, spatially congruent or disparate visual and auditory signals (Fig 1A and 1B). On each trial, visual and auditory locations were independently sampled from four possible locations along the azimuth (i. e., −10°, −3. 3°, 3. 3°, or 10°), leading to four levels of spatial disparity (i. e., 0°, 6. 6°, 13. 3°, or 20°; i. e., as indicated by the greyscale in Fig 1A). In addition, we manipulated the reliability of the visual signal by setting the horizontal standard deviation of the Gaussian cloud to a 2° (high reliability) or 14° (low reliability) visual angle. In an intersensory selective-attention paradigm, participants reported either their auditory or visual perceived signal location and ignored signals in the other modality. For the visual modality, they were asked to determine the location of the centre of the visual cloud of dots. Hence, the 4 × 4 × 2 × 2 factorial design manipulated (1) the location of the visual stimulus (−10°, −3. 3°, 3. 3°, 10°; i. e., the mean of the Gaussian), (2) the location of the auditory stimulus (−10°, −3. 3°, 3. 3°, 10°), (3) the reliability of the visual signal (2°, 14°; SD of the Gaussian), and (4) task relevance (auditory-/visual-selective report), resulting in 64 conditions (Fig 1A). To characterise the computational principles of multisensory integration, we reorganised these conditions into a 2 (visual reliability: high versus low) × 2 (task relevance: auditory versus visual report) × 2 (spatial disparity: ≤6. 6° versus >6. 6°) factorial design for the statistical analysis of the behavioural and EEG data. In addition, we included 4 (locations: −10°, −3. 3°, 3. 3°, or 10°) × 2 (visual reliability: high, low) unisensory visual conditions and 4 (locations: −10°, −3. 3°, 3. 3°, or 10°) unisensory auditory conditions. We did not manipulate auditory reliability, because the reliability of auditory spatial information is anyhow limited. Furthermore, the manipulation of visual reliability is sufficient to determine reliability-weighted integration as a computational principle and arbitrate between the different multisensory integration models (see Bayesian modelling analysis section). On each trial, synchronous audiovisual, unisensory visual, or unisensory auditory signals were presented for 50 ms, followed by a response cue 1,000 ms after stimulus onset (Fig 1B). The response was cued by a central pure tone (1,000 Hz) and a blue colour change of the fixation cross presented in synchrony for 100 ms. Participants were instructed to withhold their response and avoid blinking until the presentation of the cue. They fixated on a central cross throughout the entire experiment. The next stimulus was presented after a variable response interval of 2. 6–3. 1 s. Stimuli and conditions were presented in a pseudo-randomised fashion. The stimulus type (bisensory versus unisensory) and task relevance (auditory versus visual) was held constant within a run of 128 trials. This yielded four run types: audiovisual with auditory report, audiovisual with visual report, auditory with auditory report, and visual with visual report. The task relevance of the sensory modality in a given run was displayed to the participant at the beginning of the run. Furthermore, across runs we counterbalanced the response hand (i. e., left versus right hand) to partly dissociate spatial processing from motor responses. The order of the runs was counterbalanced across participants. All conditions within a run were presented an equal number of times. Each participant completed 60 runs, leading to 7,680 trials in total (3,840 auditory and 3,840 visual localisation tasks—i. e., 96 trials for each of the 76 conditions were included in total; apart from the four unisensory auditory conditions that included 192 trials). The runs were performed across 3 days with 20 runs per day. Each day was started with a brief practice run. Stimuli were presented using Psychtoolbox version 3. 0. 11 [69] (http: //psychtoolbox. org/) under MATLAB R2014a (MathWorks) on a desktop PC running Windows 7. Visual stimuli were presented via a gamma-corrected 30” LCD monitor with a resolution of 2,560 × 1,600 pixels at a frame rate of 60 Hz. Auditory stimuli were presented at a sampling rate of 44. 1 kHz via eight external speakers (Multimedia) and an ASUS Xonar DSX sound card. Exact audiovisual onset timing was confirmed by recording visual and auditory signals concurrently with a photodiode and a microphone. Participants rested their head on a chin rest at a distance of 475 mm from the monitor and at a height that matched participants’ ears to the horizontal midline of the monitor. Participants responded by pressing one of four response buttons on a USB keypad with their index, middle, ring, and little finger, respectively. To address potential concerns that results were confounded by eye movements, we recorded participants’ eye movements. Eye recordings were calibrated in the recommended field of view (32° horizontally and 24° vertically) for the EyeLink 1000 Plus system with the desktop mount at a sampling rate of 2,000 Hz. Eye position data were on-line parsed into events (saccade, fixation, eye blink) using the EyeLink 1000 Plus software. The ‘cognitive configuration’ was used for saccade detection (velocity threshold = 30°/sec, acceleration threshold = 8,000°/sec2, motion threshold = 0. 15°) with an additional criterion of radial amplitude larger than 1°. Individual trials were rejected if saccades or eye blinks were detected from −100 to 700 ms post stimulus. Participants’ stimulus localisation accuracy was assessed as the Pearson correlation between their location responses and the true signal source location separately for unisensory auditory, visual high reliability, and visual low reliability conditions. To confirm whether localisation accuracy in vision exceeded performance in audition in both visual reliabilities, we performed Monte-Carlo permutation tests. Specifically, we entered the subject-specific Fisher z-transformed Pearson correlation differences between vision and audition (i. e., visual–auditory) separately for the two visual reliability levels into a Monte-Carlo permutation test at the group level based on the one-sample t statistic with 5,000 permutations [70]. Continuous EEG signals were recorded from 64 channels using Ag/AgCl active electrodes arranged in a 10–20 layout (ActiCap, Brain Products GmbH, Gilching, Germany) at a sampling rate of 1,000 Hz, referenced at FCz. Channel impedances were kept below 10 kΩ. Preprocessing was performed with the FieldTrip toolbox [71] (http: //www. fieldtriptoolbox. org/). For the decoding analysis, raw data were high-pass filtered at 0. 1 Hz, re-referenced to average reference, and low-pass filtered at 120 Hz. Trials were extracted with a 100-ms prestimulus and 700-ms poststimulus period and baseline corrected by subtracting the average value of the interval between −100 and 0 ms from the time course. Trials were then temporally smoothed with a 20-ms moving window and downsampled to 200 Hz (note that a 20-ms moving average is comparable to a finite impulse response [FIR] filter with a cutoff frequency of 50 Hz). Trials containing artefacts were rejected based on visual inspection. Furthermore, trials were rejected if (1) they included eye blinks, (2) they included saccades, (3) the distance between eye fixation and the central fixation cross exceeded 2°, (4) participants responded prior to the response cue, or (5) there was no response. For ERPs (S2 and S3 Figs), the preprocessing was identical to the decoding analysis, except that a 45-Hz low-pass filter was applied without additional temporal smoothing with a temporal moving window. Grand average ERPs were computed by averaging all trials for each condition first within each participant and then across participants. For the multivariate pattern analyses, we computed ERPs by averaging over sets of eight randomly assigned individual trials from the same condition. To characterise the temporal dynamics of the spatial representations, we trained linear SVR models (LIBSVM [72], https: //www. csie. ntu. edu. tw/~cjlin/libsvm/) to learn the mapping from ERP activity patterns of the (1) unisensory auditory (for auditory decoding), (2) unisensory visual (for visual decoding), or (3) audiovisual congruent conditions (for audiovisual decoding) to external spatial locations separately for each time point (every 5 ms) over the course of the trial (S2, S3 and S4 Figs). All SVR models were trained and evaluated in a 12-fold-stratified cross-validation (12 ERPs/fold) procedure with default hyperparameters (C = 1, ε = 0. 001). The specific training and generalisation procedures were adjusted to the scientific questions (see the Shared and distinct neural representations of space across vision and audition section and the GLM analysis of audiovisual weight index wAV section for details). Combining psychophysics, computational modelling, and EEG, we addressed two questions: First, focusing selectively on the unisensory auditory and unisensory visual conditions, we investigated when spatial representations are formed that generalise across auditory and visual modalities. Second, focusing on the audiovisual conditions, we investigated when and how human observers integrate audiovisual signals into spatial representations that take into account the observer’s uncertainty about the world’s causal structure consistent with Bayesian causal inference. In the following sections, we will describe the analysis approaches to address these two questions in turn. First, we investigated how the brain forms spatial representations in either audition or vision using the so-called temporal generalisation method [21]. Here, the SVR model is trained at time point t to learn the mapping from, e. g., unisensory visual (or auditory) ERP pattern to external stimulus location. This learnt mapping is then used to predict spatial locations from unisensory visual (or auditory) ERP activity patterns across all other time points. Training and generalisation were applied separately to unisensory auditory and visual ERPs. To match the number of trials for auditory and visual conditions, we applied this analysis to the visual ERPs pooled over the two levels of visual reliability. The decoding accuracy as quantified by the Pearson correlation coefficient between the true and decoded stimulus locations is entered into a training time × generalisation time matrix. The generalisation ability across time illustrates the similarity of EEG activity patterns relevant for encoding features (i. e., here: spatial location) and has been proposed to assess the stability of neural representations [21]. In other words, if stimulus location is encoded in EEG activity patterns that are stable (or shared) across time, then an SVR model trained at time point t will be able to correctly decode stimulus location from EEG activity patterns at other time points. By contrast, if stimulus location is represented by transient or distinct EEG activity patterns across time, then an SVR model trained at time point t will not be able to decode stimulus location from EEG activity patterns at other time points. Hence, entering Pearson correlation coefficients as a measure for decoding accuracy for all combinations of training and test time into a temporal generalisation matrix has been argued to provide insights into the stability of neural representations whereby the spread of significant decoding accuracy to off-diagonal elements of the matrix indicates temporal generalisability or stability [21]. Second, to examine whether and when neural representations are formed that are shared across vision and audition, we generalised to ERP activity patterns across time not only from the same sensory modality but also from the other sensory modality (i. e., from vision to audition and vice versa). This cross-sensory generalisation reveals neural representations that are shared across sensory modalities. To assess whether decoding accuracies were better than chance, we entered the subject-specific matrices of the Fisher z-transformed Pearson correlation coefficients into a between-subjects Monte-Carlo permutation test using the one-sample t statistic with 5,000 permutations ([70], as implemented in the FieldTrip toolbox). To correct for multiple comparisons within the two-dimensional (i. e., time × time) data, cluster-level inference was used based on the maximum of the summed t values within each cluster (‘maxsum’) with a cluster-defining threshold of p < 0. 05, and a two-tailed p-value was computed. To characterise how human observers integrate auditory and visual signals into spatial representations at the behavioural and neural levels, we developed a GLM-based analysis of an audiovisual weight index wAV and a Bayesian modelling analysis that we applied to both (1) the reported auditory and visual spatial estimates (i. e., participants’ behavioural localisation responses) and (2) the neural spatial estimates decoded from EEG activity pattern evoked by audiovisual stimuli (see Fig 3 and [14,16]).

The ability to tell whether various sensory signals come from the same or different sources is essential for forming a coherent percept of the environment. For example, when crossing a busy road at dusk, seeing and hearing an approaching car helps us estimate its location better, but only if its visual image is associated-correctly-with its sound and not with the sound of a different car far away. This is the so-called binding problem, and numerous studies have demonstrated that humans solve this near-optimally as predicted by Bayesian causal inference; however, the underlying neural mechanisms remain unclear. We combined Bayesian modelling, electroencephalography (EEG), and multivariate decoding in an audiovisual spatial localisation task to show that the brain dynamically encodes multiple spatial estimates while accomplishing Bayesian causal inference. First, auditory and visual signal locations are estimated independently; next, information from vision and audition is combined. Finally, from 200 ms onwards, the brain weights audiovisual signals by their sensory reliabilities and task relevance to guide behavioural responses as predicted by Bayesian causal inference.

lay_plos

How epigenetic information is propagated during somatic cell divisions is still unclear but is absolutely critical for preserving gene expression patterns and cellular identity. Here we show an unanticipated mechanism for inheritance of DNA methylation patterns where the epigenetic mark not only recruits the catalyzing enzyme but also regulates the protein level, i. e. the enzymatic product (5-methylcytosine) determines the level of the methylase, thus forming a novel homeostatic inheritance system. Nucleosomes containing methylated DNA stabilize de novo DNA methyltransferases, DNMT3A/3B, allowing little free DNMT3A/3B enzymes to exist in the nucleus. Stabilization of DNMT3A/3B on nucleosomes in methylated regions further promotes propagation of DNA methylation. However, reduction of cellular DNA methylation levels creating more potential CpG substrates counter-intuitively results in a dramatic decrease of DNMT3A/3B proteins due to diminished nucleosome binding and subsequent degradation of the unstable free proteins. These data show an unexpected self-regulatory inheritance mechanism that not only ensures somatic propagation of methylated states by DNMT1 and DNMT3A/3B enzymes but also prevents aberrant de novo methylation by causing degradation of free DNMT3A/3B enzymes. DNA methylation is a stable gene silencing mechanism required for key biological processes including embryogenesis, genomic imprinting, X-chromosome inactivation, repression of transposons and maintenance of tissue specific gene expression patterns [1], [2]. Aberrant methylation contributes to tumorigenesis and other diseases [3], [4]. Thus, proper maintenance of DNA methylation patterns is essential for preserving cellular identity and preventing malignant cellular transformation. In mammals, DNA methylation patterns are generally thought to be established during embryonic development by de novo DNA methyltransferases 3A and 3B [5] and then stably maintained through multiple somatic divisions by the ‘maintenance activity’ of DNMT1 both during and after replication [6]. However, recent studies suggest that DNMT1 alone cannot ensure proper maintenance of methylation patterns [7] and requires co-operative activity of the de novo DNMT3A/3B enzymes [8], [9], [10], which are ubiquitously expressed in somatic cells. A revised model of inheritance was recently proposed assigning DNMT3A/3B to a maintenance role in somatic cells [11]; however, questions still remain regarding the molecular mechanisms guiding the maintenance activity of these de novo enzymes. In embryonic stem (ES) cells, DNMT3A/3B establish methylation patterns in association with DNMT3L, a regulatory factor which stimulates DNMT3A/3B de novo activity [12] and targets them to nucleosomes containing unmethylated H3K4 residues [13]. Methylated H3K4 containing chromatin regions remain refractory to such DNA methylation [14], [15]. Further, heterochromatin protein 1 (HP1) recruits DNMT3A/3B to H3K9me3 residues, established by histone methyltransferase (HMTase) Suv39h1/2, enabling de novo DNA methylation in pericentric heterochromatin [16]. In euchromatic regions, G9a, another H3K9 HMTase, recruits DNMT3A/3B for de novo methylation of early embryonic gene promoters [17]. UHRF1, which assists DNMT1 in locating to hemimethylated sites [18], also targets DNMT3A/3B for de novo methylation in ES cells [19]. However, DNMT3L is expressed only during gametogenesis and embryonic stages and not in somatic tissues [20], [21]. Further, we and others have recently shown that HP1 and UHRF1 are not required for DNMT3A/3B' s association with nucleosomes [22] and G9a does not affect maintenance of DNA methylation in somatic cells [23], [24]. Thus, other mechanisms must exist to ensure proper localization of these enzymes to silent chromatin regions in somatic cells [25], enabling faithful maintenance of methylated states. We and others have previously shown that the majority of DNMT3A/3B within a somatic cell are strongly anchored to nucleosomes containing methylated DNA with little free DNMT3A/3B proteins existing [22], [26]. Here we show that the presence of such methylated regions is essential for DNMT3A/3B' s association with chromatin and quite unexpectedly, also for maintaining the cellular levels of these enzymes. Reduction in DNA methylation levels results in reduced DNMT3A/3B binding to nucleosomes accompanied by selective degradation of the free enzymes by the cellular machinery. Restoration of DNA methylation increases DNMT3A/3B protein levels through their stabilization on nucleosomes. Further, pre-existing methylation stimulates propagation of DNA methylation in vivo by stably anchoring DNMT3A/3B to nucleosomes. DNMT3A/3B work synergistically to propagate methylation patterns with DNMT3B stimulating DNMT3A activity by promoting its association with nucleosomes, similar to DNMT3L. Taken together, these data suggest an inheritance model where DNMT3A/3B remain localized to silent methylated domains by binding to nucleosomes containing methylated DNA, enabling faithful maintenance of methylated states in cooperation with DNMT1; while non-anchored DNMT3A/3B enzymes get selectively degraded preventing spurious de novo methylation. In somatic cells, DNMT3A/3B remain bound to nucleosomes containing methylated DNA [22]. To investigate the role of DNA methylation in this binding, we used a series of HCT116 colon cancer cells with homozygous deletions for DNMT1 (DNMT1ΔE2-5; 1KO) [27], [28], DNMT3B (DNMT3B−/−; 3BKO) or both DNMT1 and DNMT3B (DNMT1ΔE2-5/DNMT3B−/−; double knockout, DKO) and consequently different levels of genomic DNA methylation [7]. For the DKO cells, which still contain residual DNMT1 activity [28], we used two clones for our analysis, DKO1 and DKO8, having lost ∼95% and ∼50% DNA methylation respectively [7]. RT-PCR analysis of DNMT3A, DNMT3B and DNMT1 transcript levels in the various HCT116 derivative cell lines showed similar or higher levels of DNMT3A1 transcripts in HCT116 knockout cell lines compared to WT HCT116; reduced levels of DNMT1ΔE2-5 hypomorph transcripts in 1KO and the two DKO clones, with relatively higher expression in the DKO8 clone and no detectable levels of DNMT3B transcripts in 3BKO and both DKO cell lines, consistent with previous data [7], [28] (Figure 1A). Next we examined DNMT protein levels in these cell lines through immunoblotting of nuclear extracts. Similar to mRNA analysis, DNMT3B and DNMT1 protein levels were severely reduced in the respective knockout cell lines (Figure 1B). Surprisingly, while DNMT3A mRNA levels were higher in both DKO clones, we found dramatically reduced DNMT3A protein in them compared to WT HCT116 cells. Similar reductions in DNMT3A protein levels were observed in whole cell lysates of both DKO cells, suggesting that the reduced nuclear levels are not the result of protein mislocalization (Figure S1). These findings were further confirmed by immunofluorescence analyses of HCT116 and DKO cells which displayed similar reduction in DNMT3A protein levels in DKO cells as observed in western blots of their nuclear extracts. Moreover, the residual DNMT3A protein displayed similar nuclear distribution in DKO cells as in WT HCT116 cells, confirming that its reduced nuclear levels in the DKO cells are not due to protein mislocalization (Figure S2). G9a, another chromatin-modifying protein, did not display such large changes in protein levels in HCT116 knockout cell lines (Figure 1B). Assessment of global DNA methylation levels using methylation-sensitive restriction enzymes revealed a direct correlation between the amount of DNMT3A protein and level of methylation retained in the knockout cells, suggesting a possible role of DNA methylation in maintaining cellular DNMT3A levels (Figure 1B, 1C). DKO8 cells, which had retained higher DNA methylation levels, showed higher DNMT3A protein compared to the minimal amount present in the severely hypomethylated DKO1 cells. Since no such decrease in DNMT3A protein was observed in the single DNMT1 and DNMT3B knockout cells (1KO and 3BKO respectively), which retained substantial levels of DNA methylation, maintenance of DNMT3A levels through possible protein-protein interactions with DNMT1 and/or DNMT3B seems unlikely. We have previously shown that DNMT3A/3B strongly associate with methylated chromatin regions [22]. To determine whether the residual DNMT3A protein in hypomethylated DKO cells retains similar affinity for chromatin as in WT HCT116 cells, we performed a salt extraction experiment as described previously [22]. Purified nuclei from HCT116 and DKO1 cells were incubated in buffers with increasing concentrations (50 mM to 400 mM) of NaCl. Nuclear pellet and supernatant fractions were independently analyzed through western blot analysis. As expected, similar amounts of core histones remained inside the extracted nuclei under all salt concentrations. In HCT116 cells, the DNMT3A protein level remained almost constant within the nuclei up to 400 mM NaCl indicating a strong binding affinity for chromatin (Figure 2A), whereas other chromatin associated proteins such as EZH2 and G9a showed relatively weaker binding affinities with substantial amounts detected in the supernatant at more than 200 mM NaCl concentrations. Interestingly, the majority of DNMT3A protein present in DKO1 cells, though greatly reduced in comparison to WT HCT116, also remained tightly associated with the chromatin at all salt concentrations (Figure 2A), possibly binding to the few methylated regions remaining in the DKO1 cells. Minimal DNMT3A protein could be detected in the supernatant fractions (50 to 300 mM NaCl) of the DKO1 cells. These data suggest that binding to methylated chromatin regions might be essential for maintaining the stability of DNMT3A protein and that any free protein unable to bind to chromatin in the absence of DNA methylation possibly gets rapidly degraded by the cellular machinery. We did observe some DNMT3A protein dissociating from the chromatin at 400 mM NaCl in DKO1 cells but not in HCT116 cells suggesting a reduction in chromatin binding affinity of DNMT3A in hypomethylated DKO1 cells compared to heavily methylated WT HCT116 cells (Figure 2A). Meanwhile, EZH2 and G9a showed weaker binding to chromatin in DKO1 cells, similar to that observed in WT HCT116. Taken together, these data suggest that binding to methylated chromatin regions may be critical for stabilization of DNMT3A protein. To assess whether the dramatic transcription-independent decrease in steady-state levels of DNMT3A protein observed in hypomethylated DKO cells was due to altered protein stability, we treated WT HCT116 and DKO8 cells with the protein synthesis inhibitor cycloheximide (CHX) [29] and measured the DNMT3A protein remaining at different time points after treatment. DNMT3A was stable in WT HCT116 cells with 93% still remaining after 6 hrs of CHX treatment (Figure 2B). However, in DKO8 cells, DNMT3A was very unstable with its level rapidly decreasing to 49% 2 hrs after treatment. The half-life of DNMT3A protein decreased dramatically from 16 hrs in WT HCT116 to 7 hrs in DKO8 cells (Figure S3). Interestingly, after a rapid initial decrease in DNMT3A protein level in DKO8 cells within the first 2 hrs of CHX treatment, a fraction of DNMT3A protein remained stable thereafter till the 8 hr time point (Figure 2B). This fraction may possibly represent the stable DNMT3A protein bound to the methylated chromatin regions in DKO8 cells, similar to that observed in DKO1 cells (Figure 2A). Taken together, these data indicate that a decrease in DNA methylation results in destabilization of DNMT3A protein, possibly due to reduced chromatin binding in the absence of methylated DNA regions, the main sites of DNMT3A/3B binding [22]. To ascertain if depletion of DNA methylation is primarily responsible for the decrease in DNMT3A protein, we sought to restore DNA methylation in the DKO cells. We expressed Myc-tagged DNMT3B1, ΔDNMT3B2 [30] or DNMT3L in DKO1 and DKO8 cells using a lentiviral system and confirmed expression of the relevant proteins by immunoblotting (Figure 3A). We did not use DNMT1 for the restoration of DNA methylation in DKO cells since expression of exogenous DNMT1 in DKO cells has previously been shown to result only in partial increase in DNA methylation [31]. More importantly, DNMT1 expression failed to restore methylation in these cells at the repetitive elements, the key sites of DNMT3A/3B binding [31]. Global DNA methylation levels in infected DKO cells were measured 8 weeks post-infection using methylation-sensitive restriction enzymes. Since DKO cells possess very low levels of a hypomorph of DNMT1 [28], the primary maintenance methyltransferase in the cell, very low levels of DNMT3A protein and no DNMT3B protein, it required a long time (∼8 weeks) to achieve restoration of DNA methylation in these cells. After 8 weeks of infection, we observed increased DNA methylation in both DKO cell lines infected with DNMT constructs compared to empty vector (E/V) controls (Figure 3B). Even though there was equivalent mRNA expression of exogenous DNMT enzymes in the two DKO clones (Figure S4), DKO8 cells, with higher baseline methylation levels, showed a greater increase in methylation compared to hypomethylated DKO1 cells for each individual construct. Moreover, the increase in methylation in the infected DKO cells was preferentially localized to loci having low-levels of pre-existing methylation and minimal de novo methylation of previously unmodified sites could be observed (De Carvalho D. and Sharma S. et. al., unpublished observations), indicating that DKO cells possess similar patterns of chromatin states as present in the parental WT HCT116 cells, including histone modifications (such as H3K4me3 and H2A. Z etc.) which are involved in guiding DNA methylation to specific genomic loci [6]. These results also indicate a stimulatory effect of pre-existing methylation [32] on DNA methylation by DNMTs in vivo, possibly through stabilization of de novo DNMT3A/3B enzymes on methylated nucleosomes as suggested by their higher protein levels in DKO8 cells (Figure 1B, Figure 3A). This process may further be enhanced by the higher levels of DNMT1 hypomorph present in DKO8 cells [33] (Figure 1B). Within each DKO clone, exogenous DNMT3L expressing cells showed the most robust increase in methylation followed by DNMT3B1 and ΔDNMT3B2 expressing cells respectively, re-emphasizing the strong stimulatory effect of DNMT3L on DNMT3A/3B activity observed in ES cells [12]. These methylation data were further confirmed through Illumina Infinium analysis [34] for each infected cell line (data not shown). Interestingly, immunoblotting of nuclear extracts revealed a substantial transcription-independent increase in DNMT3A protein level in all DNMT infected DKO cell lines (Figure 3C, Figure S5). Moreover, the increase in DNMT3A correlated with the increase in global DNA methylation levels (Figure 3C, 3B). Considering that DNMT3A primarily associates with methylated chromatin regions [22], these data suggest that presence of such methylated regions is required for maintaining its protein level in somatic cells. To examine whether the increase in DNMT3A protein observed upon restoration of DNA methylation is mediated by binding to nucleosomes, we used sucrose density gradient analysis which allows for the study of in vivo interactions between the chromatin modification enzymes and their actual nucleosomal substrates in the native state [22]. Mononucleosomal digests prepared by extensive micrococcal nuclease (MNase) digestion of nuclei from infected DKO8 cells, expressing either E/V, Myc-tagged DNMT3B1, ΔDNMT3B2 or DNMT3L, were subjected to fractionation on sucrose gradients containing 300 mM NaCl. Western blot analysis showed similar nucleosomal profile in all gradients with mononucleosomes forming a peak at fraction 6 (Figure 4). The DNMT fusion proteins displayed distinct sedimentation profiles indicating different nucleosome binding affinities. DNMT3B1 associated strongly with nucleosomes while the truncated ΔDNMT3B2 variant showed weak association with nucleosomes with a substantial amount of ΔDNMT3B2 sedimenting in nucleosome-free fractions indicating an essential role of the N-terminal region in strong nucleosomal binding, consistent with previous data [22]. However, analysis of various other truncated DNMT3B1 proteins, which contained the N-terminal region but lacked other protein regions (such as the catalytic, PHD and/or PWWP domains), revealed weak nucleosome binding for all truncated proteins (data not shown). These data suggest that DNMT3B requires a full-length protein structure and synergistic activity of its various domains for achieving strong nucleosome binding. DNMT3L showed a bimodal distribution having both nucleosome-free and nucleosome-bound protein (fractions 1–4 and 5–16 respectively). Strikingly, the increased DNMT3A protein in all of the infected cell lines remained strongly associated with nucleosomes similar to that in E/V control, independent of the nucleosome binding affinities of the exogenous proteins, suggesting a nucleosome anchorage dependent stabilization of the protein. DNMT3A formed a peak at fraction 7 in DNMT3B1 and ΔDNMT3B2 expressing cells. In DNMT3L expressing cells, the peak was shifted to fraction 9, indicating the formation of heavier DNMT3A-DNMT3L tetramer encasing the nucleosome [35]. It might also be possible that DNMT3A-DNMT3L tetramer bound nucleosomal regions may be more resistant to MNase digestion and be responsible for this shift. We did not observe any DNMT3A in the nucleosome-free fractions (1–4) co-sedimenting with the unbound pool of ΔDNMT3B2 or DNMT3L fusion proteins (Figure 4), suggesting that the increase in DNMT3A is not due to stabilization through protein-protein interactions with the exogenous proteins but is actually mediated by its binding to nucleosomes upon increase in methylation. Taken together, these data suggest that DNMT3A protein is stabilized by binding to nucleosomes containing its own product (i. e. methylated DNA), which is essential for maintaining its cellular levels. DNMT3B, like DNMT3A, also compartmentalizes to methylated regions in somatic cells via strong anchoring to nucleosomes containing methylated DNA [22], [26]. To examine whether DNMT3B also binds to nucleosomes in a DNA methylation dependent manner, we expressed Myc-tagged DNMT3B1 in three DNMT3B-knockout HCT116 cell lines, 3BKO, DKO8 and DKO1, which possess 86%, 27% and 6% of total genomic DNA methylation respectively (Figure 1C). We first tested mRNA and protein expression of the exogenous DNMT3B1 in these cell lines. Interestingly, while DNMT3B1 mRNA levels were similar in all infected cell lines, we found dramatically reduced DNMT3B1 protein, similar to DNMT3A, in severely hypomethylated DKO1 cells in comparison to 3BKO and DKO8 cells (Figure 5A, 5B). To assess whether the decrease in DNMT3B1 resulted from a reduction in binding affinity for nucleosomes in hypomethylated cells, we tested its distribution in mononucleosomal digests fractionated on 300 mM NaCl containing sucrose gradients. In 3BKO and DKO8 cells, the exogeneous DNMT3B1 showed strong association with nucleosomes similar to endogeneous DNMT3A (Figure 5C, Figure 4). However, DNMT3B1 weakly associated with nucleosomes in severely hypomethylated DKO1 cells with the bulk of the overexpressed protein sedimenting in nucleosome-free fractions (2–4), suggesting a dramatic reduction in nucleosome binding affinity upon depletion of DNA methylation. Since the increase in methylation in the infected DKO cells was preferentially localized to the same loci which were originally methylated in the parental HCT116 cells, it suggests that DKO cells possess similar patterns of histone modifications involved in guiding DNA methylation as present in WT HCT116 cells (De Carvalho D. and Sharma S. et. al., unpublished observations). Therefore, taken together, these data strongly suggest that the reduction in nucleosome binding affinity of DNMT3A/3B observed in DKO cells results from depletion of DNA methylation and is not due to clonal variation of global chromatin states. To further confirm this phenomenon, we subjected Myc-tagged DNMT3B1 expressing DKO1 cells to CHX treatment and analyzed protein stability of the nucleosome-bound and -free fractions of DNMT3B1 protein. Consistent with our previous data on endogenous DNMT3A enzyme, the overexpressed free DNMT3B1 protein underwent rapid degradation compared to the stable nucleosome-bound DNMT3B1 protein, clearly displaying the instability of the unbound protein (Figure 5D). Such degradation was inhibited by treatment with the proteosome inhibitor MG132, indicating the role of proteosomal pathway in this process. However, we could not rescue degradation of DNMT3A protein in DKO cells using MG132 treatment suggesting possible involvement of other mechanisms in its degradation (data not shown). The phenomenon of destabilization and selective degradation of unbound DNMT3A/3B proteins could also be observed in the case of Myc-ΔDNMT3B2 which showed substantially lower protein levels compared to Myc-DNMT3B1 in DKO8 cells even when both genes were expressed at similar mRNA levels (Figure 3A, Figure S4). Since Myc-ΔDNMT3B2 associated weakly with nucleosomes while Myc-DNMT3B1 bound strongly to nucleosomes in DKO8 cells (Figure 4), the reduction in Myc-ΔDNMT3B2 levels possibly results from a decrease in protein stability of the unbound Myc-ΔDNMT3B2 protein, similar to that previously observed for DNMT3A and 3B in DKO1 cells. Taken together, these data show that both DNMT3A/3B require the presence of DNA methylation for tight binding to nucleosomes and subsequent protein stabilization. Such a mechanism would enable faithful inheritance of methylated states through proper compartmentalization of DNMT3A/3B while preventing spurious de novo methylation through selective degradation of the free enzymes. In ES cells, DNMT3A/3B strongly interact and mutually stimulate each other' s activity, thus working synergistically to establish genomic DNA methylation patterns during development [36]. To ascertain whether a similar mechanism is involved in propagation of DNA methylation in somatic cells, we expressed a Myc-tagged catalytically-inactive DNMT3B1 mutant, having a cysteine to serine alteration (position 657) which destroys catalytic activity without compromising other functions [37], in DKO8 cells and confirmed its protein expression by immunoblotting (Figure 6A). To determine whether the DNMT3B1 mutant could stimulate DNMT3A activity, we measured the global DNA methylation level in the mutant expressing cells 8 weeks post-infection. We observed a substantial increase in methylation, demonstrating a stimulatory effect of DNMT3B on DNMT3A activity, independent of catalytic activity (Figure 6B). Immunoprecipitation experiments showed that the mutant DNMT3B1 strongly interacted with DNMT3A, similar to WT DNMT3B1, suggesting a DNMT3L-like stimulation mechanism which occurs through physical interaction of the two proteins [12] (Figure S6). Along with an increase in DNA methylation, we observed a substantial increase in endogenous DNMT3A protein levels in mutant DNMT3B1 expressing cells (Figure 6A), similar to WT DNMT3B1 expressing cells, suggesting DNA methylation induced stabilization of DNMT3A protein. Similar results were obtained upon expression of a catalytically-inactive ΔDNMT3B2 mutant in DKO8 cells indicating a stimulatory effect of ΔDNMT3B2 on DNMT3A activity, occurring through its physical interaction with the DNMT3A protein as shown by immunoprecipitation experiments (Figures S7, S6). In ES cells, stimulation of DNMT3A/3B activity by DNMT3L partially occurs through increased association of the enzymes with the substrate DNA, allowing these slow acting enzymes to efficiently methylate the substrate [38]. To examine whether stimulation of DNMT3A by DNMT3B in somatic cells occurs through a similar mechanism in a nucleosomal context, we analyzed mononucleosomal digests from DNMT3B1 mutant expressing cells on 300 mM sucrose density gradients. All cellular DNMT3A in infected 3BKO and DKO8 cell lines was found tightly anchored to nucleosomes suggesting that its stimulation by DNMT3B1 occurs through an increased binding to nucleosomes (Figure 6C). We could not detect DNMT3A in DKO1 cells in this assay due to its extremely low levels. In DKO8 cells expressing the mutant ΔDNMT3B2, all cellular DNMT3A protein was found strongly anchored to nucleosomes indicating that the interaction and stimulation of DNMT3A by ΔDNMT3B2 is mediated by their binding to nucleosomes (Figure S7). The DNMT3B1 and ΔDNMT3B2 mutants displayed similar binding affinity for nucleosomes as their WT counterparts suggesting that their catalytic activity has little role in nucleosome binding. Taken together, these data show that in vivo stimulation of DNMT3A by DNMT3B occurs through an increased binding to nucleosomes, similar to that observed with DNMT3L, enabling efficient methylation from these slow acting de novo enzymes and their consequent stabilization through continued association with such methylated regions. Proper maintenance of epigenetic modifications within specific chromatin domains is critical for preserving cellular identity. Recently, a common theme for inheritance of histone marks has emerged where the mark recruits and retains its own modifying enzyme and triggers renewal by stimulating that enzyme through possible allosteric activation mechanisms [39], [40], [41]. Our work suggests involvement of a similar mechanism in maintenance of DNA methylation patterns through DNMT3A/3B in somatic cells. We and others have previously shown that DNMT3A/3B, but not DNMT1, are strongly anchored to nucleosomes containing methylated DNA in somatic cells [22], [26]. Our current data shows that the presence of DNA methylation is essential for association of DNMT3A/3B with chromatin and also for maintaining the cellular levels of the DNMT3A/3B enzymes, thereby creating a homeostatic inheritance system. Such methylation directed binding stimulates DNA methylation at target loci in vivo ensuring faithful maintenance of methylation patterns, a phenomenon previously observed in inheritance of the polycomb mark [42]. Since DNMT3A/3B are slow acting enzymes compared to DNMT1 [38], stable association with their target methylated regions would be key for their ability to properly maintain methylated states. We further show that DNMT3A/3B work synergistically in this maintenance process and DNMT3B stimulates DNMT3A activity through increased association with nucleosomes, similar to DNMT3L. Thus, promotion of DNA methylation by selective binding of DNMT3A/3B to nucleosomes containing pre-existing methylation may serve as a critical positive feed-back loop mechanism essential for faithful propagation of epigenetic states through somatic cell divisions [25], [43], [44]. Another key finding of our work is the selective degradation of free DNMT3A/3B proteins which could not bind to chromatin in the absence of pre-existing DNA methylation in somatic cells. In ES cells and PGCs (primordial germ cells), DNMT3A/3B are required for establishment of global DNA methylation patterns. Therefore, in these cells, DNMT3A/3B are highly expressed at the transcriptional level and their methylation activity is strongly stimulated by DNMT3L [12], [45]. However, in somatic cells, the main role of the de novo DNMT3A/3B enzymes is to assist DNMT1 in proper maintenance of pre-established DNA methylation patterns and prevention of de novo methylation of previously unmethylated regions is required [11], [45]. Therefore, DNMT3A/3B mRNA expression is substantially downregulated and DNMT3L is not expressed in differentiated somatic tissues in order to prevent any aberrant de novo methylation [20], [45]. Our data suggest that to further regulate this maintenance process, DNMT3A/3B protein levels are post-translationally regulated by the levels of pre-existing DNA methylation in somatic cells. Selective degradation of free DNMT3A/3B enzymes may help explain how somatic cells, which still express low levels of de novo DNMT3A/3B enzymes, prevent aberrant de novo methylation of CpG islands. Our data suggests that once DNMT3A/3B are recruited to methylated chromatin domains, pre-existing methylation stabilizes their binding to such regions and enables faithful propagation of methylated states. However, in absence of DNA methylation, as would be the case with unmethylated CpG islands, these slow acting enzymes are unable to stably bind to the chromatin. The resulting free de novo enzymes, which could potentially cause spurious methylation, are then selectively degraded by the cellular machinery possibly through recognition of an altered conformation in the unbound state (Figure 7). As shown in the model, in hypomethylated DKO (DNMT1ΔE2-5/DNMT3B−/−) cells, DNMT3A loses its ability to bind to nucleosomes resulting in destabilization and selective degradation of free DNMT3A protein while the residual DNMT3A remains bound to the remaining few methylated chromatin regions (Figure 7). Since exogenous Myc-DNMT3B1 also displayed a similar DNA methylation-dependent stabilization upon nucleosomes, it suggests that a similar model might apply to the regulation of DNMT3B enzyme in somatic cells. Our data indicates that unbound DNMT3B1 is degraded through the proteosomal pathway but how DNMT3A is selectively targeted for degradation in somatic cells is still unclear. Future studies are required to further understand the exact mechanisms involved in the selective degradation of unbound DNMT3A/3B enzymes. Histone methyltransferases, however, are not regulated in such a manner and have been found to exist in both free and chromatin-bound forms within nuclei. This difference can be partially explained by the fact that histone marks are far more dynamic in nature, actively regulated by the combined action of histone methyltransferases and demethylases [46], compared to DNA methylation which is still believed to be a relatively stable mark in differentiated tissues [6]. While initial recruitment of DNMT3A/3B to methylated regions may involve other proteins, our data strongly suggests that their anchoring to chromatin primarily depends upon pre-existing DNA methylation. However, in addition to DNA methylation, certain histone modifications and accessory proteins may also help in selective compartmentalization of these enzymes. For instance, unmethylated H3K4, recently shown to bind DNMT3A [47], may assist in stable binding to silent domains. Recruitment of DNMT3A/3B to such domains may involve UHRF1 [19]. On the other hand, proteins like H2A. Z, CTCF and H3K4me3 etc. which are antagonistic to DNA methylation [14], [48], [49], may occlude binding of DNMT3A/3B to active/poised regions, thus constraining their activities to silent methylated domains only. Recently, Witcher and Emerson [50] have shown that loss of such boundary elements indeed results in aberrant spreading of DNA methylation beyond methylated domains. Our data suggests that these aberrations may involve DNMT3A/3B enzymes which remain bound to methylated regions [22]. During tumorigenesis, these de novo enzymes may progressively override the chromatin boundaries, gradually spreading methylation beyond their specific domains to the entire region [51] resulting in aberrant methylation of genes in clusters – a common feature of cancer-specific hypermethylation [52], [53]. Such a mechanism may also help explain why CpG island loci having pre-existing methylation in a normal tissue are more susceptible to undergo de novo methylation in cancer [54]. Moreover, ectopic de novo methylation, correlated with overexpression of DNMT3A/3B in several types of cancer [3], may also be maintained and propagated through continued association of DNMT3A/3B with such regions. DNA methylation inhibitors like 5-aza-CdR, widely used to inhibit aberrant methylation in cancer, target DNMTs by trapping them on DNA [55]. Since these hypomethylating drugs trap DNMTs onto the DNA, it is not feasible to use them for studying the dissociation of DNMT3A/3B from nucleosomes and destabilization upon loss in DNA methylation observed in our experiments. Nevertheless, our data suggests that destabilization of DNMT3A/3B upon removal of DNA methylation may provide another mechanism for depletion of these enzymes upon treatment with such hypomethylating drugs. However, future studies are required to further understand these mechanisms, focusing on factors determining proper compartmentalization of DNMT3A/3B to methylated regions and mechanisms responsible for selective degradation of the unbound protein. In conclusion, our data suggests a model for epigenetic inheritance of DNA methylation in somatic tissues where pre-existing methylation triggers its renewal by recruiting and stabilizing DNMT3A/3B on methylated chromatin domains, which then work synergistically to propagate DNA methylation in co-operation with DNMT1. Such a mechanism not only ensures faithful maintenance of methylated states but also guards against aberrant methylation from the de novo DNMT3A/3B enzymes. HCT116 derivative cell lines were maintained in McCoy' s 5A medium containing 10% inactivated fetal bovine serum, 100 units/ml penicillin and 100 µg/ml streptomycin. Puromycin was included in the culture medium at 3 µg/ml to maintain infected HCT116 derivative cell lines. When indicated, cycloheximide (Sigma) was added to a final concentration of 50 µg/ml. The proteosome inhibitor MG132 (Calbiochem) was used at 10 µM for 2 h prior to CHX treatment. Detailed methods are described in Text S1. Human 3B1, ΔDNMT3B2 and DNMT3L cDNA sequences having the Myc tag DNA sequence ligated to their 5′ ends were amplified from the pIRESpuro/Myc constructs [22] (a modified version of the pIRESpuro3 vector, Clontech), a generous gift from Allen Yang (USC), using polymerase chain reaction (PCR). Myc-tagged catalytically-inactive mutants of DNMT3B1 and ΔDNMT3B2, having a cysteine to serine alteration in the catalytic domain corresponding to position 657 of DNMT3B1 protein, were prepared using a site-directed mutagenesis kit (Stratagene). The mutation was confirmed by sequencing both strands of the constructs. For preparation of the constructs, the lentivirus vector pLJM1 was linearized using AgeI and EcoRI restriction enzymes and the Myc tagged DNMT cDNAs were cloned in it using In-fusion advantage PCR cloning kit (Clontech) following manufacturer' s protocol. For lentivirus production, the vesicular stomatitis virus envelope protein G expression construct pMD. G1, the packaging vector pCMV ΔR8. 91 and the transfer vector pLJM1 were used as described previously [56]. Infected HCT116 derivative cells, stably expressing various DNMTs, were selected in the presence of 3 µg/ml puromycin for three weeks. Detailed methods are described in Text S1. Nuclei from 5×106 cells were incubated in 500 µl of ice-cold RSB containing 0. 25 M sucrose, protease inhibitors and various concentrations of NaCl for 5 min at 4°C. Nuclei were then harvested by microcentrifugation, separating the supernatant and the pellet fractions. Nuclear pellets were resuspended in RIPA buffer and subjected to sonication. Proteins in the supernatant were concentrated using TCA precipitation and later resuspended in RIPA buffer. Equivalent volumes of supernatant and pellet fractions were added to SDS loading buffer and subjected to Western blotting. MNase digestion and sucrose gradients experiments were performed as described previously [22]. For details, see Text S1. Genomic DNA (10 µg) isolated from various HCT116 derivative cell lines was digested with methylation-sensitive restriction enzymes, HpaII or MspI (New England Biolabs), at 37°C over night. The digested DNA was run on an agarose gel at low voltage for 8 hrs in order to achieve good separation. The undigested DNA band in each lane was then quantified using the ImageQuant software. Percentage of genomic methylation present was calculated using the formula: where H = undigested with HpaII; M = undigested with MspI and G = genomic DNA.

Proper inheritance of DNA methylation patterns is essential for preserving cellular identity and preventing malignant cellular transformation. In mammals, DNMT3A/3B, the de novo methyltransferases, establish the DNA methylation patterns during development and then maintain them in co-operation with the maintenance methyltransferase, DNMT1, through cell divisions. However, the mechanisms by which DNMT3A/3B assist DNMT1 in faithful inheritance of methylation patterns in somatic cells while guarding against aberrant de novo DNA methylation are still unclear. In this study, we present a novel principle of enzyme regulation where the levels of the catalyzing enzymes, DNMT3A/3B, are determined by the level of their own enzymatic product, i. e. 5-methylcytosine itself. Through biochemical analyses, we have shown that binding of DNMT3A/3B to nucleosomes with methylated DNA stabilizes these proteins, enabling faithful propagation of methylation patterns through cell divisions. However, reduction in DNA methylation results in diminished nucleosome binding of DNMT3A/3B and subsequent degradation of the free DNMT3A/3B proteins. This novel self-regulatory inheritance mechanism not only ensures faithful somatic propagation of methylated states but also prevents aberrant de novo methylation by causing degradation of free DNMT3A/3B enzymes.

lay_plos

Summarize the discussion about the conclusion of experiments and questionnaire for user requirement. Project Manager: {vocalsound} So um nice to see you again. Uh. So, uh. Tod uh for this meeting I will take the notes and do the minutes. Uh so we will see our three presentations. Um we will start with the uh Manager Expert wi who will talk about uh user re requirements, whats user needs and what it desire for this devi device. Marketing: Okay, can I have the laptop over here, or? Project Manager: Yep. Oh, I don't think so. I think you have to come here. Marketing: Okay. Have to get up. {vocalsound} Project Manager: I dunno. I think it should stay. Marketing: Excuse me. Industrial Designer: Yeah, that's it. Project Manager: {gap} Marketing: {vocalsound} Okay. Project Manager: Should stay in the square here. {vocalsound} Marketing: Okay. Project Manager: Oh, maybe. Marketing: Okay, so basically I'm gonna present some findings of a study we conducted uh into uh what users want in this remote control. Project Manager: Oh, you can put it here. Oh that's okay, it's jus Marketing: Um so first of all we {disfmarker} what we did is we um conducted a an experiment with a hundred test subjects. Um we put them in a in our um usability laboratory and got them to, you know, um play with remote controls, and also to complete, after they'd done that, to complete a questionnaire uh to tell us what they like and what they don't like in remote controls. {vocalsound} {vocalsound} So basically um the major things we found out was that um basically users don't like the look and feel of of most remote controls that are currently on the market. Um they, you know, seventy five percent of the people we we uh did the experiments on, found that rem remote the remote controls that they'd used in the past were ugly. Completely ugly. Um they they didn't match the operating behaviour of the user, that is, you know, the the way users use remote controls when they're watching T_V_. Um, that the layout of the remote controls didn't match they way that they used it. Um and thirdly they say that w half of the users that we um tested said that they only used ten percent of the buttons that uh are on remote controls. Um so we collect we also some collected some usage statistics uh based on how these test subjects were using their remote control. And from this we basically came up with the figure that the channel buttons, the channel selection buttons are the most {disfmarker} by far the most used buttons on the remote control. Um and you can see they're used a hundred and sixty eight times per hour on average, um, while the user's watching T_V_. Industrial Designer: {vocalsound} Project Manager: {vocalsound} Marketing: Um the closest button that was used, well the cl the button that was used that was closest to the channel button was the teletext button, um which was used fourteen times per hour, followed by the volume button, which was four times per hour, um, all the other, all the other um buttons, such as ch um audio and picture selection um configuration buttons and things were used, you know, l approx well less than or equal to one times per hour. Um we also asked users um which buttons had the most importance to them, you know, which which buttons they felt were the most important buttons on on the remote control. And basically they came {disfmarker} they said the channel, volume, and power buttons had the highest relevance to users, um note that only power was very infrequently used, it only had a b a a fr usage frequency of about one times per hour, but users ranked it as having a very very high relevance. Um and the audio and picture settings had a very {disfmarker} that well the users thought that um w the audio and picture settings were very uh weren't very important to them um, and they used them very infrequently a as well. {vocalsound} So we asked users what what um frustrates them the most about um current remote controls. And fifty percent of the users said that uh what frustrates them is losing the remote control um somewhere in the room and not being able to find it. Um {vocalsound} they also said that it um it takes a lot of time to learn a new remote control, especially when there's many buttons and it's a, you know, a c a a unintuitive interface. Um and then thirdly, they {disfmarker} some users commented on the fact that the the you know the way that you have to hold and press buttons on a remote control ar are bad and um cause u repetitive strain injury. {vocalsound} We also asked some users about some specific features that they'd like to see in the {disfmarker} on the remote control. In particular, do they want an L_C_D_ d display, and secondly, do they think speech recognition uh is a useful feature to have on a remote control. Project Manager: {vocalsound} Marketing: Um basically our findings are that um amongst a younger age groups uh the answer is umv overwhelmingly yes. They want these features, they want these high technology features. Industrial Designer: {vocalsound} Marketing: Um for instance, ninety one percent of pe of people aged between fifteen and twenty five said yes, they want these features. Whereas um {vocalsound} the the trend was as users as users um became older and older uh they were less likely to want these sort of features in a um in a remote control. So I guess it depends on where we're focusing our our market. Um and as our company motto is putting fashion in electronics, um I think we're focusing on the younger younger um target demographic, and so maybe we should think about adding these sort of high technology features into our remote control. Project Manager: Hmm. Marketing: That's my dic that's my presentation. Thank you. Project Manager: Thank you. Industrial Designer: {vocalsound} User Interface: Okay. Project Manager: So, um maybe now we can talk about a user interface and uh about the technical function of this device. So uh Pet Peter, can you talk say something about that? Industrial Designer: Well, okay, yeah. Yeah, but the user user interface is responsible. Project Manager: No. User Interface: B you think uh I I'm User Interface Manager. Project Manager: Ah. Industrial Designer: Okay, so {gap}. Marketing: {vocalsound} Project Manager: {vocalsound} Sorry, I'm {disfmarker} Sorry. Sorry. User Interface: {vocalsound} Okay. Okay. Project Manager: {gap} User Interface: If I could go there with this cable. Industrial Designer: You're scaring me with L_C_D_ man. And speech recognition in remote unit, it will be very e expensive. Marketing: {vocalsound} Yeah, it's true, but, you know, they're features that users want, Industrial Designer: {vocalsound} Marketing: so it's {disfmarker} Industrial Designer: Yeah. Marketing: And our production cost of twelve fifty Euros per per unit is fairly high I think, User Interface: {gap} Industrial Designer: Yeah. Marketing: so I think we can afford to to add these sort of features into our remote. Industrial Designer: At least we have couple of months t to work on it so so it will be cheaper finally. {vocalsound} User Interface: {vocalsound} Sh okay. {gap} Marketing: It is true. User Interface: Where's delete button? Okay. Oh I'm sorry. {vocalsound} {gap} Industrial Designer: {vocalsound} Project Manager: {gap} User Interface: {gap} Okay. Marketing: That's the wrong one, I think. Project Manager: Yeah, it's still Bob Morris. User Interface: Oh. Presentation three? Project Manager: {vocalsound} Yeah you should have put yes. Industrial Designer: Because you cancelled it. Yeah. Project Manager: Click on yes. User Interface: Oh. {vocalsound} Project Manager: {gap} yeah. Industrial Designer: Yep. User Interface: Okay, so here is my presentation about technical function design. I will talk about different components and s of system and how they react together. Okay, uh, first what is a remote control? Simply it's a device, as you know, for uh, for sending some commands by some waves to uh another device to to tell different commands uh with this device. And the main idea we don't use any cable and we can rec react simpler with the device. Uh i it has different blocks, different blocks. Uh first is remote control have sh ha should have some electric circuits uh making interface with uh keyboard and uh r reading uh keyboard and reading the keyboard uh commands. And then inter then make uh these keyboard commands, uh in interpret these int uh keyboard commands. And then there should be uh an electronic circuit making uh electronic signals according to these commands and uh finally there is a transmitter which is a cord or a a diode making uh making uh waves to transmit through the air and uh uh this air this uh wave uh will be received by the by the other device like a television or whatever, to uh r to realise the command. Okay, {vocalsound} uh about {disfmarker} what I found about uh different uh these different blocks are uh, usually there are two different methods uh to {disfmarker} for designing a remote control. They are based on infra-red uh waves or uh uh radio waves. Industrial Designer: You still want me the presentation. User Interface: There are two different uh uh solutions I mean. This is uh this can be uh uh the the differences the th between different kind of waves, infra-red or radio waves. And uh also as uh I understood, and uh I think it was a part of uh Bob, uh uh presentation, people prefer to have uh to have uh the remote control with less button. So for the electronic part, working and interfacing, with button, we should we should try to t try to design a remote control having uh some some simpler buttons or some rolling buttons to just to just to search between different options, and showing o something on T_V_ and putting less lesser stuff on the uh on the remote control. And uh personal preferences, uh uh uh uh certainly a remote control with {disfmarker} uh working with radio waves is uh preferred because uh you can pr you can take it in any direction and you don't need to tune it any way. And uh uh again, using bigger buttons and less number of buttons are also preferred, as I see. Okay. That was my presentation. Marketing: Okay. I have a question. User Interface: Uh-huh. Marketing: Do you think radio waves um will interfere with other appliances in the home? User Interface: Uh, I don't think so, because uh we can make uh we ca we can make this wave in a specific frequency. So they can be in a range which is not inter interfering with the {vocalsound} with other devices inside the home. Marketing: Okay. Project Manager: So can we use any any frequency? Industrial Designer: Yeah, it should be okay {gap}. Project Manager: We have the right to use any frequency? User Interface: Uh no but as I know, there is a range for uh for this uh f for for uh for this stuff, for designing this circuit. We can we can we can tune our uh transmitter to work in this range, and for this range we don't need to ask any permission. Project Manager: Okay. And what happen with uh radio waves when two neighbours have the same {disfmarker} have our remote control, for example? And so do they have the same frequency, or? User Interface: Uh for this I'm not uh I I don't know the solution, but one solution can be something like putting uh p password or something inside the wave, so the only your T_V_ can understand it Project Manager: Okay. A kind of identification {gap}, User Interface: Yeah, identification code inside the {disfmarker} Industrial Designer: Yeah f {vocalsound} uh I know about this, since it's my {disfmarker} it's exactly my field, so. Project Manager: okay. So {disfmarker} Industrial Designer: It's uh kind of handshaking, uh, when starting to {disfmarker} uh when you start to communicate with the your T_V_ then then it's like an handshaking protocol with your your remote. Project Manager: Yeah. So {disfmarker} Industrial Designer: So so if two two devices are trying to go to communicate with the T_V_ set then the the the one which has the more energy in the wave is chosen. Well it can be a problem sometimes, but most of the time it works okay. Project Manager: Okay. User Interface: The password may simply {disfmarker} uh or uh i identification code may simply solve this problem. A specific uh remote control has a specific f Industrial Designer: {vocalsound} Yeah but we we don't have to think uh about this because I think as a function designer that we will use the already made uh circuits which we probably bu buy. It's worth to buy. Project Manager: Hmm. Industrial Designer: And they have these problems solved so. So we don't have to think about these. User Interface: Mm. Marketing: Okay. Project Manager: Okay. User Interface: Mm-hmm. Project Manager: So, maybe you can talk about the function, and {disfmarker} Marketing: Okay. Industrial Designer: Yes. I have only a couple of things because I had {disfmarker} I struggled a bit with the software that I'm supposed to use in this uh uh in this company. I was used to use Linux before, so. But I tried to tried to break through this {gap} too, I guess. Project Manager: Open. Industrial Designer: Mm. Ah. User Interface: {vocalsound} Industrial Designer: Okay. {vocalsound} How to make it big? Project Manager: Slide show. User Interface: Five. {gap} Industrial Designer: Slide show. Okay, thanks. {vocalsound} Project Manager: It should work, so you can {gap}. Industrial Designer: Okay. Oh so I will speak about working design. That's the first slide. Uh what uh I have to do? A look at what the other company {disfmarker} Okay so uh presently I am looking what is possible to use, what circuits to use and stuff like that because uh I didn't work uh with these uh circuits so far so I have to look what what is a v a v available on the market for for the communication {disfmarker} for the I_R_ circuits and so on, so I'm currently looking what is available on the web. And uh I wanted to ask you m maybe afterwards, after after our discussion, if we have some contacts in some companies, so, which can report on what is going on there, so, I would be glad if you can tell me about them. Project Manager: Mm. Yeah. Industrial Designer: So, you know. Uh, okay findings, that's {gap} the point that I'm working on currently but uh so far I I was uh looking what what are the blue circuit, I mean radio wave {disfmarker} radio frequency circuits are available now, Marketing: Mm-hmm. Industrial Designer: but the prices I read are high. So, I know that uh the user interface people and these speak about radio frequency waves because you can you can uh you can make the T_V_ do what you want even if you are in the bathroom or so on, User Interface: Mm-hmm. Project Manager: Hmm. Industrial Designer: but you know, when you are not close to the T_V_ you probably won't need to to change the program and so on and so I am I am voting for s to stick to the um infra-red control instead of R_W_ but we will discuss it later maybe. Uh. Components to use, I'm not exactly sure what I will use for the design of the circuit. It depends uh on whether we will use the L_C_D_ and mainly the speech recognition, User Interface: {vocalsound} Industrial Designer: because the speech rec Yeah? Marketing: Are we um are we planning to take an off the shelf an O_E_M_ component for the radio wave circuit, or are we planning to construct our own circuit board? Industrial Designer: No no no no no. This we this we buy I think, because it's rather cheap nowadays and it's not worth to construct ourselves. Marketing: Okay s So we just buy a circuit board and {vocalsound} Industrial Designer: Exactly. Marketing: Okay. Industrial Designer: But I'm not sure about the circuit which is responsible for speech recognition. This I prefer that we should make ourselves. Marketing: Okay. Industrial Designer: But it depends whether we take a decision to use it. Same thing. It's fairly expensive to use these circuits. So, speech recognition {disfmarker} well, L_C_D_ it's okay because it's common nowadays to use L_C_D_, Project Manager: {vocalsound} Industrial Designer: so I agree on using any kind of L_C_D_, less buttons good for me as a as a designer of the circuit. Project Manager: Yeah. Industrial Designer: But the speech recognition we have to compare whether the price and the {disfmarker} what does it offer, you know. User Interface: Mm. Project Manager: Yeah. So what do you think would be the price, it would be out of range? Industrial Designer: Yeah, I'm {disfmarker} Project Manager: Or it would be maybe feasible? Industrial Designer: Oh. I was not thinking too much about the price. But if we use the L_C_D_ uh even the radio frequency communication with the T_V_ set and the backlight and uh related things like the photo diodes and stuff, it should be okay. If we decide to use the speech recognition, then we probably could struggle but we'll see afterwards. Project Manager: Okay. Industrial Designer: It depends of you if if the M_E_ or U_I_D_, Project Manager: Mm. Industrial Designer: I'm sorry about the names, I don't really know, uh want to have it like in metal or in plastic, these things, it it depends on you not {disfmarker} Because the the the electronic device's price is not not so big in comparison to to the overall shape and stuff like that, so. Marketing: Okay. Industrial Designer: We will discuss it afterwards. Oh, this is nothing. This is just my notes on what to use. And uh my personal preference is yes, I would like also preferably to use R_W_ circuit, but from the point of view uh of the design and price, I would stick to I_R_s. That's my opinion. Uh I mean if infra-red uh circuit not not the radio frequency. Project Manager: Why? Because it's simpler? Industrial Designer: Because because the the range where you can use it is fair. Project Manager: Yeah. Mm. Industrial Designer: It's okay I think. And the price is fairly cheap for this. Project Manager: Okay. It's a a price matter. Industrial Designer: Well, depends. Project Manager: Yeah. Industrial Designer: Jus just the price. Project Manager: Mm. Industrial Designer: Otherwise I don't care what I put there because it's the chip which I buy or which we buy. Project Manager: Okay. Industrial Designer: So I I I think it's o y o {vocalsound}. Marketing: What how much more expensive? Are we talking three times more expensive? Project Manager: {gap} Industrial Designer: Well, three to three to five. Marketing: Or ten times more expensive? Or {disfmarker} Industrial Designer: N not ten times, Marketing: Okay. Industrial Designer: but it depends what what we {disfmarker} Marketing: Yeah yeah. That's still a lot. I think it's it's probably not worth spending the extra money, Project Manager: Hmm. Yeah. Marketing: because I mean all the other remote controls on the market have infra-red, so people don't expect anything other than infra-red. Industrial Designer: {gap} Project Manager: Yeah. Industrial Designer: Yeah. Marketing: So it's not worth spending the extra money. Project Manager: Well I {gap}, oh {disfmarker} Industrial Designer: Th they are used to use it when they can see the T_V_ so, I don't know. Project Manager: On the other side, we want to have something new. Marketing: Yeah. Project Manager: You know, where Marketing: Yeah. Project Manager: we want to to have something new and So we I think we should still thinking about it. But maybe {gap}. Marketing: But I think, based on my usability studies I th I feel that users are {disfmarker} prioritise the look and the feel and the trendiness above, you know, the difference between infra-red or radio waves. Project Manager: Okay. Marketing: So I think we're better off spending money in the usability phase. Industrial Designer: {gap} You the user interface, and management man, uh {vocalsound} {disfmarker} Project Manager: Okay. Yeah. User Interface: Mm. Marketing: Okay. Industrial Designer: Uh okay, that's it for me. Project Manager: Okay, thank you Peter. Industrial Designer:'Kay. Project Manager: So um I have to inform you I receive an email from the management bon board today and they have new requirements for the for the remote control. Um Marketing: Mm. Project Manager: first um, they say that's uh about something about t teletext. Uh apparently it becomes from {disfmarker} according to them it becomes out of date. Out-dated. And uh {disfmarker} Because of the internet popularity and everybody has internet at home, and actually it's not useful to have teletext. Um. So I think we can avoid the teletext. Um the second thing is uh they suggest that that we should uh use the remote control only for T_V_, not for D_V_D_ and other devices, because it make it {disfmarker} it makes it's too complex and uh because we have not much time for this project we should stay on T_V_ on the {disfmarker} only specific T_V_ remote control. Industrial Designer: I agree. {vocalsound} Project Manager: The third {vocalsound} the third one is uh about the the the image of the company. So um uh we should we should keep uh {disfmarker} The the product should be recognisable. Uh, uh It's {disfmarker} That mean we should use the colour of our company and maybe put somewhere the s the slogan of the company, which is uh, we put fashion of {disfmarker} in electronics. So, when people see the the remote control, they should say oh it's from Real Reaction and he s they should recognise the company. So, um {disfmarker} So now we we should take the decision what we are going to {disfmarker} what function we will have on this uh on this remote control. So, mm, are we going to use L_C_D_, speech recognition? Uh. Marketing: Well, should we start with just the core, the basic functions that we need. Project Manager: Yeah. Marketing: And then we can move on to the more advanced features. User Interface: Mm. Yes. Project Manager: Maybe, maybe. Industrial Designer: Okay, so the available things are L_C_D_, the buttons and everything. Uh radio frequency depends. And, well the recognition it depends on you guys. Project Manager: Yeah but first maybe what is {disfmarker} what are the usual function of a standard remote control? Industrial Designer: You should probably speak. Project Manager: I mean Industrial Designer: Okay. Project Manager: what do {disfmarker} Marketing: Okay, well, I mean the obvious one is changing channels. {vocalsound} Project Manager: Yeah. Yeah. {vocalsound} User Interface: Yeah. {vocalsound} Project Manager: I think we should stick on very useful functions, because we want less button. Marketing: Yeah. Project Manager: So, yeah. Marketing: Yeah, okay. Project Manager: So, turning channel, of course. Volume setting. Marketing: {gap} Industrial Designer: Uh just one note to the chan channel changing. Do we {disfmarker} will we use only two buttons, or or like numbered buttons? I mean those nine plus one or two? Project Manager: Yeah. User Interface: Mm-hmm. Project Manager: I think it would be a b {gap} Industrial Designer: Because it's many buttons and we were speaking about lowering the number. Project Manager: Yeah. User Interface: Mm. {gap} Project Manager: On the other side we have more and more channels, and if you want to pass through all the channels to get the channel you want, it's {disfmarker} Marketing: Yeah, I {disfmarker} User Interface: Yeah. Industrial Designer: Okay so so we keep all these all these buttons. Project Manager: Maybe maybe we could think of something more betweens, like User Interface: Yeah yeah, at least nine, ten button. Project Manager: uh {disfmarker} Industrial Designer: Like ten plus, five plus, one plus, one minus or something. Project Manager: Yeah. Maybe something like that. Industrial Designer: Or using the names and the keyboard {vocalsound} Marketing: {gap} Ah yeah. {vocalsound} Project Manager: {vocalsound} Uh. Industrial Designer: I dunno. User Interface: Or {disfmarker} Project Manager: Maybe {disfmarker} User Interface: Or something {gap}. Project Manager: Oh I don't know. Oh. Maybe we could have key buttons, like uh discovery channe like documentary channel, and movies channel and {disfmarker} Industrial Designer: You mean like hierarchical structure. Project Manager: And inside this this thing you can move, maybe switch. User Interface: Mm-hmm. Okay. Like categorising channels. Project Manager: If you want to see a movie you click on the movie button so you have all the movie channels and after you've you plus plus plus. Industrial Designer: Okay, so s User Interface: Mm-hmm. Industrial Designer: Oh sorry. Project Manager: I It just an idea. User Interface: Okay. Project Manager: I don't know what you think about that but. Industrial Designer: So it requires the use of L_C_D_ probably, to to tell you {disfmarker} Project Manager: Yeah, probably, yeah, yeah. Marketing: To have some feedback. Yeah. Industrial Designer: Okay, User Interface: Mm-hmm. Industrial Designer: so I'll make a note on L_C_D_. Marketing: We could maybe also c um incorporate Petre's idea of the um slider for the volume, with the channel. User Interface: Okay. Project Manager: Okay. Marketing: So we c you could quickly just {vocalsound} {vocalsound} through many channels. Industrial Designer: Yeah. Like roller for the {disfmarker} Yeah. Okay. Marketing: {vocalsound} For the channels, perhaps. Project Manager: {vocalsound} Anything else? Marketing: So we've got channel and volume. Um. Project Manager: Yeah. Industrial Designer: So we are still s speaking about the common devices User Interface: {gap} Industrial Designer: or we are inviting the new one? Marketing: I think so. Project Manager: Yeah. Marketing: I think basically the core functions we want, and then more advanced ones. Project Manager: What about the settings of the T_V_? Because it's button we don't use very often, but it's {disfmarker} we need it anyway. So {disfmarker} User Interface: So uh we don't have any uh we don't have uh {disfmarker} Marketing: Yeah. User Interface: we should just design the remote control and we sh we don't have any access to the to the T_V_ design or we can change some design. Because one solution for this um uh uh ch changing channels is to see a summary of all channels, some some preview of all channels and then you can {disfmarker} Industrial Designer: On the screen, you mean? User Interface: Yeah. Yeah b {vocalsound} Industrial Designer: Not on the control, but on the screen. User Interface: Not on the control, on the screen, on the T_V_ screen. Industrial Designer: Well, this would avoid L_C_D_, then. User Interface: And then {disfmarker} Marketing: I don't Oh, I don't but I don't think we're we're the ones. I mean this remote control we're developing is a generic control for all T_V_s, I think. Project Manager: Yeah, yeah. I don't know if it's possible to to watch something on T_V_ {disfmarker} User Interface: Mm-hmm. Industrial Designer: Oh. W I I think it it would be better to to stick to the remote control and not to bother the T_V_ to to to print these things. Project Manager: Yeah. Yeah. User Interface: Mm-hmm. Industrial Designer: Well it wi it will be still more expensive, but for the L_C_D_ and this stuff is no problem in the price. User Interface: {gap} Mm-hmm. Marketing: Okay. Project Manager: So, what are we doing with the settings? Because settings {disfmarker} if we want to do settings we need buttons for that and we want less buttons, so. Maybe with the L_C_D_ we can do something with less buttons, but {disfmarker} User Interface: Yeah. Marketing: But then you don't want to make the L_C_D_ display too complicated at the same time. Project Manager: Mm. {vocalsound} Industrial Designer: {vocalsound} Two T_V_s. Marketing: {vocalsound} I mean there's always {disfmarker} we can always have these l less often used functions hidden somewhere, under a cover or at the back of {disfmarker} under a slide or some Project Manager: Yeah. Oh yeah. Industrial Designer: Like ma Project Manager: Oh, the {gap}. Industrial Designer: We we could have for example two buttons like simple mode and advanced mode. User Interface: {vocalsound} Project Manager: {vocalsound} Industrial Designer: {vocalsound} Or I dunno. User Interface: {gap} {vocalsound} Marketing: Yeah. Industrial Designer: Or like children and grandfather's mode, Marketing: {vocalsound} Yeah. Project Manager: {vocalsound} Industrial Designer: and the, well the the user User Interface: Mm. Project Manager: {vocalsound} Industrial Designer: not the user, the man mana T_V_ manager mode {vocalsound}. Ah, I dunno. User Interface: Mm-hmm. Marketing: Okay. So we have five minutes left. Um. Project Manager: Yeah. Industrial Designer: Uh. Marketing: So I think s the settings we th we are agreeing {disfmarker} are agreed that they're required. Project Manager: Yeah. Industrial Designer: Yeah. User Interface: Mm-hmm. Marketing: It's just how to a Project Manager: {vocalsound} We should hide them somewhere. Industrial Designer: Hide them, okay. Project Manager: In the menus of the L_C_D_ or in the back of the remote control, or something like that. Marketing: Yeah. Okay. Yeah. Uh, okay, what else? Industrial Designer: Um. {gap} Yes. Marketing: I mean a power button's obviously Industrial Designer: {vocalsound} This I was thinking. Do we need a power button at all? Marketing: uh required. Industrial Designer: Should should shouldn't we do it like sleep mode after five minutes of not using it? Because generally {disfmarker} Project Manager: It's it's a kind of setting, I think. It should fit in those settings functions. User Interface: Mm. Settings. Project Manager: Because it's not a very current useful function. Marketing: Uh, well I think when they say power button they mean to turn the T_V_ on and off. Project Manager: No, I think it's after after five minutes or something {gap} a timer {disfmarker} Industrial Designer: Yes. Well I I uh Project Manager: I I think, no? Industrial Designer: {vocalsound} Marketing: But if you're watching T_V_ for two hours, you don't want your T_V_ to turn off after five five minute {vocalsound} Industrial Designer: {vocalsound} Project Manager: {vocalsound} Industrial Designer: You don't need to {disfmarker} every five minutes to keep it alive. User Interface: {vocalsound} {vocalsound} Marketing: {vocalsound} Industrial Designer: Uh probably. Okay, so we should keep this button. User Interface: Yeah. Marketing: I mean based on our usability studies again, um {vocalsound} pe um people said that the power button was v a very relevant button. Project Manager: Oh okay, yeah. User Interface: Mm-hmm. Marketing: Um, you know, it was nine out of ten Industrial Designer: Okay, so we we could p what we could probably do is to keep also to keep uh keep the L_C_D_ and all the buttons and stuff, Marketing: re relevance. Industrial Designer: we could make it like a opening opening style that if you open it you are {disfmarker} you just turn on the T_V_ and if you close it, it will Project Manager: Yeah. Yeah. Industrial Designer: turn off the T_V_. Marketing: {gap} Industrial Designer: If if you like this, {vocalsound} Marketing: Okay. B Okay. Industrial Designer: Because, well {gap} {disfmarker} it's maybe question for you t Marketing: I think we need to concentrate on the, you know, the major usage of the th of the control, User Interface: Mm-hmm. Project Manager: Yeah. Marketing: which is you sit down, you turn on your T_V_, you change channels, you change the volume, you turn the T_V_ off. Project Manager: Yeah. So s yeah. Marketing: Um and all the other f functionality is Industrial Designer: {vocalsound} User Interface: Yeah, they can yeah they can be hide somewhere by a cover or something like this. Marketing: not used very often. Project Manager: Yeah. Marketing: Yeah. User Interface: Like covering cu. Project Manager: On the back, or {disfmarker} Industrial Designer: Yeah. I mean like the {disfmarker} User Interface: Yeah, like mobile phone covering. Industrial Designer: Yeah but since we have the L_C_D_, we didn't need too much button too many buttons. Um, okay, just um the decision of the power button. User Interface: {vocalsound} Industrial Designer: Should we make it a button, or some some something which would be {disfmarker} Project Manager: For what? Industrial Designer: Uh power button. Marketing: I think a button. User Interface: A button is better. Project Manager: Ah oh yeah, yeah. Marketing: I think it should be a bu Industrial Designer: If it if it's a button or {disfmarker} User Interface: Yeah. Project Manager: Yeah a button, yeah I guess so. Industrial Designer: Okay. Marketing: Its own button on the front. User Interface: Mm. Industrial Designer: Okay, one nice big button. Marketing: {vocalsound} Industrial Designer: Old fashioned button, to satisfy the grandmothers. Project Manager: {vocalsound} User Interface: {vocalsound} Project Manager: {vocalsound} User Interface: {vocalsound} Marketing: {vocalsound} Industrial Designer: Hmm. Marketing: Okay. Project Manager: So, any other suggestions or functions? Industrial Designer: Mm. Marketing: S What about things like the clock and um timers? Industrial Designer: Do we still have the time? I I just wonder. Project Manager: Yeah, we have still one or two minutes to talk, yeah. Industrial Designer: Okay. Oh. Well what w what was the question? User Interface: Clock or {disfmarker} Marketing: Uh um, you know, some func some features on the control to display a time, or t to display {disfmarker} Project Manager: Usually it's already on T_V_ or something like that. User Interface: Yes. Industrial Designer: Yeah but since we want to control all the televisions, and and it would probably be worth to to set the timing on the remote, Project Manager: Yeah. Industrial Designer: no? Because if if the T_V_ turns on itself, it well you know, if the time The timer should be there. User Interface: Mm. Project Manager: Yeah. Yeah. If we if we add the time, we have to have maybe a bigger display or something like that, and is it very useful? I mean, are users wants to have the time on the on the remote? User Interface: Mm. Project Manager: This is the question. Marketing: Probably not. Project Manager: Is it useful? User Interface: Yes. Marketing: It's a questi yeah, it's a trade-off. Project Manager: W In my opinion we should have only useful thing and Because apparently they want {disfmarker} The simpler it's is better. Marketing:'Kay. Industrial Designer: Ah, yeah. Marketing: Okay simple. Industrial Designer: Did you did you ever use the like the timing of turning on the T_V_, User Interface: {vocalsound} Project Manager: {vocalsound} Industrial Designer: well {disfmarker} And based on your {disfmarker} User Interface: {vocalsound} Marketing: Very, yeah okay, very occasionally. Industrial Designer: {vocalsound} Marketing: But I do use the I do use the display of the time quite often, but {disfmarker} User Interface: Mm. Industrial Designer: Yeah but it can be on the display in in a corner all the time on the remote. Marketing: Yeah. Project Manager: {vocalsound} Marketing: Yeah that's okay, that's true. Okay, so no time button {gap}. Okay. Project Manager: No time on {gap}. Industrial Designer: {gap} User Interface: And uh do we need to include anything about the speech recognition and the speech commands, or? Industrial Designer: Yes yes. Project Manager: I think {disfmarker} User Interface: I I thin I think it will not take lots of place, we just need a microphone and the software, so, and it sh and it will be a little bit new and interesting, so people may be attracted to buy this stuff and it's not very difficult to uh put a software inside the the electronic device and put a microphone. It doesn't take that much place and also that much {disfmarker} It doesn't cost that much. Project Manager: Yeah. Industrial Designer: Yeah but we we should be careful about the battery life, then. If we use the speech recognition. Project Manager: {vocalsound} So maybe we think {disfmarker} we can think more about that, and discuss that maybe last time {disfmarker} next time. User Interface: Mm-hmm. Industrial Designer: {vocalsound} Yeah. Project Manager: And uh yeah, now the meeting room is busy. Somebody booked the meeting room just for {disfmarker} at one P_M_, and so we should leave. Uh. So, um. So now we are going for for a small lunch. It's uh funded by the company. And uh after we have uh thirteen minutes to to do indevel individual works. And uh I will do the minutes. And uh you are going to work on your individual works. And uh you will receive as usual your specific instruction and so on. Marketing: Okay, Project Manager: Thank you everybody. Marketing: cool. Okay User Interface: Thanks. Marketing: Thank you.

Users didn't like the look and feel of most remote controls currently on the market. And most users only used ten percent of buttons on the remote control. Channel selection button was the most commonly used button, followed by the volume button. Young users might prefer to have high technology features such as LCD display or speech recognition function on the remote control.

qmsum

--[[--------------------------------------------------------- Name: Jill Help Functions Desc: Heres where you'll find the help functions -----------------------------------------------------------]] JILL = JILL or {} util.AddNetworkString( "Jill::SavePosition" ) net.Receive( "Jill::SavePosition", function( _, ply ) if ( not ply:IsSuperAdmin() ) then return end local data = net.ReadTable() if ( not file.IsDir( "jilldir/entities/", "DATA" ) ) then ply:MessageClient( "bad", JILL.Error_Three ) return end if ( not file.IsDir( "jilldir/entities/" .. data[ 1 ][ "ent" ]:GetClass(), "DATA" ) ) then file.CreateDir( "jilldir/entities/" .. data[ 1 ][ "ent" ]:GetClass(), "DATA" ) ply:MessageClient( "good", "'" .. data[ 1 ][ "ent" ]:GetClass() .. "' does not exist, but was created successfully." ) end local files, folders = file.Find( "jilldir/entities/" .. data[ 1 ][ "ent" ]:GetClass() .. "/*", "DATA" ) for k, v in pairs( files ) do file.Delete( "jilldir/entities/" .. data[ 1 ][ "ent" ]:GetClass() .. "/" .. v ) end for k, v in pairs( data ) do file.Write( "jilldir/entities/" .. v[ "ent" ]:GetClass() .. "/" .. k .. "_pos.txt", tostring( v[ "pos" ] ) ) v[ "ent" ]:Remove() end ply:MessageClient( "good", "Successfully saved all the positions for '" .. data[ 1 ][ "ent" ]:GetClass() .. "'" ) ply:MessageClient( "good", "Removed all " .. data[ 1 ][ "ent" ]:GetClass() .. "'s. Next time someone takes this quest the " .. data[ 1 ][ "ent" ]:GetClass() .. "'s will spawn at the saved locations." ) end ) util.AddNetworkString( "Jill::SpawnEntity" ) net.Receive( "Jill::SpawnEntity", function( _, ply ) if ( not ply:IsSuperAdmin() ) then return end local Vec = net.ReadVector() local ent = string.lower( net.ReadString() ) local SpawnedEntity = ents.Create( tostring( ent ) ) if ( not IsValid( SpawnedEntity ) ) then return end SpawnedEntity:SetPos( Vector( Vec.x, Vec.y, Vec.z ) ) SpawnedEntity:Spawn() end ) util.AddNetworkString( "QuestSystem::SaveNPC" ) net.Receive( "QuestSystem::SaveNPC", function( _, ply ) if ( not ply:IsSuperAdmin() ) then return end local Header = net.ReadString() local NModel = net.ReadString() local ID = net.ReadString() local QNPC = ents.Create( "jill" ) QNPC:SetPos( ply:GetEyeTrace().HitPos ) QNPC.Header = Header QNPC.NModel = NModel QNPC.ID = ID QNPC:SetNPCHeader( Header ) QNPC:SetNPCUniqueID( ID ) QNPC:Spawn() QNPC:SetModel( NModel ) end ) util.AddNetworkString( "QuestSystem::EditNPC" ) net.Receive( "QuestSystem::EditNPC", function( _, ply ) if ( not ply:IsSuperAdmin() ) then return end local npc = net.ReadEntity() local Header = net.ReadString() local NModel = net.ReadString() local ID = net.ReadString() if ( not ID or ID == nil ) then ply:MessageClient( "bad", "Skipping NPC as custom ID does not exist" ) return end local oldID = IsValid( npc ) and npc.ID or 0 file.Delete( "jilldir/" .. oldID .."/header.txt", "DATA" ) file.Delete( "jilldir/" .. oldID .."/model.txt", "DATA" ) file.Delete( "jilldir/" .. oldID .."/npc_id.txt", "DATA" ) file.Delete( "jilldir/" .. oldID .."/pos.txt", "DATA" ) file.Delete( "jilldir/" .. oldID .."/ang.txt", "DATA" ) file.Delete( "jilldir/" .. oldID, "DATA" ) file.CreateDir( "jilldir/" .. ID, "DATA" ) if ( IsValid( npc ) ) then local pos = npc:GetPos() local ang = npc:GetAngles() npc:SetNPCHeader( Header ) npc:SetNPCUniqueID( ID ) npc:SetModel( NModel ) npc.ID = ID npc.Header = Header npc.NModel = NModel file.Write( "jilldir/" .. ID .. "/pos.txt", tostring( pos ) ) file.Write( "jilldir/" .. ID .. "/ang.txt", tostring( ang ) ) file.Write( "jilldir/" .. ID .. "/header.txt", tostring( Header ) ) file.Write( "jilldir/" .. ID .. "/model.txt", tostring( NModel ) ) file.Write( "jilldir/" .. ID .. "/npc_id.txt", tostring( ID ) ) end ply:MessageClient( "good", "Successfully edited: " .. ID ) end ) util.AddNetworkString( "QuestSystem::DeleteNPC" ) net.Receive( "QuestSystem::DeleteNPC", function( _, ply ) if ( not ply:IsSuperAdmin() ) then return end local ent = net.ReadEntity() local id = net.ReadString() if ( not file.IsDir( "jilldir/" .. id, "DATA" ) ) then return end file.Delete( "jilldir/" .. id .."/header.txt", "DATA" ) file.Delete( "jilldir/" .. id .."/model.txt", "DATA" ) file.Delete( "jilldir/" .. id .."/npc_id.txt", "DATA" ) file.Delete( "jilldir/" .. id .."/pos.txt", "DATA" ) file.Delete( "jilldir/" .. id .."/ang.txt", "DATA" ) file.Delete( "jilldir/" .. id, "DATA" ) if ( IsValid( ent ) ) then ent:Remove() end ply:MessageClient( "good", "Successfully deleted NPC: " .. id ) end )

The code includes network functions for managing entities and NPCs in a game, accessible only to superadmin players. It also includes file operations for managing directories, files, and data writing.

stacksmol

Immunizing human volunteers by mosquito bite with radiation-attenuated Plasmodium falciparum sporozoites (RAS) results in high-level protection against infection. Only two volunteers have been similarly immunized with P. vivax (Pv) RAS, and both were protected. A phase 2 controlled clinical trial was conducted to assess the safety and protective efficacy of PvRAS immunization. A randomized, single-blinded trial was conducted. Duffy positive (Fy+; Pv susceptible) individuals were enrolled: 14 received bites from irradiated (150 ± 10 cGy) Pv-infected Anopheles mosquitoes (RAS) and 7 from non-irradiated non-infected mosquitoes (Ctl). An additional group of seven Fy- (Pv refractory) volunteers was immunized with bites from non-irradiated Pv-infected mosquitoes. A total of seven immunizations were carried out at mean intervals of nine weeks. Eight weeks after last immunization, a controlled human malaria infection (CHMI) with non-irradiated Pv-infected mosquitoes was performed. Nineteen volunteers completed seven immunizations (12 RAS, 2 Ctl, and 5 Fy-) and received a CHMI. Five of 12 (42%) RAS volunteers were protected (receiving a median of 434 infective bites) compared with 0/2 Ctl. None of the Fy- volunteers developed infection by the seventh immunization or after CHMI. All non-protected volunteers developed symptoms 8–13 days after CHMI with a mean pre-patent period of 12. 8 days. No serious adverse events related to the immunizations were observed. Specific IgG1 anti-PvCS response was associated with protection. Immunization with PvRAS was safe, immunogenic, and induced sterile immunity in 42% of the Fy+ volunteers. Moreover, Fy- volunteers were refractory to Pv malaria. Identifier: NCT01082341. Although there has been a decrease in malaria incidence globally during the past 15 years (~37%) [1], this infection remains a major public health problem with 214 million cases and 438,000 deaths estimated in 2015 [1]. Plasmodium falciparum (Pf) causes the greatest malaria burden particularly in Africa, and is the focus of most attention, including the search for a vaccine. Recently, a vaccine based on the Pf circumsporozoite (CS) protein (RTS, S) received a positive decision by the European Medicines Agency (EMA) for potential use in African children to reduce episodes of clinical malaria, based on the results of phase 3 studies, while the World Health Organization (WHO) recommended feasibility and pilot effectiveness implementations [2]. Protection afforded by RTS, S is limited to reduction of clinical disease in infants and young children; the vaccine is not intended for older children or adults, for use in Europe or the USA, or to block infection or prevent transmission. Plasmodium vivax (Pv) is the second most abundant malaria parasite, posing a serious threat in Asia, Oceania, and Latin America and also requires a specific and effective vaccine. Progress in developing Pv vaccines lags far behind that for Pf. Acquisition of clinical immunity to malaria is a slow process and sterile immunity is never achieved under natural conditions, although it can be reproducibly induced by immunization via mosquito bite with radiation-attenuated sporozoites (RAS), the parasite stage transmitted by mosquitoes to humans [3–5]. This approach induces immune responses that block the sporozoite (SPZ) invasion of hepatocytes and subsequent schizogonic development in the liver, thereby preventing the pathogenic asexual blood stage infection that causes malaria disease. Such responses also prevent the development of gametocytes (sexual blood stages); thus, RAS immunization could serve as a vaccine to interrupt malaria transmission. Pre-erythrocytic stage vaccines such as RAS, therefore represent an ideal approach for vaccine development [6] as has been reported previously for Pf [7]. In the 1970s, sterile immunity against malaria was first demonstrated in humans vaccinated using RAS [3,4, 8]. Since then, multiple studies have confirmed the high reproducibility of this vaccination model [9,10]. Significant efforts are now being invested and good progress has been achieved in developing a parenterally injectable vaccine based on cryopreserved PfRAS [7]. Several PfRAS phase 1 and 2 trials have been conducted by Sanaria Inc. and collaborators, using a PfSPZ vaccine, a GMP product consisting of aseptic, purified, radiation-attenuated, cryopreserved PfSPZ. This vaccine has shown high-level efficacy in naïve adults [7]. Additionally, several parasite antigens found to be active in RAS immunization and possibly associated with protection have been the subject of intense research on the development of subunit vaccines (reviewed in [11]). Despite the epidemiological importance of Pv, the PvRAS model has not been reproduced since the early 1970s, when two volunteers were immunized by receiving >1000 mosquito infectious bites; both were protected from infectious Pv spz challenge [12]. This lag is partly explained by the lack of Pv in vitro culture methods, promoting the development of alternative, more complex infection methods that rely on obtaining fresh, gametocytemic blood from Pv-infected donors. Anopheles mosquito colonies have been established [13] and methods to routinely infect mosquitoes using blood from acutely ill Pv malaria patients have now been standardized [14], resulting in safe, reliable and reproducible infection of human volunteers through mosquito bites [15–17]. The purpose of the study described here was first to establish a solid proof-of-principle that humans could be protected by immunization via the bites of PvRAS-infected mosquitoes and second, to obtain sera and cells to study the mechanisms of protective immunity and identify the antigenic targets of immune responses. A phase 2 trial was conducted in healthy adult Colombian volunteers without previous exposure to malaria. This trial was conducted according to ICH E-6 Guidelines for Good Clinical Practices [18]. Institutional Review Boards of the Malaria Vaccine and Drug Development Center (MVDC, CECIV), and Centro Médico Imbanaco (CEICMI), Cali, approved the protocol. Written informed consent (IC) was obtained from all volunteers, with a separate IC for HIV screening. The clinical trial was registered on clinicaltrials. gov, registry number NCT01082341. The protocol for this trial and supporting CONSORT checklist are available as supporting information (S1 Checklist and S1 Protocol). A phase 2 controlled randomized, single-blinded clinical trial was conducted at the MVDC, Cali, Colombia. A total of 89 malaria-naïve volunteers (18–45 years old) were assessed for eligibility (Fig 1). Two approaches to immunization were used in this study. First, Duffy-positive (Fy+) individuals were assigned to RAS or mock-immunized control groups using a single-blinded design (volunteers but not investigators blinded) to assess the safety, tolerability, immunogenicity and protective efficacy of PvRAS immunization. Second, taking advantage of the fact that Fy- erythrocytes are refractory to Pv invasion, a third group of Fy- volunteers was immunized with bites from infected non-irradiated mosquitoes to assess the impact of exposure to PvSPZ developing fully in the liver (as opposed to arresting early in liver stage development, as in the case of RAS). Immunization was performed by direct exposure to bites of irradiated (Fy+ volunteers) or non-irradiated (Fy- volunteers) Pv-infected mosquitoes, and mock immunization by exposure to the bites of non-irradiated, non-infected mosquitoes. After the immunization schedule, volunteers were subjected to a Pv controlled human malaria infection (CHMI), carried out by exposing volunteers to the bites of non-irradiated, Pv-infected mosquitoes. Clinical outcome, parasitemia as measured by thick blood smear microscopy (TBS), and clinical laboratory and immunological parameters were assessed. Antimalarial treatment was provided to all volunteers becoming TBS-positive or completing the study to day 60 post-CHMI. Volunteers were informed about the risks of participation and were provided sufficient opportunity to read the IC forms. Before signing the IC, volunteers had to pass an oral or written exam concerning the trial and its risks as described elsewhere [16]. In addition, all participants were informed about their right to voluntarily withdraw from the study at any time for any reason. Exclusion criteria included pregnancy, abnormal clinical hematology, and chemistry test results, glucose-6-phosphate dehydrogenase deficiency (G6PDd), and infectious diseases (syphilis, HIV, Chagas disease, HTLV 1–2, hepatitis B and hepatitis C; S1 Table, S2 Table, S1 text). Anopheles albimanus mosquitoes reared at the MVDC insectary in Cali were infected with blood from Pv-infected patients (18–45 years old) recruited at outpatient clinics in malaria-endemic areas of Colombia. TBS was performed on all volunteers seeking care for malaria diagnosis as required by the National Malaria Control Program. Only volunteers who tested positive by this method were invited to participate in the study and were informed of the research aims, potential risks, and benefits. After signing the IC and before the antimalarial treatment, whole blood (35 mL) was collected by venipuncture. All samples were confirmed to be Pv malaria mono-infections by quantitative PCR (qPCR) and negative for other infectious agents (syphilis, HIV, Chagas disease, HTLV 1–2, hepatitis B and hepatitis C; S2 Table). Mosquitoes were membrane-fed with infected blood as described previously [19]. Batches with >50% mosquitoes harboring spz in their salivary glands were used for immunization and CHMI. For both procedures, individual screen-meshed boxes containing infected mosquitoes were used. Mosquitoes were allowed to feed on the volunteer for a 5–10 minute period as previously standardized [14]. After biting, all mosquitoes were dissected and microscopically examined to confirm the presence of blood meal and spz in the salivary glands. CHMI of all volunteers was carried out on the same day by exposing volunteers to bites of 2–4 mosquitoes infected with the same parasite isolate [15–17]. Infected bites were calculated as the number of fed mosquitoes times the percentage infected. Sporozoite attenuation was performed by exposure of Pv-infected mosquitoes to 150 ± 10 cGy of gamma radiation using a Varian Clinac IX Series 927 linear accelerator at the radiotherapy unit of Hospital Universitario del Valle in Cali as previously described [20]. The primary objective of the study was the immunization and CHMI of all volunteers using mosquitoes as described above. Fy+ volunteers were assigned to either RAS (n = 14) or Ctl (n = 7) groups, and Fy- volunteers to the Fy- group (n = 7). A total of seven immunizations were carried out using for each immunization a mean of 65 infectious mosquito bites. Two weeks after the last immunization, all volunteers were treated orally with curative doses of chloroquine (600 mg on day one and 450 mg on days two and three) and primaquine (30 mg daily for 14 days) to eliminate any subpatent malaria infections that may have developed during the immunization period, so that incident infections from CHMI could be accurately determined. Plasma levels of chloroquine and primaquine were measured by high-performance liquid chromatography (HPLC; [21]) two weeks prior to CHMI, to ensure drug clearance. Eight weeks after the last immunization, and one month after completing antimalarial treatment, all volunteers received CHMI using 2–4 Pv-infected mosquito bites. Physical examination, clinical laboratory, and immunological tests were performed after every immunization and CHMI (Fig 2). Adverse events (AE) were recorded, graded and classified according to FDA recommendations [22]. Whole blood was collected by venipuncture of the arm at inclusion (baseline), ten days after each immunization, before CHMI, and six months post-CHMI for clinical laboratory and immunological tests. After each immunization, volunteers were followed-up on days 1,2 and 10 in person for a physical examination and by phone on days 7 and 14. Likewise, after CHMI, volunteers were followed up every day by phone until day 5 and malaria infection monitored daily in an outpatient clinic from day 6–28 post-CHMI; thereafter twice a week from day 29–60 post-CHMI for volunteers who did not develop fever or patent infection within 28 days post-CHMI as determined by TBS microscopy and qPCR. Additionally, volunteers were encouraged to visit the Centre for medical consultation at any moment if they developed any symptom or had any concern. Treatment was initiated immediately after parasitemia was confirmed by TBS and the volunteers followed-up until three consecutive TBS resulted negative. Afterward, volunteers had TBS assessed on days 7,14,21 and 45 post-treatment to confirm cure and absence of relapse [15–17]. Serum and plasma were stored at -20°C until use. Peripheral blood mononuclear cells (PBMC) obtained by Ficoll density gradient centrifugation were stored in liquid nitrogen until use. Vaccine efficacy was assessed by prevention of patent parasitemia. Infection was diagnosed by TBS examination by two independent experienced microscopists, and parasitemia determined by counting the number of asexual Pv parasites per 400 white blood cells (WBC), assuming normal WBC counts (8,000 cells/μL). Samples were considered negative after observation of 200 microscopic fields and qPCR was performed subsequently for retrospective analyses. Clinical laboratory tests were periodically performed during immunizations and as required by clinical judgment after the CHMI to ascertain health status (same methods as recruitment screening tests, S1 Table). A secondary outcome was the evaluation of humoral immune responses. Specific antimalarial antibodies (Ab) were determined by enzyme-linked immuno-sorbent assay (ELISA). The presence of IgG to PvCS (NRC and N peptides) and to merozoite surface protein-1 (PvMSP-1) was assessed in sera diluted 1: 200 as previously described [17]. PvCS corresponded to a chimeric synthetic polypeptide composed of the amino (N) flank, the VK210 and VK247 natural repeat variants (R), and the carboxyl (C) flanking sequences of the protein [23]; PvMSP-1 corresponded to a recombinant fragment from the N region of the protein, namely r200L [24]. IgG isotypes against PvCS-NRC peptide were detected using mouse monoclonal Abs to specific to human IgG1, IgG2, IgG3 and IgG4 (Sigma-Aldrich), followed by HRP-conjugated anti-mouse. In all cases, the optical density (OD) was measured using a BioTek ELISA Reader (BioTek, Winooski, VT). Cut-off values were calculated as three SD above the mean OD value of negative control sera. Results were expressed as reactivity index (RI), defined as optical density (OD) values of test sample divided by the cut-off value. Immunofluorescence tests (IFAT) were used to assess the Ab reactivity with PvSPZ. To determine the frequency of T cells responding to P. vivax antigens, IFN-γ production was quantified using an ELISpot assay. Briefly, the assay was performed in multiscreen 96-well plates (MAHAS 4510, Millipore) coated with anti-human IFN-γ capture antibody (1-D1K; Mabtech AB). Fresh PBMC collected 12 days previous to the CHMI were plated into duplicate wells at 4 x 105 cells in complete RPMI-1640 medium (cRPMI; Gibco, Invitrogen) supplemented with 10% FBS. The PBMC were stimulated for 40 h at 37°C with 10 μg/mL of PvSPZ lysate, PvCS-NRC or PvTRAP (thrombospondin-related adhesive protein). cRPMI medium-only and PHA controls were used in all assays. Biotinylated anti-IFN-γ antibody (7-B6-1; Mabtech AB) was added followed by alkaline phosphatase-streptavidin conjugate (Mabtech AB). Spots were visualized by adding BCIP/NBT (Sigma-Aldrich), scanned and counted using the AID ELISpot reader (AID Autoimmun Diagnostika GmbH, Germany) to determine the number of spots/well. Results were expressed as spots per 106 PBMC, normalized by the antigen-stimulated spots less cRPMI medium. Data were collected and managed using REDCap (Nashville, TN, USA) electronic data capture tools, analyzed using SPSS version 16. 0 software (SPSS Inc., Chicago, IL, USA), and plotted using GraphPad Prism version 6. 0 (GraphPad Software, San Diego, California, USA). We estimated a sample size of 21 Fy+ individuals (2: 1, RAS to Ctl) at a 5% significance level and 80% power to assess the protective efficacy of immunization. Nominal variables were analyzed using descriptive statistics. Mann-Whitney U or the Kruskal-Wallis tests were used as needed. Fisher' s exact test was used to compare proportions. Spearman’s rank correlation (rs) was used to assess the correlation between numeric variables. Incubation and pre-patent periods were determined by TBS and qPCR and visualized using Kaplan–Meier estimator. A p value < 0. 05 was considered statistically significant. A total of 28 of the screened volunteers were enrolled and began the immunization schedule between Sept 26,2013, and Feb 15,2014. However, only 19 completed the schedule (Fig 1 and Fig 2). Mean age at enrollment was 30,29 and 25 years, and the male/female ratio was 5: 9,5: 2,0: 7 for the RAS, Ctl, and Fy- groups, respectively (Table 1). A total of seven immunizations were carried out at mean intervals of nine weeks (range 3–25 weeks) in volunteers who then continued to complete the CHMI. The RAS and Fy- groups received a median of 434 (range 362–497) and 476 (range 358–487) total infective bites over the seven immunizations, respectively, whereas the Ctl group received 954 (range 945–963) non-infective (placebo) bites during the immunization protocol. The total number of infective bites, non-infective bites, fed mosquitoes, and spz in salivary glands per volunteer were determined by post-feeding salivary gland dissection and microscopy examination (S3 Table). No volunteer developed clinical malaria or parasitemia by TBS during the immunization phase, although low levels of parasite DNA were detected in peripheral blood by qPCR from day 8–16 after immunizations in the Fy- group, which declined after every subsequent immunization (Fig 3A). At the time of the CHMI, all volunteers had cleared both primaquine and chloroquine in plasma, although two volunteers in the RAS group had low detectable levels of chloroquine two weeks prior to the CHMI. Notably, both volunteers developed malaria infection. Seven to nine days after the first immunization, 1/14 and 5/7 volunteers of the RAS and Fy- groups, respectively, developed fever, chills, headache and profuse sweating consistent with malaria, which lasted 1–2 days. All five symptomatic Fy- volunteers had negative TBS but positive qPCR that resolved spontaneously, whereas the RAS volunteer was negative by TBS and qPCR. Headache and local reaction in the immunization site were the most common AE during initial immunizations with decreasing frequency throughout the immunizations (S4 Table). After CHMI headache, chills, fever, and malaise were common AE (Fig 4). In the RAS and Ctl groups, a mean of 11 and 16 AE per individual were reported after CHMI, respectively. In contrast, in the Fy- group a mean of two AE was reported. No serious AE related to immunizations were observed, although one female developed severe elevation of hepatic transaminases after CHMI (>10 times upper limit of normal [xULN]) and lactic dehydrogenase (2. 5xULN) with abdominal pain, nausea, and vomiting during Pv malaria mono-infection. This patient was observed in the emergency room and completely recovered without sequelae. No alternative etiologies for the elevated transaminases were identified (volunteer was negative for hepatitis C, hepatitis B, HIV and hepatitis A, and she was not consuming any medications). At day 60 post-CHMI (last day of follow-up), the total protective efficacy in the RAS group was calculated at 42% (5/12 RAS, 0/2 Ctl) as determined by TBS and confirmed by qPCR; all protected subjects were women (Table 1). All malaria-positive volunteers presented with low parasitemia, with median density values lower by TBS than by qPCR (TBS: 140 parasites/μL; IQR 95–210, and qPCR: 220 parasites/μL; IQR 29. 2–361). Mean incubation period was 9. 9 days (range 8–13); mean prepatent period was 12. 8 days (range 12–13) by TBS; and 9. 0 days (range 8–11) by qPCR. No significant differences were found between the Ctl and positive RAS subjects in prepatent period or density of parasitemia by TBS or qPCR (Fig 3B). However, survival analysis showed a significantly greater incubation period in RAS than in Ctl volunteers (S1 Fig). These results were compared with the parasite dynamics of a previous CHMI trial carried out in naïve volunteers using the same infection protocol. Those volunteers who did not develop malaria were followed up until day 60 post-CHMI after which antimalarial treatment was administered. Volunteer 001 of the RAS group developed malaria-related symptoms at day 58 post-CHMI, but parasitemia was only detected on day 66 by TBS. This prepatent period was considered as partial protection induced by vaccination. Seroconversion using the PvCS-NRC peptide was observed in all 12 RAS volunteers, mostly after the second immunization (10/12) and in all Fy- volunteers between the second and fifth immunizations. In both groups, IgG reactivity was low (RI < 6); all Ctl volunteers remained seronegative during the immunization phase (Fig 5A and 5B). A positive correlation between the RI for PvCS-NRC and number of infective bites was observed for the Fy- group but not for the RAS group (Fig 5C). No significant association between total anti PvCS-NRC RI and protection was found (Fig 5D); however, the specific IgG1 response was significantly higher in protected individuals (Fig 6A and 6B). All Fy- volunteers and one in RAS group developed anti-PvMSP-1 IgG response after seven immunizations. In contrast, all Ctl volunteers remained negative for all antigens tested (S2 Fig). After immunization, 11/12 of RAS and 4/5 of Fy- volunteers had IFAT Abs to Pv spz, respectively, but no association with protection was found (S5 Table). Moreover, all RAS and Fy- sera recognized PvCS by Western blot (S3 Fig). After seven immunizations and before to CHMI, PMBCs of the RAS group were able to produce IFN-γ after stimulation with the tested antigens Pv spz lysate, PvCS-NRC, and PvTRAP (Fig 7A) at significantly higher levels than the other two groups (p <0. 05 for all antigens). In the Fy- group, PvCS-NRC and PvTRAP induced IFN-γ production but was not significantly higher than the observed in the Ctl volunteers (Fig 7B). No significant differences were observed between protected and not protected volunteers in the RAS group (Fig 7C). This trial has allowed the establishment of the PvRAS immunization model with protection in an unprecedented number of volunteers. To our knowledge, only two volunteers had been previously reported to be protected from CHMI by PvRAS immunization [12]. As is true for PfRAS, immunization by mosquito bite with the PvRAS is safe, immunogenic and able to induce sterile protection. A series of clinical trials conducted with PfRAS indicated high protective efficacy (~90%) and protection lasting up to 42 weeks with a dose-dependent efficacy based on ten immunization sessions and a total of ~1000 RAS mosquito bites [3–5,8, 9,12]. This study could not reproduce those conditions due to the difficulty of obtaining PvRAS, which include the need of regular P. vivax infected donors from malaria endemic areas, willing to participate and complying with all inclusion criteria. In addition, not all P. vivax samples are successfully infective to An. albimanus mosquitoes due to numerous biological factors [25]. Nevertheless, seven immunization sessions provided a median of 434 PvRAS bites for an efficacy of 42%. This is similar to what has been found with Pf immunization, where the protective efficacy against CHMI in volunteers receiving < 1000 infectious bites was 40% [9]. Despite the high number of volunteers that withdrew from the Ctl group, the two remaining showed a trend in the parasite dynamics similar to that observed in a total of 29 naïve Fy+ volunteers infected with 2–4 An. albimanus mosquito bites in three previous CHMI trials [15–17]. In one of these trials [17], even semi-immune volunteers from endemic areas previously exposed to natural malaria infection developed similar parasite patency, indicating the relevance of the sterile protection induced here by PvRAS immunization. Therefore, given that both controls turned positive following CHMI, we are confident that PvRAS immunization induced sterile protection as described in RAS group. A summary of parasitological data for naïve volunteers participating in previous CHMI carried out in our Centre, which demonstrate the consistency of this procedure, is shown (Table 2). Since no detectable levels of chloroquine were observed in most of RAS volunteers, and the two volunteers with low detectable levels had undetectable levels immediately previous to CHMI and developed malaria infection, we concluded that lack of parasitemia was not dependent of chloroquine antiplasmodial activity. Significant progress has been achieved regarding the development of a practical approach for Pf immunization based on whole SPZ [7]. Intravenous administration of aseptic, purified, cryopreserved, radiation-attenuated PvSPZ [7] has shown the highest efficacy, protecting up to 100% of study subjects. Based on the results of our study, we can anticipate that a similar Pv product would be equally protective, and could potentially be combined with Pfspz to induce potent immunity to the two major species of human malaria. The use of the whole SPZ approach may therefore be an effective route to solving the malaria problem, given that subunit vaccines appear to have a longer development trajectory. The RTS, S vaccine, the most advanced Pf malaria subunit vaccine, has been assessed as meeting EMA standards, setting the stage for potential licensure in African countries, and has subsequently been recommended by WHO for testing in pilot implementations in Africa [2]. Several other vaccine candidates are also under development [26]. RTS, S is to be licensed for reducing the incidence of clinical malaria but not preventing malaria infection, and is insufficiently potent for use in elimination campaigns. Progress in subunit vaccines for Pv has been especially limited due to the lack of an in vitro culture method and the scarce funding. In order to fill this gap, we approached the development of PvRAS by accessing to fresh gametocytemic blood from patients to assess the PvRAS model’s feasibility and reproducibility under controlled conditions, and to generate immune reagents to determine correlates of protection. Additionally, this study took the novel approach of immunizing Fy- volunteers by repeated exposure to viable PvSPZ. Because most Fy- individuals are refractory to blood infection by Pv, this allowed evaluation of immune responses elicited specifically against liver-stage parasites. Although there have been reports from Madagascar and Cameroon-endemic areas that some of these subjects may develop the blood cycle when infected by Pv [27,28], this did not happen in our study with different natural parasite isolates. To our knowledge, this was the first time that Fy- volunteers have been used as a model for a better understanding of the immune responses to Pv liver stages. The presence of symptoms in Fy- following only the first immunization, and the diminishing qPCR positivity as immunizations continued, indicate that the Fy- volunteers developed sterile immunity to Pv infection based on immunity targeting the pre-erythrocytic stages. Reagents generated in this study allow the use of both classic and high throughput methods to analyze the immune response to PvRAS, and comparison of responses to the early liver stages in the PvRAS and Fy- groups. Sera and cells are currently being studied using high throughput systems in an attempt to determine correlates of immune protection. Interestingly, all protected volunteers were women, whereas all men developed malaria despite receiving similar parasite doses. No covariates, such as numbers of immunizing bites, were identified to explain this finding. This is consistent with other studies where women mounted a more vigorous immune response than men (reviewed in [29]), although this was not evident here at least for the parameters evaluated, which may or may not serve as correlates of protection. We achieved the doses necessary to protect almost all challenged women (5/7) but not men (0/5). Immunization with both PvRAS and viable Pv spz induced a measurable although weak ELISA antibody response to PvCS, and there was no association between total IgG Ab levels to PvCS and protection. Nonetheless, the protected volunteers had a greater IgG1 response against PvCS-NRC peptide, which is in agreement with studies describing associations between higher levels of IgG1 and IgG3 Abs and protection against severe Pf malaria episodes [30] as well as predominant markers for exposure to Pv malaria [31]. However, the borderline p value (p = 0. 048) for the association between PvCS-NRC peptide ELISA titer and protection was possibly due to the relatively small number of individuals tested. All Fy- individuals and only one PvRAS developed Ab levels against PvMSP-1 protein after seven immunizations. This appears to be in agreement with the fact that Pv is able to completely develop the liver cycle and release merozoites into circulation in Fy- volunteers as demonstrated by qPCR, whereas RAS appears to arrest development in early phases of the liver cycle [32]. It is also consistent with the fact that Fy- volunteers developed fever and other malaria symptoms during the first immunizations and the fact that parasite DNA was detected up to the fourth immunization. The decrease in anti-CS Ab levels after the third and fourth immunizations, when there was a pause of several months in immunizations, indicates that these Abs are short-lived, although memory cell responses were present as demonstrated by the rapid boosting of specific Abs after the fifth immunization. This trial confirms the reproducibility of the RAS vaccination model in Pv malaria. Despite the lack of correlation between protection and the tested immune responses, high throughput analyses of cells and sera, i. e., transcriptomics and anti-parasite Ab microarray profiles, may offer a better understanding of the parasite targets involved and the immune effector mechanisms associated with protection.

Despite the advances in Plasmodium falciparum (Pf) vaccine development, progress in developing P. vivax (Pv) vaccines lags far behind. Immunization via mosquito bites with Pf radiation-attenuated sporozoites (RAS) has been the gold standard model for induction of sterile protection against malaria infection and has allowed the study of the complex mechanisms of immunity. The first trials using PfRAS were performed in the late 1960's, and thereafter greatly contributed to the development of vaccines against Pf. However, PvRAS immunization in humans has only been carried out in two volunteers since 1974. To our knowledge, this is the first clinical trial using significant numbers of volunteers for PvRAS immunization. Our findings confirm that immunization with PvRAS is safe, immunogenic and induces sterile immunity in 42% of the volunteers. It demonstrates that it is possible to induce sterile protection with PvRAS as seen with PfRAS and confirms that immunity against the PvCS protein (IgG1 levels) correlates with protection. Research findings and reagents generated in this study are expected to yield insights on key immune determinants of sterile protection against Pv, which may guide the development of a cost-effective vaccine against this parasite species.

lay_plos

They go to sleep several hours after sunset and typically awaken before sunrise They do not sleep more than “modern” humans, with average durations of 5.7–7.1 hr How did humans sleep before the modern era? Because the tools to measure sleep under natural conditions were developed long after the invention of the electric devices suspected of delaying and reducing sleep, we investigated sleep in three preindustrial societies []. We find that all three show similar sleep organization, suggesting that they express core human sleep patterns, most likely characteristic of pre-modern era Homo sapiens. Sleep periods, the times from onset to offset, averaged 6.9–8.5 hr, with sleep durations of 5.7–7.1 hr, amounts near the low end of those industrial societies []. There was a difference of nearly 1 hr between summer and winter sleep. Daily variation in sleep duration was strongly linked to time of onset, rather than offset. None of these groups began sleep near sunset, onset occurring, on average, 3.3 hr after sunset. Awakening was usually before sunrise. The sleep period consistently occurred during the nighttime period of falling environmental temperature, was not interrupted by extended periods of waking, and terminated, with vasoconstriction, near the nadir of daily ambient temperature. The daily cycle of temperature change, largely eliminated from modern sleep environments, may be a potent natural regulator of sleep. Light exposure was maximal in the morning and greatly decreased at noon, indicating that all three groups seek shade at midday and that light activation of the suprachiasmatic nucleus is maximal in the morning. Napping occurred on <7% of days in winter and <22% of days in summer. Mimicking aspects of the natural environment might be effective in treating certain modern sleep disorders. T.BlackwellS.Ancoli-IsraelS.RedlineK.L.Stone Osteoporotic Fractures in Men (MrOS) Study Group Factors that may influence the classification of sleep-wake by wrist actigraphy: the MrOS Sleep Study. J. Clin. Sleep Med. 7 : 357-367 Among Tsimane, summer wake times were earlier and sleep onset times were later than in the winter, accounting for their reduced sleep duration; however, in the San, despite their shorter sleep duration in the summer, as in the Tsimane, the time of awakening was significantly later, with the decreased sleep times being entirely the result of later sleep onset. The San the participants awakened, on average, 1.0 hr after sunrise in the summer. The Tsimane awakened 1.4 hr before sunrise in the summer ( Table S2 ). The 2.4 hr difference in awakening times, with respect to sunrise, was significant (t = 8.4, df = 22, p = 1.2E-08). This was not due to differences in day/night length at the two recording locations. The summer observation period in the San had 11 hr nights and 13 hr days. The summer observation period in the Tsimane had 11.1 hr nights and 12.9 hr days. The difference between the sleep offset times in these two populations, despite the similar light conditions, may be due to the much cooler morning temperatures (by 6°C on average) and the shifting of the temperature nadir into the light period ( Figure 4 ) in the San’s environment, paralleling the effect of winter on changes in sleep duration. Because we noticed that the Hadza, Tsimane, and San did not initiate sleep at sunset and that their sleep was confined to the latter portion of the dark period, we investigated the role of temperature. We found that the nocturnal sleep period in the Hadza was always initiated during a period of falling ambient temperature ( Figure S1 ), and we saw a similar pattern in the Tsimane. Therefore, we precisely measured ambient temperature at the sleeping sites along with finger temperature and abdominal temperature in our studies of the San []. Figures 4 and S1 show that sleep in both the winter and summer occurred during the period of decreasing ambient temperature and that wake onset occurred near the nadir of the daily temperature rhythm. A strong vasoconstriction occurred at wake onset in both summer and winter ( Figures 4 and S2 ), presumably functioning to aid thermogenesis in raising the brain and core temperature for waking activity. See the Supplemental Experimental Procedures for a discussion of the use of iButtons to measure vasoconstriction and vasodilation. The presence of vasoconstriction at awakening indicates that the subjects were not vasoconstricted prior to awakening. Sleep offset, averaged across all subjects and all days, consistently occurs near the nadir of daily environmental temperature, in both summer (A) and winter (B). For the San recorded in the summer, the temperature nadir occurred after sunrise, as did awakening. In the winter, the nadir occurred near sunrise with awakening preceding sunrise. Note that the ambient temperature has a gradual fall at night and a rapid rise starting at sunrise, with sleep occurring during the period of slowly falling temperature. Vasoconstriction is seen upon awakening in both summer and winter. In the winter, there are additional vasoconstrictions occurring during the day. These are most likely related to food preparation or other similar activities exposing the hands to cold. Violet lines, environmental temperature; red lines, abdominal temperature; and blue lines, finger temperature. See also Figure S2 for an example of individual subject data. All temperatures recorded by iButtons are synchronized to the Actiwatch time ±2 min. Black bars, night; orange bars, waking; and blue bars, sleep. Vertical lines at top of the figures indicate light-dark transitions; those at bottom indicate sleep-wake transitions. Sleep measures are the averages of the 15 participants recorded in the summer and the 13 of these participants recorded in winter (see Figure 1 and Table S1 ). Red arrows indicate onset of drop in finger temperature starting near the temperature nadir, indicative of peripheral vasoconstriction, serving to warm proximal regions with awakening. See also Figure S1 (D and E) Averaged data across all San recorded in summer and winter. Note the consistent pattern across groups and seasons. Time is local clock time. (C) Staying out of the midday sun. Plots are centered at noon. Light levels recorded by the Actiwatch drop steeply and consistently at midday, despite the increase of the ambient light level from morning (9 a.m.) to noon levels. The figure shows the average of 60 days of data from the ten Hadza recorded in Tanzania. It shows the reduction in light exposure during the afternoon; a lack of reduction in afternoon activity to sleep levels, consistent with the lack of regular napping; and the reduction in activity throughout the sleep period. No regular period of activity was seen in the night, consistent with the lack of a “second sleep” scored by the algorithm (see also Figure S1 ). (A and B) Average light and activity level in plots centered at midnight. Two San participants (T3 and T5) are shown. Both have shorter sleep time in the summer despite later awakening. Participant data are the average sleep parameters over the summer or winter recording periods. The yellow line indicates subject light exposure as measured by the Actiwatch. Sunset, identifiable by the vertical interrupted black line, is not tightly linked to sleep onset. Interrupted blue bars indicate sleep periods. The red line at the bottom of each graph plots average of 1 min epochs with (+1) and without (0) activity. Note the maintained and even increased activity (black) with sunset, location of inactivity linked to sleep at the end of the dark period, awakening before dawn in winter, lack of period of activity within sleep, and differences between the duration of summer and winter nighttime inactivity period. The durations of these inactivity epochs are used in the algorithm that identifies sleep ( Figure 1 ). Sleep onset occurs from 2.5 to 4.4 hr after sunset in all of the groups examined (mean = 3.3 hr). Average sleep onset across groups occurred between 2.5 and 4.4 hr after sunset (mean = 3.3 hr) ( Table S2 and Figure 3 ). At the latitudes of the participant populations, the duration of evening and morning civil twilight ranges from 24–28 min. Therefore, the participants remained awake long after darkness had fallen. The three groups often had small fires, but the Actiwatch-measured light levels remained below 5.0 lux (the lower limit of the Actiwatch-2 sensor) throughout the night ( Figure 3 ). Awakening occurred on average 1 hr before sunrise in the Tsimane and Hadza, well before civil twilight ( Table S2 ), but awakening was much closer to sunrise than sleep onset was to sunset. Awakening was also well before civil twilight in the winter in the San ( Table S2 ). But in the summer, awakenings in the San participants occurred 1 hr after sunrise, on average (t = 2.4, df = 20, p = 0.02) ( Figures 2 3, and 4 ). The shorter sleep duration in the summer was completely a result of later sleep onset, not of earlier awakening (mean sleep onset time = 22:44 summer versus 21:16 winter) (t = 5.0, df = 20, p = 6.8E-5]. So, neither sleep onset nor offset were tightly linked to solar light level. A striking feature of the light exposure in all three groups was that it decreased from a maximum level at approximately 9 a.m. to a lower level at noon, despite the doubling of ambient light levels over this period. This occurred in winter as well as in summer, indicating that all three groups sought shade from the midday sun ( Figure 3 ). Since insomnia is a complaint and does not closely correspond to sleep time [], we investigated the prevalence of this complaint in the Tsimane and San groups. At the time of application of Actiwatches on the Tsimane, G.Y. and a Tsimane translator visited the participants in their homes early in the morning to conduct an interview on fatigue and sleep quality. A similar interview was done by J.M.S. on the San group. Neither group has a word for insomnia in their language, so we explained the concept in terms of sleep onset insomnia and sleep maintenance insomnia not due to illness. Five percent of the participants said they sometimes had sleep onset problems and 9% sometimes had sleep maintenance problems. Less than one-third of these participants said that they had these problems regularly, i.e., more than once a year (1.5% and 2.5% of the total number of participants). These numbers are far lower than the 10%–30% chronic insomnia rate reported in industrial societies []. It has long been known that “modern” humans experience an extended dip in midafternoon alertness, which is not due to food intake []. It has been speculated that under “natural” conditions, a nap would occur during this period and that this nap has been suppressed by industrial lifestyles. An automated Actogram analysis using the Actogram program (see “Actiwatch-2 devices” in the Supplemental Information ) on the data from the San scored no afternoon naps in 210 days of recording in the winter. It scored ten naps on 364 days in the summer (3% of days) (see Table S3 ). Nocturnal awakenings were also infrequent (see Table S3 ). The Actograms of the Tsimane and Hadza participants showed a similar dearth of potential napping and nocturnal waking intervals ( Figure 1 B). Because Actiwatches have not been as thoroughly validated against polygraphic recording for naps as they have for nighttime sleep [], we conducted a second quantification of naps using visual scoring of the Actiwatch records to identify periods with motor activity reduced to levels seen within the nighttime sleep for periods of 15 min or longer. We saw such episodes in only 7% of the recording afternoons in the San winter data. This should be considered the maximum incidence of napping, since we cannot exclude the possibility that some or all of these were waking rest periods. In the summer, 22% of days had potential naps (comparing summer and winter frequencies: t = 3.5, df = 25, p = 0.0007) ( Figure 1 B). Nap duration using the longer summer visually scored putative nap periods averaged 32 min. Thus, if all potential napping time was considered sleep time, it would raise the average daily sleep duration in summer by 7 min. It remains possible that naps shorter than 15 min occur, but electroencephalogram recording would be necessary to identify them. Psychological and behavioral changes during confinement in a 520-day simulated interplanetary mission to mars. PLoS ONE. 9 : e93298 T.BlackwellS.Ancoli-IsraelS.RedlineK.L.Stone Osteoporotic Fractures in Men (MrOS) Study Group Factors that may influence the classification of sleep-wake by wrist actigraphy: the MrOS Sleep Study. J. Clin. Sleep Med. 7 : 357-367 (B) Change in sleep onset and offset times across the seasons. The same San individuals were sampled for a 28-day period in summer and a 21-day period in winter (a total of 1,260 sleep onsets and offsets). Note the much later sleep onset in the summer and the later wake onset in the summer relative to winter, despite the shorter sleep times. Bin size is 0.4 hr (24 min). The blue vertical line marks solar noon. (A) Sleep duration decreased from winter to summer. (Note that the Hadza, San, and Tsimane live in the southern hemisphere.) The Tsimane data were from six separate groups recorded over the 4-month period. Each group consisted of seven to 12 individuals recorded for 7 days. A parallel study in the San recorded from ten individuals, each for 21 days in May–June. An additional five San individuals were recorded for 11 days in August, and 13 of the initial 15 were recorded for 28 days in Jan–February of the next year (two of the original ten participants had migrated out of Den/ui). The Tsimane and San live far enough south of the equator to have substantial seasonal changes in day length and temperature. Tsimane participants recorded in the winter slept 56 min longer than those in summer ( Figure 2 A) (t = 2.1, degrees of freedom [df] = 19, p = 0.05). In the San, we recorded the same group of 13 participants in winter and summer. Sleep times in the winter were longer than in the summer by an average of 53 min ( Figure 2 B and Table S2 ) (t = 3.7, df = 20, p = 0.001). Mean body mass indices (BMIs) of the three groups were between 18.3 and 26.2 ( Table S2 ), with none of the participants having BMIs >30, in keeping with prior anthropological observations of a lack of obesity in these populations []. Sleep time in the Hadza, San, and Tsimane groups ( Figure 1 B) was similar, between 5.7 and 7.1 hr, with the sleep period duration (time between sleep onset and offset) of from 6.9 to 8.5 hr ( Table S1 ). Sleep parameters were determined with Actiwatch-2 devices, which have been extensively validated with polysomnography ( Figure 1 B; see the Supplemental Experimental Procedures ). The SD of sleep onset times exceeded the SD of sleep offset times in all San individuals (N = 27, p = 7.4E-5, binomial test) and in all Tsimane individuals (N = 45, p = 2.0E-08), with a similar trend in the more limited dataset from the Hadza. Therefore, sleep duration was much more strongly correlated with sleep onset time than with sleep offset time in both summer and winter. Sleep onset and offset times were very weakly correlated with each other ( Table S1 ). In these societies, electricity and its associated lighting and entertainment distractions are absent, as are cooling and heating systems. Individuals are exposed, from birth, to sunlight and a continuous seasonal and daily variation in temperature within the thermoneutral range for much of the daylight period, but above thermoneutral temperatures in the afternoon and below thermoneutrality at night. By examining three such groups in two continents over long periods of time, we were able to evaluate common elements and differences that provide insights into the nature of human sleep under natural conditions. In the current paper, we examine sleep duration, timing, and relation to natural light, ambient temperature, and seasons in three preindustrial human societies ( Figure 1 A). The Hadza live in northern Tanzania, 2° south of the equator, in woodland-savannah habitats around Lake Eyasi. The Hadza in this study were wholly dependent on hunting and gathering each day for wild foods. Until the recent past, the Kalahari San were also nomadic hunter-gatherers. The Ju/’hoansi (Ju/’hoan language group) San that we studied live in the Den/ui village, 20° south of the equator, are currently not migratory, but they are isolated from surrounding villages and continue to live as hunter-gatherers. Genetic studies indicate that the Kalahari San genome is the most variable of those yet sequenced, being much more variable within this group than in the descendants of the small groups that migrated out of Africa to populate Europe, Asia, and the Americas []. The Tsimane, living near the furthest reaches of the human migration out of Africa, close to the Maniqui River in Bolivia and 15° south of the equator, are hunter-horticulturalists. Extensive health studies of the Tsimane have found that although child mortality is higher than in “modern” societies, largely due to infectious diseases, adults have lower levels of blood pressure and atherosclerosis and higher levels of physical fitness than industrial populations []. Many live into their 60s, 70s, 80s, and beyond. Similar health findings have been reported among Hadza [] and San []. (B) Representative Actograms from Hadza, Tsimane, and San subjects (the bottom two sets show the same San participant in summer [upper set] and winter [lower set]). Sleep onset time is highly variable and occurred several hours after sunset in all groups. Awakening time was relatively regular and occurred before sunrise, except in the San in summer. Naps may have occurred on up to 7% of days in winter and up to 22% of days in summer. Extended periods of nocturnal waking were rare. Yellow, log plot of light level; red, 1 min intervals with movement; black, number of movements in each 1 min interval; light blue, Actogram-scored rest; and dark blue, Actogram-scored sleep period. Sleep period, defined as the interval between sleep onset and offset, is greater than sleep time, defined as the sleep period minus waking after sleep onset (WASO). Sleep efficiency (sleep time divided by “bed” time) was between 81% and 86%, similar to that in industrial populations. See Table S1 ). Transition from a hunter-gatherer to a settled lifestyle in the! Kung San: effect on iron, folate, and vitamin B12 nutrition. Am. J. Clin. Nutr. 40 : 1295-1303 Inflammation and infection do not promote arterial aging and cardiovascular disease risk factors among lean horticulturalists. PLoS ONE. 4 : e6590 It has been argued that the invention of the electric light, followed by the development of television, the Internet, and related technologies, along with increased caffeine usage, has greatly shortened sleep duration from “natural” levels and disrupted its evolved timing. The purported reduction in sleep duration has been linked to obesity, mood disorders, and a host of other physical and mental illnesses thought to have increased recently ( http://www.healthypeople.gov/2020/topics-objectives/topic/sleep-health#eight ), although complaints about reduced sleep time in the “modern world” were made at least as far back as the 1880s []. Discussion A striking finding is the uniformity of sleep patterns across groups despite their ancient geographic isolation from each other. This suggests that the observed patterns are not unique to their particular environmental or cultural conditions but rather are central to the physiology of humans living in the tropical latitudes near the locations of the San and Hadza groups, where our species evolved. 1 Marlowe F. The Hadza Hunter-Gatherers of Tanzania. University of California Press, ; 4 Blackwell T. Ancoli-Israel S. Redline S. Stone K.L. T.BlackwellS.Ancoli-IsraelS.RedlineK.L.Stone Osteoporotic Fractures in Men (MrOS) Study Group Factors that may influence the classification of sleep-wake by wrist actigraphy: the MrOS Sleep Study. J. Clin. Sleep Med. 7 : 357-367 5 Heeren M. Sojref F. Schuppner R. Worthmann H. Pflugrad H. Tryc A.B. Pasedag T. Weissenborn K. Active at night, sleepy all day--sleep disturbances in patients with hepatitis C virus infection. J. Hepatol. 60 : 732-740 6 Natale V. Léger D. Martoni M. Bayon V. Erbacci A. The role of actigraphy in the assessment of primary insomnia: a retrospective study. Sleep Med. 15 : 111-115 7 Robillard R. Naismith S.L. Smith K.L. Rogers N.L. White D. Terpening Z. Ip T.K. Hermens D.F. Whitwell B. Scott E.M. Hickie I.B. Sleep-wake cycle in young and older persons with a lifetime history of mood disorders. PLoS ONE. 9 : e87763 22 Evans D.S. Snitker S. Wu S.H. Mody A. Njajou O.T. Perlis M.L. Gehrman P.R. Shuldiner A.R. Hsueh W.C. Habitual sleep/wake patterns in the Old Order Amish: heritability and association with non-genetic factors. Sleep. 34 : 661-669 23 Worthman C.M. After dark: the evolutionary ecology of human sleep.: 291-313 Oxford University Press, ; 24 Kleitman N. Sleep and Wakefulness. University of Chicago Press, ; 25 Lehnkering H. Siegmund R. Influence of chronotype, season, and sex of subject on sleep behavior of young adults. Chronobiol. Int. 24 : 875-888 In some ways, the sleep in these traditional human groups is more similar to sleep in industrial societies than has been assumed. They do not sleep more than most individuals in industrial societies []. The traditional groups do not regularly awaken for extended periods in the middle of the night (see the Supplemental Experimental Procedures ), despite anecdotal reports []. Sleep is strongly modulated by the seasons, averaging 53–56 min longer in the winter, coincident with a 1.2 or 2 hr increase in the night duration in the San and Tsimane, respectively ( Table S2 and Figure 2 ). In contrast, no seasonal effect on sleep durations has been reported in most studies in industrial societies. Kleitman [] summarizes some of the early, conflicting data on seasonal changes in sleep (p. 192). A recent large-scale study of seasonal sleep changes reported an 18 min difference between summer and winter. This study investigated participants in Berlin (latitude +53°), where night duration changes from 7 hr 39 min in summer to 16 hr 21 min in winter) []. Of course, the Berlin participants were not as directly exposed to changes in light and temperature as were our participants. 19 Buysse D.J. Insomnia. JAMA. 309 : 706-716 26 de la Iglesia H.O. Fernández-Duque E. Golombek D.A. Lanza N. Duffy J.F. Czeisler C.A. Valeggia C.R. Access to Electric light is associated with shorter sleep duration in a traditionally hunter-gatherer community. J. Biol. Rhythms. 30 : 342-350 27 Santhi N. Thorne H.C. van der Veen D.R. Johnsen S. Mills S.L. Hommes V. Schlangen L.J.M. Archer S.N. Dijk D.J. The spectral composition of evening light and individual differences in the suppression of melatonin and delay of sleep in humans. J. Pineal Res. 53 : 47-59 28 Wright Jr., K.P. McHill A.W. Birks B.R. Griffin B.R. Rusterholz T. Chinoy E.D. Entrainment of the human circadian clock to the natural light-dark cycle. Curr. Biol. 23 : 1554-1558 27 Santhi N. Thorne H.C. van der Veen D.R. Johnsen S. Mills S.L. Hommes V. Schlangen L.J.M. Archer S.N. Dijk D.J. The spectral composition of evening light and individual differences in the suppression of melatonin and delay of sleep in humans. J. Pineal Res. 53 : 47-59 26 de la Iglesia H.O. Fernández-Duque E. Golombek D.A. Lanza N. Duffy J.F. Czeisler C.A. Valeggia C.R. Access to Electric light is associated with shorter sleep duration in a traditionally hunter-gatherer community. J. Biol. Rhythms. 30 : 342-350 28 Wright Jr., K.P. McHill A.W. Birks B.R. Griffin B.R. Rusterholz T. Chinoy E.D. Entrainment of the human circadian clock to the natural light-dark cycle. Curr. Biol. 23 : 1554-1558 29 Piosczyk H. Landmann N. Holz J. Feige B. Riemann D. Nissen C. Voderholzer U. Prolonged sleep under Stone Age conditions. J. Clin. Sleep Med. 10 : 719-722 30 Wehr T.A. Aeschbach D. Duncan Jr., W.C. Evidence for a biological dawn and dusk in the human circadian timing system. J. Physiol. 535 : 937-951 Light has been shown to be a major factor in human sleep and circadian rhythm control, partially mediated by light’s effects on the melanopsin system []. Consistent with this, we show here that sleep occurs almost entirely during the dark period in these traditional societies. In contrast, sleep typically continues well after sunrise in industrial populations []. A recent study has shown a striking difference in the sleep onset and offset times as a function of light exposure in a comparison of two closely related traditional Argentinian hunter-gatherer populations []. Three other studies showed the rapid regularization of human sleep patterns created by moving “modern” subjects into more natural lighting situations []. 31 Kripke D.F. Elliott J.A. Welsh D.K. Youngstedt S.D. Photoperiodic and circadian bifurcation theories of depression and mania. F1000Res. 4 : 107 32 Uzoma H.N. Reeves G.M. Langenberg P. Khabazghazvini B. Balis T.G. Johnson M.A. Sleemi A. Scrandis D.A. Zimmerman S.A. Vaswani D. et al. Light treatment for seasonal Winter depression in African-American vs Caucasian outpatients. World J. Psychiatry. 5 : 138-146 Our finding that hunter-gatherers get maximal light exposure in the morning, rather than at noon, is consistent with behavioral thermoregulation to avoid afternoon heat. It also may explain the greater effectiveness of morning light [] in the reversal of depression, since such treatments tend to restore the evolved pattern of human exposure to light. Of the ten groups we studied, the only group in our study that awakened after sunrise was the San in the summer. The Tsimane always arose before dawn. At the end of November to the beginning of December (2 weeks from the summer solstice), they awakened more than 1 hr 20 min before sunrise, whereas the San, at approximately the same season with nearly indistinguishable seasonal light levels (13 versus 12.9 hr of light), awakened nearly 1 hr after sunrise. Our data suggest that ambient temperature might be responsible for the difference between these groups and might be a major determinant of sleep timing and duration, independent of light level. 33 Ekirch A. At Day’s Close, Night in Times Past. W.W. Norton and Company, ; 30 Wehr T.A. Aeschbach D. Duncan Jr., W.C. Evidence for a biological dawn and dusk in the human circadian timing system. J. Physiol. 535 : 937-951 Historical evidence suggests that “until the close of the early modern era, Western Europeans experienced two major intervals of sleep bridged by up to an hour or more of quiet wakefulness” [] (see also []). Our results suggest that the bimodal sleep pattern that may have existed in Western Europe is not present in traditional equatorial groups today and, by extension, was probably not present before humans migrated into Western Europe. Rather, this pattern may have been a consequence of longer winter nights in higher latitudes. In this view, the “recent” disappearance of bimodal sleep was not a pathological development caused by restricted sleep duration, but rather a return to a pattern still seen today in the groups we studied, enabled by the electric lights and temperature control that restored aspects of natural conditions in the tropical latitudes. 34 Kräuchi K. The thermophysiological cascade leading to sleep initiation in relation to phase of entrainment. Sleep Med. Rev. 11 : 439-451 35 Fronczek R. Overeem S. Lammers G.J. van Dijk J.G. Van Someren E.J. Altered skin-temperature regulation in narcolepsy relates to sleep propensity. Sleep. 29 : 1444-1449 36 Raymann R.J.E.M. Swaab D.F. Van Someren E.J.W. Skin deep: enhanced sleep depth by cutaneous temperature manipulation. Brain. 131 : 500-513 37 Van De Werken M. Giménez M.C. De Vries B. Beersma D.G. Van Someren E.J. Gordijn M.C. Effects of artificial dawn on sleep inertia, skin temperature, and the awakening cortisol response. J. Sleep Res. 19 : 425-435 27 Santhi N. Thorne H.C. van der Veen D.R. Johnsen S. Mills S.L. Hommes V. Schlangen L.J.M. Archer S.N. Dijk D.J. The spectral composition of evening light and individual differences in the suppression of melatonin and delay of sleep in humans. J. Pineal Res. 53 : 47-59 We found that nocturnal sleep in all groups occurred toward the end of the night, during the period of lowest ambient temperatures. In nature, the daily rhythm of environmental temperature is tightly locked to the rhythm of sunset and sunrise. However, in most industrial societies, the seasonal and circadian temperature rhythms are greatly attenuated by insulated buildings and artificial heating and cooling. The synchronization that we observed between the reduction in ambient temperature at night and sleep under traditional conditions, with its associated decline in core temperature [], may have evolved to save energy by reducing the temperature differential between body and environment and consequent heat loss. Being active during the late night period of lowest temperatures would be metabolically costly. Individuals in groups like those we observed may be less vulnerable to insomnia because they are exposed to a falling ambient temperature at the time of sleep onset and do not have to actively shed heat to achieve the body temperature reduction that accompanies sleep onset []. The daily reduction in light is followed by the daily reduction in temperature. The delayed melatonin response to darkness is adaptive in facilitating sleep after darkness [], bringing the entire sleep period in synchrony with the lowest nighttime temperatures. Our findings indicate that sleep in industrial societies has not been reduced below a level that is normal for most of our species’ evolutionary history. Recreating aspects of the environments that we observed in preindustrial societies might have beneficial effects on sleep and insomnia in industrial populations. The team asked 94 people from these groups to wear Actiwatch-2 devices, which automatically recorded their activity and ambient-light levels. The data revealed that these groups all sleep for nightly blocks of 6.9 and 8.5 hours, and they spend at least 5.7 to 7.1 hours of those soundly asleep. That’s no more than what Westerners who have worn the same watches get; if anything, it’s slightly less. They don’t go to sleep when it gets dark, either. Instead, they nod off between 2 and 3 hours after sunset, well after it becomes pitch-black. And they napped infrequently: The team scored “naps” as periods of daytime inactivity that lasted for at least 15 minutes, and based on these lenient criteria, the volunteers “napped” on just 7 percent of winter days and 22 percent of summer ones. The volunteers also slept continuously. They would toss and turn like everyone does, but they almost never woke up for a concerted window in the middle of the night. This contradicts a growing idea, popularized by historian Roger Ekirch, that sleeping in eight-hour chunks is a modern affectation. Ekirch combed through centuries of Western literature and documents to show that Europeans used to sleep in two segments, separated by an hour or two of wakefulness. Siegel doesn’t dispute Ekirch’s analysis; he just thinks that the old two-block pattern was preceded by an even older single-block one. “The two-sleep pattern was probably due to humans migrating so far from the equator that they had long dark periods,” he says. “The long nights caused this pathological sleep pattern and the advent of electric lights and heating restored the primal one.” Of course, the Hadza, San, and Tsimane are not ancestral humans. They might live traditional lifestyles, but they are modern people living in today’s world. That’s why Siegel studied all three of them. “If I recorded from just one group, an obvious criticism would be: How do you know this group is typical of our ancestors?” he says. “But given that these groups have different cultures and are isolated from each other, the commonality suggests that this is the basic human wiring, and reflects how humans sleep in the natural environment.” Horacio de la Iglesia from the University of Washington disagrees that these short sleep durations are a “signature of human ancestral sleep,” noting that some other hunter-gatherer communities sleep for up to 9 hours. And “there is plenty of evidence that industrialization has indeed reduced sleep,” he adds. In one of his own studies, a group of Argentinian hunter-gatherers with access to electric lights got an hour less sleep every day than a neighboring community that relied only on natural lighting. (By contrast, Siegel’s study lacked a similar control group.) Even if Siegel is right, that doesn’t mean that our sleeping patterns have been unaffected by modern lifestyles. After all, his team found that insomnia, a common affliction of Western society, is almost non-existent in the three groups. Neither the San nor Tsimane even have a word for insomnia in their language. Why? Up Late? Looks Like Our Paleo Ancestors Didn't Sleep Much Either Enlarge this image Oivind Hovland/Getty Images/Ikon Images Oivind Hovland/Getty Images/Ikon Images In America, it seems only unicorns get seven or eight hours of sleep a night, and the rest of us suffer. But people may be meant to sleep as little as 6 1/2 hours nightly and were doing so long before the advent of electricity and smartphones, researchers say. To find that out, they consulted with some of the few people on the planet who live roughly the same lifestyle humans did in the Paleolithic. Psychiatrist and sleep researcher Jerome Siegel at UCLA's Semel Institute of Neuroscience and Human Behavior started studying three different hunter-gatherer groups in Africa and South America. "All three don't have any electricity, don't have any of the sort of modern electronic developments that many think have reduced our sleep," he says. Those hunter-gatherers spent about seven or eight hours a night in bed, but they slept for just five to seven of those hours, according to the study, published Thursday in Current Biology. "It's clear that the amount of sleep that all of these groups get is at the low end of what we'd see in the United States today," Siegel says. Sleeping that little has been linked to everything from shorter life span to stomach problems and weight gain in industrial societies. But unlike many people in the United States or Europe who sleep less than seven hours a night, members of the Hadza in Tanzania, San in Namibia, and Tsimane in Bolivia tend to be very healthy. There's virtually no obesity, many have very long lives, and nearly everyone in these societies does not have trouble sleeping. "Approximately 20 percent of our population complains of chronic insomnia at some point," Siegel says. "The two groups we quizzed on this don't have a word for insomnia." That raises a lot of questions about why we think we need eight hours of shuteye. "That classic teaching that adults need seven or eight hours of sleep has to do with population-based evidence," says Dr. Indira Gurubhagavatula, a sleep expert at the University of Pennsylvania Perelman School of Medicine who was not involved with the study. "This paper questions, is that data flawed? And if so, how or why? Or it could be that the sleep we're getting is lower quality, and we need more of it to feel restored?" Siegel thinks that might be because we evolved in the environment's natural 24-hour pattern of light and temperature, but we're cut off from that rhythm now. By contrast, these hunter-gatherers go to sleep a few hours after sunset, when the night gets chilly. They wake up when the day begins warming from the sunrise. Following Earth's natural tempo in this way could improve the quality of their sleep, says Kristen Knutson, a sleep researcher and biomedical anthropologist at the University of Chicago. Our bodies' core temperature also cycles this way, regardless of air conditioning or heating. "If their sleep is following the environment's temperature rhythm more closely and naturally, then their sleep quality may indeed be better than what is happening in the United States," she says. Researchers already know that light and temperature play an important role in sleep. Light can reverse jet lag and help set internal clocks, and people fall asleep more easily when their core body temperature falls. This all could contribute to why hunter-gatherers' sleep less than we do on average, Gurubhagavatula says. And it could also mean that many non-hunter-gatherers may not need to sleep eight or more hours a night. "I think the beauty of this current study is that maybe we shouldn't be ramming this requirement down [every person's] throat so to speak," she says. That's not to say that there aren't lots of people who are incredibly sleep-deprived, Gurubhagavatula says. Light and temperature aren't the only things dictating how much we sleep. "It's our activity and diet and stress level. I see patients who are single parents and have three jobs, and they'll be lucky to have five hours of sleep and are tired all the time." Those people need more sleep. There are other habitual short sleepers in our society — truck drivers, graduate students, and idiot reporters who should know better — with lifestyles vastly different from a hunter-gatherer. "[They're] not the same as someone in our society who only sleeps 6 1/2 hours," says Dr. Elizabeth Klerman, a sleep researcher at the Harvard Medical School and Brigham and Women's Hospital in Boston. So it could be unhealthy for people in industrial societies to sleep that little. What's natural for a hunter-gatherer might not be natural for everyone, Siegel agrees. "I don't think we could just fling someone back into an equatorial lifestyle, and that'll be entirely beneficial," he says. But he's excited about other possibilities. If hunter-gatherers are sleeping better because they're more in tune with the daily temperature cycle, maybe we can do the same by programming thermostats to echo conditions outside. "That's a specific aim of my next grant," he says. Angus Rohan Chen is a reporter and radio producer living in New York City. He has a dry wit and no hobbies. Please be his friend on Twitter @angRchen.

You've heard of the Paleo diet, but the next big thing in health may well be the Paleo sleep schedule. A UCLA researcher studied three hunter-gatherer and hunter-farmer groups-the Hadza in Tanzania, San in Namibia, and Tsimane in Bolivia, "who live roughly the same lifestyle humans did in the Paleolithic," as NPR reports-and determined our ancient ancestors may not have slept nearly as much we thought, and may have actually slept less than modern Westerners. "People like to complain that modern life is ruining sleep, but they're just saying: Kids today!" Jerome Siegel tells the Atlantic. "It's a perennial complaint but you need data to know if it's true." Siegel found that members of the three aforementioned groups sleep between 5.7 hours and 7.1 hours per night. That's less than is recommended for our health, yet the groups seemed very healthy indeed. Siegel tells NPR members of those three groups have long lives, very little obesity, and no trouble sleeping. "Two groups we quizzed on this (the San and Tsimane) don't have a word for insomnia," he says. Contrary to popular belief, Siegel found these groups don't go to bed when it gets dark and wake up with the sun. Instead they go to bed a few hours after dark when the night starts cooling off and begin waking up when the night is at its coldest point, just as the sun begins to warm things up. "The daily cycle of temperature change, largely eliminated from modern sleep environments, may be a potent natural regulator of sleep," states Siegel's study, published Thursday in Biology. Siegel points out that what works for hunter-gatherers may not work for the rest of us, but it can point us in the direction of getting better sleep, NPR reports.

multi_news

The insulin/IGF signaling pathway is a highly conserved regulator of metabolism in flies and mammals, regulating multiple physiological functions including lipid metabolism. Although insulin signaling is known to regulate the activity of a number of enzymes in metabolic pathways, a comprehensive understanding of how the insulin signaling pathway regulates metabolic pathways is still lacking. Accepted knowledge suggests the key regulated step in triglyceride (TAG) catabolism is the release of fatty acids from TAG via the action of lipases. We show here that an additional, important regulated step is the activation of fatty acids for beta-oxidation via Acyl Co-A synthetases (ACS). We identify pudgy as an ACS that is transcriptionally regulated by direct FOXO action in Drosophila. Increasing or reducing pudgy expression in vivo causes a decrease or increase in organismal TAG levels respectively, indicating that pudgy expression levels are important for proper lipid homeostasis. We show that multiple ACSs are also transcriptionally regulated by insulin signaling in mammalian cells. In sum, we identify fatty acid activation onto CoA as an important, regulated step in triglyceride catabolism, and we identify a mechanistic link through which insulin regulates lipid homeostasis. The insulin/IGF signaling (IIS) pathway is a highly conserved and critical regulator of metabolism in mammals and in flies, where it senses organismal nutrient levels to regulate multiple physiological functions including carbohydrate metabolism, tissue growth and longevity [1]–[3]. Insulin regulates carbohydrate metabolism by controlling expression and activity of a number of metabolic enzymes such as phosphofructokinase-2, PEPCK, Glycogen synthase and Glycogen phosphorylase [4]. Conditions of altered insulin signaling are associated not only with changes in carbohydrate metabolism, but also with abnormal lipid metabolism, as in the cases of Type 2 Diabetes-associated obesity and Non-Alcoholic Hepatic Steatosis [5], [6]. A large body of evidence suggests that insulin resistance plays a central, causal role in the development of the lipid imbalances observed in both of these conditions [5], [6], however the molecular mechanisms leading to these lipid imbalances are not completely understood. This raises the need to better understand the molecular connections between insulin signaling and lipid metabolism. The molecular relationship between insulin signaling and lipid homeostasis is complex, as dyslipidemia is considered to be both a cause and a consequence of insulin resistance [6]. That said, IIS clearly plays a causative role in regulating the balance of lipid production versus breakdown in animals, since mice and flies in which IIS has been specifically manipulated have altered lipid metabolism ([7], [8] and reviewed in [9]–[11]). The molecular mechanisms by which IIS regulates lipid metabolism are only partially understood. On the one hand, IIS promotes fatty acid biosynthesis [12], [13]. On the other, IIS regulates fatty acid catabolism [13], [14]. Fatty acid catabolism is a multi-step process (Figure 1A). First, fatty acids are mobilized from stored triacylglycerols (TAG) via the activity of lipases to yield free fatty acids. Second, the free fatty acids are activated by coupling to Coenzyme A (CoA). This step is catalyzed by the acyl-CoA synthetase (ACS) family of enzymes. Third, the free fatty acids are imported into mitochondria. Finally, in mitochondria, the fatty acids are oxidized, yielding energy. Some of the steps in this catabolic pathway are known to be regulated by IIS. For instance, IIS inhibits expression and activity of lipases such as adipose triglyceride lipase and hormones sensitive lipase [15], [16]. IIS also decreases the rate of fatty acid entry into mitochondria [17] in part via a FoxO-dependent process [18]. A complete molecular understanding of how IIS regulates fatty acid catabolism, however, is currently lacking. The upstream signaling events of the IIS pathway are fairly well characterized. Activation of insulin/IGF receptor (s) leads to a relay of phosphorylation events activating a number of kinases including PI3K, Akt/PKB, TOR-C1 and S6K, thereby inhibiting a key transcription factor FOXO (for review [19]). A challenge in the field remains, however, to obtain a complete understanding of how these upstream ‘signaling’ components of the IIS pathway link to, and regulate, the metabolic biochemical pathways controlling cellular metabolism. Discovering the connections between the signaling components of the insulin pathway and the metabolic enzymes controlling cellular biochemical pathways remains an important step in understanding how IIS controls metabolism generally, and lipid metabolism in particular. We identify here an ACS which we term pudgy, as a gene that is strongly upregulated upon fasting in Drosophila. We find that pudgy is a target of the insulin signaling pathway, as its expression is suppressed by insulin signaling, as a consequence of direct regulation by FOXO. We find that animals with reduced levels of pudgy expression are hyper-triglyceridemic and have defects in their lipid usage upon fasting. This suggests that in order to effectively channel fatty acids towards beta-oxidation upon fasting conditions, organisms need to induce both the lipolysis of fatty acids from TAG, as well as the activation of fatty acids at mitochondria for beta-oxidation. Finally, we show that expression of multiple mammalian ACSs are also regulated by insulin signaling in mouse muscle, liver and adipose cells. In sum, this work uncovers fatty acid activation by ACSs as a novel and important insulin-regulated step in TAG catabolism. We previously studied the transcriptional output of insulin signaling in Drosophila by performing microarray analyses on fasted versus fed animals [20]. By comparing wildtype versus FOXO mutant animals, we pinpointed genes that are regulated in a FOXO-dependent manner [20]. In this and similar studies by other groups [21]–[24], a number of acyl-CoA synthetases (ACSs) were found to be regulated by nutrient status. In particular, the ACS gene CG9009 emerged in our analysis as a strongly regulated gene, which we characterize further here. Quantitative RT-PCR analysis on wildtype larvae shows that expression of CG9009, which we term here pudgy (pdgy), is very strongly up-regulated in the fat body upon 18 hours of fasting, increasing 110-fold (Figure 1B). (The Drosophila fat body performs the functions of mammalian adipose tissue and liver combined.) In contrast, in FOXO21/25 null mutant larvae, expression of pudgy only increases 3. 4-fold in the fat body upon fasting, indicating that the up-regulation of pudgy is strongly FOXO dependent (Figure 1C, note different scale compared to Figure 1B). Pudgy expression behaved similarly in muscle (Figures 1B and 1C). Pudgy expression could either be regulated directly or indirectly by FOXO. To distinguish these possibilities, we performed a bioinformatic scan of the promoter region of the pudgy gene, as we previously showed that functional FOXO binding sites in Drosophila are usually clustered within a few kilobases of the transcription start site of regulated genes [20]. The pudgy promoter region had a significant number of consensus FOXO binding sites – 3 perfect (GTAAACAA) and 3 imperfect (1 mismatch in the 1st or 2nd position) (indicated by asterisks in Figure 1D). We first tested whether this region is able to serve as a FOXO-responsive cis-regulatory enhancer element. Test genomic regions were linked to a basal promoter directing luciferase expression in S2 cells. As a positive control, a genomic region of the 4E-BP gene, an established direct target of FOXO [25], [26], was able to induce luciferase activity in response to FOXO expression (Figure 1E). Likewise, an 800 bp fragment of the pudgy region, containing 3 of the 6 FOXO binding sites, induced luciferase activity in response to FOXO expression (Figure 1E), suggesting it is a bona fide FOXO response element. Next, to test whether endogenous FOXO binds these sites in vivo, we performed chromatin immunoprecipitations (ChIP) of endogenous FOXO from 3rd instar larvae. We performed two negative controls: a mock ChIP using pre-immune serum on wildtype larval lysates, as well as a ChIP using anti-FOXO antibody [26] on lysates of FOXO21/25 null mutant larvae (Figure 1F) [23]. Quantitative PCR on the immunoprecipitated material revealed that the promoter region of 4E-BP was strongly enriched in the FOXO ChIP from wildtype larvae compared to the negative control ChIPs (ttest<0. 001, Figure 1F, black bars versus grey bars). Likewise, two test regions in the first intron of pudgy, P1 and P2 (Figure 1D), were also significantly enriched in the FOXO ChIP compared to the negative control ChIPs (ttest<0. 05 for P1 and ttest<0. 01 for P2, Figure 1F). As negative controls, the genomic regions of mir-278 and sty were not enriched in the FOXO ChIP compared to control ChIPs (Figure 1F). Together, these data indicate that FOXO binds the pudgy promoter region in vivo. In sum, this identifies pudgy is a bona fide direct FOXO target. Since FOXO activity is repressed by insulin signaling, pudgy expression should also be repressed by insulin. Indeed, pudgy expression was reduced in explants of both fat body tissue and muscle tissue when they were treated with insulin (ttest<0. 001, Figure 1G). Moreover, in vivo, insulin signaling drops when larvae have terminated feeding and start wandering out of the food. Consistent with this, pudgy expression was 4-fold higher in wandering 3rd instar larvae (wL3) compared to feeding 3rd instar larvae (fL3) (Figure 1H). Previous computational analyses identified CG9009/pudgy as a gene encoding an acyl-CoA synthetase (ACS) [27]. ACSs are a family of enzymes which activate free fatty acids for subsequent anabolic or catabolic reactions by loading them onto CoA. Each member of this family has distinct substrate specificity, loading fatty acid molecules of different lengths or saturation onto CoA [27]. In addition, each member of the ACS family has a distinct intracellular localization. This is particularly relevant in lieu of that fact that subsequent reactions involving activated acyl-CoA molecules take place in distinct subcellular compartments. Fatty acid oxidation occurs either in mitochondria in the form of beta-oxidation, or in peroxisomes. In contrast, anabolic reactions take place predominantly in the cytoplasm or endoplasmic reticulum. Thus the subcellular localization of each member of the ACS family may influence the fate of the acyl-CoA molecules that it generates [28]. By channeling fatty acids towards downstream anabolic or catabolic processes, ACSs such as Pudgy have the potential to influence the fate of the fatty acids and the overall balance of organismal lipid homeostasis [29], a hypothesis which we test here. To confirm that the protein encoded by pudgy is indeed an ACS, we recombinantly expressed and purified His-tagged pudgy from E. coli and found that it has acyl-CoA synthetase activity in vitro (Figure 2A). Pudgy is expressed in all tissues of the larva that we tested (Figure 2B). Since the localization of ACSs influences their function, we investigated the subcellular localization of pudgy. Expression of a C-terminal epitope-tagged version of pudgy in S2 cells revealed that it co-localizes with a GFP construct marking mitochondria (mitoGFP) (Figure 2C), suggesting pudgy may load fatty acids onto CoA for mitochondrial beta-oxidation (see below). To study the physiological role of pudgy, we obtained flies containing a transposon insertion in the 5′ UTR of pudgy (P{GT1}BG02662, “pdgy[BG]”, Figure 1D). The pdgy[BG] mutation was back-crossed into the w1118 background for five generations (via females) in order to obtain two stocks with similar genetic backgrounds, differing by presence or absence of the pdgy[BG] mutation. The resulting stock carrying the pdgy[BG] mutation in the w1118 background was used for all subsequent experiments described here, and will be referred to as pdgy[BG] mutant flies, whereas the w1118 flies will be referred to as controls. pdgy[BG] homozygous larvae and adults have strongly reduced expression of pudgy, measured by quantitative RT-PCR (Figure 2D and 2D′ respectively). We believe this animal model may not represent a complete pudgy null situation, but is a good model for studying the physiological effects of strongly reduced pudgy function. To test whether pudgy is involved in fatty acid oxidation, we measured oxygen consumption in control and pdgy[BG] mutant larvae using a Clark electrode. In the absence of drugs, oxygen consumption in pdgy[BG] mutant larvae was significantly reduced compared to controls (Figure 2E). Subsequent addition of etomoxir, a specific inhibitor of Carnitine palmitoyltransferase I (CPTI) [30], required for the transport of fatty acids into mitochondria where beta-oxidation takes place, causes this difference in oxygen consumption to be abrogated (300 µM etomoxir, Figure 2E). This indicates that the difference in oxygen consumption between pdgy[BG] mutants and controls is due to differential mitochondrial lipid oxidation. Subtraction of the basal rate of oxygen consumption in the presence of 300 µM etomoxir from the oxygen consumption in the absence of etomoxir, yields the rate of CPTI-dependent oxygen consumption, revealing that pdgy[BG] mutants have significantly reduced ß-oxidation levels compared to controls (Figure 2E′). Conversely, overexpression of pudgy in larvae was sufficient to increase the rate of fatty acid beta-oxidation (Figure S1). The above-mentioned data indicate that insulin/IGF signaling modulates pudgy expression in vivo. Therefore, we asked whether modulation of pudgy expression has an impact on organismal lipid homeostasis. We first tested the effect of increasing pudgy expression. Ubiquitous over-expression of pudgy from a transgene using the GAL4/UAS system [31] was sufficient to cause a significant reduction in organismal triglyceride levels both in larvae and in adults (Figure 3A and 3A′ respectively). pdgy[BG] homozygous mutants are viable, fertile, and normally patterned (Figure S2A). Conversely to pudgy gain-of-function, pdgy[BG] mutant larvae and adults have significantly elevated triglyceride levels compared to controls (Figure 3B and 3B′ respectively). This phenotype was fully rescued in larvae and partially rescued in adults by introducing UAS-pudgy into the pdgy[BG] mutants, since the pdgy[BG] insertion is a GAL4 gene trap resulting in both pudgy loss-of-function as well as GAL4 expression (Figure 3B and 3B′). A comprehensive lipidomic analysis using Ultra Performance Liquid Chromatography coupled to mass spectrometry (UPLC-MS) of molecular lipid species in pdgy[BG] mutant versus control flies revealed that many, but not all, triglyceride species were significantly elevated in pdgy[BG] mutant adults (Table S1). The results for the 20 most abundant TAGs are shown in Figure 3C. In addition, levels of some other complex lipids, such as cholesteryl ester (19∶0), were also elevated in pdgy[BG] mutants (Table S1). The increased adiposity of pdgy[BG] mutants is consistent with the reduced levels of fatty acid oxidation observed in these animals (Figure 2E and 2E′). Furthermore, Pudgy mutants do not ingest more than control animals (Figure S3A and S3A′) and have reduced expression of key lipogenic genes such as Acetyl-CoA Carboxylase (ACC) and Fatty Acid Synthase (FAS) (Figure S3B and S3B′), suggesting that mutant animals may be trying to compensate for their increased adiposity. These results are analogous to those observed in ACSL1 knockout mice, which have elevated fat mass [32]. Together, they indicate that the level of expression of ACSs is important for setting steady-state lipid levels both in flies and in mammals. We next studied the physiological consequences of impaired pudgy expression in flies upon fasting. Upon complete food withdrawal, pdgy[BG] mutants survived significantly longer compared to controls (Figure 4A and Figure S3C). This is likely due in part to the increased adiposity of pdgy[BG] mutants, as starvation survival is known to correlate with lipid levels in the fly [25], [33]–[35]. Additionally, this could also be due in part to a reduced rate of lipid catabolism which is nonetheless sufficient to support viability. We therefore tested whether lipid catabolism might also be impaired in pdgy[BG] mutants, as they have reduced fatty acid oxidation. Upon food removal, control flies progressively catabolized their triglyceride stores. After 6 hours of fasting, both control larvae and control adult flies significantly reduced their triglyceride stores (Figure 4B and 4B′, grey curves). Control larvae reproducibly displayed an unexpected transient increase in stored triglycerides after 2 hours of fasting before starting to deplete them (Figure 4B). In contrast, pdgy[BG] mutants did not show any reduction in triglyceride levels the first 6 hours of starvation (Figure 4B and 4B′, black curves). Only as of 8 hours of starvation did pdgy[BG] mutants start depleting their triglycerides stores, completely depleting them by 36 hours of fasting (Figure 4B, 4B′ and Figure S3D), indicating that after an initial period, they were nonetheless able to catabolize lipids. Similar defects could also be observed by staining fat bodies of control and pdgy[BG] mutants with Nile Red (Figure 4C). Interestingly, both the extended survival upon food withdrawal as well as the delay in triglyceride consumption the first 6 hours of fasting are also observed in mutants for another gene involved in lipid catabolism - the fly homolog of adipocyte triglyceride lipase, brummer [24], [36]. To study lipid catabolism in pdgy[BG] mutants in more detail, we performed quantitative lipidomic profiling of fed versus fasting flies. Since the direct substrates of ACS action are free fatty acids, we first quantified free fatty acids in pdgy[BG] mutant and control animals (Figure 4D). Upon starvation, levels of free C14: 0, C16: 0 and C16: 1 drop in control animals (Figure 4D). Since levels of free fatty acids reflect the balance between fatty acid lipolysis and fatty acid ligation to CoA (Figure 1A), this indicates that upon starvation ACSs become activated in order to handle the increased production of free fatty acids coming from triglyceride lipolysis. In contrast, in pdgy[BG] mutants, levels of free C14: 0 and C16: 0 remained aberrantly high (Figure 4D), as expected from impaired ACS activity in the pudgy mutants. Defects were only apparent in a subset of free fatty acids (Figure 4D) suggesting that the metabolism of all fatty acids might not be affected equally by loss of pudgy in vivo. We next performed quantitative lipidomic profiling to detect all TAG species in fed versus fasting control and pdgy[BG] flies. Although many TAG species are normally catabolized in pdgy[BG] mutants (Table S2), some species are not. For instance, levels of TAG (39∶1) dropped in control animals upon fasting but remained elevated in pdgy[BG] mutants (Figure 4E), whereas TAG (53∶3) remained constant in control animals but dropped in pdgy[BG] mutants (Figure 4E′). Therefore, pudgy mutants display an altered profile in the catabolism of lipid species. Consistent with this, pdgy[BG] mutants have aberrant expression of a large number of putative lipases, elongases and ACSs (Figure S4) suggesting that lipid catabolic pathways may be readjusting in response to loss of pudgy. In sum, our data indicate that pudgy mutants are initially defective in the catabolism of fatty acids, but after an initial period are able to catabolize all triglycerides, albeit with a different pattern compared to controls. Interestingly, although Pudgy is an enzyme involved in lipid metabolism, we found that pudgy mutants also have a number of other non-lipid phenotypes. Pudgy mutants had significantly reduced expression of insulin-like peptides (Figure 5A and 5A′). Correspondingly, they had elevated expression of 4E-BP, a direct FOXO target, consistent with reduced insulin signaling in these animals (Figure 5A and 5A′). Pudgy mutants also have two phenotypes associated with reduced insulin signaling: they are mildly, but significantly reduced in size compared to controls (Figure 5B and 5B′) and they are long-lived (Figure 5E). In addition, pudgy mutants also have reduced glycogen stores (Figure 5C and 5C′) and increased circulating sugars (Figure 5D and 5D′) suggesting elevated mobilization of carbohydrates. Conversely, pudgy overexpression leads to reduced circulating sugars (Figure 5F). Although these phenotypes are not the focus of this story, and we do not know their underlying molecular mechanisms, they are worth noting as they probably represent crosstalk mechanisms in pdgy[BG] animals caused by their elevated lipid stores and reduced lipid oxidation, of interest for future studies. We next asked whether our two central observations from Drosophila—that insulin signaling regulates ACS expression and that ACS expression levels are important for lipid homeostasis—can also be observed in a mammalian context. To this end, we treated three different cell types, 3T3-L1 adipocytes, Hepa1. 6 hepatocytes and C2C12 myotubes, representing three different tissues of metabolic importance, in the presence or absence of insulin, and measured by quantitative RT-PCR the expression of all medium-chain, long-chain and very-long-chain ACSs. Reported in Figure 6 are the ACSs who' s transcription was regulated in a manner similar to that of pudgy, i. e. repressed by insulin. In addition, other ACSs were either not transcriptionally regulated by insulin, or were induced by insulin (Table S3A and S3B). In 3T3-L1 adipocytes, expression of six different ACSs was up-regulated upon removal of serum (Figure 6A). This up-regulation was suppressed if insulin was supplied upon serum removal, indicating that the up-regulation was specific for insulin signaling (Figure 6A). In particular, expression of ACSL4 increased very strongly, 12-fold, within the short 1-hour time window of serum removal (Figure 6A). Likewise, expression of 6 different ACSs increased in an insulin-dependent manner in Hepa1. 6 hepatocytes upon serum removal, with ACSVL5 increasing 13-fold (Figure 6B). Although some ACSs are similarly regulated by insulin in both cell types, such as ACSL1, other ACSs are specifically regulated in one cell type or the other, probably reflecting the specific function of each tissue. Finally, a number of ACSs were also regulated by insulin in C2–C12 myotubes (Figure 6C). (Since C2–C12 myoblasts are differentiated into myotubes by culturing in low-serum conditions, the ‘control’ and ‘serum-deprived’ conditions are similar in gene expression.) To test whether the level of expression of ACSs in 3T3-L1 adipocytes affects lipid homeostasis, we knocked down expression of three different ACSs – ACSL1, ACSL3 and ACSL4. Knockdown of ACSL1 and ACSL3 using siRNAs caused reduced triglyceride storage in differentiated 3T3-L1s (Figure 6D) in a manner that correlated with relative knock-down efficiency (Figure S5A, S5B). This is consistent with previous reports that ACSL1 promotes fatty acid uptake and incorporation into TAG in 3T3-L1s [37], [38]. Using a different approach, 3T3-L1s expressing an shRNA targeting ACSL4 also had reduced triglyceride storage (Figure 6E and Figure S5C). Fatty acid (FA) catabolism represents an important energy yielding mechanism for cells and organisms, contributing up to 50–60% of a person' s energy expenditure under aerobic exercise conditions [39]. Fatty acid catabolism can be envisioned in two steps (Figure 6F). First, fatty acids are mobilized from stored triacylglycerols (TAG) via the activity of lipases to yield free fatty acids. Second, the free fatty acids are oxidized, yielding energy. Traditionally, textbook knowledge considers the first step – mobilization via lipases – to be the important regulated step. However, several lines of reasoning suggest that lipolysis cannot be the only important regulated event in the fatty acid catabolic pathway. Firstly, liberation of free fatty acids from TAG does not necessarily channel them towards beta-oxidation. Free fatty acids can have several fates, including not only beta-oxidation but also fatty acid elongation (yielding very long chain fatty acids) and re-esterification to generate complex lipids including TAG [40], [41]. In fact, a large fraction of FAs liberated from TAG participate in a ‘futile’ cycle, being re-esterified to generate new TAG [42]. Quantitative estimates of the triglyceride/fatty acid cycle in humans and in animals show that only a small fraction of the FFA released as a result of lipolysis in adipose tissue are oxidized, and the majority are re-esterified to triglycerides in various tissues [43]. Secondly, elevated levels of free FA are believed to be deleterious to animals, causing lipotoxicity and contributing towards insulin resistance [40]. Therefore, increased FA levels due to increased lipolysis without concurrent upregulation of downstream biochemical pathways might actually be noxious to the animal. We identify here the subsequent step in fatty acid catabolism - coupling of fatty acids to CoA via ACSs - as an additional, important regulated step in lipid catabolism. A priori, it was possible that the level of expression of pudgy in vivo was not limiting for lipid oxidation, and that lipid catabolism in Drosophila is only regulated by availability of free fatty acids via lipolysis. However, our data suggest this is not the case. Both a reduction and an increase in pudgy levels effects total lipid levels in the fly (Figures 3A, 3A′, 3B, 3B′), indicating that regulation of pudgy levels contributes significantly to total body lipid homeostasis. This makes sense in light of the fact that free fatty acids can have multiple different fates once released from triglycerides, such as beta-oxidation or re-esterification to form triglycerides. Therefore the relative activities of biochemical reactions downstream of lipolysis are important for determining the fate of the released fatty acids. In particular, the balance in expression and activity of ACSs that activate fatty acids for beta-oxidation versus lipid biosynthesis may be of particular importance. In Drosophila, upon starvation, FOXO upregulates expression of the fly adipocyte triglyceride lipase homolog, brummer [24]. By upregulating expression of both brummer and pudgy, FOXO mounts a concerted effort towards channeling fatty acids from their stored form towards beta-oxidation. It may appear surprising that blocking fatty acid ß-oxidation via mutation of pudgy leads to increased TAG levels in the animal, since lipolysis is often considered to be the key step in regulating TAG levels. Indeed, via the actions of lipases and acyl-transferases, fatty acids cycle between a free form and a stored TAG form (Figure 6F), however neither of these enzymatic activities either creates or destroys fatty acids. The steady-state level of fatty acids in an organism depends only on the relative balance of fatty acid synthesis/uptake versus fatty acid oxidation. Therefore, reducing ß-oxidation increases total organismal fatty acids. Since free fatty acids are in equilibrium with the stored TAG form, this entails an increase in TAG levels (Figure 6F). An alternate interpretation of our data is that the observed delay in TAG consumption reflects a reduced global metabolic rate caused indirectly by lack of pudgy activity. We believe this interpretation is unlikely, because a global redution in metabolic rate would be expected to lead to a concomitant increase in the levels of both stored lipids and stored carbohydrates (ie glycogen). Pudgy mutants, however, have elevated lipids levels but reduced glycogen levels, suggesting a lipid-specific defect in accordance with pudgy' s ACS function. Insulin/IGF signaling is known to control lipid biosynthesis in part via SREBP1, and lipid catabolism via regulation of lipases such as hormone sensitive lipase and via decreasing the rate of fatty acid entry into mitochondria [15], [44], [45]. We identify here the ACS CG9009/pudgy as one molecular link between the insulin signaling pathway and lipid catabolism in Drosophila. We find that pudgy is a transcriptional target gene of the insulin pathway which is directly regulated by FOXO. By repressing pudgy expression, insulin blocks the channeling of fatty acids towards the beta-oxidative pathway. Insulin has been reported to induce expression of two ACSs in mammals - ACSL5 via a mechanism involving SREBP1c [46], and ACSL6 via an unknown mechanism [47] – however to our knowledge pudgy is the first example of an ACS which is repressed by insulin. Likewise, although pudgy belongs to a clade of ACSs that does not also include human paralogs, we identify a number of human ACSs that are transcriptionally repressed by insulin in mammalian cells, analogously to pudgy. We find that pudgy mutants have a significant number of metabolic alterations. For instance, in addition to the changes in lipid metabolism, we find that pudgy mutants have reduced glycogen stores and increased circulating sugars. Although the underlying mechanism is unclear, one plausible explanation is that pudgy mutants need to rely more on glucose mobilization to maintain cellular energy levels, to compensate for reduced fatty acid beta-oxidation, which is normally a significant energy source. We also find that pudgy mutants have a different profile of lipid homeostasis and starvation-induced catabolism compared to controls. Under fed conditions, some lipid species in pudgy mutants are highly elevated, such as TAG (50∶1) which is almost 3-fold the normal levels, whereas others such as TAG (42∶0) are unperturbed (Figure 3C). Likewise, during starvation, the catabolism of lipid species is altered, with some TAGs being catabolized more readily and some less readily compared to controls (Figure 4E and 4E′). Fatty acid species are linked to each other via a complex network of biochemical pathways involving saturases, desaturases, elongases, ACSs, lipases, etc. This ‘landscape’ of lipid species is clearly perturbed by removal of pudgy. This perturbation might be partly a direct consequence of loss of pudgy, and partly an attempt of the system to compensate. Indeed, at the gene expression level, a very large proportion of genes with putative functions in fatty acid metabolism are altered in pudgy mutants, suggestive of compensatory mechanisms (Figure S4). For instance, the elongase eloF is more than 2-fold up-regulated in the pudgy mutant, and the ACS CG6432 is dramatically down-regulated. In sum, we identify here the ACS pudgy as a transcriptional target of insulin signaling, and show that modulation of pudgy expression levels causes changes in steady-state lipid levels in the fly. Mammalian tissue culture experiments suggest similar mechanism may be at work in mammalian cells. A list of oligos used for clonings and quantitative PCRs can be found in Supplemental Materials & Methods (Text S1). Additional oligos sequences are available upon request. UAS-pudgy was generated by cloning the CG9009 coding sequence, obtained by RT-PCR as an XhoI-XbaI fragment, into the XhoI-XbaI sites of pUAST. The mito-GFP ORF, encoding the 31 amino acid mitochondrial import sequence from human cytochrome C oxidase subunit VIII fused to the N terminus of GFP, was amplified from flies carrying mito-GFP (Bloomington Stock Center, [48]) and cloned into pCasper4 carrying a tubulin promoter. For luciferase assays, the FOXO enhancer region of pudgy intron 1 was amplified as a KpnI-KpnI fragment and cloned into the KpnI site of a luciferase plasmid containing the Adh basal promoter, described in [20]. Remaining constructs for the FOXO luciferase assay were described previously [20]. FOXO21 and FOXO25 flies [23]; P{GT1}BG02662 flies and actin-GAL4 flies (Bloomington Stock Center). For all metabolic, respiratory, and longevity analyses, animals were reared under strictly controlled growth conditions. Eggs were collected on apple plates, and newly hatched L1 larvae were seeded in vials at a density of 60/vial and grown at 25°C without yeast supplementation. Adult flies were then aged 3 days for analysis. All assays were done in triplicate. Our fly food recipe is as previously reported [49]. Metabolic, starvation and longevity assays were performed as in [40] and as detailed in Text S1 (Supplemental Materials and Methods). Growth controlled w1118 and pdgy[BG] mutant males were aged 3 days, and then fed normal food or starved on 0. 8% agarose/PBS overnight (16 hours). The flies were frozen in liquid nitrogen and cryo-dried. The samples were then analyzed by UPLC-QTof-MS using an Acquity BEH C18 (1. 7 µm 2. 1×100 mm) column and electrospray ionization in positive ion mode. Details are provided in the Supplemental Materials & Methods (Text S1). His-tagged pudgy protein was obtained by cloning the coding sequence into pET23d, expressing it in BL21 E. coli, and purifying it using Ni-NTA Agarose beads (Qiagen). 4. 2 µg of recombinant pudgy-His, or an equivalent amount of eluate from a parallel purification using bacteria not expressing pudgy-His (circa 4. 3 mg), were added into reaction buffer (50 mM Tris–HCl pH 7. 8,10 mM sodium acetate, 4 mM ATP, 0. 15 mM CoA, 1 mM magnesium chloride, 10 mM DTT) with 10 nmol free fatty acid. After incubated at 37C for 30 min, the synthesized acyl-CoA was detected using the Free Fatty Acids Quantification Kit (Biovision), omitting the ACS incubation step. As a positive control, 4. 2 µg of ACS supplied with the kit was used. Growth-controlled, wandering third instar larvae were cleaned in cold PBS, dried on filter paper and weighed. Larvae were then dissected into ice-cold BIOS buffer (2. 77 mM CaK2EGTA, 7. 23 mM K2EGTA, 5. 77 mM Na2ATP, 6. 56 mM MgCl2·6H2O, 20 mM Taurine, 15 mM Na2Phospho-creatine, 20 mM Imidazole, 0. 5 mM DTT, 50 mM MES) and subsequently permeabilized with 4 mM digitonin in BIOS buffer for 15 min at 4°C in a shaker. Tissues were then resuspended in ice-cold FAO medium (110 mM NaCl, 4. 7 mM KCl, 2 mM MgSO4,1. 2 mM Na2HPO4,2. 5 mM glucose adjusted to pH 7. 4, supplemented with 0. 5 mM carnitine). Oxygen consumption was measured using a Clark electrode and normalized to animal body weight. Etomoxir was added (50 µM or 300 µM) to block acyl-CoA transport via CPTI. Detailed procedures of methods used are included in the Supplemental Materials & Methods (Text S1).

Type 2 diabetes, which is reaching epidemic proportions worldwide, is often associated with obesity and an imbalance in organismal lipid homeostasis. Therefore, understanding how insulin regulates lipid biosynthesis and breakdown is necessary. Surprisingly, the molecular mechanisms by which insulin regulates fatty acid catabolism are not entirely understood. We show here that insulin signaling regulates expression of acyl-CoA Synthetases (ACS). ACSs couple fatty acids to Coenzyme A, thereby activating them for subsequent biochemical reactions. In Drosophila, we find that insulin signaling modulates expression of one ACS called Pudgy, which activates fatty acids for beta-oxidation. Modulation of pudgy expression leads to changes in overall organismal lipid homeostasis. Likewise, we show that in mammalian cells insulin signaling regulates expression of a number of ACSs and that ACS expression modulates steady-state lipid levels.

lay_plos

SECTION 1. SHORT TITLE. This Act may be cited as the ``Extended Disaster Mental Health Services Act of 2001''. TITLE I--MENTAL HEALTH SERVICES PURSUANT TO PUBLIC HEALTH EMERGENCIES SEC. 101. GRANTS TO STATES AND POLITICAL SUBDIVISIONS FOR MENTAL HEALTH SERVICES AS RESPONSE TO PUBLIC HEALTH EMERGENCIES. Subpart 3 of part B of title V of the Public Health Service Act (42 U.S.C. 290bb-31 et seq.) is amended by adding at the end the following section: ``SEC. 520K. GRANTS TO STATES AND POLITICAL SUBDIVISIONS FOR MENTAL HEALTH SERVICES AS RESPONSE TO PUBLIC HEALTH EMERGENCIES. ``(a) In General.--The Secretary, acting through the Director of the Center for Mental Health Services, may make grants to States and political subdivisions of States for the purpose of providing the mental health services described in subsection (b) in response to public health emergencies, including diseases or disorders that present such emergencies, natural disasters, major transportation accidents, technological disasters, and disasters resulting from terrorism. ``(b) Services.--The mental health services referred to in subsection (a) with respect to a public health emergency are the following: ``(1) Crisis counseling in the aftermath of such emergency. ``(2) In the case of children, adolescents, and adults at risk of developing mental health disorders as a result of such emergency-- ``(A) outreach and screening programs to identify such individuals; and ``(B) early intervention services, including counseling. ``(3) Mental health services beyond such crisis counseling (referred to in this section as `extended therapeutic services') that-- ``(A) are provided to individuals with diagnosed mental health disorders resulting from or exacerbated by the emergency, including disaster survivors, family members of victims, first responders, and others with such disorders; and ``(B) are provided by mental health professionals who are licensed or otherwise regulated by a State agency. ``(4) Assessments of the need for extended therapeutic services. ``(5) Casefinding and other outreach services to inform the public of the availability of crisis counseling and extended therapeutic services. ``(c) Relation to Other Sources of Funding.--A condition for the receipt of a grant under subsection (a) is that the applicant involved agree as follows: ``(1) With respect to activities for which the grant is authorized to be expended, the applicant will maintain expenditures of non-Federal amounts for such activities at a level that is not less than the level of such expenditures maintained by the applicant for the fiscal year preceding the first fiscal year for which the applicant receives such a grant. ``(2) The grant will not be expended to make payment for the provision of extended therapeutic services for an individual to the extent that payment has been made, or can reasonably be expected to be made, for the services-- ``(A) under a State compensation program, under an insurance policy, or under a Federal or State health benefits program; or ``(B) by an entity that provides health services on a prepaid basis. ``(3) The grant will not be expended to make payment for the provision of mental health services to the extent that such services are available pursuant to responses to the public health emergency involved by the Federal Emergency Management Agency, or by other Federal or State agencies or programs that provide for emergency medical services. ``(d) Statewide Mental Health Disaster Plan.-- ``(1) In general.--For fiscal year 2003 or any subsequent fiscal year, a condition for the receipt of a grant under subsection (a) by a State or a political subdivision is that, in accordance with criteria established by the Secretary, the State has developed a statewide plan for the provision of mental health services in response to public health emergencies. The preceding sentence applies without regard to whether the State receives a grant under section 520L. ``(2) Certain criteria of secretary.--The criteria of the Secretary under paragraph (1) shall include criteria for coordinating the program under this section with programs of the Federal Emergency Management Agency and with other Federal or State programs regarding the provision of emergency medical services, including mental health services. ``(e) Administration of Grant Through State and Local Mental Health Agencies.--A condition for the receipt of a grant under subsection (a) is that the applicant involved agree that the grant and activities under the grant will be administered through the agency of the State or political subdivision (as the case may be) that has the principal responsibility for carrying out mental health programs. ``(f) Certain Requirements.--With respect to an application that, pursuant to section 501(l), is submitted to the Secretary for a grant under subsection (a), the Secretary may make the grant only if the application contains-- ``(1) a description of the purposes for which the applicant intends to expend the grant; ``(2) an assurance that the activities to be carried out under the grant are consistent with the State plan referred to in subsection (d)(1), as applicable, together with a description of the manner in which the grant activities will be coordinated with the State plan; ``(3) an assurance that the applicant will coordinate activities under the grant with other public or private providers of mental health services, together with a description of the manner in which the grant activities will be so coordinated; and ``(4) in the case of an application from a political subdivision, an assurance that the application was developed in consultation with the State agency referred to in subsection (e). ``(g) Duration of Grant.--The period during which payments are made to an applicant from a grant under subsection (a) may not exceed three years. The provision of such payments are subject to annual approval by the Secretary of the payments and to the availability of appropriations for the fiscal year involved to make the payments. This subsection may not be construed as establishing a limitation on the number of grants under such subsection that may be made to an applicant. ``(h) Technical Assistance.--The Secretary may, directly or through grants or contracts, provide technical assistance to grantees under subsection (a) in carrying out the purpose described in such subsection. ``(i) Funding.-- ``(1) Authorization of appropriations.--For the purpose of carrying out this section, there are authorized to be appropriated such sums as may be necessary for each of the fiscal years 2002 through 2006. ``(2) Allocation.--Of the amounts appropriated under paragraph (1) for a fiscal year, the Secretary may obligate not more than 7 percent for the administrative expenses of the Secretary in carrying out this section.''. TITLE II--STATEWIDE MENTAL HEALTH DISASTER PLANS SEC. 201. GRANTS TO STATES FOR STATEWIDE MENTAL HEALTH DISASTER PLANS. Subpart 3 of part B of title V of the Public Health Service Act, as amended by section 101 of this Act, is amended by adding at the end the following section: ``SEC. 520L. GRANTS TO STATES FOR STATEWIDE MENTAL HEALTH DISASTER PLANS. ``(a) In General.--The Secretary, acting through the Director of the Center for Mental Health Services, may make grants to States for the purpose of-- ``(1) developing, and periodically reviewing and as appropriate revising, statewide plans for providing mental health services in response to public health emergencies (including emergencies referred to section 520K(a)); ``(2) training personnel to implement such plan effectively; and ``(3) carrying out other activities determined appropriate by the Secretary to prepare for the provision of mental health services in response to such emergencies. ``(b) Certain Requirements.--A condition for the receipt of a grant under subsection (a) is that the State involved agree that the statewide plan under such subsection will with respect to public health emergencies include provisions for each of the following: ``(1) Providing the mental health services described in section 520K (relating to crisis counseling, outreach and screening programs, early intervention services, extended therapeutic services, needs assessments, and casefinding and other outreach services), taking into account the need for increased capacity to provide services pursuant to such emergencies. ``(2) As necessary, carrying out paragraph (1) with respect to special populations such as children, the elderly, individuals with disabilities, and individuals with pre- existing mental health disorders. ``(3) Coordinating the provision of mental health services with appropriate public and private providers of emergency medical services and with Federal, State, and local programs that provide funding for such services. ``(4) Coordinating with local educational agencies. ``(5) Providing information and education to the public during public health emergencies. ``(6) Providing, at times other than public health emergencies, information and education to the public regarding the statewide plan. ``(7) Designation of the State official who will have the principal responsibility for administering such plan, including the initial implementation of the plan. ``(c) Authorization of Appropriations.--For the purpose of carrying out this section, there is authorized to be appropriated $25,000,000 for each of the fiscal years 2002 through 2006.''. TITLE III--NATIONAL MENTAL HEALTH CRISIS RESPONSE TECHNICAL ASSISTANCE CENTER SEC. 301. NATIONAL MENTAL HEALTH CRISIS RESPONSE TECHNICAL ASSISTANCE CENTER. Subpart 3 of part B of title V of the Public Health Service Act, as amended by section 201 of this Act, is amended by adding at the end the following section: ``SEC. 520M. NATIONAL MENTAL HEALTH CRISIS RESPONSE TECHNICAL ASSISTANCE CENTER. ``(a) In General.--The Secretary, acting through the Director of the Center for Mental Health Services, shall establish within such center an administrative unit to be known as the National Mental Health Crisis Response Technical Assistance Center (referred to in this section as the `Technical Assistance Center'). ``(b) Duties.--The purpose of the Technical Assistance Center is to carry out, in accordance with policies of the Director of the Center for Mental Health Services, the following functions: ``(1) Provide consultation and technical assistance to the Director, and to State and local governmental providers of mental health services, on developing and implementing plans for providing appropriate mental health services in response to public health emergencies, including statewide plans under section 520L. ``(2) Provide technical expertise on planning, preparedness, and response evaluation activities. ``(3) Develop policy guidelines on mental health concerns related to crisis incidents and develop recommendations for proposed regulations and or legislative proposals. ``(4) Develop and conduct training events and conferences on mental health needs of disaster victims and witnesses. ``(5) Serve as the principal clearinghouse operated by the Secretary for the collection and dissemination of information concerning the mental health aspects of public health emergencies, including information in published documents, information on technical assistance resources, and information on relevant Internet sites. ``(6) Assist States in preparing for the behavioral health consequences of terrorism. ``(7) Provide onsite technical expertise during public health emergencies, when requested by a State. ``(c) Certain Authority.--The Technical Assistance Center may carry out the functions under subsection (b) directly or through grant or contract, subject to the approval of the Director of the Center for Mental Health Services. ``(d) Authorization of Appropriations.--For the purpose of carrying out this section, there is authorized to be appropriated $2,000,000 for each of the fiscal years 2002 through 2006.''. TITLE IV--TRAINING GRANTS SEC. 401. TRAINING GRANTS. Subpart 3 of part B of title V of the Public Health Service Act, as amended by section 301 of this Act, is amended by adding at the end the following section: ``SEC. 520N. TRAINING GRANTS. ``(a) In General.--The Secretary, acting through the Director of the Center for Mental Health Services, shall award grants to eligible entities to enable such entities to provide for the training of mental health professionals with respect to the treatment of individuals who are victims of disasters. ``(b) Eligibility.--To be eligible to receive a grant under subsection (a) an entity shall-- ``(1) be a-- ``(A) regional center of excellence; or ``(B) a mental health professional society; and ``(2) prepare and submit to the Secretary an application at such time, in such manner, and containing such information as the Secretary may require. ``(c) Use of Funds.--An entity that receives a grant under this section shall use amounts received under the grant to provide for the training of mental health professionals to enable such professionals to appropriately diagnose individuals who are the victims of disasters with respect to their mental health and to provide for the proper treatment of the mental health needs of such individuals. ``(d) Training Materials and Procedures.--The Director of the Center for Mental Health Services, in consultation with the Director of the National Institute of Mental Health, the National Center for Post- Traumatic Stress Disorder, the International Society for Traumatic Stress Studies, and the heads of other similar entities, shall develop training materials and procedures to assist grantees under this section. ``(e) Definition.--In this section, the term `mental health professional' includes psychiatrists, psychologists, psychiatric nurses, mental health counselors, marriage and family therapists, social workers, pastoral counselors, school psychologists, licensed professional counselors, school guidance counselors, and any other individual practicing in a mental health profession that is licensed or regulated by a State agency. ``(f) Authorization of Appropriations.--There are authorized to be appropriated to carry out this section such sums as may be necessary for each of fiscal years 2002 through 2005. ``(g) Program Management.--In carrying out this section, the Secretary may use amounts appropriated under subsection (f) for the administration of the program under this section.''.

Extended Disaster Mental Health Services Act of 2001 - Amends the Public Health Service Act to authorize the Secretary of Health and Human Services to make three-year grants to States and their subdivisions for mental health services in response to public health emergencies, including disease, natural, technological, or terrorism-related disasters and major transportation accidents. Includes post-emergency crisis counseling, outreach, intervention, and extended therapeutic services.Sets forth grant requirements, including the development of a statewide plan and coordination with other governmental programs (including those of the Federal Emergency Management Agency) and providers of mental health services.Authorizes the Secretary to make grants to States to develop their statewide plans, requiring such plans to address the need: (1) for increased capacity for emergency response; (2) of special populations such as children, the elderly, the disabled, and those with pre-existing mental health disorders; and (3) for informing the public and coordinating with other mental health service providers. Requires such plans to designate a primarily responsible State official.Requires the Secretary to establish within the Center for Mental Health Services a National Mental Health Crisis Response Technical Assistance Center to provide technical assistance during emergencies and for developing and implementing plans and policy guidelines. Requires such Center to conduct training and serve as the principal clearinghouse for information concerning the mental health aspects of public health emergencies.Directs the Secretary to award grants for training mental health professionals to treat individuals who are victims of disasters.

billsum

Although host genetics influences susceptibility to tuberculosis (TB), few genes determining disease outcome have been identified. We hypothesized that macrophages from individuals with different clinical manifestations of Mycobacterium tuberculosis (Mtb) infection would have distinct gene expression profiles and that polymorphisms in these genes may also be associated with susceptibility to TB. We measured gene expression levels of >38,500 genes from ex vivo Mtb-stimulated macrophages in 12 subjects with 3 clinical phenotypes: latent, pulmonary, and meningeal TB (n = 4 per group). After identifying differentially expressed genes, we confirmed these results in 34 additional subjects by real-time PCR. We also used a case-control study design to examine whether polymorphisms in differentially regulated genes were associated with susceptibility to these different clinical forms of TB. We compared gene expression profiles in Mtb-stimulated and unstimulated macrophages and identified 1,608 and 199 genes that were differentially expressed by >2- and >5-fold, respectively. In an independent sample set of 34 individuals and a subset of highly regulated genes, 90% of the microarray results were confirmed by RT-PCR, including expression levels of CCL1, which distinguished the 3 clinical groups. Furthermore, 6 single nucleotide polymorphisms (SNPs) in CCL1 were found to be associated with TB in a case-control genetic association study with 273 TB cases and 188 controls. To our knowledge, this is the first identification of CCL1 as a gene involved in host susceptibility to TB and the first study to combine microarray and DNA polymorphism studies to identify genes associated with TB susceptibility. These results suggest that genome-wide studies can provide an unbiased method to identify critical macrophage response genes that are associated with different clinical outcomes and that variation in innate immune response genes regulate susceptibility to TB. TB, a leading cause of death worldwide, is characterized by different clinical forms including latent TB (LTB), localized pulmonary infection, and various forms of extrapulmonary TB including TBM. 90% of people infected with Mtb have latent infection with no symptoms and an immune response that contains the bacilli. In 10% of infected individuals, symptoms develop and most commonly manifest as pulmonary disease, which accounts for 80% of all forms of TB disease [1]. TBM develops in around 1% of all cases of active TB [1] and is the most severe form with mortality rates of 20–25% and high rates of neurological sequelae in many of those who survive [2], [3]. Although only 10% of individuals who are infected with Mtb develop active disease, it is not known which immune responses are associated with susceptibility or resistance. In addition, it is not known why some individuals have disseminated TB that spreads to the meninges and central nervous system, while most people have localized disease in the lungs. Although environmental exposures, pathogen virulence traits, and host genetics have the potential to influence the different clinical manifestations of TB, it is not currently understood which factors are the most important [4]. Several lines of evidence, including twin and genome-wide linkage studies, suggest that host genetics strongly influences susceptibility to TB [5]–[9]. Candidate gene association studies have implicated common polymorphisms in genes that may influence the development of TB [10], [11]. Although there is potential for candidate gene study designs to be successful when sample sizes are sufficient and case and control groups are accurately defined, candidate genes are usually selected from lists of genes with known functions. A fundamental problem with this strategy is an inherent selection bias dominated by well-characterized genes. Furthermore, many genes are selected based on phenotypes identified from in vivo murine studies. Although mouse studies have provided powerful methods to dissect TB immunopathogenesis, the murine system models primary, progressive disease, which is only one of several phenotypes observed in humans. There are no well-established murine models of latent infection or the various types of disseminated disease, including TBM. Mtb intrathecal infection of rabbits recapitulates some of the inflammatory pathology but does not provide insight into the steps in immunopathogenesis involved in dissemination and invasion of the central nervous system [12]. To identify genes involved in TBM pathogenesis and to avoid gene selection bias, we chose to directly examine humans with different clinical types of TB with an array-based method to identify candidate genes. Macrophages mediate the host innate immune response to Mtb through pathogen recognition and activation of an inflammatory response. Mtb resides in the macrophage phagolysosome, where it evades the immune response in the majority of infected individuals. Successful containment of Mtb replication results in LTB with no clinical symptoms, which depends on stimulation of innate and adaptive immune responses that lead to macrophage activation, formation of granulomas and elimination of the bacilli. In contrast, failure to contain bacilli replication is associated with active pulmonary disease and/or the development of disseminated disease. We hypothesized that different macrophage responses to Mtb are associated with distinct clinical outcomes that are genetically regulated. Expression microarrays have been previously used to examine gene expression profiles in the immune response to TB [13]–[17]. None of these studies attempted to distinguish different clinical forms of active TB such as pulmonary and meningeal disease. In addition, the sample sizes were generally small and the findings were often not validated in independent sample sets. Finally, these previous approaches were not coupled with human genetic studies to examine the clinical significance associated with variation in the identified genes. In this manuscript, we examined ex vivo Mtb-stimulated monocyte-derived macrophages (MDMs) from subjects with pulmonary, meningeal and latent infection. We attempted to find unique gene expression profiles to determine whether clinical phenotypes in TB are associated with distinct early macrophage responses to Mtb stimulation. We then used a case-control genetic association study to examine whether genetic variation of these selected genes was associated with susceptibility to Mtb. TBM subjects were recruited as part of a larger clinical study at the Hospital for Tropical Diseases, in Ho Chi Minh City (HCMC), Vietnam [18]. All subjects were >14 years of age and HIV-negative. TBM patients were described as having clinical meningitis (defined as nuchal rigidity and abnormal cerebrospinal fluid parameters) in addition to having a positive Ziehl-Neelsen stain for acid-fast bacilli and/or Mtb cultured from the cerebrospinal fluid. Subjects were treated for TBM and were clinically well (recovered for >3 years) when samples for this study were taken. For PTB subjects, samples were taken from individuals who had been previously treated and had recovered from uncomplicated PTB (no evidence of miliary or extrapulmonary TB). LTB subjects were defined as highly exposed individuals who had no history of active TB disease. LTB subjects were healthy nursing staff members who had worked at Pham Ngoc Thach Hospital for Tuberculosis and Lung Disease, HCMC, Vietnam for more than 20 years. They were tested for Mtb exposure using an ESAT-6 and CFP-10- specific IFN-γ ELISPOT assay using a previously described method [19]. For the initial microarray study, twelve subjects were enrolled with three clinical forms of TB; TBM (n = 4), PTB (n = 4) and LTB (n = 4). All of the LTB subjects tested positive in the ESAT-6 and/or CFP-10- specific IFN-γ ELISPOT assay, suggesting previous or current infection with Mtb. An extended sample set containing 34 subjects with TBM (n = 10), PTB (n = 12) and LTB (n = 12) was used in validation experiments. Of the 12 LTB subjects, 10 were IFN-γ ELISPOT positive according to our defined cut-off [at least 10 spot forming units (SFU) more than the negative PBS control and at least twice as many SFU as the negative PBS control]. The 2 IFN-γ ELISPOT indeterminate LTB subjects had borderline responses (6. 7 SFU with a ratio of 2 and 6 SFU with a ratio of 2. 5) which were considerably higher than an unexposed population (average of −2. 8 SFU with a ratio of 0. 8). For the case-control genetic association study the cohort of TBM (N = 114) and PTB (N = 159) patients, and population controls (cord blood; N = 188) has been previously described [20]. All samples came from unrelated individuals who were ethnic Vietnamese Kinh, as assessed by questionnaire. Written informed consent was obtained from each patient. Protocols were approved by human subjects review committees at the Hospital for Tropical Diseases and Pham Ngoc Thach Hospital for Tuberculosis and Lung Disease, Ho Chi Minh City, Vietnam. Ethical approval was also granted by the Oxford Tropical Research Ethics Committee, UK (OXTREC), The University of Washington Human Subjects Committee (USA) and the Western Institutional Review Board (USA). Peripheral blood mononuclear cells (PBMCs) were separated from heparinized whole blood by Lymphoprep (Asix-Shield, Norway) gradient centrifugation according to the manufacturer' s protocol. From 20 ml of blood we obtained approximately 1–1. 5×107 PBMCs. To derive monocytes, PBMCs were plated in Nunclon Suface 6-well plates (Nunc, Denmark) containing RPMI-1640 (Sigma, Germany) with 10% heat-inactivated fetal calf serum (FCS; Sigma, Germany), 2 mM L-glutamine and 100 units of penicillin for 2 hours at 37°C. Non-adhered cells were removed by washing with phosphate buffered saline (PBS) containing 3% FCS and adhered cells were incubated for 5 days at 37°C, 5% CO2 to obtain MDMs. Cells were subsequently stimulated with PBS or 5 µg/ml of an irradiated, soluble, whole cell lysate of Mtb H37Rv [obtained from the Mycobacteria Research laboratories at Colorado State University, USA (http: //www. cvmbs. colostate. edu/microbiology/tb/top. htm) ] for 4 hours before RNA extraction. Pilot studies indicated that 5 µg/ml was an optimal dose for stimulating TNF-α production. RNA was extracted from macrophages using Trizol according to the manufacturer' s protocol (Invitrogen, USA), dissolved in RNase-free water and stored at −70°C until use. Total RNA (100 ng) was reverse transcribed to cDNA, amplified, labeled, and hybridized to the Human Genome U133 Plus 2. 0 Array (Affymetrix, USA), according to the manufacturer' s instructions. This array contains probe sets to measure the expression level of 47,000 transcripts, including 38,500 well-characterized human genes. Twelve Mtb-stimulated (TBM n = 4, PTB n = 4, and LTB n = 4) and 12 PBS-stimulated (hereafter called unstimulated) samples were hybridized to the array. The microarray data is publicly available at ArrayExpress, EMBL-EBI (Submission in progress, awaiting Accession number; http: //www. ebi. ac. uk/microarray-as/aer/? #ae-main0). After normalization of the expression values, the data from 12 Mtb-stimulated samples were compared with the 12 unstimulated samples. Data were considered significant when (1) the false discovery rate (FDR) from the Significance Analysis of Microarray (SAM) analysis for the comparison of stimulated and unstimulated expression values was <0. 05, and (2) the P value of the comparison between stimulated versus unstimulated expression values by Student' s t-test was <0. 05. In order to focus on highly regulated genes, we also restricted the majority of the analysis to genes with changes in expression levels of at least 2-fold. To compare gene expression levels among the three different clinical types of TB, we first calculated the fold stimulation of each gene for each individual by dividing the Mtb–stimulated value by the unstimulated control values. The averages of the 4 samples in each clinical group were calculated and then compared to the other groups by calculating the ratios of expression levels. The pair-wise comparisons included TBM vs. PTB, TBM vs. LTB, and PTB vs. LTB. SAM [20] was used to derive the FDR for microarray data, which is the proportion of genes likely to have been identified as significant by chance. Student' s t-test and analysis of variance (ANOVA) were used to compare mean expression levels. To analyze expression patterns in multiple genes simultaneously we used Hierarchical Clustering [21]. Analyses were performed using MultiExperiment Viewer (MeV version 4. 0, USA) [22] and SPSS (version 14. 0, USA). Taqman real time PCR was used to validate microarray gene expression results. cDNA was synthesized from total RNA samples using reverse transcription with Superscript II following the manufacturer' s protocol (Invitrogen, USA). A commercial Low Density Array (LDA) format with Taqman probes and primers was then used for PCR validation (Applied Biosystems, USA). Expression levels in 88 genes [86 selected genes and 2 controls (GAPDH; Hs00237184_m1 and Hs00266705_g1) ] were examined in each sample according to the manufacturer' s instructions. CCL1 gene expressions on human and mice were examined by using Taqman probes and primers (Applied Biosystems, USA). Samples were normalized to GAPDH and analyzed by using either Applied Biosystems SDS 2. 1 Relative Quantification software or an Excel spreadsheet to perform relative quantification analysis. PBMC were isolated from whole blood and cytokine assays were prepared by plating 105 cell per well with RPMI (Life Technologies) in a 96-well dish, stimulating for 24 hours, and then harvesting supernatants. Stimuli included: Ultrapure lipopolysacharide (LPS) at 100 ng/ml, from Salmonella minnesota R595 (List Biological Labs, Inc.), Mtb H37Rv whole cell lysate, Mtb H37Rv cell wall fraction and Mtb H37Rv cytosol fraction (TB Vaccine Testing and Research Materials Program at Colorado State University). Chemokine levels were determined with a sandwich ELISA technique (Duoset, R&D Systems, Minneapolis, MN). SNPs in the CCL1 and CCR8 genes were genotyped in patients with TBM (N = 114), PTB (N = 159), and in Vietnamese Kinh population controls (N = 188). This genotyping was performed as part of a larger genome-wide genetic association study of TB using the Affymetrix 250K NspI Chip (unpublished). The whole genome SNP genotyping was performed according to the manufacturer' s specifications and the data obtained was analyzed following rigorous quality control. Briefly, data quality control was performed using DM, BRLMM, RELPAIR, and manual viewing of cluster plots prior to statistical analysis. STRUCTURE and Eigentstrat were also used to analyse the population structure of the sample set. Genomic DNA quality was first assessed with 50 control SNPs and only samples with a call rate of greater than 93% were studied further. For each polymorphism in the full dataset, filter criteria were applied that included <5% missing values and HWE P value>10−5. Power for this study was calculated by using Power Calculator for Genetic Studies, CaTS version 0. 0. 2 (http: //www. sph. umich. edu/csg/abecasis/CaTS). With a sample size of controls = 188 and PTB = 159 we have 82% power to detect an effect with an odds ratio of 2 for SNPs with an allele frequency of 10% and significance level of 0. 01. With a sample size of controls = 188 and TBM = 114, we have a power of 71% to detect the same effects. Genotyping was also carried out on selected CCL1 SNPs using a larger sample set TBM (N = 162), PTB (N = 175), and in Vietnamese Kinh population controls (N = 380). This was performed by a MassARRAY™ technique (Sequenom, San Diego, USA) using a chip-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometer as previously described [18]. All of the CCL1 SNPs genotyped by Sequenom were in Hardy Weinberg Equilibrium (HWE) (P>0. 05) in population controls. Univariate analysis was performed for categorical variables with a Chi-Square test. Two-sided testing was used to evaluate statistical significance. We hypothesized that macrophages from individuals with different TB clinical phenotypes have distinct gene expression profiles in response to Mtb stimulation. All subjects with pulmonary and meningeal disease had been treated and were free of symptoms at the time of venipuncture. Gene expression of MDMs from subjects with three clinical forms of TB including LTB, PTB, and TBM (n = 4 in each group) was examined by microarray. MDMs were stimulated either with a whole cell lysate of Mtb H37Rv or PBS for 4 hours. RNA expression was analyzed using a Human Genome U133 Plus 2. 0 Array (Affymetrix, USA) which contains probe sets for 47,000 transcripts including 38,500 well-characterized human genes. We compared RNA transcription levels in Mtb-stimulated (n = 12) versus PBS-stimulated (n = 12) MDMs. 1,608 genes with a FDR of <5% and a P value of <0. 05 by Students' s t-test were differentially expressed by greater than 2-fold (Table 1). Of these genes, 1,260 were up-regulated and 348 genes were down-regulated. A list of the 1,608 genes that were differentially expressed in the two groups (n = 24) with their mean expression intensities, FDR and P values are presented in Table S1. 74 genes were up-regulated more than 10-fold, whereas only one gene was down-regulated by greater than 10-fold (Table 1). We used PANTHER (Protein Analysis Through Evolutionary Relationships; http: //www. pantherdb. org/) to analyze the molecular functions and biological processes of genes induced and repressed in Mtb-stimulated MDMs. The changes in gene expression induced after stimulation contained 144 (8. 4%) immunity and defense genes, including cytokines, chemokines, and receptors. Thirty six of these genes (25%) were up-regulated more than 10-fold. In contrast, no immunity and defense genes were repressed more than 10-fold. Other categories included; development (6. 7%), protein and nucleic metabolism (19. 2%) and signal transduction (11. 9%). By comparison to the entire human genome, the proportion of immunity and defense genes is 5. 2%. Percentages of other categories include: development (8. 5%), protein and nucleic metabolism (25. 1%) and signal transduction (13. 4%). To examine whether individuals with different clinical forms of TB have distinct gene expression profiles, we calculated the fold stimulation of each gene for each individual (dividing Mtb stimulated value by the unstimulated value) and then calculated the ratios of gene expression levels in each pair of TB forms. Six pair-wise comparisons in Table 1 show the change of gene expression between disease types (in fold change). 33 genes were differentially expressed between disease types with a ratio >10 and 228 genes had a ratio from 5 to 10. In Table 2, half of the genes with a ratio >10 (16/33) were immunity genes including chemokines, cytokines and immune receptors. Others such as MMP1 and HAS1 are involved in degrading the extracellular matrix [23]. When all 3 clinical groups were compared, 16 genes had expression values that were significantly different (CXCL5, EREG, TNIP3, INHBA, HAS1, MGC10744, CCL1, KCNJ5, SERPINB7, HS3ST2, APOBEC3A, MYO10, SLC39A8, CXCL11, F3, and DUSP5, ANOVA <0. 05). We then compared expression values of pairs of clinical groups. There were 11 genes highly expressed in TBM in comparison to other forms of TB (Table 2). 6/11 genes (IL1B, CXCL5, EREG, TNIP3, CCR2, and INHBA) were significantly induced in TBM in comparison to PTB (t test, P<0. 05), and all are genes related to immune function. 5/11 genes were highly expressed in TBM in comparison to LTB (IL12B, PTGS2, MMP1, IL23A, and CCL20) however this did not reach statistical significance due to a consistent outlier in the LTB group (L2 which does not cluster with the other samples; see below). Twelve genes were highly expressed in PTB in comparison to LTB and TBM (PTB/LTB; MMP1, IL23A, HAS1, PTGS2, MGC10744, CCL20, CCL1, and IL12B, PTB/TBM; HAS1, KCNJ5, SERPINB7, and HS3ST2). 6/12 had significantly different expression levels (t test, P<0. 05; Table 2). Nine genes were induced in LTB more than in other TB and 7 of these reached statistical significance (LTB/TBM; APOBEC3A, LTB/PTB P2RY13, MYO10, SLC39A8, CXCL11, F3, APOBEC3A, DUSP5). Together these results suggest that gene expression profiles in Mtb-stimulated macrophages may distinguish between the 3 different clinical forms of TB, LTB, PTB, and TBM. We used real-time PCR using a TaqMan Low Density Array technique to confirm microarray results in 86 genes in an extended sample set which included 12 LTB, 12 PTB, and 10 TBM individuals. Fifty-eight of the 86 genes were selected from the microarray data based on high levels of induction (>15 fold) or repression (>5 fold) following Mtb stimulation. Forty six genes were selected based on array expression differences among the 3 clinical groups (>5 fold). We first assessed whether the expression patterns of the 58 up and down-regulated genes were replicated in the independent sample set using RT-PCR. In total, 90% (52/58) of the microarray results were confirmed by RT-PCR when assessing Mtb and PBS-stimulated expression values in the validation sample set (Table 3 and Table S2). The RT-PCR results showed that 5/58 genes (IFIT1, CXCL6, MERTK, CD36, and MS4A6A) were not significantly induced or repressed by Mtb stimulation (n = 34; P>0. 05 by t-test) and the expression pattern of one gene, CCR2, was reversed (Table 3). In addition, the majority of the genes in the validation group (n = 34) had a higher induction level in comparison to the microarray group (n = 12; Table 3). We next compared gene expression levels in the 3 clinical groups in the validation sample set. The RT-PCR results showed that 2/46 genes (CCL1 and HS3ST3B1) were differentially expressed in groups with different TB phenotype (P<0. 05 by t-test; Table 4). CCL1 was up-regulated in PTB when compared to LTB in both the RT-PCR LDA validation samples (P = 0. 02 by t-test; 1. 9-fold) and the initial microarray analysis (12. 8-fold; Table 4 and Table S3). HS3ST3B1 was down regulated in LTB when compared to TBM in the RT-PCR LDA validation samples (P = 0. 008 by t-test; ratio = 0. 4) but this pattern of expression was reversed in the initial microarray analysis (ratio = 12. 8) (Table 4). Scatter plots of CCL1 and HS3ST3B1 are shown in Figure 1 along with 3 other representative genes. Seven other genes (INHBA, TSLP, LY6K, IL12B, MMP1, CCL20 and HAS1) had a greater than 2-fold change in expression ratios of the validation samples in each pair-wise comparison, but these differences did not reach statistical significance (P>0. 05; Table 4). These results suggest that the different TB clinical phenotypes cannot easily be distinguished by examining expression levels of single genes. We next hypothesized that expression profiles from multiple genes would need to be combined to detect patterns that could distinguish the different clinical disease phenotypes. We selected 1,608 highly induced or repressed genes from the microarray data set (Table S1) and used an unsupervised, hierarchical clustering algorithm [21] of 12 individual samples to attempt to distinguish the profiles of the 3 groups (Figure S1). These results show that, (1) there was more relatedness between expression levels of samples from the same clinical group, i. e. L1 and L3 are very similar, P1, P2 and P3 are very similar, and M1 and M4 are very similar, and (2) one large cluster containing data from all TBM subjects, all PTB subjects and one LTB subject (L4) is very distinct to data from subjects L2, L1 and L3. Together, these findings suggest that cluster analysis can partially distinguish different clinical forms of TB. CCL1 was the only gene whose expression was up-regulated in both the microarray and validation data sets when comparing clinical forms of TB (PTB vs LTB). We next examined whether genetic variants of CCL1 were associated with susceptibility to TB in a case-control study with TBM (N = 114) and PTB patients (N = 159), and population controls (N = 188) by using gene chip mapping assays. Forty nine SNPs were genotyped across a 200 kb region of the chromosome 17 CCL gene family cluster. Eight of the forty nine SNPs were associated with TB. To further locate the region associated with TB, we arbitrarily divided the whole region into four 50 kb sections. The first section containing CCL2 had 1/9 associated SNPs, the second containing CCL7 and CCL11 had 1/9 associated SNPs, the third containing CCL8 and CCL13 had 1/7 associated SNPs and the fourth containing CCL1 had 4/23 associated SNPs (Figure 2). To investigate this further we genotyped 10 SNPs nearby and in the coding region of CCL1 using Sequenom. Two more SNPs in the CCL1 gene were significantly associated with TB by genotypic comparison (Table 5). Together these results suggest that polymorphisms near and within the CCL1 genomic region are associated with susceptibility to different TB phenotypes. To further investigate the role of CCL1 in Mtb pathogenesis, we examined regulation of its expression. We found that CCL1 mRNA expression was cell-specific and highly induced in monocytic (THP-1, U937, & PBMCs) cells stimulated with Mtb lysates or TLR ligands (LPS, PAM2, PAM3) (Figure 3A). In contrast, no expression was found in epithelial cell lines (HeLa & A549, data not shown). We also found that CCL1 protein secretion was induced in THP1 cells and PBMCs by Mtb, including whole cell lysates, cell wall and cytosolic fractions [Figure 3B and data not shown; PBS vs TB whole cell lysate (TBWCL; P = 0. 01), PBS vs TB cell wall (TBCW; P = 0. 006) and PBS vs TB cytosol (P = 0. 02) ]. Finally, we examined CCL1 expression in murine bone-marrow derived macrophages stimulated with PBS, LPS or Mtb from wild-type (WT) and Myd88−/− mice. CCL1 expression was highly induced by LPS and Mtb in WT bone marrow macrophages (BMMs). However, CCL1 expression was decreased in MyD88-deficient BMMs stimulated with LPS (P = 0. 03) or Mtb (P = 0. 002) (Figure 3C). Together, these results suggested that CCL1 expression is highly enriched in monocytes and induced by Mtb components in a MyD88-dependent manner. In this study we examined macrophage transcriptional profiles in individuals with different clinical forms of TB. The majority of reported TB microarray studies have examined healthy donors, cell lines or murine cells [13]–[17]. Only one previous study has compared gene expression profiles of individuals with different clinical forms of TB [24]. Mistry et al obtained whole blood from individuals with active, latent, cured (following 1 disease episode) and recurrent TB (following 2–3 episodes) [24]. Discriminant analysis suggested that 9 genes could distinguish the 4 clinical TB groups [24]. We examined these 9 genes in our data set and found these genes could not differentiate our latent and cured TB groups. These differences may be attributable to the study design, which was substantially different from the current investigation with regard to cell population (whole blood vs MDMs), stimuli (none vs whole cell Mtb lysate), ethnic background (South African vs Vietnamese) and comparison of different clinical phenotypes. Despite these methodologic differences, both studies suggest that host gene expression profiles uniquely identify groups of individuals with different types of TB. Our study further illustrates that macrophages, the primary host cell involved in TB pathogenesis, are a key source of the unique transcriptional profile that distinguishes clinical forms of TB. One limitation of our study was the small sample size. Although this is the largest number of individuals ever studied in a TB microarray study, comparable only to the study by Mistry et al [24], the sample size remains small for this statistically challenging question. To overcome some of the limitations of a small sample size for microarrays (n = 12), we included an independent set of samples for validation (n = 34). We also chose to use a whole cell lysate of a standardized Mtb strain rather than live organisms and a relatively short stimulation time (t = 4 hours) to minimize variation in our stimulation conditions and to enhance the detection of early innate immune response genes. We examined these cells in an ex vivo environment to avoid variability that is attributable to complex in vivo environments. For example, we studied individuals after they had been treated for TB to avoid detecting gene expression changes that are attributable to stimulation of in vivo inflammatory pathways from active disease. We also chose to study macrophages rather than whole blood in order to concentrate on a single cell population that is most relevant for Tb pathogenesis. A number of studies have shown that the strain of Mtb induces different immune responses [25], [26]. Although the choice of Mtb strain could stimulate different gene expression profiles, we chose to study the commonly used laboratory strain (Mtb H37Rv). Each of these experimental conditions was selected to maximize the opportunities of detecting differences attributable to genetic variation in the macrophage innate immune response to TB. Comparison of gene expression results with alternative experimental conditions (such as different cell types, Mtb strains, Mtb growth conditions, and time points) could further illuminate the role of these genes in Tb pathogenesis. In addition to comparing expression profiles among people with different types of TB, our study contributes further data on the set of genes that are activated in response to Mtb stimulation of macrophages. Our results demonstrated that 1,608 genes in macrophages were stimulated (up or down-regulated) by Mtb. Furthermore, 90% of a subset of these genes (n = 58 genes induced >15 fold by Mtb stimulation) in a second round validation also showed altered expression. Many genes identified in our study have also been detected in previous studies investigating the host response to Mtb infection [13], [16], [17]. Ragno et al studied THP-1 cells stimulated with live TB and measured the expression of 375 genes after 6 or 12 hours of stimulation. Our data confirmed 15 genes significantly induced following 6 hr stimulation in their data set (MIP-1α, MIP-1β, MIP-3α, MPIF-1, PARC, RANTES, IL-8, GRO-α, GRP-β, GRO-γ, CCL1, CCR3, IL-1β, TNFα, and VEGF) [17]. Nau et al studied primary human MDMs stimulated with live Mtb [16]. Eleven genes were highly expressed in both data sets (TNFAIP6, CXCL3, CXCL1, CCL4, PTGS2, SERPINB2, PTX3, INHBA, TRAF1, JAG1, and SOD2) and 3 genes were inhibited (MERTK, GLUL, and DAB2). These gene lists include cytokines, chemokines and immune receptors, which may be involved in inflammatory responses in the early phases of defense against Mtb. All of the up-regulated genes identified by Nau et al were found in our dataset [16]. In contrast, only 50% (24/50) of highly expressed genes in our dataset were identified by Nau et al, a difference that is likely due to the array sizes that were utilized (38,000 vs. 980 genes). Although these microarray studies have important methodologic differences (e. g primary cells vs cell lines, healthy subjects vs. TB patients, live versus dead Mtb stimulation, stimulation times, arrays and genes analyzed), all of these studies have identified novel genes potentially related to the host macrophage response to Mtb. Our study compares transcriptional profiles of individuals with TBM with individuals with other forms of TB. We identified genes that were distinctly expressed in macrophages from individuals with a history of TBM. After bacilli invade the host lung within the pulmonary alveolar macrophage, they replicate and disseminate to the regional lymph nodes. During this early stage of infection, before the development of adaptive immunity, the bacteria can spread haematogenously to other organs in the body and cause extrapulmonary disease, such as TBM [27], [28]. This step may be determined by the nature and extent of the innate immune response activated by infected macrophages. We found that several macrophage immune response genes (IL1B, IL12B, TNF, TNIP3, CXCL10, CXCL11, CCL12, and CCL1) were up-regulated in TBM subjects in comparison to those with PTB and LTB. In addition, some genes, such as MMP1 and HAS1, were found with differing expression in PTB and TBM patients. These genes are involved in degrading the extracellular matrix and could mediate a role in granuloma formation and bacillus containment, which could influence dissemination and development of TBM [23]. Although the relationship between the inflammatory response and TBM pathogenesis is only partially understood, excessive immune activation may be intimately associated with disease severity and outcome. Case-control genetic association studies of biologically plausible candidate genes have been performed with the hope to identify genes involved in susceptibility to, and clinical outcome of, TB. However it has always been challenging to identify potential candidate genes in an unbiased manner. The expression profiling study we describe here can serve as a hypothesis generating, unbiased methodological approach to identify genes for potential association studies. Despite this advantage, gene regulation is not the only mechanism for genetic resistance or susceptibility and non-synonymous coding region SNPs which alter protein structure and function also play an important role. From the genes that were differentially expressed between TB disease types, as assessed by microarray, we tested 46 genes in a separate, larger sample set by RT-PCR. The expression of only one of these genes, CCL1, remained significantly different between patients with different clinical TB outcomes. To test our selection approach we performed a case-control genetic association study and found that SNPs near CCL1 were associated with susceptibility to PTB. The fact that SNPs near CCL1 were significantly associated with PTB in our study highlights the feasibility of this unbiased selection approach. Even though the associated SNPs are not within the CCL1 coding region, it is a likely candidate gene due to it' s proximity to the cluster of associated SNPs and its functional relevance. CCL1, like other members of the CC chemokine family, is an inflammatory mediator that stimulates the migration of human monocytes [29]. CCL1 is produced by monocytes (as well as other cells) and binds its receptor CCR8, which is present on lymphocytes and monocytes [30]. Interestingly, CCR8 has enriched expression on Th2 and regulatory T cells and may influence the development of Th2 type T cell responses in vivo [31], [32]. In addition, CCR8 regulates migration of dendritic cells to lymph nodes [33]. Hoshino et al [34] found that the expression of CCR8 was specifically up-regulated by CCL1 stimulation of peritoneal macrophages, which may lead to cell aggregation at a site of tissue damage. In the lungs, CCL1 expression was up-regulated in Mycobacterium bovis purified protein derivative (PPD) induced granulomas [35]. In this study, we found that CCL1 expression was induced by Mtb and TLR ligands in several monocyte/macrophage lineages. Furthermore, we found that its expression was MyD88-dependent when cells were stimulated with LPS or Mtb. Genetic variation leading to the loss or alteration of CCL1 function may influence the ability of T cells, monocytes and dendritic cells to migrate to the site of infection, aggregate into granulomas and develop an effective immune response. This may result in inadequate containment of the bacterium and allow unimpeded bacterial growth leading to pulmonary disease. With currently available tools, clinicians are unable to identify the subset of latently infected patients who will develop active disease. Furthermore, there are no techniques available to prospectively identify individuals at risk for the devastating consequences of TBM versus more treatable forms of TB such as localized pulmonary disease. Further studies in this area could lead to tests that could alter treatment algorithms with more accurate prognostic information. In addition, such studies may lead to novel molecular insight into TB pathogenesis.

Although TB is a leading cause of death worldwide, the vast majority of infected individuals are asymptomatic and contains the bacillus in a latent form. Among those with active disease, 80% have localized pulmonary disease and 20% have disseminated forms. TB meningitis (TBM) is the most severe form of TB with 20-25% of sufferers dying, and of the survivors, many have disability. We currently do not understand the host factors that regulate this diverse spectrum of clinical outcomes. We hypothesized that variation in innate immune gene function is an important regulator of TB clinical outcomes. We measured the mRNA expression levels of >38,500 genes in macrophages taken from people with a history of latent, pulmonary, or meningeal TB and found genes with unique activation patterns among the clinical groups. Furthermore, we studied one of these genes further and found that CCL1 polymorphisms were associated with pulmonary TB (PTB) but not other types of TB disease. To our knowledge, this is the first study to combine mRNA expression studies with genetic studies to discover a novel gene that is associated with different clinical outcomes in TB. We speculate that this approach can be used to discover novel strategies for modulating immune function to prevent adverse outcomes in TB.

lay_plos

More bad news for folks who used their credit cards at Target during the holiday shopping season. Many of the 40 million credit cards that the company says were part of the data breach are already for sale on black markets around the world. That report comes from KrebsonSecurity, the website run by cyber-security reporter Brian Krebs, who initially broke the story about the Target breach. On Friday, Krebs posted another story detailing how he had tracked down phony cards made using information that was stolen as part of the Target data breach: STORY: Target data theft fuels new worries on cybersecurity "Credit and debit card accounts stolen in a recent data breach at retail giant Target have been flooding underground black markets in recent weeks, selling in batches of one million cards and going for anywhere from $20 to more than $100 per card, KrebsOnSecurity has learned." On Thursday, Target confirmed that someone had hacked into its systems and had stolen 40 million debit and credit cards from stores across the country. The breach apparently lasted from Black Friday to Dec. 15. As expected, the thieves are using that information obtained from those credit cards to make phony copies that are being sold on black market stores around the world, Krebs found. At just one site that sells such counterfeit cards, Krebs helped one bank find 100 cards for sale that were made using information obtained from customers who were affected by the Target breach. ALSO: 5 things you can do to protect yourself after Target data breach Target shoppers vent concerns about credit data theft on Twitter Credit and debit card accounts stolen in a recent data breach at retail giant Target have been flooding underground black markets in recent weeks, selling in batches of one million cards and going for anywhere from $20 to more than $100 per card, KrebsOnSecurity has learned. Prior to breaking the story of the Target breach on Wednesday, Dec. 18, I spoke with a fraud analyst at a major bank who said his team had independently confirmed that Target had been breached after buying a huge chunk of the bank’s card accounts from a well-known “card shop” — an online store advertised in cybercrime forums as a place where thieves can reliably buy stolen credit and debit cards. There are literally hundreds of these shady stores selling stolen credit and debit cards from virtually every bank and country. But this store has earned a special reputation for selling quality “dumps,” data stolen from the magnetic stripe on the backs of credit and debit cards. Armed with that information, thieves can effectively clone the cards and use them in stores. If the dumps are from debit cards and the thieves also have access to the PINs for those cards, they can use the cloned cards at ATMs to pull cash out of the victim’s bank account. At least two sources at major banks said they’d heard from the credit card companies: More than a million of their cards were thought to have been compromised in the Target breach. One of those institutions noticed that one card shop in particular had recently alerted its loyal customers about a huge new batch of more than a million quality dumps that had been added to the online store. Suspecting that the advertised cache of new dumps were actually stolen in the Target breach, fraud investigators with the bank browsed this card shop’s wares and effectively bought back hundreds of the bank’s own cards. When the bank examined the common point of purchase among all the dumps it had bought from the shady card shop, it found that all of them had been used in Target stores nationwide between Nov. 27 and Dec. 15. Subsequent buys of new cards added to that same shop returned the same result. On Dec. 19, Target would confirm that crooks had stolen 40 million debit and credit cards from stores nationwide in a breach that extended from Nov. 27 to Dec. 15. Not long after that announcement, I pinged a source at a small community bank in New England to see whether his institution had been notified by Visa or MasterCard about specific cards that were potentially compromised in the Target breach. This institution has issued a grand total of more than 120,000 debit and credit cards to its customers, but my source told me the tiny bank had not yet heard anything from the card associations about specific cards that might have been compromised as a result of the Target breach. My source was anxious to determine how many of the bank’s cards were most at risk of being used for fraud, and how many should be proactively canceled and re-issued to customers. The bank wasn’t exactly chomping at the bit to re-issue the cards; that process costs around $3 to $5 per card, but more importantly it didn’t want to unnecessarily re-issue cards at a time when many of its customers would be racing around to buy last-minute Christmas gifts and traveling for the holidays. On the other hand, this bank had identified nearly 6,000 customer cards — almost 5 percent of all cards issued to customers — that had been used at Target stores nationwide during the breach window described by the retailer. “Nobody has notified us,” my source said. “Law enforcement hasn’t said anything, our statewide banking associations haven’t sent anything out…nothing. Our senior legal counsel today was asking me if we have positive confirmation from the card associations about affected cards, but so far we haven’t gotten anything.” When I mentioned that a big bank I’d spoken with had found a 100 percent overlap with the Target breach window after purchasing its available cards off a particular black market card shop called rescator[dot]la, my source at the small bank asked would I be willing to advise his fraud team on how to do the same? CARD SHOPPING Ultimately, I agreed to help in exchange for permission to write about the bank’s experience without actually naming the institution. The first step in finding any of the bank’s cards for sale was to browse the card shop’s remarkably efficient and customer-friendly Web site and search for the bank’s “BINs”; the Bank Identification Number is merely the first six digits of a debit or credit card, and each bank has its own unique BIN or multiple BINs. A quick search on the card shop for the bank’s BINs revealed nearly 100 of its customers’s cards for sale, a mix of MasterCard dumps ranging in price from $26.60 to $44.80 apiece. As one can imagine, this store doesn’t let customers pay for purchases with credit cards; rather, customers can “add money” to their accounts using a variety of irreversible payment mechanisms, including virtual currencies like Bitcoin, Litecoin, WebMoney and PerfectMoney, as well as the more traditional wire transfers via Western Union and MoneyGram. With my source’s newly registered account funded via wire transfer to the tune of USD $450, it was time to go shopping. My source wasn’t prepared to buy up all of the available cards that match his institution’s BINs, so he opted to start with a batch of 20 or so of the more recently-issued cards for sale. Like other card shops, this store allows customers to search for available cards using a number of qualifications, including BIN; dozens of card types (MasterCard, Visa, et. al.); expiration date; track type; country; and the name of the financial institution that issued the card. A key feature of this particular dumps shop is that each card is assigned to a particular “base.” This term is underground slang that refers to an arbitrary code word chosen to describe all of the cards stolen from a specific merchant. In this case, my source at the big bank had said all of the cards his team purchased from this card shop that matched Target’s N0v. 27 – Dec. 15 breach window bore the base name Tortuga, which is Spanish for “tortoise” or “turtle.” Indeed, shortly after the Target breach began, the proprietor of this card shop — a miscreant nicknamed “Rescator” and a key figure on a Russian-language cybercrime forum known as “Lampeduza” — was advertising a brand new base of one million cards, called Tortuga. Rescator even created a graphical logo in the Lampeduza forum’s typeface and style, advertising “valid 100% rate,” and offering a money-back guarantee on any cards from this “fresh” base that were found to have been canceled by the card issuer immediately after purchase. In addition, sometime in December, this shop ceased selling cards from other bases aside from those from the Tortuga base. As the month wore on, new Tortuga bases would be added to shop, with each base incrementing by one with almost every passing day (e.g., Tortuga1, Tortuga2, Tortuga3, etc.). Another fascinating feature of this card shop is that it appears to include the ZIP code and city of the store from which the cards were stolen. One fraud expert I spoke with who asked to remain anonymous said this information is included to help fraudsters purchasing the dumps make same-state purchases, thus avoiding any knee-jerk fraud defenses in which a financial institution might block transactions out-of-state from a known compromised card. The New England bank decided to purchase 20 of its own cards from this shop, cards from Tortuga bases 6-9, and Tortuga 14 and 15. The store’s “shopping cart” offers the ability to check the validity of each purchased card. Any cards that are checked and found to be invalid automatically get refunded. A check of the cards revealed that just one of the 20 had already been canceled. The bank quickly ran a fraud and common point-of-purchase analyses on each of the 19 remaining cards. Sure enough, the bank’s database showed that all had been used by customers to make purchases at Target stores around the country between Nov. 29 and Dec. 15. “Some of these already have confirmed fraud on them, and a few of them were actually just issued recently and have only been used at Target,” my source told me. Incredibly, a number of the cards were flagged for fraud after they were used to make unauthorized purchases at big box retailers, including — wait for it — Target. My source explained that crooks often use stolen dumps to purchase high-priced items such as Xbox consoles and high-dollar amount gift cards, goods that can be fenced, auctioned or otherwise offloaded quickly and easily for cash. My source said his employer isn’t yet sure which course of action it will take, but that it’s likely the bank will re-issue some or all of the 5,300+ cards affected by the Target breach — most likely sometime after Dec. 25. The bank is unconcerned that its cards compromised in the Target breach might be used for online shopping fraud because the stolen data does not include the CVV2 — the three digit security code printed on the backs of customer cards. Most online merchants require customers to supply the CVV2 as proof that they posses the legitimate, physical card for the corresponding account that is being used to fund the online purchase. Update, 5:20 p.m. ET: In a message to consumers, Target CEO Gregg Steinhafel said Target would be offering free credit monitoring for affected customers. Not sure how credit monitoring helps with this specific breach, but at any rate here’s the rest of his statement: “Yesterday we shared that there was unauthorized access to payment card data at our U.S. stores. The issue has been identified and eliminated. We recognize this has been confusing and disruptive during an already busy holiday season. Our guests’ trust is our top priority at Target and we are committed to making this right. We want our guests to understand that just because they shopped at Target during the impacted time frame, it doesn’t mean they are victims of fraud. In fact, in other similar situations, there are typically low levels of actual fraud. Most importantly, we want to reassure guests that they will not be held financially responsible for any credit and debit card fraud. And to provide guests with extra assurance, we will be offering free credit monitoring services. We will be in touch with those impacted by this issue soon on how and where to access the service. We understand it’s been difficult for some guests to reach us via our website and call center. We apologize and want you to understand that we are experiencing unprecedented call volume. Our Target teams are working continuously to build capacity and meet our guests’ needs. We take this crime seriously. It was a crime against Target, our team members, and most importantly, our guests. We’re in this together, and in that spirit, we are extending a 10% discount – the same amount our team members receive – to guests who shop in U.S. stores on Dec. 21 and 22. Again, we recognize this issue has been confusing and disruptive during an already busy holiday season. We want to emphasize that the issue has been addressed and let guests know they can shop with confidence at their local Target stores.” [EPSB] Have you seen: Non-US Cards Used At Target Fetch Premium”…An underground service that is selling millions of credit and debit card accounts stolen in a recent data breach at retail giant Target has stocked its virtual shelves with a new product: Hundreds of thousands of cards issued by non-U.S. banks that were used at Target across the United States during the retailer’s 19-day data breach. It’s not clear how quickly the non-U.S. cards are selling, but they seem to be fetching a much higher price than those issued by U.S. banks. [/EPSB] Tags: ATM, BINs, Bitcoin, card shop, dumps, Litecoin, mastercard, moneygram, PerfectMoney, PIN, target, target credit card hack, target data breach, tortuga, tortuga base, Visa, webmoney, western union

As you might expect, credit card hackers move fast. Many of the 40 million cards compromised in the recent Target breach are up for sale (or already scooped up) on the black market, reports Krebs on Security. Reporter Brian Krebs gets into the nitty gritty of how these underground "card shops" work and finds that the Target cards are going for $20 to more than $100 apiece. One bit of good news: It doesn't appear that the stolen data includes the three-digit number on the rear of the cards, meaning they probably won't be used for online shopping. Still, the hackers have access to customer names and all the data that was embedded on the cards' magnetic stripes, so making dupes to sell wouldn't be all that difficult, notes the LA Times. It's a mess, writes Kashmir Hill at Forbes. "Ideally, banks and credit card companies would just go ahead and cancel all affected cards, but that's expensive for them and a headache for last minute Christmas shoppers."

multi_news

The investigation of RNA-based regulation of cellular processes is becoming an increasingly important part of biological or medical research. For the analysis of this type of data, RNA-related prediction tools are integrated into many pipelines and workflows. In order to correctly apply and tune these programs, the user has to have a precise understanding of their limitations and concepts. Within this manuscript, we provide the mathematical foundations and extract the algorithmic ideas that are core to state-of-the-art RNA structure and RNA–RNA interaction prediction algorithms. To allow the reader to change and adapt the algorithms or to play with different inputs, we provide an open-source web interface to JavaScript implementations and visualizations of each algorithm. The conceptual, teaching-focused presentation enables a high-level survey of the approaches, while providing sufficient details for understanding important concepts. This is boosted by the simple generation and study of examples using the web interface available at http: //rna. informatik. uni-freiburg. de/Teaching/. In combination, we provide a valuable resource for teaching, learning, and understanding the discussed prediction tools and thus enable a more informed analysis of RNA-related effects. Bioinformatics analyses have become indispensable to biological research. While platforms like Galaxy enable the setup of tool pipelines without expert knowledge [1,2], one requires a general understanding of underlying concepts and algorithms to be able to successfully apply and adapt these pipelines to biological data [3,4]. Thus, bioinformatics is taught n both computer science and biology studies. It has been established that, when teaching mathematics, a combination of reflective example study and problem solving by hand fosters learning. This learning effect is heightened when done iteratively with increasing difficulty [5]. Thus, diverse examples covering different aspects of the topic have to be provided to guide the learning process. This is even more important in an e-learning or self-study context, in which the study of examples that show different aspects of a problem might compensate for the missing interaction with a teacher [6,7]. Here, we focus on RNA-related bioinformatics and especially on approaches for RNA structure and RNA–RNA interaction prediction. Both are essential when investigating the vast amount of regulatory RNA that is common to all kingdoms of life [8,9]. The function of many RNA species is guided by their structure that is defined by the formation of intramolecular base pairs. For instance, prokaryotic small RNAs show evolutionary conserved unstructured regions that regulate the expression of their target mRNAs via intermolecular base pairing [10,11]. Thus, the prediction of both functional intramolecular structures of RNAs as well as their intermolecular (RNA–RNA) interaction potentials are central bioinformatics tasks. Most computational methods for RNA structure or RNA–RNA interaction prediction are based on thermodynamic models and provide an efficient computation, since Richard Bellman' s principle of optimality [12] can be applied. This means that optimal solutions of a problem can be composed of optimal solutions of (independent) subproblems. This is used by dynamic programming approaches that decompose a problem into smaller problems and tabularize partial solutions. Robert Giegerich and colleagues developed a rigorous framework, namely Algebraic Dynamic Programming (ADP) [13,14], to systematically study and develop dynamic programming approaches in a computer science context. In addition, they provided an online platform to study ADP programs for various problems also covering RNA related topics [15]. The central idea of ADP is to separate the strategy of how a problem is decomposed into subproblems from the evaluation strategy, i. e., the objective of the optimization. We use the counting of structure alternatives for a given RNA to illustrate how dynamic programming can be applied to prediction problems. In particular, we introduce the decomposition strategy for (nested) RNA structure models. The teaching of dynamic programming approaches is typically split into a theoretical introduction by the lecturer showing individual examples and a subsequent manual application by students in which the methods are implemented or applied to solve small-scale problems for exercise. This leads often to a very small set of examples discussed due to the high amount of work needed for manual application and the limited gain of knowledge by iterated usage of once-understood solution strategies. To increase the number of examples, e. g., to focus on different aspects of an individual method or to compare different approaches, either partial solutions have to be provided or implementations made available. Besides single instances like the Nussinov algorithm, most state-of-the-art methods and their underlying algorithmic ideas are not covered by textbooks, e. g., [16–18]. Resorting to the original literature for teaching these algorithms, however, is complicated, as most approaches are introduced for very sophisticated energy models. While these advanced energy models are required for a successful application of these tools in real-world scenarios, they often mask the basic and transferable algorithmic ideas for the nonexpert reader since they require a high level of background knowledge. We approach the aforementioned problems in two ways. First, we have stripped the model-specific energy details from the state-of-the-art methods for RNA structure prediction and RNA–RNA interaction prediction and present their underlying (or basic) algorithmic ideas. For that purpose, we use the most simple energy model available. State-of-the-art energy models take the structural context of base pairs into account. To this end, RNA structures are decomposed into loops (i. e., a region that is enclosed by one or more base pairs) to calculate their overall energy. However, the algorithmic principles are essentially the same when using an energy model that considers base pairs without their structural context as basic units. Since all methods are presented using the same mathematical nomenclature, relationships and differences are easy to understand. Second, we provide a web interface that provides interactive implementations of all algorithms discussed with extensive visualizations. This interface (i) helps to understand and follow the algorithms, (ii) eases the generation of interesting examples for different aspects to teach, and (iii) provides master solutions for comparison with your own calculations or implementations. Each section closes with a list of advanced questions that exemplify what can be studied and answered using the provided web interfaces available at http: //rna. informatik. uni-freiburg. de/Teaching/. RNA structure prediction topics covered within this manuscript are the formalization of RNA secondary structures and simplified energy models, computation of the number of structures with regards to the given model [19,20], identification of the minimum free energy structure [21,22], computation of partition functions [23], probability calculation for single base pairs and unpaired regions [23,24], and identification of the maximum expected accuracy structure [25,26]. RNA–RNA interaction prediction approaches are grouped according to their algorithmic idea, as in [27], into hybrid-only interaction prediction [28–30], concatenation-based/cofolding interaction prediction [31,32], and accessibility-based interaction prediction [24,33,34]. Ribonucleic acid (RNA) is a linear molecule built from nucleotides. The ribose sugars of the nucleotides are bound via interlinking phosphate groups. Furthermore, each sugar is connected to a nitrogenous base, typically one of adenine (A), guanine (G), cytosine (C), or uracil (U). The bases can form hydrogen bonds between two (nonconsecutive) nucleotides, which is then called a base pair. Although other forms are possible, the typically considered base pairs are G−C, A−U, and G−U in both orientations. Pairing between nucleotides of the same molecule (intramolecular) defines its three-dimensional structure. In order to fulfill a certain regulatory function, typically a stable structure is needed. Thermodynamic analyses have identified base (pair) stacking as the major stabilizing force within RNA structures [35], and according energy estimates have been identified experimentally, e. g., refer to [36]. The functional structure of an RNA can regulate, e. g., other RNA molecules by direct (intermolecular) base pairing, i. e., forming base pairs between two RNAs, called RNA–RNA interactions. While the probability of an initial contact is dependent on many factors, such as concentration or location, the subsequent formation of a stable RNA–RNA interaction is assumed to follow the same thermodynamic principles as single structure formation. Thus, most ideas and parameters from RNA structure prediction are transfered to RNA–RNA interaction prediction approaches. It is important to note that thermodynamics-based approaches are again models that do not consider all factors that influence structure/interaction formation, e. g., already bound molecules, specific solution conditions, or kinetics of structure formation. Nevertheless, they typically allow for accurate predictions for the majority of RNA molecules [37]. In the following, we provide the mathematical framework needed to define and solve RNA-related problems. The primary structure of an RNA molecule can be described by its sequence of bases. That is, an RNA molecule of length n is defined by its sequence S∈{A, C, G, U}n of respective International Union of Pure and Applied Chemistry (IUPAC) single-letter codes [38]. The secondary structure P of an RNA S is defined as a set of (ordered) base pairs, i. e., P⊂[1, n]×[1, n] with (i, j) ∈P→i<j. Typically, it is assumed that each nucleotide can pair with at most one other nucleotide, i. e., ∀ (i, j) ≠ (p, q) ∈P: {i, j}∩{p, q} = ∅, and that only the introduced Watson–Crick or G−U base pairs are allowed, i. e., ∀ (i, j) ∈P: {Si, Sj}∈{{A, U}, {C, G}, {G, U}} extraneous to order. Such base pairs are said to be complementary. Furthermore, to restrict computational complexity of prediction algorithms, structures are constrained to be noncrossing (nested), i. e., ∄ (i, j), (p, q) ∈P: i<p<j<q. Using noncrossing structures generally allow a good estimate of the overall structure stability. However, it is important to note that crossing base pairs do exist, albeit not as abundant as noncrossing base pairs, and contribute to the final stability of the three-dimensional shape. It is typically assumed that first noncrossing structural elements are formed that subsequently are linked via few crossing base pairs [39]. Thus, the majority of the structure can be modeled/predicted via nested base pairing, which strongly reduces the computational complexity. Finally, it is commonly enforced that pairing bases have a minimal sequence distance of l, also called minimal loop length, to incorporate steric constraints of structure formation. In the following, we will denote with P the set of all possible structures (also referred to as structural ensemble or structure space) that can be formed by a given sequence S. It has been shown that the size of the structure space P grows exponentially with sequence length n. For a minimal loop length l of 3, the growth is about 2. 3n [40]. Nested secondary structures can be visualized as outerplanar graphs in which nucleotides are represented by nodes, and edges represent base pairs or sequential backbone connections. Furthermore, dot-bracket strings can be used that encode for each position i whether it is unpaired “. ”, it is the smaller index (opening) of a base pair “ (, ” or the larger (closing) index “) ”. As motivated by Ruth Nussinov and coworkers [21], we relate the stability of an RNA structure directly with its number of base pairs. Since some algorithms require explicit energy contributions of individual base pairs (e. g., McCaskill' s algorithm to compute base pair probabilities), we set the energy of any base pair Ebp to −1 for simplification purposes. Thus, the energy of a structure is given by E (P) = |P|∙Ebp. Note, this is in stark contrast to state-of-the-art RNA structure prediction approaches (e. g., using Zuker' s algorithm [22]), which typically apply a Nearest Neighbor energy model [41,42] and experimentally derived energy contributions [36]. Furthermore, all algorithms for RNA–RNA interaction prediction ignore concentration dependence and other factors influencing the duplex formation, which is typically modeled within the Nearest Neighbor model by an “initiation” energy term [24,33,34]. Nevertheless, the use of the simplified base pair-focused model enables a much clearer presentation of the algorithms, which is better suited (and sufficient) to understanding their ideas and mechanisms. The transfer from the simple base pair maximization to the advanced energy models, as done by Michael Zuker and Patrick Stiegler [22], is generic and can be applied to all problems discussed within this manuscript. References to extended versions and implementations are provided for each approach. A first task that introduces the general structure of dynamic programming approaches used for RNA structure prediction is to compute the number of structures a sequence S can form, i. e., |P|. Since the structure space P grows exponentially, explicit enumeration is inefficient. In order to apply dynamic programming, we first have to have a strategy of how to decompose such a problem into independent subproblems. Let us consider the subsequence Si.. Sj. We can easily split the problem into two independent problems by introducing a case distinction for its last position Sj; case (1) Sj is not involved in any base pairing, and case (2) Sj is paired with some position Sk (i≤k<j). Both cases are depicted in Fig 1. The first case can be easily reduced to a smaller problem, namely to Si.. Sj−1, since the unpaired position Sj does not allow any structural alternatives. Thus, the reduced problem directly provides a count for case 1. On the contrary, each possible base pairing of Sj in the second case decomposes the problem into two smaller independent problems (one to the left of and one enclosed by the base pair (k, j) ), since no base pair is allowed to cross (k, j) (nestedness condition, see section on RNA secondary structure). Since any structural alternative of the left subproblem can be combined with any of the enclosed ones, we have to multiply the numbers from these smaller subproblems to get the overall count for case 2. Michael S. Waterman and Temple S. Smith applied this idea to solve the counting problem using a table C [19,20]. An entry Ci, j provides the number of structures for a subsequence Si.. Sj. Thus, we initialize Ci, i = 1 for all positions i, since any subsequence of length one is confined to the unpaired structure. The recursion for longer subsequences is given by Ci, j=Ci, j−1+∑i≤k< (j−l) Sk, Sjcompl. Ci, k−1⋅Ck+1, j−1 (1) which combines the two discussed cases to consider all possible “states” of nucleotide Sj in valid structures. The first (Ci, j−1) covers all cases where Sj is unpaired, and the second counts all cases where Sj is paired with an Sk within the subsequence (second case). Note, the base pair (k, j) has to respect the minimal loop length l. The overall number of structures is accessed by |P|=C1, n. Given l and an RNA sequence, our user interface computes and depicts the filled matrix C. Example questions Ruth Nussinov and coworkers introduced in 1978 [21] a first algorithm that efficiently predicts a nested structure with the maximal number of base pairs for a given RNA sequence S, i. e., argmaxP∈P (|P|). The corresponding recursion Ni, j=max{Ni, j−1Sjunpairedmaxi≤k< (j−l) Sk, Sjcompl. (Ni, k−1+Nk+1, j−1+1) Sk, Sjpair (2) is strongly related to the counting approach from Eq 1. Here, an entry Ni, j stores the maximal number of base pairs that can be formed by the subsequence Si.. Sj. Thus, summation in Eq 1 is replaced by maximization and multiplication with summation, while the second case considers the formed base pair with “+1. ” N is initialized with 0 and can be filled in O (n3) time while using O (n2) memory. A depiction of the recursion is given in Fig 2. The maximal number of base pairs formed by any structure can be found in N1, n, and a respective optimal structure P can be identified via traceback starting in N1, n. Thus, for a given cell Ni, j, the traceback discovers how the value of Ni, j was obtained. To this end, the case distinctions of the (filling) forward recursion (e. g., from Eq 2) are considered. If it holds Ni, j = Ni, j−1 (first case), position j is found to be unpaired, and the traceback proceeds with cell Ni, j−1. Otherwise, position j has to form a base pair with some position i≤k<j, which is identified in accordance to the second case of Eq 2. The base pair (k, j) is stored as part of the final structure P and the traceback proceeds for both subintervals represented by Ni, k−1 and Nk+1, j−1. For the identification of functional structures or the study of structural alternatives, the enumeration of suboptimal structures is of interest. A generic approach was introduced by Stefan Wuchty and coworkers [43] that enables the enumeration of all structures that are in a certain range of the minimal energy. An implementation is also available in our web interface. Our interactive user interface enables the computation of both optimal and suboptimal structures. For a user defined sequence as well as recursion and traceback parameters, the dynamic programming table is provided along with a list of (sub) optimal structures. On selection, the according traceback is highlighted within the matrix. This is complemented with a graphical representation of the structure using Forna [44]. Different recursions can be chosen to examine the effects of ambiguous recursions versus the original one. In the following, such an ambiguous variant from [17] is presented. Ni, j=max{Ni+1, jSiunpairedNi, j−1SjunpairedNi+1, j−1+1ifSi, Sjcompl. andi+l<jmaxi<k< (j−1) Ni, k+Nk+1, jdecomposition (3) While this recursion also computes the same entries of N and thus maximal number of possible base pairs (N1, n), it is not using a unique decomposition of the structure, i. e., the same structural variant is considered by different recursion cases. This causes duplicated enumeration of (sub) optimal structures when using Wuchty' s traceback algorithm, which can be studied in our web server for different recursions. Furthermore, it is not possible to use variants of ambiguous recursions like Eq 3 to count structures (consider relation of Eqs 2 and 1) or to compute the partition function of the structural ensemble (as discussed next), since both requires a unique consideration of each structure. In 1981, Michael Zuker and Patrick Stiegler introduced a dynamic programming approach that efficiently computes minimum free energy structures using a Nearest Neighbor energy model [22]. Using further restriction, the same time and space complexity compared to Nussinov' s algorithm is kept. The approach with according decomposition depictions and how it relates to Nussinov' s algorithm is introduced in detail, e. g., in [45]. Implementations like UNAFold [46] (formerly mfold [47]) or RNAfold [31,37] are the current state-of-the-art tools for RNA secondary structure prediction. Example questions To estimate the probability of a given structure P within the structural ensemble P, statistical mechanics typically dictate a Boltzmann distribution when using minimal assumptions [48]. Thus, the probability of a structure P is directly related to its energy E (P) by Pr (P) =exp (−E (P) /kBT) ∑P′∈Pexp (−E (P′) /kBT) (4) given the Boltzmann factor kB and the system' s temperature T. Note, when using an energy model with units “per mole, ” which is typically the case when using a Nearest Neighbor model with measured energy contributions, one has to replace kB with the gas constant R. Note further, the structure with minimal free energy, e. g., predicted with algorithms discussed above, will always have maximal probability according to Eq 4. Thus, the most stable structure is automatically the most likely structure. The nominator of Eq 4 is called Boltzmann weight (of structure P). The denominator is called canonical partition function Z, which is the sum of the Boltzmann weights of all structures in P. Since P grows exponentially, its exhaustive enumeration to compute Z is impracticable. Nevertheless, it is possible to compute Z efficiently using a variant of the counting algorithm. This approach was first introduced for the Nearest Neighbor energy model by John S. McCaskill (1990) [23], and we rephrase a variant for the simplified base pair model. First, we have to note that the Boltzmann weight of a structure P can be computed based on the energy of its base pairs Ebp, as follows exp (−E (P) /kBT) =exp (−∑ (i, j) ∈PEbp/kBT) =∏ (i, j) ∈Pexp (−Ebp/kBT). (5) That is, the structure' s weight is computed by the product of individual base pair weights. To simplify notation in the following, qbp = exp (−Ebp/kBT) refers to the Boltzmann weight of a single base pair. Given this, we can alter the counting recursion from Eq 1 to Qi, j=Qi, j−1+∑i≤k< (j−l) Sk, SjpairQi, k−1⋅Qk+1, j−1⋅qbp. (6) This directly provides the partition function Z =Q1, n in O (n3) time. For some approaches and research questions, probabilities of individual base pairs Prbp (i, j) are of interest. This is the probability that a base pair (i, j) is formed by some structure, which can be calculated by summing up the probabilities of all structures containing (i, j), i. e., Prbp (i, j) =∑P∈P (i, j) ∈Pexp (−E (P) /kBT) Z. (7) As for counting, the base pair (i, j) decomposes all structures into the enclosed and outer subsequence that are independent concerning base pairing. Thus, the partition functions of the according subsequences can be used to compute Prbp (i, j) efficiently. To do so, we need an auxiliary matrix Qbp. Each entry Qi, jbp holds the partition function for the subsequence Si.. Sj, with the side constraint that i and j form the base pair (i, j). If this is not possible due to noncomplementarity or the minimal loop constraint, the entry is 0. Given this, we can rewrite Eq 6 as follows Qi, j=Qi, j−1+∑i≤k< (j−l) Qi, k−1⋅Qk, jbp (8) Qi, jbp={Qi+1, j−1⋅qbpifSi, Sjcomplementary0otherwise (9) and compute the base pair probability using Prbp (i, j) =Q1, i−1⋅Qi, jbp⋅Qj+1, nQ1, n+∑p<i, j<qPrbp (p, q) ⋅qbp⋅Qp+1, i−1⋅Qi, jbp⋅Qj+1, q−1Qp, qbp. (10) The first term in Eq 10 covers structures where (i, j) is an external base pair, i. e., not enclosed by any other base pair. The second term considers all structures in which (i, j) is directly enclosed by a base pair (p, q) and corrects the respective base pair probability Prbp (p, q) by the probability of the structure subensemble that contains both base pairs and no “in-between spanning” base pair (k, l) with p<k<i<j<l<q. The latter probability is defined by the fraction within the second term. Note (again) that by using a simple energy model, we omit all the complex case distinctions, which allows one to concentrate on the main cases of algorithmic importance. In the full model, the first case would have been the same, whereas the second one would have been split to consider specifically each structural context a base pair can have. In analogy to base pair probabilities, it is also possible to define and compute the unpaired probability Prss (i, j) of a subsequence Si.. Sj (Eq 11), i. e., the probability of all structures that show no base pairing in the single-stranded subsequence. Prss (i, j) =∑P∈Pi.. jssexp (−E (P) /kBT) Z (11) withPi.. jss={P|∄ (k, l) ∈P: k∈[i, j]∨l∈[i, j]}⊆P (12) The unpaired probability is also sometimes termed “accessibility, ” as an unpaired region in an RNA is accessible for pairing to another RNA. For the computation of Prss (i, j), we only have to replace Qi, jbp with 1 in Eq 10, since only the unpaired structure with energy zero has to be considered for Si.. Sj, which has a Boltzmann weight of 1. Stephan H. Bernhart and coworkers provide in [49] details for the extension of the introduced recursions to the Nearest Neighbor model, which is also nicely detailed in [45]. Implementations are for instance available in the Vienna RNA package [37]. The authors also show how to reduce the time complexity of the probability computation from O (n4) to O (n3). To this end, they introduce another auxiliary matrix Q^bp that provides the “outer” partition function, which reflects only base pairs not enclosed by respective subsequences. Our web implementation enables the computation of both base pair probabilities as well as unpaired probabilities. To provide insights into how the temperature and energy model influence structure and base pair probabilities, the user can alter the used temperature as well as Ebp. Besides a visualization of the partition function tables Q and Qbp, the user is provided with a visualization of the base pair and unpaired probabilities using the established dot plot format (e. g., used also by UNAfold/mfold [46,47] or RNAfold [37,50]). Within this matrix-like illustration, each base pair probability is represented by a dot of proportional size, i. e., the higher the probability, the larger the dot and small probabilities are not visible. With a bit of visual practice, dot plots enable an easy identification of highly probable substructures and the study of structural alternatives. Example questions The fastest class of RNA–RNA interaction prediction approaches focuses only on the identification of the interaction site, i. e., only on the intermolecular base pairs, without considering the intramolecular structures of the interacting RNAs. To this end, the prefix-based decomposition scheme of global sequence alignment [52] can be adapted. Given two RNA sequences S1 and S2 of lengths n and m, respectively, we denote with S←j2 the reversely indexed S2 to simplify the index notation, since RNA molecules interact in antiparallel orientation. The latter applies to both intra- and intermolecular base pairing. When considering S1 and S←j2, we can design a dynamic programming approach for the simplified energy model using a two-dimensional matrix H. An entry Hi, j will provide the maximal number of intermolecular base pairs for the prefixes S1.. i1 and S←1.. j2. The decomposition scheme for the recursion of Eq 16 to compute Hi, j is visualized in Fig 3. As already mentioned, Eq 16 is a variant of the global sequence alignment approach introduced by Saul B. Needleman and Christian D. Wunsch [52] using an adapted scoring scheme (base pair instead of match/mismatch scoring for Si1, S←j2 and no gap cost). Thus, initializing all Hi, 0/H0, j with 0, the entry Hn, m provides the maximal number of intermolecular base pairs that can be formed, and a traceback starting at Hn, m yields the respective interaction details. This approach enables very low runtimes (O (nm) ), as observed by Brian Tjaden and coworkers, who presented in [30] a variant of Eq 16. When computing hybridization-only interactions via minimizing a more sophisticated energy model, the strategy has to be altered to follow a scheme similar to local sequence alignment as defined by Temple Smith and Michael S. Waterman [53], which is detailed in [30]. The web interface of our implementation identifies and reports all optimal interaction sites. For each, an American Standard Code for Information Interchange (ASCII) visualization of the intermolecular base pairs is provided. Note, to reduce code redundancy, we do not use an implementation of Eq 16 but use a base pair-maximization variant of Eq 19, which is discussed in the next section. Adaptations of this approach to the Nearest Neighbor model have been discussed in [28] and, e. g., implemented in the tools TargetRNA [30], RNAhybrid [29], or RNAplex [54]. While such methods have been successfully applied for target site identification of very short RNAs, they often overestimate the length of target sites since intramolecular base pairing is ignored [33,54]. These problems are tackled by concatenation- and accessibility-based approaches discussed next. Example questions Among the first approaches to predict the interacting base pairs for two RNA molecules are concatenation-based or cofolding approaches [31,32]. Here, two or more RNA sequences are concatenated into a single sequence with special interspacing linker sequences. The resulting hybrid sequence is used within an adaptation of a standard structure prediction that takes special care of the linker sequences. The linked sequences are forbidden to form base pairs, and the structural elements containing linker sequences are treated energetically as external, as discussed by Ivo L. Hofacker and colleagues [31]. The extension of standard structure prediction approaches to RNA–RNA interaction prediction directly yields the possibility to compute according probabilities of interaction sites or intermolecular base pairs [55]. A first implementation of concatenation-based prediction using the Nearest Neighbor energy model was reported for mfold [47] and later implemented in, e. g., the tools MultiRNAFold [56] and RNAcofold [55]. Our implementation extends the Nussinov recursion from Eq 2 with a special handling for linker sequence characters “X. ” Base pairs (case 2) are not allowed to involve a linker position. No special energy treatment is necessary for the simplified energy model since we treat intra- and intermolecular base pairs equally and without considering their context. The input is restricted to two RNA sequences that are concatenated by a linker of length l+1 (where l is the minimal loop size) to ensure the presence of a linker and that the concatenated sequence ends can form a base pair. Our interactive cofolding web interface lists (sub) optimal hybridization structures using our generic suboptimal traceback implementation. Within the reported dot-bracket strings, intramolecular base pairs are encoded using parentheses “ (), ” intermolecular base pairs (spanning the linker) are represented by brackets “[], ” and the linker itself is depicted by linker characters “X. ” For each hybridization structure, a traceback is visualized on selection along with a Forna 2D structure graph visualization. Furthermore, an ASCII visualization of only the intermolecular base pairs is provided. Concatentation-based approaches do incorporate the competition of intra- and intermolecular base pairing, which is a central weakness of hybridization-only prediction algorithms. Still, not all important interaction patterns can be predicted using cofolding approaches since the hybrid structure has to be nested. For instance, common kissing stem–loop or kissing–hairpin interactions cannot be predicted because they form a crossing structure in the concatenated model (see Fig 4). To predict such patterns, accessibility-based approaches, discussed next, can be applied. Example questions The previously introduced concatenation-based approaches directly reflect the competition of intra- and intermolecular base pairing by optimizing both at the same time. Nevertheless, they are neglecting that the intramolecular structure is established before an intermolecular interaction is formed. That is, intramolecular base pairs (might) have to be opened/broken such that intermolecular base pairs can form a stable interaction. To be favorable, the interaction energy must outweigh the energy needed to make the subsequences accessible. This two-step process is modeled by accessibility-based interaction prediction approaches. The following formula, depicted in Fig 5, is used to compute the final interaction energy values Ij, li, k that incorporate both the hybridization/duplex energy D as well as the penalties ΔE1, ΔE2 for inaccessible sites of the RNAs S1, S2, respectively. Note, ΔEj.. l2 is computed for the reversely indexed sequence S←2 to ease the notation. This reversal has to be taken into account for hybridization energy computations, since Nearest Neighbor models have to incorporate the chemical 5′- to 3′-end orientation of RNAs. The entry of I with minimal energy is used to traceback the interaction details of the optimal interaction. Only entries in I with an energy lower than zero mark favorable interactions since here, the duplex energy D outweighs the ΔE penalties to make the respective subsequences accessible. The energy penalties ΔEi.. j resemble the free energy needed to make the interaction site Si.. Sj accessible, i. e., to unfold the site' s intramolecular base pairs [24,33]. To reflect the structural flexibility of RNAs, the terms are based on the structure ensembles that can be formed rather than individual structures. The penalties can be computed from the energy difference of the structure ensemble with accessible site that is single stranded, Ei.. jss, versus the whole structure ensemble, Eens. Both energies can be computed from the respective partition functions Zi.. jss (for Pi.. jss from Eq 12) and Z using the inverse Boltzmann weight. In the following, we show the relation of ΔE and the unpaired probability Prss. ΔEi.. j=Ei.. jss−Eens=− (RT⋅log (Zi.. jss) −RT⋅log (Z) ) =−RT⋅log (Zi.. jss/Z) =−RT⋅log (Prss (i, j) ). (18) Note, since Prss (i, j) is ≤1, all ΔEi.. j penalties are ≥0. To add such site-specific terms to duplex energies, we cannot simply use the prefix-based recursion from Eq 16, since Hi, j only provides the optimal value for all interaction sites with right ends Si1 and S←j2 and not for individual sites. Thus, for exact results, we have to relate to a subsequence-based computation that explicitly stores values for all subsequence combinations. To further simplify the recursions, we use dedicated calculations (and matrices) for the duplex energy (matrix D, Eq 19) and the overall interaction energy including inaccessibility penalties (matrix I, Eq 17). Both matrices are four-dimensional, in which an entry Dj, li, k provides the duplex energy of the interacting sites Si.. k1 and S←j.. l2 under the assumption that the boundaries form the intermolecular base pairs (i, j) and (k, l); otherwise, the entry is set to ∞. Dj, li, k=min{EbpSi1, S←j2compl., i=k, j=lmini<p≤kj<q≤l (Ebp+Dq, lp, k) Si1, S←j2compl., i<k, j<l+∞otherwise. (19) The first case represents the initiation of a new interaction that covers only the intermolecular base pair (i, j) with according energy Ebp. The second case extends an already-computed interaction of Sp.. k1, S←q.. l2 with a new base pair (i, j), while the third case is applied if the base pair (i, j) cannot be formed or the indices violate order constraints. Note, the given recursion has an O (n6) time complexity due to arbitrarily large gaps in the second case. Given the typically applied thermodynamic model and statistics from known interactions, the sequential distance between neighbored intermolecular base pairs is normally restricted to a small constant <30 [24], which reduces the time complexity to O (n4). The space complexity can be reduced to O (n2), as shown in [33], by interactively computing parts of D for a fixed right-boundary base pair (k, l). Our implementation provides the list of all optimal interactions and visualizes the selected interaction details using an ASCII chart. Due to the four-dimensionality of the matrices D and I, only the value Ij, li, k for the current selection as well as the penalty tables ΔE1+ΔE2 used for computation are shown. The interactive web interface enables a straightforward comparison of the effects and restrictions of the three different interaction prediction approaches introduced. For instance, using the simple example sequences S1 = CCC and S2 = CCCGGGGGG, the hybridization-only optimization reports (as expected) any interaction patterns of S1 with G nucleotides of S2. In contrast, intermolecular base pairs predicted by the cofolding approach are restricted to the 3′-end of S2 since the central G nucleotides are blocked by an intramolecular hairpin structure (similar to Fig 4A). Both approaches neglect that RNA S2 will first (most probably) fold into a hairpin structure (with unpaired/accessible nucleotides in the center) before both interact. Thus, it is most likely this central unpaired region of S2 where interaction formation with S1 will start. The growing interaction would have to break the already-formed intramolecular base pairs for larger interaction patterns, which is not necessarily favorable. This scenario is modeled by accessibility-based approaches, which predict interactions to be restricted to the loop region only. The resulting interaction resembles a kissing stem–loop pattern (see Fig 4B). Note, while accessibility-based approaches are well suited to predict interaction patterns like stem–loop or kissing hairpin interactions, they are still not able to model arbitrary interaction patterns. For instance, double kissing hairpin interactions can not be modeled correctly [57]. The first accessibility-based approach RNAup for the Nearest Neighbor model was introduced by Ulrike Mückstein and colleagues [24]. While it is still among the state-of-the-art prediction tools [27], its vast runtime requirements of O (n4) render it inapplicable for large-scale data analyses, such as genome wide target screens. This problem was tackled by Anke Busch and coworkers with IntaRNA [33,34], which implements a heuristic version of an accessibility-based approach that extends fast hybridization-only recursions with ΔE penalties. IntaRNA results in a much lower O (n2) time complexity [33] when using precomputed or approximate ΔE terms, as introduced in [58]. A detailed introduction is also given in [45]. A similar heuristic extension was recently reported for TargetRNA2 [59]. Current versions of the initially hybridization-only approach RNAplex [54] and its webserver RNApredator [60] incorporate an approximate, position-specific accessibility model to increase prediction quality [61]. Example questions All discussed algorithms and visualizations have been implemented in JavaScript. This enables client-side computation (no backend server hardware needed) as well as local download and application (from GitHub repository) for offline usage. Since all algorithms are dynamic-programming approaches, a generic inheritance hierarchy was implemented to reduce code redundancy and to simplify maintenance and extensibility. We use Knockout. js as the controller to bind input/output elements from within the HTML pages with the JavaScript data structures and computations. The understanding of RNA structure and RNA–RNA interaction prediction approaches is central to ensure correct result interpretation and an awareness of their limitations, both essential to avoid wrong conclusions. Furthermore, it ensures proper embedding in RNA-related analysis pipelines or their extension to new fields of applications. To gain this level of understanding, the original literature is often of limited didactic value, since scientific articles are typically not meant for educational use. Thus, approaches are either represented on a very detailed expert level or sketched briefly, since the manuscript focuses on the biological results rather than algorithmic details. Here, we provide a compact summary of the relevant theoretical background for the most common algorithmic approaches and their state-of-the-art instances currently used. Algorithms are stripped from complicating energy model details to enable an easy understanding of the underlying concepts and the resulting limitations. Furthermore, we provide web-based implementations and visualizations of all presented approaches for their ad hoc use. The latter is of importance, since example-driven (self-) study is known to significantly foster learning and understanding. To further support such self-learning efforts based on our manuscript and web service, we provide small exemplary tasks for each algorithm group that can be tackled using our web implementations. The web service [62] is being continually extended with the implementation and visualization of additional methods. Planned implementations cover pseudoknotted (crossing) structure prediction approaches as well as comparative approaches for RNA structure and RNA–RNA interaction prediction, e. g., discussed in [57]. Eventually, we provide both a comprehensive review of current RNA thermodynamic-focused prediction approaches to spark ideas for new approaches and interactive teaching material, which will help ensure that available tools are correctly applied and interpreted.

RNA molecules are central players in many cellular processes. Thus, the analysis of RNA-based regulation has provided valuable insights and is often pivotal to biological and medical research. In order to correctly select appropriate algorithms and apply available RNA structure and RNA-RNA interaction prediction software, it is crucial to have a good understanding of their limitations and concepts. Such an overview is hard to achieve by end users, since most state-of-the-art tools are introduced on expert level and are not discussed in text books. Within this manuscript, we provide the mathematical means and extract the algorithmic concepts that are core to state-of-the-art RNA structure and RNA-RNA interaction prediction algorithms. The conceptual, teaching-focused presentation enables a detailed understanding of the approaches using a simplified model for didactic purposes. We support this process by providing clear examples using the web interface of our algorithm implementation. In summary, we have compiled material and web applications for teaching-and the self-study of-several state-of-the-art algorithms commonly used to investigate the role of RNA in regulatory processes.

lay_plos

Influenza A NS1 and NS2 proteins are encoded by the RNA segment 8 of the viral genome. NS1 is a multifunctional protein and a virulence factor while NS2 is involved in nuclear export of viral ribonucleoprotein complexes. A yeast two-hybrid screening strategy was used to identify host factors supporting NS1 and NS2 functions. More than 560 interactions between 79 cellular proteins and NS1 and NS2 proteins from 9 different influenza virus strains have been identified. These interacting proteins are potentially involved in each step of the infectious process and their contribution to viral replication was tested by RNA interference. Validation of the relevance of these host cell proteins for the viral replication cycle revealed that 7 of the 79 NS1 and/or NS2-interacting proteins positively or negatively controlled virus replication. One of the main factors targeted by NS1 of all virus strains was double-stranded RNA binding domain protein family. In particular, adenosine deaminase acting on RNA 1 (ADAR1) appeared as a pro-viral host factor whose expression is necessary for optimal viral protein synthesis and replication. Surprisingly, ADAR1 also appeared as a pro-viral host factor for dengue virus replication and directly interacted with the viral NS3 protein. ADAR1 editing activity was enhanced by both viruses through dengue virus NS3 and influenza virus NS1 proteins, suggesting a similar virus-host co-evolution. Influenza A viruses are the causative agents of seasonal and pandemic infections and are responsible for the death of at least half a million people worldwide each year. The genome of influenza A viruses is composed of eight negative-sense single-stranded RNAs encoding 13 proteins. NS1 and NS2 are derived from alternatively spliced RNAs that are transcribed from the eighth RNA segment. The segments are encapsidated by binding to nucleoproteins (NP) and the polymerase complex (PA, PB1 and PB2) forming the viral ribonucleoproteins (vRNPs). The viral particle contains eight vRNPs, the surface glycoproteins haemagglutinin (HA) and neuraminidase (NA), the matrix proteins (M1 and M2) and the NS2 protein. Some strains express the pro-apoptotic PB1-F2 protein and two additional virulence factors, PB1-N40 and PA-X, have been recently identified [1]–[3]. The NS1 protein is not incorporated in the virus. It exerts a large spectrum of functions through interactions with a variety of cellular components residing either in the cytoplasm or in the nucleus. NS1 is a pleiotropic virulence factor repressing innate antiviral mechanisms e. g. by interfering with the type I interferon system through direct interaction with PKR and TRIM25, or through the sequestration of double-stranded RNA [4]–[8]. NS1 is also known to perturb the mRNA processing by interacting with CPSF4 and PABPN1 to inhibit nuclear export of cellular mRNA [9] and is suspected to hijack the RNA translation machinery in favor of translation of viral protein e. g. by interacting with STAU1 [10]–[11]. In contrast to NS1, NS2 protein is a structural component of the viral particle and it associates with the viral matrix M1 protein [12]. NS2 mediates the export of vRNPs from the nucleus to the cytoplasm through export signal [13] via its interaction with XPO1 [14]. In addition, NS2 interacts with nucleoporins and was suggested to serve as an adaptor between vRNPs and the nuclear pore complex [13]. A role of NS2 in the regulation of influenza virus transcription and replication has also been proposed [15]. However, many functions of NS2, in particular its transit through the cytoplasm and its incorporation into the viral particle, are not understood. Several screens have been performed to identify host factors involved in the influenza virus replication cycle, mainly focusing on interactors of vRNPs or of the polymerase by using affinity purification or yeast two-hybrid techniques [16]–[18]. A proteome-wide screen of virus-host protein-protein interactions has provided an important resource of 135 interactions [19]. However, the weak overlap of the public datasets suggests that they are far from being complete. The impact of cellular proteins on the influenza virus replication has been extensively studied using RNAi screens [19]–[24]. Although poorly overlapping at the gene level, these screens better converge at the level of biological processes [25]–[27]. Hence, more than the identification of host factors, these studies highlighted major cellular functions that are essential for the virus replication. However, for the majority of identified host factors, the mode of action remains to be determined. Furthermore, comparisons of strain-specific virus-host interactomes are clearly missing, which is required to reveal general principles governing infection mechanisms and to identify common therapeutic targets as well as broad-spectrum antivirals. In the present study we conducted stringent yeast two-hybrid screens to identify human proteins interacting with NS1 and NS2 from 9 influenza A virus strains representative of the variability in nature. The functional impact of all NS1 and NS2 interactors on viral replication was systematically addressed by RNA interference. In combination with published datasets, our new results offer a comprehensive view of NS1 and NS2 interactomes and corresponding targeted cellular functions. The global analysis of the NS1 and NS2 host cell targets reveals an enrichment of double-stranded RNA binding domain (DRBD) containing proteins for the 9 tested influenza virus strains. A focus was put on ADAR1 since this protein is critical for the replication of other viruses [28], is highly expressed in human lung cells [29], is induced by type I interferon [30], is interfering with interferon signalling production [31] and is interacting with all tested NS1 proteins. In addition, we also observed in another screen that ADAR1 interacts with the dengue virus NS3 protein which is a bifunctional enzyme containing protease and helicase activity [32]. We show that ADAR1 is a pro-viral host factor favoring replication of influenza virus and dengue virus and that these viral proteins can control ADAR1 editing activity. To identify all cellular proteins interacting with influenza virus NS1 and/or NS2 proteins, yeast two-hybrid screens (Y2H) were carried out using NS1 and NS2 proteins from 9 different virus strains as baits (Table S1) and three cDNA libraries (from human spleen, fetal brain and respiratory epithelium). Key features of the virus strains are provided in Text S1. NS1 and NS2 proteins selected for this study are representative of the natural diversity since they are distributed all along the phylogenetic trees of known NS1 and NS2 sequences (Text S1, Figures S1 and S2 in Text S1, Alignments of NS1 and NS2 protein sequences are presented in Figures S3 and S4 in Text S1). Seventy nine non-redundant cellular proteins were identified to interact with NS1, NS2 or both and were individually retested in a pairwise array (Figure 1A). From a total of 1422 possible interactions tested (79 cellular proteins tested against 9 NS1 and 9 NS2 proteins), 562 tested positive. In this way, we identified 33 cellular proteins interacting exclusively with NS1,28 exclusively with NS2, and 18 with both NS1 and NS2. The vast majority (97. 5%) of the NS1 and NS2 interactors are known to be expressed in the respiratory epithelium (Table S2). Twelve out of the 79 host interactors have already been reported (AIMP2, SCRIB, CPSF4, the kinases PIK3R1, PIK3R2, MAPK9, CRK and proteins with a double-stranded RNA-binding domain STAU1, PRKRA, ADAR1, TARBP2, ILF3) [9], [11], [19], [33]–[38]. 21. 5% of host interactors are targeted by all virus strains (Figure 1B) and 5% appear to be strain specific. 80% of the cellular interactors bind to more than 50% of the tested NS1 and NS2 proteins indicating that the dataset is more appropriate to the identification of common rather than differential interaction profiles. Together with previously published data available in the VirHostNet database [39], we now provide a list of 111 non-redundant cellular proteins interacting exclusively with NS1,32 exclusively with NS2 and 18 with both proteins (a complete list of influenza virus interactors is given in Table S3). Consistent with observations from previous virus-host interactome studies, NS1 and NS2 proteins tend to interact with highly central proteins in the human interactome [40]–[43]. Indeed, the degree distribution of targeted human proteins was significantly higher than the degree distribution in the human interactome (U-test, p-value<2. 2×10−16) (Figure 1C). Similarly, the betweenness distribution of targeted human proteins was significantly higher than the betweenness distribution in the human interactome (U-test, p-value<2. 2×10−16) (Figure 1D). This suggests that influenza NS1 and NS2 proteins preferentially target pleiotropic cellular proteins [44]. Finally, an assessment of Gene Ontology categories revealed a significant enrichment (p-value = 3. 3×10−14) for DRBD-containing proteins (DRBPs) in the interaction dataset. Strikingly, DRBPs were exclusively targeted by NS1 proteins. All virus strains interacted with most of the DRBPs suggesting that the direct targeting of DRBDs is of special importance for influenza A viruses. Among the 79 NS1 and NS2 interactors identified here, 12 have been previously identified in recent genome-wide siRNA screens as modulators of viral replication - ATP6V1G1, RPL13A [21], [23], GMEB1, PIK3R2 [19], SON, EEF1A1 [23], CHCHD5, RPL23A [24] and NUP214 [22]. However, since these genome-wide siRNA screens are weakly overlapping, it is very likely that numerous modulators of viral replication have been missed or remain to be confirmed [25]–[27]. We have therefore performed a systematic siRNA-based screen in A549 human lung epithelial cells to explore the functional contribution of the 79 cellular NS1 and NS2 interactors to virus replication. The silencing phenotype was first tested by measuring replication of the A/H1N1/Puerto Rico/8/34 virus strain which was used in the yeast two- hybrid screen. The complete replication cycle was first probed by measuring the neuraminidase activity in the supernatant 48 h post-infection. The assay was calibrated by using siRNAs against ATP6V1G1 and CSNK2B that have been previously described as pro-viral host factor and anti-viral host factor respectively. ATP6V1G1 is a subunit of the vacuolar ATPase proton pump required for influenza A virus replication [21] while CSNK2B gene silencing increases virus replication in A549 infected cells [45]. As expected, siRNAs targeting ATP6V1G1 and CSNK2B respectively reduced and increased the neuraminidase activity in the supernatant, thus validating the assay (Figure 2A and 2B). By comparison with these controls, virus replication should be altered by at least 35% according to the threshold defined by König et al. in their genome-wide siRNA screen [22]. This threshold together with a silencing efficiency greater than 60% for each siRNAs without detectable cytotoxicity were used for a stringent selection of the pro-viral and anti-viral host factors (Table S4). These criteria are in the range applied in earlier siRNA-based screens [23], [46], [47] (information on individual silencing efficiency is also provided in Table S4). In this way, we identified the two pro-viral host factors, ADAR1 and RPSA, and confirmed ATP6V1G1, RPL13A, EEF1A1 and SON (Figure 2A). In addition, one new anti-viral host factor, N-PAC, was identified (Figure 2A). These results were confirmed by using plaque assays, revealing a broader range of inhibition or activation of virus replication (Table S4). In conclusion, out of the 79 cellular interactors of NS1 and NS2 identified in this study, 7 were identified as possible direct modulators of A/H1N1/Puerto Rico/8/34 virus replication. Importantly, these results were confirmed with the A/H1N1/New Caledonia/2006 influenza virus strain that was not used in the yeast two-hybrid screen (Figure 2B), although RPL13A only scored positive 72 h post infection (Figure S5 in Text S1). As it is a critical component of virus-host interaction, production of type I interferon was quantified in the supernatant of infected cells transfected with siRNAs (Table S4). Silencing of ADAR1, ATP6V1G1, BCLAF1, RPL13A and SON increased interferon production in infected cells. These proteins interact with NS1 and, except for BCLAF1, are pro-viral host factors for the virus. Therefore, 4 of the 6 pro-viral host factors are implicated in interferon production. This is consistent with the role of NS1 in interfering with the type I interferon system. The function and subcellular localization of cellular interactors identified in the present and published studies indicate that both NS1 and NS2 are pleiotropic proteins required for several essential steps of the viral life cycle (Figure 3). Although NS2 function in the cytoplasm remains elusive, it is shown here that NS2 mostly targets proteins of the cytoskeleton and involved in intracellular transport (Figure 3). Given that NS2 also interacts with the vRNPs, it might also mediate their transport to the plasma membrane or to the nucleus. In case of the latter, NS2 is implicated in the export of vRNPs, consistent with the observed interaction of NS2 with the NPC [13]. The targeting of transcription-regulating proteins by NS2 is much less documented. A role of NS2 in regulating influenza virus RNA genome transcription via its interaction with vRNPs has been previously proposed [15], [48]. Although such a direct interaction with the components of the vRNPs is not ruled out, our data indicate a direct targeting of the cellular transcriptional machinery by NS2 (Figure 3, box regulation of transcription). Interestingly, since NS1 also targets this process, a potential cooperation between NS1 and NS2 for the control of the cellular transcription machinery can be speculated. NS1 proteins target several DRBPs either localized in the nucleus or in the cytoplasm [49]. These proteins are critical transcriptional or translational checkpoints. NS1 is known to inhibit the activation of PKR, one of the major interferon-inducible antiviral effectors, through direct interaction [6]. More recently, SON has been described to be important for the trafficking of influenza virions [23]. Here, we confirmed that SON is essential for viral replication and suggest that this activity could be related to the NS1 protein. ADAR1 and PKR have an opposite effect on virus replication although they are both induced by type I interferon. ADAR1 is a type I interferon-induced protein that is expressed in human lung [29], [30] and interacts with all tested NS1 proteins. The 150 kDa interferon-inducible ADAR1 isoform is expressed in A549 cells upon influenza A virus infection and by type I interferon. The constitutive 110 kDa ADAR1 isoform was only induced upon infection indicating that ADAR1 expression can also be controlled by an interferon-independent mechanism, at least in the setting of an influenza A virus infection (Figure 4A). ADAR1-specific siRNAs efficiently reduced the expression of ADAR1 isoforms and blocked their induction upon infection (Figure 4B). The silencing of ADAR1 inhibited virus release from 15% at 8 h post-infection to 90% at 48 h post infection (Figure 4C). Expression of viral proteins (here HA, NP, M1 and NS1) was also significantly reduced as early as 8 h post infection. NS1, NP and M1 expression was delayed while HA expression remained very low until 24 h post infection (Figure 4B). Thus, ADAR1 is a pro-viral host factor for virus protein expression and virus production. Immunofluorescence revealed that ADAR1 is diffusely distributed in the nucleus and relocalized in nuclear structures in influenza virus-infected cells (Figure 5A). In these structures ADAR1 colocalized with NS1 but not with HA for which no interaction with ADAR1 could be detected. As NS1 interacts with several DRBD-containing proteins, the NS1 binding site in ADAR1 could be a DRBD. Amino acid sequence alignment of DRBDs revealed a conserved region of 47 amino acid residues within the two firsts DRBD of ADAR1 (Figure S6 in Text S1). A set of 4 ADAR1 deletion mutants, differing in their number of DRBDs, and a plasmid encoding the 47 amino acid residues of the first DRBD were constructed (Figure 5B). In a yeast two-hybrid array, ADAR1 interacted with NS1 even in the absence of its first DRBD while interaction was completely abrogated when the first two DRBDs were deleted. The peptide of 47 amino acid residues also interacted with NS1 (Figure 5C) in the array and in GST pull-down assays (Figure 5D). Thus, ADAR1 displays two potential NS1 interaction sites located on the first two double-stranded RNA-binding domains. To validate these results the NS1 RNA-binding domain (RBD) and effector domain fused to GST were used in pull-down experiments for the mapping of NS1 interaction with 3×Flag tagged ADAR1 after co-expression in HEK293T cells (Figure 5E). Full-length NS1 and NS1 RBD domain efficiently co-precipitated ADAR1 but not the effector domain (Figure 5F) indicating that NS1 interacts with ADAR1 through its RBD. GST pull-down and RNAse A treatment showed that RNA is marginally involved in the NS1-ADAR-1 interaction (Figure S7 in Text S1). A mutant of NS1 that lacks double-stranded RNA-binding activity still interacts with ADAR1, albeit with reduced efficiency (Figure S8 in Text S1) confirming that RNA is not strictly required for NS1 interaction with ADAR1. To evaluate the functional impact of ADAR1-NS1 interaction on the catalytic activity of the enzyme, an original editing reporter system was constructed. This reporter system consists of a 24 nucleotide-long minimal ADAR1 substrate derived from the sequence of the antigenome of the hepatitis delta virus that is edited by this enzyme [50]. In this sequence, ADAR1 editing activity changes a stop codon into a tryptophane codon (Figure 6A) [51]. The reporter plasmid contains the ADAR1 substrate sequence inserted in frame in-between the Renilla and the Firefly luciferase genes (Figure 6A). In this configuration, the Firefly luciferase activity reflects the extend of editing and thus ADAR1 activity, leading to the conversion of the stop codon into the tryptophane codon. ADAR1 was co-expressed in HEK293T cells with NS1 or its RBD and with the editing reporter construct. The NS1 effector domain or DLG4, which does not bind to ADAR1 (data not shown), was used as negative control in analogous co-transfection experiments. NS1 RBD and full-length NS1 increased the Firefly luciferase signal by 30% and 60% respectively (Figure 6B) suggesting that NS1 can cooperatively interact with ADAR1 via its RNA-binding domain to promote ADAR1 editing activity (Figure 6B). Editing activity was also analyzed in the context of influenza virus infection after expression of the editing reporter construct (Figure 6C). H1N1 influenza virus infection increased the editing activity of ADAR1 by 70% and this was completely reversed when ADAR1 expression was silenced by RNA interference. To validate these observations, a catalytically inactive ADAR1 (E912A) mutant was constructed [52]. Unfortunately, A549 cells became refractory to plasmid DNA transfection after siRNA transfection, thus precluding functional tests of the mutant in this cell line (not shown). As an alternative, we tested a potential transdominant negative effect of the ADAR1 mutant on influenza virus growth. The catalytically inactive ADAR1 (E912A) mutant construct was therefore transfected into A549 cells and virus growth in these cells was compared to the one achieved with mock-transfected cells or in wild type ADAR1-transfected cells. Viral protein expression was reduced in A549 cells expressing the ADAR1 mutant compared to control cells (Figure 6D). Consistent with this result, neuraminidase activity in the supernatant was also significantly reduced (Figure 6E). Importantly, since influenza A virus infection induces endogenous ADAR1 expression, the impact of the ADAR1 mutant is most likely underestimated in this experimental system. We therefore concluded that the RNA editing function is required for the pro-viral activity of ADAR1. During the course of a systematic screening for virus-host protein-protein interactions with a yeast two-hybrid system, we also identified ADAR1 as an interactant of the NS3 protein of dengue virus type 2. This interaction was confirmed in a yeast two-hybrid pairwise array (Figure 7A). As for NS1 of influenza A virus, interaction between NS3 and ADAR1 was validated by GST pull-down experiments (Figure 7B) and also in this case, RNA contributed to this interaction only to a very minor extent (Figure S9 in Text S1). Both ADAR1 isoforms were induced upon dengue virus infection as well as upon type I interferon treatment of Huh-7 cells (Figure 7C). Silencing of ADAR1 expression by RNA interference (Figure S10 in Text S1) resulted in a strong decrease of dengue virus replication (Figure 7D). This result was confirmed with a subgenomic dengue virus replicon stably replicating in Huh-7 cells, indicating that ADAR1 acts at a post-entry step in the dengue virus life cycle (Figure S11 in Text S1). Likewise, as observed for influenza virus, dengue virus infection strongly increased the editing activity of ADAR1 (Figure 7E). In fact, full-length NS3 and the helicase domain increased the Firefly signal by 24% and 44% respectively, suggesting that NS3 cooperatively interacts with ADAR1 to enhance its editing activity (Figure 7F). In conclusion, both influenza virus and dengue virus (i) induce over-expression of ADAR1, (ii) interact with ADAR1 through the RNA-binding domain of influenza virus NS1 and the helicase domain of dengue virus NS3, (iii) enhance the editing activity of ADAR1 and (iv) are dependent on ADAR1 expression for efficient virus replication. This study describes an exhaustive interaction profile for NS1 and NS2 proteins of 9 influenza virus strains. More than 560 interactions between 79 cellular proteins and NS1 and NS2 were identified. Thirty-three cellular proteins interacted exclusively with NS1,28 exclusively with NS2, and 18 with both NS1 and NS2. Since NS1 and NS2 are the products of alternatively spliced RNAs, shared interactions may reflect binding to the common N-terminal 10 amino-acid residues long sequence. This result suggests that influenza viruses have evolved two proteins to interact with cellular proteins that are potentially essential for them. Twelve out of the 79 NS1 and NS2 cellular interactors have already been reported in the literature, demonstrating the reliability and robustness of our screening approach. For NS1, there is a strong overlap with hits published by others (11 of the 51 interactors identified in the present study, which is well above the average overlap) [41], [53], suggesting that the NS1 interactome dataset is now close to completion. In case of NS2, only 4 cellular interactors have been published and one of them, AIMP2, has been confirmed in our screens. Although 46 new NS2 interactors have been identified, it is difficult at this stage to estimate the completion level of the NS2 interactome due to the lack of published interaction data. Overall, most of the cellular targets interacted with the majority of NS1 or NS2 proteins of the different influenza viruses arguing that we have identified highly relevant and evolutionary conserved interactions. Interestingly, a significant proportion of these proteins is also targeted by other viruses (44. 7%, exact Fisher test, p-value<2. 2×10−16) indicating that these cellular proteins are likely to be involved in a generic process of viral infection [39]. Our interaction dataset indicates that NS1 and NS2 proteins are likely to be involved in multiple steps of the viral replication cycle, paving the way for challenging functional explorations. This was largely unexpected for NS2, which is known to be involved in the nuclear export of the vRNPs. Its interaction with the cytoskeleton appears particularly interesting for further studies. Although the pleiotropic nature of NS1 is well established [54], our study provides new insights into the breadth of interactions and activities of this regulatory protein. In addition to the 67 new interactors, the current dataset also provides additional information on previously known interactors and related targeted functions. For instance, the CPSF4 interaction with NS1 has been described as a potential therapeutic target [55] and is confirmed in our study. Three NS1 proteins also interacted with CPSF3L, a protein participating in the endonuclease activity of CPSF, suggesting that the corresponding viruses evolved alternative strategies to interfere with the cellular 3′end mRNA processing [56]. The phenotypic analysis of the cellular targets of NS1 and NS2 by RNA interference revealed an enrichment in modulators of influenza virus replication, further validating the interaction dataset. Indeed, out of the 79 cellular interactors of NS1 and NS2 identified in this study, 7 revealed to control positively or negatively the replication of two influenza virus strains. Interaction profiles suggest that the data could be extrapolated to other strains with the noticeable exception of RPL13A, an exclusive target of A/H1N1/Puerto Rico/8/34 NS1. The validation rate of cellular interactors by RNAi reached about 9% (15. 2% when data from the literature are included) and is similar to that of Shapira et al. [19] while the validation rate of virus replication modulators identified from genome-wide siRNA screens ranges from 0. 75 to 1. 5%. Therefore, combining interactomic screens with genetic screens drastically enhances the rate of functional validation, providing lists of cellular proteins strongly enriched in pro- and anti-viral host factors (exact Fisher test, p-value = <2. 1 10−4, Text S1). Interaction of NS1 with some members of the DRBD protein family have been sporadically documented [6], [10], [19]. Here we observed a massive enrichment of the DRBD protein family in our NS1 interactome for which we used 9 different influenza virus strains. One hundred and sixty five independent screens have been performed with other viral baits using the same cDNA libraries (45 with the fetal brain cDNA library, 31 with the respiratory epithelium library and 89 with the spleen library). The GO term “Double-stranded RNA-binding domain (DRBD) containing proteins” has never been enriched in any of these screens while it was enriched for the 9 tested influenza virus strains. Reciprocally, a large diversity of other GO terms was enriched in these different screens and in screens performed by other laboratories using the same libraries. Therefore, we could be confident that the DRBD containing proteins enrichment reflects a real propensity of NS1 to interact with this protein family. This is most likely reflecting the ability of NS1 to interact with the double-stranded RNA-binding domain of cellular partners through its own RNA-binding domain. Two DRBD-containing proteins, SON and ADAR1, were found to be essential for virus replication. Conflicting results on the role of ADAR1 for virus replication have been published. Initially suspected to have an antiviral activity because of its induction by interferon, ADAR1 appears to promote the replication of several viruses (measles virus, vesicular stomatitis virus, hepatitis delta virus, human immunodeficiency virus type 1 and Kaposi' s sarcoma-associated virus). In contrast ADAR1 was reported to display an antiviral activity against hepatitis C virus and lymphocytic choriomeningitis virus [28], [57]. Concerning influenza A virus, two studies provided evidence for an antiviral role of ADAR1. Mice lacking IKKε become highly susceptible to influenza virus infection, express ADAR1 only to low amounts and show a reduced editing of matrix M1 mRNA isolated from infected lung. However, since IKKε knock-out also strongly affects the expression of other type I interferon-stimulated genes, the susceptibility of these mice to infection could not be attributed to a unique defect in ADAR1 activity [58]. An increased cytopathic effect of influenza A virus has been observed in mouse cells derived from non-viable embryos unable to express the p150 isoform of ADAR1. However, this effect was not correlated to an increased virus replication [59]. In the present study, we show that inhibiting ADAR1 expression by RNA interference reduced viral protein expression and drastically impaired virus replication. Thus, ADAR1 appeared as an important host dependency factor for influenza viruses. Several studies have demonstrated a role of ADAR1 in modulating interferon signaling. Inducible ADAR1 disruption in mice causes a global interferon response [31]. Mutations in ADAR1 responsible for Aicardi-Goutières syndrome in humans are associated with upregulation of interferon-stimulated genes [60]. ADAR1 also suppresses measles virus-induced production of interferon-β mRNA [61]. Here, we show that interferon-β is enhanced in ADAR1-deficient cells after infection with influenza A virus. NS1 is a well-known antagonist of the antiviral response. Its mode of action is pleiotropic including interference with signaling induced by RIG-I like receptors (RLRs) [62]. A combined action of ADAR1 and NS1 protein is suggested by our results. The double-strand RNA editing activity of ADAR1 produces double-strand RNA with I: U pairs instead of A: U pairs. Interestingly, I: U-containing double-strand RNA can suppress the induction of interferon-stimulated genes [63]. Conceivably NS1 might potentiate the hyperediting of an as yet unknown double-strand RNA substrate and thus interfere with interferon induction. A similar mechanism can be expected for dengue virus NS3 protein. Both NS1 and ADAR1 also interfere with PKR activity [6], [64]. ADAR1 and PKR are recognized by non overlapping domains of NS1 (respectively the RNA binding domain and the effector domain [7]). Thus, both NS1 and ADAR1 could sequester double-strand RNA or could form inactive complexes, suppressing PKR-mediated proapoptotic and interferon-mediated amplification activities. Influenza A NS1 protein is considered as a valid target for the development of antiviral drugs. The druggability of NS1 has been demonstrated in a proof-of-concept study with an inhibitory peptide derived from CPSF30, a cellular protein that interacts and interferes with the effector domain of NS1 [55]. Such a strategy can be extended to other NS1 interactors once the interacting sequences have been mapped and the 3D structure is solved. The interacting sequences, e. g. the ADAR1-derived 47 amino acid peptide, could then be used for the design of low molecular weight compounds. In this respect, the systematic screening for protein-protein interactions between a virus and its host cell identifies cellular proteins promoting or restricting virus replication. Interference with these interactions may offer new alternatives to enlarge the diversity of potential therapeutic targets and prevent the emergence of resistance caused by rapid viral adaptation. Small molecules targeting these host interaction surfaces and developed for other therapeutic purposes could now be tested for their ability to control virus replication. Concerning ADAR1, new inhibitors of the RNA editing activity are being screened and could be tested for their capacity to block the replication of influenza A virus or anti-dengue virus [65]–[67]. The dual luciferase editing reporter described in this study is well suited for screening RNA editing inhibitors at a high throughput level. Human HEK293T and human lung adenocarcinoma A549 cells were maintained in Dulbecco' s modified Eagle' s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 50 IU/ml penicillin G, 50 µg/ml streptomycin, at 37°C under 5% CO2. Huh-7 cells were grown in DMEM supplemented with 2 mM L-glutamine, non-essential amino acids, 100 IU/ml of penicillin, 100 µg/ml of streptomycin and 10% fetal calf serum. Influenza ORFs (Text S1) were transferred from pDONR207 into bait vector (pPC97, Lifetechnologies) to be expressed as Gal4-DB fusions in yeast. Bait vectors were transformed into AH109 (bait strain, Clontech [68]), and human spleen, fetal brain and respiratory epithelium Gal4-AD-cDNA libraries (each containing more than 106 primary clones) were transformed into Y187 (prey strain, Clontech). Single bait strains were mated with prey strains and diploids were plated on SD-W-L-H+ 10 mM 3-AT medium. Each screen has covered more than one time the libraries. Positive clones were maintained onto this selective medium for 15 days to eliminate any contaminant AD-cDNA plasmid. AD-cDNAs were PCR-amplified, sequenced and analyzed using pISTil [69]. Cellular ORFs (interacting domains found in Y2H screens) were amplified from a pool of human cDNA libraries or from a plasmid encoding the corresponding cDNA from the MGC collection (IMAGE consortium) using KOD polymerase (Toyobo) and cloned by recombinational cloning into pDONR207 (Invitrogen). Primers contained the attB1. 1 and attB2. 1 gateway recombination sites. All entry clones were sequence-verified and individually transferred by recombinational cloning into a prey vector (pPC86, Invitrogen) to be expressed as Gal4-AD (activating domain) fusion in yeast. Pairwise yeast two-hybrid interaction analyses were also performed by yeast mating using Y187 and AH109 yeasts strains (Clontech [68]), as described in [70]. Bait and prey strains were mated in an all-against-all array (together with negative controls, either empty bait vector or empty prey vector) and plated on a selective medium lacking histidine and supplemented with increasing concentrations of 3-AT (0,5, 10,15 mM) to test the interaction-dependent transactivation of HIS3 reporter gene. Interactions were scored as positive if observed in at least 2 out of 3 independent arrays. When yeasts containing an empty bait vector and a prey vector were still able to grow, the corresponding proteins were rejected as being auto-activators and thus false positives. The R statistical environment was used to perform statistical analysis and the igraph R package to compute network topology measures [71]. Protein-protein interaction networks are formed by a set of N nodes (or vertices) representing proteins connected by E edges representing physical interactions between these proteins. The topology of protein-protein interaction networks can be described by a set of measures: The degree or connectivity (k) of a node v in a graph is a local centrality measure which summarizes the number of edges that are incident to this node v. The betweenness (b) of a node v in a graph is a global centrality measure which can be defined by the number of shortest paths going through this node v and is normalized by twice the total number of protein pairs in the graph. The equation used to compute betweenness centrality, b (v), for a node v is: where gij is the number of shortest paths going from node i to j, i and j ∈ V and gij (v) the number of shortest paths from i to j that pass through the node v. DAVID database was used for functional annotation [72]. DAVID functional annotation chart tool was used to perform Gene Ontology categories analysis. Gene Ontology terms with a Benjamini-Hochberg corrected p-value smaller than 5. 102 were considered as significantly overrepresented. 5 pmoles of each siRNA (stealth select RNAi, Invitrogen) were arrayed in 96 plates in 10 µl of OptiMEM (2 siRNAs per gene). After 20 minutes of room temperature incubation with a transfection agent (0. 2 µl of lipofectamine RNAiMAX in 10 µl of OptiMEM), siRNA-transfection agent mix was added to 3. 104 A549 suspension cells. Cells were incubated for 48 hours at 37°C and 5% CO2 before influenza A virus infection at MOI 0. 5. At indicated time post-infection, supernatants were titered. siRNA-transfected cells were washed twice with DMEM and infected with the A/H1N1/Puerto Rico/8/34 strain or the A/H1N1/New Caledonia/2006 strain at indicated MOI in infection medium (DMEM supplemented with 0. 2 µg. ml−1 TPCK-trypsin (Sigma) ). After 1 h at 37°C, the inoculum was discarded and cells were washed again and incubated in infection medium at 37°C and 5% CO2. Standard fluorimetric assay was used to measure influenza virus neuraminidase activity [73]. Influenza virus neuraminidase is able to cleave the methyl-umbelliferyl-N-acetylneuraminic acid (4-MUNANA, Sigma) yielding a fluorescent product that can be quantified. In 96-black plate, 25 µl infection supernatants were diluted in 25 µl D-PBS containing calcium and magnesium and the reaction was started with 50 µl of 20 µM 4-MUNANA. After 1 h incubation at 37°C, the reaction was terminated by adding 100 µl of glycine 0. 1 M, 25% ethanol pH 10. 7. Fluorescence was recorded with TECAN infinite M1000 instrument at 365 nm excitation and 450 nm emission wavelengths. ADAR1 was transferred from pDONR207 to pCIneo3×Flag (kind gift of Dr Y. Jacob, Pasteur Institute, Paris, France). NS1 and NS3 constructs were transferred in pDEST27 (Invitrogen). Plasmids coding for mutant NS1 (pCAGGS-NS1-R38AK41A) and control NS1 (pCAGGS-NS1) are kind gifts from A. Garcia-Sastre (Mount Sinai School of Medicine, New York). HEK293T cells were transfected in 6-well plates using JetPEI transfection reagent (Polyplus Transfection). 48 h post-transfection, cells were lysed in a cold extract buffer (20 mM Tris-HCl pH 8. 0,150 mM NaCl, 1 mM EDTA, 0. 5% Igepal and a protease inhibitor cocktail (Roche) ). Protein extracts (300 µg) were incubated overnight with Glutathione Sepharose 4B beads (GE Healthcare) at 4°C. Beads were then extensively washed with the cold extract buffer, proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. GST-tagged viral proteins and 3×FLAG-tagged cellular proteins were detected using standard immunoblotting techniques with a mouse peroxidase-conjugated anti-GST monoclonal antibody (Sigma) or a mouse peroxidase-conjugated anti-FLAG M2 monoclonal antibody (Sigma). When indicated, pull-downs were treated with 2 µg of RNAse A (Invitrogen) in a buffer containing 100 mM NaCl for 30 min at 4°C. Proteins bound and released in the supernatants were then detected by immunoblotting using anti-ADAR (Sigma), anti-influenza A virus (Chemicon) and anti-NS1 antibodies (Abcam). Anti-actin antibody was purchased from Sigma. HEK293T were transfected in 24-well plates with a total of 1 µg plasmid DNAs (editing reporter plasmid, 3XF-ADAR1 and plasmids coding for indicated viral proteins) using the JetPEI. 24 h post-transfection, cells were seeded in 96-well plates in DMEM and incubated for 24 h. The Dual-Glo Luciferase Assay System (Promega) was then added to measure both Firefly and Renilla luminescence activities using the TECAN infinite M1000 instrument. Relative Light Unit (RLU) is the ratio of luminescence from FLUC to luminescence from RLUC. For influenza virus, HEK293T cells were seeded at 20,000 cells/well in 96-well plate and were transfected or not with anti-ADAR1 or control siRNAs, 24 h prior transfection with the editing reporter plasmid. 24 h post transfection, cells were infected influenza A virus at MOI 10 in DMEM supplemented with 10% FCS. 24 h post-infection cells were subjected to the procedure of editing assay described above. For dengue virus, Huh-7 cells were seeded at 3. 105 cell/well in 12-well plates and were transfected or not with anti-ADAR1 or control siRNAs, 24 h day prior transfection with the editing reporter. 24 h later, cells were infected with dengue virus type 2 with an MOI of 20. Luciferase values were measured as described above. A549 cells were infected with influenza A virus at MOI 3 in DMEM supplemented with 50 IU/ml penicillin, 50 µg/ml streptomycin and 0. 25 µg/ml TPCK-trypsin. Eight hours post-infection cells were fixed with 4% formaldehyde for 30 min and permeabilized with 0. 5% Triton X100. Double staining were performed by incubation with mouse monoclonal antibodies anti-NS1 (clone 1A7, kindly provided by Robert G. Webster) or anti-HA (Abcam) and rabbit anti-ADAR (Sigma) in combination with Alexa 488-labeled anti-rabbit F (ab) ′2 fragment and Alexa 546-labeled anti-mouse F (ab) ′2 fragment (Molecular Probes). Analyzes were performed with a laser-scanning confocal microscope (Axioplan LSM510 v3. 2 (Zeiss) ) and images were processed using LSM Image Browser (Zeiss).

Viruses are obligate intracellular parasites that rely on cellular functions for efficient replication. As most biological processes are sustained by protein-protein interactions, the identification of interactions between viral and host proteins can provide a global overview about the cellular functions engaged during viral replication. Influenza viruses express 13 viral proteins, including NS1 and NS2, which are translated from an alternatively spliced RNA derived from the same genome segment. We present here a comprehensive overview of possible interactions of cellular proteins with NS1 and NS2 from 9 viral strains. Seventy nine cellular proteins were identified to interact with NS1, NS2 or both NS1 and NS2. These interacting host cell proteins are potentially involved in many steps of the virus life cycle and 7 can directly control the viral replication. Most of the cellular targets are shared by the majority of the virus strains, especially the double-stranded RNA binding domain protein family that is strikingly targeted by NS1. One of its members, ADAR1, is essential for influenza virus replication. ADAR1 colocalizes with NS1 in nuclear structures and its editing activity is enhanced by NS1 expressed on its own and during virus infection. A similar phenomenon is observed for dengue virus whose NS3 protein also interacts with ADAR1, suggesting a parallel virus-host co-evolution.

lay_plos

By. Daily Mail Reporter. PUBLISHED:. 15:17 EST, 1 April 2013. |. UPDATED:. 15:22 EST, 1 April 2013. Taking precautions: Harris County District Attorney Mike Anderson now has a round-the-clock security detail because other lawmakers have been targeted by assassins. The district attorney for Houston is now being trailed by a 24-hour security team after a fellow Texas lawmaker and his wife were assassinated on Saturday. Authorities have said little about the investigation into the deaths of Kaufman County District Attorney Mike McLelland and his wife, whose bodies were found Saturday. The couple's slayings came less than two weeks after Colorado's prison chief was shot to death at his front door, apparently by an ex-convict, and a couple of months after Kaufman County Assistant District Attorney Mark Hasse was killed in a parking lot near his courthouse office. No arrests have been made in Hasse's January 31 killing. The highest-profile official to accept the protection has been Mike Anderson, the district attorney for Harris County, which includes Houston. 'Mike has accepted additional security offered by Sheriff Garcia, just in case there are any immediate threats in Harris County,' his spokesperson Sara Marie Kinney told The Houston Chronicle. 'This danger is present every day. They put bad people behind bars everyday, and prosecutors and investigators carry this with them every day. 'It's sad, it's tragic. It weighs heavy on Mike, but it's not going to let it stop him, or anyone in this office, from doing their job.' Kaufman County district attorney Mike McLelland and his wife, Cynthia. were found murdered in their home Saturday. Authorities have yet to prove a connection the killings but are assuming one exists until evidence shows otherwise. Deputies escorted some Kaufman County employees into the courthouse Monday as other officers were seen patrolling one side of the courthouse, one holding a semi-automatic weapon, while others walked around inside. 'I don't want to walk around in fear every day... but on the other hand, two months ago, we wouldn't be having this conversation,' County Judge Bruce Wood, the county's top administrator, said Monday at a news conference. Assistant DA Mark Hasse was gunned down in January, and had recently pursued cases involving the Aryan brotherhood. Law enforcement agencies throughout Texas were on high alert, and steps were being taken to better protect other DAs and their staffs. In Harris County, which includes Houston, District Attorney Mike Anderson said he accepted the sheriff's offer of 24-hour security for him and his family. Anderson said he also would take precautions at his office, the largest of its kind in Texas, with more than 270 prosecutors. 'I think district attorneys across Texas are still in a state of shock,' Anderson said Sunday. McLelland, 63, was the 13th prosecutor. killed in the U.S. since the National Association of District Attorneys. began keeping count in the 1960s. Kaufman. County Sheriff David Byrnes would not give details Sunday of how the. killings unfolded and said there was nothing to indicate for certain. whether the DA's slaying was connected to Hasse's. El. Paso County, Colorado, sheriff's spokesman Sergeant Joe Roybal said. investigators had so far found no evidence connecting the Texas killings. to the Colorado case, but added: 'We're examining all possibilities.' Colorado's. corrections director, Tom Clements, was killed March 19 when he. answered the doorbell at his home outside Colorado Springs. Evan. Spencer Ebel, a white supremacist and former Colorado inmate suspected. of shooting Clements, died in a shootout with Texas deputies two days. later about 100 miles from Kaufman. Crime scene: Law enforcement officers investigate the scene of Mark Hasse's killing in January. In an Associated Press interview shortly after the Colorado slaying, McLelland himself raised the possibility that Hasse was gunned down by a white supremacist gang. McLelland, elected in 2010, said his office had prosecuted several cases against such gangs, particularly one known as the Aryan Brotherhood. The groups have a strong presence around Kaufman County, a mostly rural area dotted with subdivisions, with a population of about 104,000. After Hasse's slaying, McLelland said, he carried a gun everywhere around town, even when walking his dog. Suspect: Evan Spencer Ebel (left) is the main suspect in the murder of Colorado prison's chief Tom Clements (right) and he was killed in a shootout with police in Texas two days after the murder. He figured assassins were more likely to try to attack him outside. He said he had warned all his employees to be constantly on the alert. 'The people in my line of work are going to have to get better at it,' he said of dealing with the danger, 'because they're going to need it more in the future.' The number of attacks on prosecutors, judges and senior law enforcement officers in the U.S. has spiked in the past three years, according to Glenn McGovern, an investigator with the Santa Clara County, California district attorney's office who tracks such cases. For about a month after Hasse's slaying, sheriff's deputies were parked in the district attorney's driveway, said Sam Rosander, a McLelland neighbor. The FBI and the Texas Rangers joined the investigation into the McLellands' deaths

Kaufman County, Texas, district attorney Mike McLelland and his wife Cynthia were found murdered in their home Saturday. Now Harris County district attorney Mike Anderson has round-the-clock security detail due to threats. Kaufman County assistant district attorney Mark Hasse was ambushed by at least one gunman as he walked from his car to the county courtroom in January. Hasse was involved in the investigation of members of the Aryan Brotherhood and police think there may be a link between the two murders.

cnn_dailymail

Beards in the operating room have sparked controversy because of their potential to retain and transmit pathogenic organisms.1–5 Even after it is washed with soap and water, facial hair may retain enough microorganisms to establish infection.1 In an extreme example, an outbreak of 16 severe surgical site infections was linked to a surgeon whose hair was colonized by Staphylococcus aureus despite negative throat and nose culture findings.2 McLure et al6 compared rates of bacterial shedding in bearded and clean-shaven medical personnel who wore surgical masks and performed a series of facial motions. This study found that bearded men shed more colony-forming units (CFUs) than both clean-shaven men (P<.01) and women (P<.001) and concluded that facial hair may be unsafe in a sterile environment. Despite the potential risk of infection posed by beards, they are common among surgeons and operating room staff. At the authors' institution, many surgeons, including those with and without beards, elect to wear nonsterile surgical hoods to minimize the risk of surgical site infection. These hoods have been shown to be superior to surgical masks alone and as efficacious as sterile ventilated hoods in decreasing the number of bacterial CFUs in wounds by 98% to 99%.7 Multiple studies have shown that facial hair can act as a reservoir for pathogenic organisms. However, the study by McLure et al6 is the only investigation that has compared the degree of bacterial shedding over a sterile field in bearded and clean-shaven men.1–5 The goal of the current study was to determine whether non-sterile surgical hoods reduce the risk of bacterial shedding posed by beards. Materials and Methods After institutional review board approval was obtained, 10 bearded and 10 clean-shaven subjects at the study institution were identified and provided consent to participate in the study. The clean-shaven group consisted entirely of orthopedic surgeons. Because of a limited number of bearded orthopedic surgeons, the bearded group included 6 orthopedic surgeons, 2 general surgeons, and 2 orthopedic surgical ancillary staff. Beard length was measured by placing a ruler perpendicular to the underside of the chin, specifically, 1 cm posterior to the apex of the chin in the midline. The average of 3 measurements, rounded to the nearest half centimeter, was recorded as the final beard length. Average beard length (±SD) was 12±7 mm (range, 5–25 mm). Three beards were shorter than 10 mm (mean length, 5±0 mm), 4 were between 10 and 19 mm (12.5±2.9 mm), and 3 were 20 mm or longer (21.6±2.8 mm) (Figure 1). Figure 1: Photographs of 1 tested subject. Clean-shaven. The mustache shown was not present during testing (A). Beard between 10 and 20 mm (B). Beard of 20 mm or more (C). The clean-shaven subjects had no facial hair present. The bearded subjects had full beards. No mustaches, goatees, or other styles of facial hair were included. The experimental protocol described by McLure et al6 was used to compare the results. To minimize bacterial contamination, participants were tested in an empty room in the presence of a single investigator. Bacterial shedding was assessed with 2 standard blood agar plates placed 15 cm below the chin of the seated subject. Two plates placed 50 cm from the subject acted as the control. Subjects performed a series of facial motions in a standardized pattern for 90 seconds. To minimize contamination by oral flora, subjects were asked not to open their mouths. All subjects wore clean disposable surgical caps and a surgical scrub top. Each subject was evaluated 3 times: while unmasked, while masked, and while masked with a nonsterile surgical hood. Flat pleated, tie-on surgical masks (Model 1818; 3M, St Paul, Minnesota) and polypropylene spunbound surgical hoods (Filtration Systems Products, Inc, Pevely, Missouri) (Figure 2) were studied. One plate each was incubated at 37°C aerobically and anaerobically for 48 hours. Colonies were counted and identified with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI BioTyper; Bruker Daltonics, Billerica, Massachusetts). Figure 2: Photograph showing the surgical mask and nonsterile surgical hood used in the experimental protocol. Based on the data of McLure et al,6 power analysis determined that inclusion of 10 subjects per group was sufficient to detect a 40% difference between the number of CFUs in masked bearded and clean-shaven groups. The Wilcoxon signed rank-sum test was used to evaluate differences within groups. The Kruskal-Wallis rank-sum test was used to determine differences between groups. All analysis was performed with the JMP statistical software package, version 8 (SAS Institute Inc, Cary, North Carolina). P<.05 was considered statistically significant. Results A mean of 6.5 CFUs (anaerobic and aerobic) were isolated per subject while unmasked, and this number was significantly higher than the number isolated while masked (1.4 CFUs, P=.02) or hooded (1.1 CFUs, P=.01). The addition of a surgical hood, compared with the use of masks alone, did not lower the number of CFUs isolated (P=.5). Bearded and clean-shaven subjects shed similar amounts of bacteria while unmasked (9.5 vs 3.3 CFUs, P=.1), masked (1.6 vs 1.2 CFUs, P=.9), or hooded (0.9 vs 1.3 CFUs, P=.6) (Figure 3). Figure 3: Mean total (both anaerobic and aerobic) colony-forming units (CFUs) isolated from each subject. Bearded and clean-shaven subjects shed similar amounts of bacteria while unmasked (9.5 vs 3.3 CFUs, P=.1), masked (1.6 vs 1.2 CFUs, P=.9), or hooded (0.9 vs 1.3 CFUs, P=.6). Error bars represent standard error. The number of aerobic CFUs isolated was similar in bearded and clean-shaven subjects while unmasked (4.6 vs 2.5 CFUs, P=.4), masked (0.4 vs 0.6 CFUs, P=.9), or hooded (0.8 vs 0.5 CFUs, P=.9). The organisms isolated from aerobic cultures are shown in Table 1. Table 1: Organisms Isolated From Aerobic Culture in Order of Prevalence The number of anaerobic CFUs isolated was similar in bearded and clean-shaven subjects while unmasked (4.9 vs 0.8 CFUs, P=.07), masked (1.2 vs 0.6 CFUs, P=.5), or hooded (0.8 vs 0.5 CFUs, P=.9). The organisms isolated from anaerobic cultures are shown in Table 2. Table 2: Organisms Isolated From Anaerobic Culture in Order of Prevalence When participants were stratified by beard length (0 mm, <10 mm, 10–19 mm, and ≥20 mm), subjects with beards that were 2 cm or longer shed more than clean-shaven subjects (18 vs 3.3 CFUs, P=.03). No differences in shedding were observed between any group while masked (P=.4) or hooded (P=.3) (Figure 4). Figure 4: Mean total (both anaerobic and aerobic) colony-forming units (CFUs) isolated from each subject after stratification by beard length (0 mm, <10 mm, 10-19 mm, and ≥20 mm). No differences in shedding were observed between any of the groups while masked (P=.4) or hooded (P=.3). Participants with beards that were 2 cm or longer showed more shedding than clean-shaven subjects (18 vs 3.3 CFUs, P=.03) while unmasked. Error bars represent standard error. Discussion McLure et al6 previously reported that bearded men wearing surgical masks and performing facial motions showed more aerobic bacterial shedding than clean-shaven subjects. The current study replicated the experimental protocol of McLure et al6 to compare data. McLure et al6 found that 10 bearded men shed a total of 29 CFUs compared with 8 CFUs shed by women (P<.0001) and 5 CFUs shed by clean-shaven men (P<.01). In comparison, the 10 bearded and 10 clean-shaven men in the current study shed a total of 4 and 6 aerobic CFUs, respectively. Further, the current study found no difference in bacterial shedding between bearded and clean-shaven subjects while unmasked, masked, or hooded. There was a trend for bearded subjects to shed more bacteria while unmasked, but this difference did not reach statistical significance. This trend was eliminated with the addition of surgical masks, with or without hoods. It is unclear why the current study did not find the significant difference in bacterial shedding between bearded and clean-shaven groups that was reported by McLure et al.6 Contributing variables may include the brand of surgical masks used; however, the current study used the same soft tie-on variety that McLure et al6 reported using. The current study also cultured the plates for 48 vs 24 hours, which theoretically would have increased the number of CFUs observed. An important variable may be beard length, which was not reported by McLure et al.6 In the current study, average beard length was 12±7 mm (range, 5–25 mm), and all participants had a full beard. Mustaches, goatees, soul patches, and other beard variants were excluded. In this study, participants with beards that were 2 cm or longer showed significantly more shedding than the clean-shaven subjects, but this difference was eliminated with the addition of a mask. No notable difference was found in bacterial shedding between beards of different lengths while the participants were masked. However, there was a trend for the longest beards to shed the least when the participants wore a mask and hood. A possible explanation for this finding may be that longer hair creates a less abrasive articulation with the material and therefore decreases shedding, although a greater number of subjects would be needed to demonstrate this finding. At the study institution, all personnel who enter the operating room wear a non-sterile surgical hood for arthroplasty procedures. The goal of the current study was to reevaluate the risk of bacterial shedding posed by beards and the ability of masks and nonsterile surgical hoods to reduce this risk. This is an important consideration for surgeons who want to eliminate variables that are responsible for surgical site infections but also wish to wear beards. According to the social science literature, beards convey superior health, maturity, attractiveness, and dominance.8–14 The current findings are tempered by the experimental design, which attempted to isolate bacterial shedding posed by beards alone. This study likely underestimated the protective ability of both masks and hoods by eliminating confounding factors, such as talking and movement of the head and neck. A study by Friberg et al7 found nonsterile hoods to be superior to surgical masks in simulated laminar flow opera tive conditions. Conclusion Bearded surgeons did not appear to have an increased likelihood of bacterial shedding compared with their nonbearded counterparts while wearing surgical masks. The addition of nonsterile surgical hoods did not decrease the amount of bacterial shedding observed compared with the use of surgical masks alone. References Barbeito MS, Mathews CT, Taylor LA. Microbiological laboratory hazard of bearded men. Appl Microbiol. 1967; 15(4):899–906. Dineen P, Drusin L. Epidemics of postoperative wound infections associated with hair carriers. Lancet. 1973; 2(7839):1157–1159. doi:10.1016/S0140-6736(73)92933-4 [CrossRef] Summers MM, Lynch PF, Black T. Hair as a reservoir of staphylococci. J Clin Pathol. 1965; 18:13–15. doi:10.1136/jcp.18.1.13 [CrossRef] Black WA, Bannerman CM, Black DA. Carriage of potentially pathogenic bacteria in the hair. Br J Surg. 1974; 61(9):735–738. doi:10.1002/bjs.1800610916 [CrossRef] Carpel CG. Forbid beards among health personnel [in Swedish]. Lakartidningen. 1990; 87(44):3591. McLure HA, Mannam M, Talboys CA, Azadian BS, Yentis SM. The effect of facial hair and sex on the dispersal of bacteria below a masked subject. Anaesthesia. 2000; 55(2):173–176. doi:10.1046/j.1365-2044.2000.055002173.x [CrossRef] Friberg B, Friberg S, Ostensson R, Burman LG. Surgical area contamination: comparable bacterial counts using disposable head and mask and helmet aspirator system, but dramatic increase upon omission of head-gear. An experimental study in horizontal laminar air-flow. J Hosp Infect. 2001; 47(2):110–115. doi:10.1053/jhin.2000.0909 [CrossRef] Urban JA. Cost analysis of surgical site infections. Surg Infect (Larchmt). 2006; 7(suppl 1):S19–S22. doi:10.1089/sur.2006.7.s1-19 [CrossRef] Li GQ, Guo FF, Ou Y, Dong GW, Zhou W. Epidemiology and outcomes of surgical site infections following orthopedic surgery. Am J Infect Control. 2013; 41(12):1268–1271. doi:10.1016/j.ajic.2013.03.305 [CrossRef] de Lissovoy G, Fraeman K, Hutchins V, Murphy D, Song D, Vaughn BB. Surgical site infection: incidence and impact on hospital utilization and treatment costs. Am J Infect Control. 2009; 37(5):387–397. doi:10.1016/j.ajic.2008.12.010 [CrossRef] Whitehouse JD, Friedman ND, Kirkland KB, Richardson WJ, Sexton DJ. The impact of surgical-site infections following orthopedic surgery at a community hospital and a university hospital: adverse quality of life, ex cess length of stay, and extra cost. Infect Control Hosp Epidemiol. 2002; 23(4):183–189. doi:10.1086/502033 [CrossRef] Dixson BJ, Brooks RC. The role of facial hair in women's perceptions of men's attractiveness, health, masculinity and parenting abilities. Evol Hum Behav. 2013; 34(3):236–241. doi:10.1016/j.evolhumbehav.2013.02.003 [CrossRef] Neave N, Shields K. The effects of facial hair manipulation on female perceptions of attractiveness, masculinity, and dominance in male faces. Pers Individ Dif. 2008; 45(5):373–377. doi:10.1016/j.paid.2008.05.007 [CrossRef] Reed JA, Blunk EM. The influence of facial hair on impression formation. Soc Behav Pers. 1990; 18(1):169–175. doi:10.2224/sbp.1990.18.1.169 [CrossRef] Table 1 Organisms Isolated From Aerobic Culture in Order of Prevalence Organism No. Bearded Clean-shaven Staphylococcus epidermidis 45 9 Staphylococcus capitis 7 4 Staphylococcus aureus 4 6 Corynebacterium pseudodiphtheriticum 1 6 Micrococcus luteus 1 4 Staphylococcus species 0 3 Dietzia cinnamea 1 2 Rothia mucilaginosa 1 0 Paenibacillus species 0 1 Pantoea agglomerans 0 1 Staphylococcus pasteuri 1 0 Staphylococcus lugdunensis 1 0 Staphylococcus caprae 1 0 Micrococcus terreus 1 0 Acinetobacter baumannii 1 0 Neisseria flavescens 1 0 Pinterest Fox Searchlight / Everett Collection Don’t want to show some skin? Good news! You don’t have to. Everyone knows that beards are dope as hell, despite what's currently happening with Kit Harington's face. Some proper scruff provides a touch of organized chaos, and can dramatically elevate your look with only minimal input. (All you have to do is stop shaving, really.) But with Memorial Day in the rearview and summer hot on the horizon, chances are that soon you might find yourself reaching instinctively for a razor. And we understand why: heat, humidity, and an increased likelihood of exposed skin help reinforce the idea that having a beard between June and September would be rather uncomfortable. But that’s simply not the case—in reality, sporting one not only may make no difference, it can actually enhance your summer. Let us explain. EDITOR’S PICK First off, we’re here to debunk that whole warmth issue. Now, you might think that the added bulk of a beard would increase the average temperature on your face, but that’s simply not true. On the contrary, while beards do protect from elements such as wind and rain, they don’t actually provide much insulation. So you can scratch that off your list of summer beard concerns. Furthermore, as it turns out, many bearded men say that beards can actually cool down the jawline, keeping sweat and moisture close to the face and subsequently chilling the skin when the beard catches a breeze. Next, let’s talk about appearances. A lot of guys shave off their face manes come June, simply because a clean-shaven face has this socially constructed air of freshness about it—as if there’s something inherently dirty about sporting a chinful of follicles. Perhaps it’s a visual thing; a clean swath of skin simply appears far more tidy. But as any hairy man who’s worth his salt knows, it doesn’t take much to give even the wildest beards a similar impression of order. All you need is to shear away some bushiness, plus take care along the jawline, and your mane will be summer-ready in no time. Finally, on to the true benefit of having a beard during beach season: sun protection. You see, according to a study in Radiation Protection Dosimetry, beards can protect skin from 50 to 95 percent of UV radiation—UVB rays in particular—depending on the thickness and angle of the hair compared to the sun. Now, does this mean you can go ahead and forget applying sunscreen to your mug? Of course not. But it does mean that your jaw’s got a lesser chance of getting burned should you happen to forget. It is unknown whether healthcare workers' facial hair harbours nosocomial pathogens. We compared facial bacterial colonization rates among 408 male healthcare workers with and without facial hair. Workers with facial hair were less likely to be colonized with Staphylococcus aureus (41.2% vs 52.6%, P = 0.02) and meticillin-resistant coagulase-negative staphylococci (2.0% vs 7.0%, P = 0.01). Colonization rates with Gram-negative organisms were low for all healthcare workers, and Gram-negative colonization rates did not differ by facial hair type. Overall, colonization is similar in male healthcare workers with and without facial hair; however, certain bacterial species were more prevalent in workers without facial hair. Copyright © 2014 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Go ahead, grow a beard: It's just as hygienic as having a clean face, and possibly more so, according to two recent studies. In stark contrast to earlier research that found beards could be as dirty as toilets, researchers who swabbed the faces of 409 male hospital workers found clean-shaven men were three times more likely to carry bacteria on their faces compared to men with beards. And that's not all. "The clean-shaven men actually had higher rates of certain bacterial species" and were 10% more likely to carry antibiotic resistant bacteria MRSA on their faces, study author Carrie Kovarik explains in a release. Though Penn Medicine researchers didn't examine why this was the case, Kovarik says shaving causes tiny cuts on the skin where bacteria can fester. "In general, we have no need to fear the beard," she says. In a second study, researchers took a closer look at the beards of surgeons, which are "controversial because of their potential to retain and transmit pathogenic organisms." After 10 surgeons with full beards and 10 without shook their faces over a petri dish, researchers concluded "the bearded group did not shed more than the clean-shaven group while unmasked, masked, or hooded." "It's not unthinkable that the cleanliness of my own beard may pale in comparison to the immaculate whorls of a surgeon's tress or nurse's sterile chops," Joshua A. Krisch writes at Vocativ. "But, at the very least, these studies suggest that all facial hair deserves to be judged on a case by case basis." More good news for men with beards: Facial hair keeps sweat close to the face for a nice cooling effect in summer, per GQ.

multi_news

Mycetoma is a chronic infectious disease of the subcutaneous tissue with a high morbidity. This disease has been reported from countries between 30°N and 15°S since 1840 but the exact burden of disease is not known. It is currently unknown what the incidence, prevalence and the number of reported cases per year per country is. In order to estimate what the global burden of mycetoma is, a meta-analysis was performed. In total 50 studies were included, which resulted in a total of 8763 mycetoma cases. Most cases were found in men between 11 and 40 years of age. The foot was most commonly affected. Most cases were reported from Mexico, Sudan and India. Madurella mycetomatis was the most prevalent causative agent world-wide, followed by Actinomadura madurae, Streptomyces somaliensis, Actinomadura pelletieri, Nocardia brasiliensis and Nocardia asteroides. Although this study represents a first indication of the global burden on mycetoma, the actual burden is probably much higher. In this study only cases reported to literature could be used and most of these cases were found by searching archives from a single hospital in a single city of that country. By erecting (inter) national surveillance programs a more accurate estimation of the global burden on mycetoma can be obtained. Mycetoma is a chronic infectious disease, characterized by the formation of tumor like-swellings and the formation of grains. This disease usually begins with a small trauma on the foot which introduces the causative agent into the subcutaneous tissue. Inside this tissue the causative agent will organize itself into small granules called grains. A small nodule will arise which gradually will grow into a large subcutaneous mass with sinuses which will secrete pus and grains. Eventually the bone will be invaded and a large mutilating lesion will be formed and the foot will be amputated [1]. Although mycetoma is usually found in the foot, other body sides can be affected as well [2]. Although, the first documented clinical cases of mycetoma were already described in 1842 by Gill, uncertainty remains about the total number of cases world-wide as already mentioned by Gokhale in 1981 [3]. A first attempt to map the number of mycetoma cases in a certain region was made by Abbott in the early 1950s [4]. Abbott, studied 1321 mycetoma cases in the Sudan in a 2. 5 year period and published his findings in 1956 [4]. Many scientists were surprised by the amount of cases to be retrieved in such a small period, which could indicate that the burden of mycetoma was higher than previously thought. Therefore, others started to determine the burden of mycetoma in other countries known to be endemic for mycetoma, such as Congo [5], Somalia [6], [7], [8], [9], Argentina [10], [11] and Mexico [12], [13]. Although several studies have been performed to determine the prevalence of mycetoma in a certain region or country, no overall study has been performed to determine the prevalence of mycetoma world-wide. Furthermore mycetoma is not a reportable disease; therefore it is currently still not known what the global burden of mycetoma is. In the surveys performed in the past and based on case-studies it appeared many different species, both bacteria and fungi, are able to cause mycetoma. In the review written by Ahmed et al. 48 species were listed as causative agents [1]. Some of these agents were considered to be common causative agents of mycetoma while others were found only rarely. A definition on common or rare was not given. Recently, based on sequencing either the 16S region (for bacteria) or the ITS region (for fungi) more causative agents were added to this list [14], [15], [16]. Since molecular identification is not used in the endemic regions, the total number of species able to cause mycetoma is still not known. Furthermore, no clear definition is given which species are common causative agents of human mycetoma and which are only rarely implicated. The distribution of the mycetoma causative agents is not equal around the globe. In overall, actinomycetoma – mycetoma caused by bacteria – is more commonly found in Middle and South-America while eumycetoma – mycetoma caused by fungi – is more commonly found in Africa [4], [13]. But within a country, this could also differ per region [17], [18]. In order to estimate what the true burden of mycetoma is globally, a meta-analysis was performed in which all studies in which the epidemiology of the mycetoma causative agents was studied were reviewed. Studies with more than 10 cases were included. The burden of mycetoma was determined in terms of prevalence and the number of reported cases per year per country. Furthermore we determined which species were most commonly associated with mycetoma and prepared definitions based on the ∼8000 cases to determine which species were commonly, occasionally or rarely associated with mycetoma development. A systematic review of available literature on the epidemiology of mycetoma was searched using the electronic database PubMed with the use of the following search terms: Mycetoma AND epidemiology or Madura foot AND epidemiology. Studies published in languages other than English, French, Spanish, Portuguese, German or Dutch were excluded. The search was supplemented by reviewing the reference lists of all selected studies. Studies were excluded if the number of patients studied was <10 patients, if a study was already published before and if the paper was written as a review. To determine the prevalence for each country, the number of reported cases for each year for that country were divided through the total population of that country for that year. Population figures were derived from IndexMundi (http: //www. indexmundi. com/facts/indicators/SP. POP. TOTL/compare#country=ma). This site only gives data from 1960 onwards. If studies had data from older years, the population size of 1960 was used to calculate the prevalence. To determine the prevalence within individual Indian states, population figures were derived from http: //www. citypopulation. de/India. html. From each study, the sampling period, the region of sampling, the sex distribution, age distribution and species isolated were recorded. To determine which species were most prevalent the percentages of each species present in a certain study was calculated. The percentage was used for comparison since some studies reported on >2000 cases while others reported only 11. To determine the global prevalence, the sum of the means for each country for a certain species was taken and then divided by the total of countries included. To determine which species were most prevalent the following definitions were used: common: >5% of the reported cases world-wide was caused by this species; occasional: 1–5% of the reported cases world-wide was caused by this species; rare: <1% of the reported cases world-wide was caused by this species. In total 8763 mycetoma cases were included in this study. Most of the cases were reported from Mexico (2607 cases) [12], [13], [53], Sudan (2555 cases) [4], [34], [70] and India (1392 cases) [17], [18], [35], [42], [44], [51], [59], [60], [65], [66], [67]. Countries with only limited cases reported are Uganda (11 cases) [64], Rumania (13 cases) [68], Nigeria (15 cases) [55], Bulgaria (16 cases) [58] and Thailand (17 cases) [56]. In order to estimate the prevalence of mycetoma the number of reported cases for each year for that country were divided through the total population of that country for that year as shown in figure 2A. As you can see in this figure, countries with the highest prevalence include Mauritania (prevalence of 3. 49 cases per 100,000 inhabitants) and Sudan (prevalence of 1. 81 cases per 100,000 inhabitants). Also Mexico, Senegal, Niger and Somalia have a relatively high prevalence for mycetoma. In order estimate the number of mycetoma cases reported per year the following calculation was made: the total number of cases was divided through the number of years in which they were gathered (figure 2B). So if we take as an example Mali. In Mali 54 cases were reported in a 10 year period between 1985 and 1994 [45]. So in total there were 54/10 = 5. 4 cases/year seen. According to figure 2B, Sudan reported the highest number of cases yearly, namely 106 per year. For Mexico and Mauritania, this was 80. 7 and 69. 7 respectively. World-wide most cases were found in men: 4060 cases in men versus 1175 cases in women. Exceptions were Thailand and Tunisia. In Thailand, men and women were equally affected (8 man and 9 women), while in Tunisia actually mycetoma was reported more in women (16 cases) than in men (12 cases). As is seen in figure 3, of the 5240 cases in which age was reported, 70% of the cases (3664 cases) were found in people with an age between 11 and 40 (3664 cases). Furthermore the most affected body site was the foot (68. 7%), followed by the leg (9. 9%), trunk (6. 1%) and arm (4. 0%) (Figure 4). Although these observations were based on all the cases reported world-wide, there are some regional differences. In all studies, the foot was the most reported lesion site, but in South-American patients the trunk was more often the site of infection than in African or Asian patients. For example, in Sudan the trunk as lesion site was only reported in 1. 4% of the cases, while in Mexico it was reported in 18. 7% of all cases. As is seen in table 1, most cases were actinomycetoma cases, but the total number of actinomycetes able to cause mycetoma is less. In total, 7 different actinomycetes were identified in the studied actinomycetoma cases, but for some cases the causative agents was not identified to the species level. Some were classified as Nocardia spp. and others as actinomycetoma spp. Based on our criteria the species Actinomadura madurae, Streptomyces somaliensis, Actinomadura pelletieri, Nocardia brasiliensis and Nocardia asteroides were considered to be common causative agents. Each of them was found in >5% of the studied cases. Nocardia otidiscaviarum was only found occasionally, and Actinomyces israeli rarely. For the eumycetoma cases, 18 different fungal species were identified as causative agents. Furthermore, for some cases the fungus could not be identified to the species level, but only to the genus level. These belonged to the genera Fusarium, Cladosporium or Exophiala. For 219 fungi, the fungus was not even identified to the genus level. Only the fungus Madurella mycetomatis was considered to be a common causative agent. In fact it appeared the most common causative agent of all mycetoma cases since 24. 3% of all studied cases were caused by this fungus. Scedosporium boydii, Falciformispora senegalensis and Trematosphaeria grisea were occasionally isolated from mycetoma cases, and Acremonium falciforme, Aspergillus fumigatus, Exophiala jeanselmei, Geotrichum candidum, Neotestudina rosatii, Medicopsis romeroi, Biatriospora mackinnonii, Aspergillus flavus, Microsporum audouini, Cochliobolus lunatus, Rhinocladiella atrovirens, Aspergillus nidulans and Neoscytalidium dimidiatum only rarely. The identified species were not evenly distributed throughout the world (Figure 5). M. mycetomatis, S. somaliensis and A. pelletieri were highly prevalent in Africa and Asia but hardly found in South-America. In contrast, the most prevalent species encountered in South America, N. brasiliensis, was hardly found in Africa, Europe and Asia. The only species which was found on all continents in equal amount was A. madurae. Until now, we generalized all studies per country, but within a country there can be large differences. As an example India is taken, since from this country we had data from 11 different studies. These studies originated from different parts of the country, namely Punjab, Rajasthan, Madhya Pradesh, Adhra Pradesh, Tamil Nadu and West-Bengal. Most of these states have roughly the same population size at the time most studies were performed (∼50,000,000 inhabitants/state in 1981), only Punjab had a smaller population (16,788,915 inhabitants/state in 1981). As is seen in figure 6, in Rajasthan most cases were reported per year (33. 3), followed by Tamil Nadu (16. 8 reported cases/year) and West-Bengal (13. 2 reported cases/year). The reported species per state also differed, in Rajasthan 62. 5% of all mycetoma cases was caused by fungi, while in the other states most cases were caused by actinomycetoma' s (54. 3%–83. 3% of all mycetoma cases). Not surprisingly, the species encountered also differed per state (figure 6). Since it is currently not known what the global burden of mycetoma is in terms of prevalence and incidence, we tried to make a rough estimation by performing a meta-analysis on the published literature from 1955 onwards. The number of papers published around the globe was relatively little. Only 50 papers could be used, which resulted in a total number of 8763 mycetoma cases documented since 1944. The globally reported number of cases is therefore 127 cases/year. The 8763 mycetoma cases were reported from 23 different countries. For some countries we only had data on 11 cases (e. g. Uganda), while for others we had data on 2607 cases (Mexico). The more cases reported per country the more reliable the data are. Since for most studies we knew in which years the cases were seen, a rough prevalence figure was calculated by dividing the number of cases seen in each year through the total population of that country in that year. This resulted in prevalence numbers ranging from <0. 01 cases per 100,000 inhabitants (several countries) to 1. 8 cases per 100,000 inhabitants (Sudan) and an average of cases reported per year ranging from 0. 9 (Tunesia) to 106 (Sudan) (Figures 2A and 2B). These numbers are gross underestimations of the true prevalence, since only cases reported to literature could be used. Most of these cases were found by searching archives from a single hospital in a single city of that country. Only those studies which searched multiple centres throughout the country, such as the studies performed by Abbott in Sudan [4] and by Lopez Martinez in Mexico [13], more than 1000 patients were documented. Based on these short-comings we estimate that the total number of mycetoma cases will be much higher. Some evidence of a higher prevalence exists, although not reported to literature. For instance, in 1991 the Mycetoma Research Centre in Khartoum, Sudan was established. Since its founding, 6334 patients have been seen. In 2012 only, 5158 patients were seen of which 402 were new cases. This means that there were (5158 cases/year) seen in Sudan only, which were not reported to literature (prof A. Fahal, personal communication). Furthermore, the incidence calculated on the figures from the Mycetoma Research Centre are most probably also an underestimation, since a cohort study performed in the endemic village Abu Gumri in Sudan performed in 1960, indicated that there was a prevalence of 6. 2 per 1000 inhabitants in that particular village [72]. Although the reported number of cases seen per year in this study is an underestimation, they are comparable with the number of cases reported for Buruli Ulcer and human African trypanosomiasis in many countries [73]. For instance, the number of reported Buruli Ulcer cases in 2009 were below 100 cases for Australia, Sudan, Gabon, Nigeria, and Guinea, while there were between 100 and 500 cases reported in Cameroon, Congo and the Democratic republic for the Congo in that year [73]. The number of reported Human African Trypanosomiasis (HAT) from surveillance studies in 2009 were below 100 reported cases for Kenya, Tanzania, Malawi, Zambia, Zimbabwe, Congo, Cameroon and Gabon [74]. The countries in which between 100 and 500 HAT cases were reported included Sudan, Angola and Uganda [74]. The data obtained for both Buruli Ulcer and HAT were from surveillance data, which makes these numbers more reliable than, the number of mycetoma cases/year reported here were derived from single center studies, and represent an underestimation. Therefore the prevalence of mycetoma is probably higher than that of either Buruli Ulcer or HAT. Still, Buruli Ulcer and HAT are reported on the list of neglected diseases while mycetoma is not. In this study it was shown that mycetoma could be caused by many different micro-organisms, both bacteria and fungi. Globally, most cases were caused by bacteria (50. 8%) and a smaller percentage by fungi (41. 7%) (Table 1), although this differed per country. In many African countries actually more eumycetoma cases were found as actinomycetoma cases. In table 1 it was also shown that there are many different bacteria and fungi able to cause mycetoma. There were 7 different bacterial species and 16 different fungal species reported. The total list of species able to cause mycetoma is actually longer. Since we only focused on studies reporting more than 10 cases per study, we missed the many species named in case reports. Other bacterial species implicated in mycetoma are Actinomadura latina, Gordonia terrae, Nocardia farcinica, Nocardia harenae, Nocardia mexicana, Nocardia transvalensis, Nocardia veterana, Nocardia yamanashiensis, Nocardiopsis dassonvillei and Streptomyces sudanensis [1], [14], [75], [76], [77], [78], [79], [80], [81], [82], [83]. Other fungal species implicated in mycetoma are Acremonium recifei, Cladophialophora bantiana, Corynespora cassiicola, Curvularia geniculata, Diaporthe phaseolorum, Fusarium oxysporum, Gibberella fujikuroi (synonym: Fusarium monoliforme), Haematonectria haematococca (synonym: Fusarium solani), Ilyonectria destructans (synonym: Cylindrocarpon destructans), Falciformispora tompkinsii (synonym: Leptosphaeria tompkinsii), Madurella fahalii, Madurella pseudomycetomatis, Madurella tropicana, Microsporum canis, Phaeoacremonium parasiticum, Phialophora cyanescens (synonym: Cylindrocarpon cyanescens), Phialophora verrucosa, Pleurostomophora ochracea, Pseudochaetosphaeronema larense, Rhinocladiella atrovirens, Sarocladium kiliense (synonym: Acremonium kiliense), Setosphaeria rostrata (synonym: Exserohilum rostrata) [1], [15], [16], [84], [85], [86], [87], [88], [89], [90], [91], [92], [93], [94], [95], [96], [97], [98]. Since most of these species are only found in case studies, these are probably only rarely associated with mycetoma, if associated at all. Although most species represented in Table 1 were isolated from multiple cases, some species were only isolated from 1–5 cases, which makes one wonder if this was the true causative agent. Only for Nocardia asteroides [99], Nocardia brasiliensis [99], [100], [101], [102], Nocardia caviae [99], [100], Nocardia transvalensis [103], Madurella mycetomatis [104], [105] the pathogenicity in animal models has been demonstrated. In all these animal models pathology resembling mycetoma and the formation of grains was demonstrated. The only attempt ever recorded for Actinomadura madurae, Actinomadura pelletieri and Streptomyces somalienis failed [104]. So, one could wonder if all species listed in Table 1 and in the discussion are true causative agents for mycetoma. The causative agents presented in Table 1 were identified based on histology only or on histology combined with culture. Therefore, the possibility arises that some of the causative agents were misidentified. Although widely used, the limitations of histological diagnosis have been known since the early 1960s. It is impossible to differentiate Nocardia spp. to the species level based on histology only, all species form white to yellow, small spherical grains [106], [107]. Also S. boydii, Acremonium spp and Fusarium spp are difficult to differentiate [106], [107]. These species produce white to yellow grains with a dense mass of slender, septate, hyaline hyphae with occasional vesicles or swollen hyphae [107]. The black-grain causative agents F. senegalensis, F. tompkinsii, M. romeroi, Exophiala jeanselmei and T. grisea also produce similar type of grains [107]. These species produce black grains, which can be tubular or hollow with a darker periphery [107]. The Falciformispora grains usually contain larger vesicles than the other species [107]. Some causative agents can cause multiple grain types in histological slides. Madurella mycetomatis is known to produce three structural forms of the fungal grain: the filamentous type, the vesicular type and a mixture of both [2]. In the vesicular type of grain the center is light colored and the hyphae in the periphery are brown, just as the ones described above for F. senegalensis, F. tompkinsii, M. romeroi, Exophiala jeanselmei and T. grisea [2]. For some of the studies culturing of the causative agents was also included. But identification of species based on culturing only, has known to be troublesome too, especially for the fungi. Identification of the fungi is usually achieved by observation of the growth rate, colony morphology, production of conidia and assimilation patterns. Most of the black-grain eumycetoma causative agents only rarely produce conidia and therefore misidentifications are known to occur frequently [108]. A well-known example is T. grisea, previously known as Madurella grisea. Due to the numerous misidentifications McGinnis suggested in 1996 that this species should be considered a complex of different fungi, classified together because of their sclerotial color and architecture and colony characteristics [109]. Some isolates of B. mackinnonii are known to be misidentified as T. grisea, since some of these isolates did not form pycnidia at standard media, but only after stimulation [109]. Furthermore, with classical culture methods, not all species can be differentiated. An example is Streptomyces sudanensis, which morphologically is equal to Streptomyces somaliensis. Only by sequencing the 16S gene of these species, it appeared that out of 9 previously identified S. somaliensis strains, 5 were actually misidentified and were renamed S. sudanensis [14]. The same was true for M. mycetomatis, in 2012 it appeared that M. mycetomatis had some close relatives which could only be identified by ITS sequencing, not on morphology [15]. Even when the species was already known, misidentifications have occurred in the past. N. brasiliensis has always been considered the most common causative agent of actinomycetoma in Mexico. Sánchez-Herrara demonstrated in 2012, that from the 18 previously identified N. brasiliensis isolates obtained from mycetoma patients in Mexico City between 1947 and 1959, only 7 were N. brasiliensis, the other 11 were proven to be N. farcinica based on sequencing and phenotypic profiles [110]. Therefore it remains doubtful that the data derived from the studies used for this meta-analysis were based on correctly identified species. Even with all these short-comings listed of this meta-analysis, it still gives a good overview of our current knowledge on the burden of mycetoma world-wide. In order to better estimate the burden, global surveillance programs should be erected, like for instance the surveillance program for Buruli Ulcer. This program is the result of the Global Buruli Ulcer initiative established in 1998 by the WHO and recognized by the 57th World Health Assembly in 2004 [111]. For this surveillance program, standardized forms (BU02) were generated which are now used in the endemic countries. If such a system was developed for mycetoma, better understanding on the global burden, and the epidemiology of this disease will be gained.

Mycetoma is a chronic infection resulting in large masses of the subcutaneous tissue of mainly the foot. It can be caused by bacteria or fungi. Treatment for most mycetoma cases is poor and amputations are common. Although this disease was already described almost 200 years ago, it is currently not known how many people over the globe actually suffer from this disease and which countries are mostly affected. These data are useful because they can be used to concentrate medical help in places where it is really needed and to focus to search on new medication on the most common causative agents. Since mycetoma is not a reportable disease, a meta-analysis was performed based from reports in literature, in order to estimate what the global burden of mycetoma is. In total 8763 mycetoma cases were analysed. It appeared that most cases were reported from Mexico, Sudan and India and that the fungus Madurella mycetomatis was the most prevalent causative agent world-wide. Although this study represents a first indication of the global burden on mycetoma, the actual burden is probably much higher. By erecting (inter) national surveillance programs a more accurate estimation of the global burden on mycetoma can be obtained.

lay_plos

Background The Package Delivery Market Is Complex and Changing Rapidly The package delivery market is growing and changing rapidly. In 2013, U.S. businesses and consumers reportedly spent more than $68 billion to ship packages domestically using the three largest national package delivery providers in the U.S.—United Parcel Service (UPS), Federal Express Corporation (FedEx), and USPS. This spending is driven in large part by recent growth in electronic commerce, which is forecast to grow in the double digits year-over-year to bring electronic commerce’s share of overall U.S. retail sales to almost 9 percent, or $490 billion, by the end of 2018. According to USPS, revenues from its Shipping and Packages services—which includes Parcel Select—have grown from about $11.6 billion in fiscal year 2012 to over $13.7 billion in fiscal year 2014, and generate about 20 percent of USPS’s total operating revenues. USPS is placing increased emphasis on growth in these services to partially offset a continued decline in First-Class Mail. Mailers have developed new delivery options to grow their businesses as the package delivery market evolves. For example, Amazon and Google started offering same-day package delivery service in select metropolitan areas. Some mailers have also introduced services that allow consumers to avoid package deliveries when they are not at home by picking-up packages themselves at alternative delivery locations. This development could reduce the need to provide last-mile delivery to individual residences. For example, Amazon allows customers to retrieve packages at self-service lockers in shopping centers, retail stores, transit stations, Similarly, in October 2014 and other access points in selected locations.UPS announced that consumers in two cities may retrieve packages from nearby locations—primarily neighborhood convenience and grocery stores—and it plans to expand the service to cover all major U.S. metropolitan markets in 2015. In addition to large national mailers, smaller regional companies compete for business in the package delivery market. For example, LaserShip is a regional package delivery company that provides last-mile delivery to major East Coast markets. Similarly, OnTrac provides regional overnight package delivery service within several states on the West Coast. As the package delivery market has changed, USPS must manage complex business relationships with its competitors. For example, although UPS and FedEx both pay USPS to deliver packages the last mile under certain circumstances, they also compete with USPS for end- to-end package delivery business—a concept that USPS officials refer to as “coopetition”. Similarly, FedEx is USPS’s largest contractor providing air transportation for Priority Mail Express (formerly Express Mail), Priority Mail, and First-Class Mail. UPS also is one of USPS’s largest contractors providing long-distance mail transportation. Parcel Select NSAs Provide Increased Volume and Revenue to USPS and Discounted Prices to Mailers USPS uses Parcel Select NSAs to encourage additional mail volume and revenue by providing mailers with discounted prices in exchange for meeting the contract’s terms and conditions. USPS data show that the Parcel Select product, including Parcel Select NSAs, is an increasingly important source of additional volume and revenue (see fig. 1). While USPS uses Parcel Select NSAs to generate additional revenue, among other benefits, mailers use them to lower delivery costs and to take advantage of USPS’s extensive “last mile” network that serves more than 140 million residential delivery points 6 days a week. Contracts may require the mailer to ship a minimum volume of packages each contract year (minimum volume requirement) to qualify for discounted prices. Some contracts may require the mailer to pay USPS the difference between the discounted price and published price for Parcel Select if the mailer fails to meet this minimum volume requirement. Shipping packages under Parcel Select lowers mailers’ delivery costs by allowing them to enter bulk shipments of packages into USPS’s network—generally at Destination Delivery Units (DDU)close to the final delivery point (see fig. 2). As a result, these packages generally bypass most of USPS’s mail processing and transportation network. Prices vary, depending on package weight, entry point, and the total volume shipped. USPS must ensure that all Parcel Select NSAs comply with statutory requirements under the Postal Accountability and Enhancement Act (PAEA). Notably, each Parcel Select NSA is required to generate enough revenue to cover its attributable costs. For Parcel Select, these costs primarily include USPS labor involved in sorting packages at postal facilities and delivering them to the final delivery point. PRC reviews each Parcel Select NSA to ensure that it is projected to comply with this and other requirements before USPS can implement it. According to PRC, this pre-implementation review is justified to preserve fair competition.also reviews each contract after implementation to ensure compliance with statutory criteria—including the contract’s attributable cost coverage—in its Annual Compliance Determination Report. USPS’s Recently Established Standard Procedures for Parcel Select NSAs Have Gaps USPS Established Standard Procedures in June 2014 USPS’s Sales Department (Sales) has lead responsibility for managing Parcel Select NSAs and recently established standard procedures for managing NSA contracts. Specifically, in June 2014, Sales created the Standard Procedure for Managing NSA Contracts (Standard Procedures), which is an internal document that provides guidance that USPS departments should follow to manage all NSAs. Sales officials told us that they created the guidance in an effort to incorporate best practices and to improve USPS’s NSA contract management procedures by balancing standardization with flexibility. The Standard Procedures address, in part, some leading contract management practices. For example, as we discuss below, the procedures define some contract management responsibilities, such as performance monitoring and evaluation activities. Previously, USPS officials told us that no document or policy outlined USPS’s contract management procedures. Absent such guidance, USPS managers told us that they relied primarily upon their professional expertise and experience to manage the contracts. Standard Procedures Define Some Contract Management Responsibilities The Standard Procedures define some responsibilities for USPS departments involved in developing, monitoring, and evaluating the performance of Parcel Select NSAs, as indicated below. USPS officials told us about other responsibilities that are not specifically included in the Standard Procedures. Contract Development The Standard Procedures delineate contract development responsibilities for various departments within USPS, including Sales, USPS’s Finance Department (Finance), and USPS’s Legal Department (Legal) (see fig. 3). After PRC issues an order approving a Parcel Select NSA, USPS begins to monitor and evaluate the performance of the contract. These responsibilities include internal performance reporting by Sales and Finance, submission of an annual compliance review to the PRC by Finance, periodic business reviews between USPS and mailers, and following contract termination or renewal procedures, as described below. Internal performance reporting: The Standard Procedures call for Sales and Finance to use internal reports to monitor and evaluate performance under each Parcel Select NSA. Thus, Sales or Finance creates monthly, quarterly, and annual reports that analyze data that include the volume of packages shipped and revenue generated under the contract. For example, Finance develops a quarterly performance report that is used internally by Sales. This report communicates the Parcel Select NSA’s contractual requirements, actual and projected volume and revenue data, as well as descriptive information such as the contract’s effective and expiration dates. Submission of compliance review to the PRC: In addition to internal reporting, USPS reports each NSA’s performance externally to the PRC. Specifically, Finance files USPS’s Annual Compliance Report to the PRC; this report covers the extent to which USPS finds that each NSA has met statutory requirements including whether each contract has covered its attributable costs. According to PRC officials, all Parcel Select NSAs have complied with these statutory requirements each year. However, PRC officials noted that they do not review and are not statutorily required to review the extent to which mailers or USPS have complied with other contractual requirements, such as minimum volume requirements and any payments to USPS for failure to meet these requirements as part of its Annual Compliance Determination Report. Business reviews: USPS officials said that they use performance reports to conduct business reviews with mailers. Business reviews are meetings where USPS and the mailer discuss business opportunities, contract terms—such as volume requirements and pricing—and contract performance. The Standard Procedures define additional responsibilities USPS should perform if a mailer is at risk of failing to meet contractual conditions. For example, if Parcel Select NSA performance is below contractual terms, Sales should establish a plan and timeline for improved performance. Furthermore, Sales should schedule additional monthly or bi-monthly business reviews to ensure performance improves in accordance with the established plan. Contract termination or renewal: According to Sales officials, the final activity for managing a Parcel Select NSA is to terminate, renew, or allow the contract to expire. Each Parcel Select NSA describes how the contract can be terminated. Contract terms and conditions typically allow USPS and mailers to terminate a Parcel Select NSA by mutual agreement in writing for convenience. According to the PRC, USPS should promptly notify the PRC if a Parcel Select NSA is terminated prior to the scheduled expiration date. Sales officials said that they generally contact mailers 3 to 6 months before a Parcel Select NSA expires to explore whether renewing the contract would be mutually beneficial. If both parties agree, Sales officials told us that USPS and the mailer should follow the same procedures used to create the original contract to execute the renewal. If either party does not wish to renew the contract, the Parcel Select NSA expires on its contractually specified date. Standard Procedures Have Gaps in Contract Monitoring and Evaluation While the Standard Procedures address some responsibilities involved in managing Parcel Select NSAs, some gaps exist. Specifically, the procedures lack documentation requirements and clearly defined management responsibilities for some contract monitoring and evaluation activities. A senior USPS official acknowledged that the Standard Procedures contained gaps when the document was initially established and that Sales had not reviewed or updated the procedures, which could have provided USPS with an opportunity to identify and address such gaps. Documenting contract monitoring and evaluation activities: USPS’s Standard Procedures lack documentation requirements for some contract monitoring and evaluation activities such as the occurrence and results of business reviews for active Parcel Select NSAs. Each of the seven contracts that were effective in October 2014 requires USPS to conduct business reviews to discuss contract performance semiannually. However, Sales officials told us they did not know the extent to which they conduct business reviews for each contract, because they do not always document that the meetings actually happen. Sales officials verified—through emails, meeting agendas, and other documents—that staff scheduled or conducted at least one business review for five of these seven contracts. Similarly, the Standard Procedures lack requirements for documenting some key management decisions pertaining to contract monitoring and evaluation. For example, USPS did not document management decisions about an underperforming contract. Specifically, according to Sales officials, when one mailer did not reach its minimum volume requirement, Sales did not document any discussions with the mailer to address the issue. Moreover, USPS did not require the mailer to pay the difference between the discounted price and the published price, as called for under the contract. Sales officials told us that they decided not to require the payment for business reasons, including maintaining this mailer as a source of package volume and allowing the mailer time to adjust to changing market conditions that influenced its performance. Although we recognize that USPS should have flexibility to reach management decisions based upon the facts and circumstances of each contract, Sales officials did not document the reasons for their decision to leave the performance issue unresolved and forgo the additional revenue. Documenting such information would help ensure that future decisions are based upon past results and enhance accountability for the effective and efficient use of USPS resources. Internal control standards state that information should be documented in a form and within a time frame that enables individuals to carry out their internal control and other responsibilities and that the documentation should be properly managed and maintained. Reviewing and updating the Standard Procedures to include documentation requirements would provide additional assurance that USPS’s Parcel Select NSAs are effectively managed. Inconsistent management and maintenance of documentation also increases the risk that USPS may not retain important institutional knowledge. As previously mentioned, management responsibility for Parcel Select NSAs has transitioned among three different USPS entities since calendar year 2008 (see fig. 4). USPS officials told us that documentation of key management decisions was lost or discarded when lead responsibility for managing the contracts transitioned from Domestic Products to Sales in 2012. The projected importance of package shipping and Parcel Select NSAs to USPS’s financial future highlights the importance for USPS to document contract management activities to retain institutional knowledge and inform future decision-making. Defining management responsibilities for contract monitoring and evaluation activities: USPS’s Standard Procedures do not define who is responsible for addressing unresolved contract performance issues and how staff should resolve them. Internal control standards state that organizations should clearly define key areas of responsibility and establish procedures to ensure that management promptly resolves performance issues. Without clearly defining who is responsible for promptly addressing performance issues and the procedures these individuals should follow, USPS is at risk of leaving performance issues unresolved—such as the example of a mailer’s failure to reach minimum volumes discussed above—issues that could impact future contract performance. In addition, the Standard Procedures do not clearly define who is responsible for reporting contract changes, such as rate adjustments and amendments, to PRC. Internal control standards and leading contract management practices we identified state that organizations should establish appropriate lines of reporting. Without clearly defining who is responsible for reporting information to PRC in the Standard Procedures, USPS is at risk of not reporting contract changes to PRC as required. For example, although PRC requested that USPS file an amendment to a Parcel Select contract in November 2014, USPS did not file the requested amendment as ordered. Similarly, according to PRC notices, on several occasions USPS did not report discretionary rate adjustments for Parcel Select NSAs as required. USPS Could Improve Its Method to Estimate Attributable Costs for Parcel Select NSAs Each competitive product, including each Parcel Select NSA, must earn sufficient revenues to cover its attributable costs; however, USPS’s costing method to estimate attributable costs for individual Parcel Select contracts does not account for all key cost factors. USPS compiles attributable costs for the Parcel Select product by using data from various information systems—notably financial accounting systems—that collect data on employee compensation and benefit costs. USPS then estimates how much employee time is spent handling Parcel Select packages by collecting additional data.USPS then estimates the total attributable costs for the Parcel Select product. However, USPS officials told us that they do not collect or study some information that could improve these estimates for individual Parcel Select contracts. Specifically, while USPS does collect data on the weight of all Parcel Select packages, it does not collect information on the size of NSA packages and has not studied the impact of either of these factors on USPS’s delivery costs for specific contracts. This limits USPS’s analysis of attributable costs for Parcel Select NSAs. USPS uses three basic delivery modes: door, “curbline,” and centralized delivery. Door delivery includes delivery to mail slots in the door as well as mailboxes attached to houses. Curbline delivery includes delivery to curbline mailboxes that are typically unlocked mail receptacles on a post and commonly used on routes serving residential customers. Centralized delivery is provided to centrally located mail receptacles, such as apartment house mailboxes and cluster box units. As we reported in May 2014, USPS estimated that its delivery costs in fiscal year 2012 ranged from about $380 annually for the average door delivery point to about $240 for curbline delivery and about $170 for centralized delivery. See GAO, U.S. Postal Service: Delivery Mode Conversions Could Yield Large Savings, but More Current Data Are Needed, GAO-14-444 (Washington, D.C.: May 12, 2014). or split routes, resulting in decreased operational efficiency and additional operating costs (see fig. 5). Moreover, private sector mailers use package weight and size information to inform their own business decisions. For example, UPS and FedEx— both of which pay USPS to deliver packages the last mile under certain circumstances and compete with USPS for end-to-end package delivery business—recently began basing the ground delivery rates they charge on both the size and weight of packages, a concept called “dimensional Representatives from another mailer told us that in serving weighting.”their own customers, they continually assess the characteristics of each package—including weight and size—to determine the costs of handling each package and to make cost-effective decisions regarding the delivery method they choose. If mailers choose to route larger or heavier packages via Parcel Select, collecting and studying information on the weight and size of packages could position USPS to better understand how this affects its delivery costs and the extent to which revenues for individual Parcel Select NSAs cover their attributable costs. In addition to not collecting detailed cost information that accounts for key package characteristics, USPS’s method to estimate attributable costs uses national averages for Parcel Select packages instead of contract- specific cost estimates for each NSA. This estimating further limits USPS’s analysis of the extent to which each contract covers its attributable costs. Specifically, because USPS does not have contract- specific cost estimates, USPS compares (a) the average cost per piece for Parcel Select packages with (b) the average revenue per piece for each Parcel Select NSA. PRC officials told us that based on currently available information, they had not observed significant variations in the characteristics of packages shipped under individual Parcel Select NSAs that would suggest that using average costs is not reasonable. However, as we describe above, USPS does not collect or study information that could help inform the reasonableness of its method to estimate attributable costs. Finance officials stated that because the weight of packages shipped under the Parcel Select NSAs did not vary significantly from the average weight, they had no reason to believe that heavier Parcel Select packages delivered under the NSAs would be more costly to process and deliver than the average cost to deliver packages that are not under the NSAs. However, these officials noted that they had not studied the extent to which package characteristics—including package weight and dimension—varied under individual Parcel Select NSAs or whether they deviated from the characteristics of packages that USPS used to estimate the national average. USPS officials questioned whether the time and expense required to develop contract-specific attributable cost estimates that account for package weight and dimension would significantly improve decision- making. Specifically, USPS officials said that for packages that mailers enter at DDUs, developing such estimates would require USPS to determine the costs that each DDU in its network would accrue— including costs associated with package size, package weight, delivery mode, and exact handling and delivery personnel—for each contract. For packages entered higher in USPS’s delivery stream (at sectional center facilities and network distribution centers), USPS officials noted that obtaining contract-specific information would require USPS to fully integrate the entire USPS mail data system and entirely replace its existing cost system, which would cost many hundreds of millions of dollars. We recognize that USPS must carefully balance the costs and benefits associated with developing contract-specific attributable cost estimates for Parcel Select NSAs. However, without taking steps to collect and study key cost information to develop these estimates, limitations on USPS’s analysis of attributable costs will persist. In the past, USPS has used other less intensive methods to improve its cost estimates for other products. For example, USPS has used “special studies” to improve cost information by examining operations at a sample of USPS facilities over a limited period of time. According to USPS officials, such special studies have generally cost hundreds of thousands of dollars to conduct in the past, with costs varying depending on the study’s scope. For example, in 2008 PRC noted that disaggregated Parcel Select and Parcel To disaggregate these costs, USPS Return Service costs were needed.conducted a field study at sampled DDUs in the summer of 2008 and used the data it collected to isolate transportation and mail processing costs specifically for Parcel Select and Parcel Return Service products. As another example, USPS recently conducted a comprehensive study of city carrier street time activities and costs, which involved collecting data on sampled city routes to help attribute city carrier costs to various types of mail, including parcels. Conclusions USPS continues to face significant financial challenges. Parcel Select helps to address these challenges by providing USPS with a revenue source that has increased from $466 million in fiscal year 2009 to over $2.5 billion in fiscal year 2014. Over time, USPS has taken important steps to standardize the procedures it uses to develop, monitor, and evaluate the performance of its Parcel Select NSAs, including developing its Standard Procedures in June 2014. However, the Standard Procedures lack documentation requirements and clearly defined management responsibilities for some contract monitoring and evaluation activities. Reviewing and updating the standard procedures to address these gaps would provide additional assurance that USPS’s Parcel Select NSAs are effectively managed. In addition, collecting and studying information on the size and weight of Parcel Select NSA packages could improve USPS’s analysis of attributable cost coverage for each contract and position USPS to better understand how mailers’ business decisions affect these costs. This may prove to be important, as mailers have developed more discrete costing methods based on both the size and weight of packages, which may influence the characteristics of packages that they provide to USPS for last-mile delivery. Recommendations for Executive Action To provide additional assurance that the procedures USPS uses to develop, monitor, and evaluate the performance of its Parcel Select NSAs are effective, the Postmaster General should direct executive leaders in Sales to: review the Standard Procedures to identify gaps in contract management responsibilities, including documentation requirements and assigning clearly defined management responsibilities for contract monitoring and evaluation activities, and update the procedures to address the identified gaps. To better understand attributable costs for individual Parcel Select NSAs, the Postmaster General should direct the appropriate staff to: identify and implement cost-effective methods, such as using a sample, to collect and study information on the costs of delivering Parcel Select packages of varying characteristics in order to develop contract-specific attributable cost estimates. Agency Comments and Our Evaluation We provided a draft of this report to USPS and PRC for review and comment. USPS and PRC provided written comments, which are summarized below and reproduced in appendix II and appendix III, respectively. USPS and PRC also provided technical comments, which we incorporated, as appropriate. In its comments, USPS agreed to update its Standard Procedures and concurred in principle to develop contract-specific attributable cost estimates for Parcel Select NSAs. USPS noted that it did not believe that package size and weight significantly affect the costs of Parcel Select NSAs, because most packages are entered at the DDU and thus bypass much of USPS’s processing and transportation network. However, as the report notes, while USPS does collect data on the weight of all Parcel Select packages, it does not collect information on the size of NSA packages and has not studied the impact of either of these factors on USPS’s delivery costs for specific contracts. This limits USPS’s analysis of attributable costs for Parcel Select NSAs. USPS stated it would explore obtaining additional mailer characteristics and ascertaining their relationships to costs for specific Parcel Select NSAs. USPS noted in its comments that it has already taken steps to obtain contract-specific characteristics for Sunday delivery of NSA packages. Sunday delivery, however, currently constitutes a relatively small percentage of Parcel Select volume. As the report notes, collecting and studying information on the size and weight of Parcel Select packages is important, because larger and heavier packages can increase USPS’s delivery costs. The report also points out that private sector mailers use package weight and size information to inform their own business decisions, such as making cost- effective decisions regarding the delivery method they choose. As the report concludes, collecting and studying information on the size and weight of Parcel Select NSA packages could improve USPS’s analysis of attributable cost coverage for each contract and position USPS to better understand how mailers’ business decisions affect these costs. This understanding will be important given the rapidly changing package delivery market. In its comments, PRC said that it found our draft report well-researched and balanced and agreed with both of our recommendations. PRC supported the cost-effective development of more accurate attributable cost estimates for Parcel Select NSAs. PRC also clarified that USPS’s method to estimate average costs for Parcel Select packages excludes Parcel Select mail weighing less than one pound. We modified the relevant text to note this clarification. As agreed with your office, unless you publicly announce the contents of this report earlier, we plan no further distribution until 30 days from the report date. At that time, we will send copies to the appropriate congressional committees, Postmaster General, Acting Chairman of PRC, USPS Office of Inspector General, and other interested parties. In addition, the report will be available at no charge on the GAO website at http://www.gao.gov. If you or your staff have any questions about this report, please contact me at 202-512-2834 or rectanusl@gao.gov. Contact points for our Offices of Congressional Relations and Public Affairs may be found on the last page of this report. GAO staff making key contributions to this report are listed in appendix IV. Appendix I: Scope and Methodology To examine the procedures that the U.S. Postal Service (USPS) has established to develop, monitor, and evaluate the performance of its Parcel Select negotiated service agreements (NSA), including implementation, we reviewed relevant laws and regulations such as the Postal Accountability and Enhancement Act (PAEA). We also examined USPS documents such as the Standard Procedure for Managing NSA Contracts that describe the procedures. We also reviewed copies of all 13 Parcel Select NSAs executed by USPS along with their supporting file documentation, such as contract performance and business review reports. This includes eight Parcel Select and five Parcel Select & Parcel Return Service contracts. For purposes of our report, we refer to both of these contract categories as Parcel Select NSAs, since each includes the Parcel Select product. We also examined Postal Regulatory Commission (PRC) documents, such as orders and notices. We did not review the extent to which USPS complied with all of its management procedures because this did not fall within the scope of this review. We also did not report volume, revenue, or mailer-specific information for individual NSAs because USPS and mailers consider the information proprietary, and according to USPS officials, public disclosure would violate contractual confidentiality provisions. In addition, we conducted interviews with USPS officials from the Sales, Finance, and Legal departments, who are responsible for developing, monitoring, and evaluating the performance of Parcel Select NSAs; PRC officials responsible for reviewing the contracts; and representatives from eight mailers that signed 12 of the 13 Parcel Select NSAs to determine how USPS implements its procedures. The number of mailers is less than the number of implemented contracts because some mailers signed more than one contract. We did not interview representatives from one of the mailers that signed a Parcel Select NSA, because the company was sold and knowledgeable representatives were no longer available to meet with us. According to USPS, no packages were ever shipped under that contract. We compared USPS’s procedures for managing the contracts against selected leading contract management practices. To select these practices, we: Identified criteria sources that included leading contract management practices and then confirmed that these sources were appropriate through consultation with internal stakeholders. These sources included: (a) our standards for internal control in the federal government; (b) guidance documents and regulations for federal agencies, such as best practices issued by the Office of Federal Procurement Policy; and (c) documents from contract management and administration organizations including the National Contract Management Association and the Institute for Supply Management, which were recognized for their expertise in this area. Reviewed information included in each criteria source to identify practices that are relevant to either (a) buyers and sellers or (b) just sellers. Because the Postal Service is the service provider (or seller) under Parcel Select NSAs, we excluded leading practices that are only pertinent to buyers. In addition, we identified practices that were applicable to USPS and its unique statutory and regulatory requirements as an independent establishment of the executive branch of the federal government. Exercised professional judgment to select practices that we identified as particularly relevant to the managing Parcel Select NSAs. Based upon these steps, we selected the following leading contract management practices: defining roles and responsibilities; establishing procedures for the contract management process; planning and negotiation; managing performance; communicating effectively; and documenting management activities and decisions. USPS agreed that these practices were relevant and reasonable. To determine revenue and volume trends for Parcel Select, which we describe in the background of our report, we reviewed USPS Revenue, Pieces, & Weight reports, which present official USPS estimates. We assessed the reliability of these data by reviewing related documentation, such as a December 2007 USPS Office of Inspector General report on and by collecting information from knowledgeable USPS’s data system,USPS officials, and determined that the data were sufficiently reliable for our reporting purpose. To examine the method that USPS uses to determine whether each Parcel Select NSA covers its attributable costs, we reviewed relevant laws and regulations that establish requirements for product categories and contracts such as PAEA. We also analyzed agency documents that describe or discuss the methodology that USPS uses, such as USPS’s fiscal year 2014 Annual Compliance Report and PRC’s Annual Compliance Determination Reports for fiscal years 2008 through 2013, the most recent reports available. We also reviewed relevant federal agency, academic, and GAO reports that describe USPS’s costing approach and related issues, such as the USPS Office of Inspector General’s audit reports. In addition, we conducted interviews with USPS and PRC officials to obtain their views on USPS’s cost coverage methodology. Finally, we made written requests for information regarding the methodology used to calculate attributable cost to USPS and PRC officials, who provided written responses on the subject. We did not review USPS’s postal costing methodology generally, such as methods to divide costs into attributable and institutional costs and methods to distribute attributable costs to various USPS products and services. We conducted this performance audit from April 2014 through April 2015 in accordance with generally accepted government auditing standards. Those standards require that we plan and perform the audit to obtain sufficient, appropriate evidence to provide a reasonable basis for our findings and conclusions based on our audit objectives. We believe that the evidence obtained provides a reasonable basis for our findings and conclusions based on our audit objectives. Appendix II: Comments from the United States Postal Service Appendix III: Comments from the Postal Regulatory Commission Appendix IV: GAO Contact and Staff Acknowledgments GAO Contact Staff Acknowledgments In addition to the individual named above, key contributors to this report were Lorelei St. James (Director); Derrick Collins (Assistant Director); Amy Abramowitz; Teresa Anderson; William Colwell; Aaron Colsher; Caitlin Dardenne; Colin Fallon; Kenneth John; DuEwa Kamara; Kimberly McGatlin; SaraAnn Moessbauer; Josh Ormond; Amelia Shachoy; Crystal Wesco; and Bill Woods.

USPS faces significant financial challenges. Parcel Select is a key USPS package shipping product to help address these challenges, with revenues growing from $466 million to over $2.5 billion from fiscal year 2009 through fiscal year 2014. As of April 3, 2015, USPS has executed 13 Parcel Select NSAs-customized contracts that lower mailers' shipping prices in exchange for meeting volume targets and other requirements. GAO was asked to review how USPS manages these contracts. GAO examined (1) USPS's procedures to manage Parcel Select NSAs and (2) its method to determine attributable cost coverage for each contract. To perform this work, GAO reviewed relevant laws and regulations; analyzed documents for all 13 contracts; compared USPS's procedures against selected leading contract management practices most applicable to USPS's role; and interviewed USPS and PRC officials and representatives from 8 mailers that signed 12 of the 13 contracts. In June 2014, the U.S. Postal Service (USPS) established standard procedures that departments should follow to manage all negotiated service agreement (NSA) contracts, including Parcel Select NSAs. Although the procedures, in part, address some leading contract management practices, such as defining performance management activities, they lack documentation requirements and clearly defined management responsibilities for some activities. For example, the procedures do not require USPS to document some key management decisions, such as USPS's decision to forego additional revenue when a mailer did not ship a minimum volume of packages, as contractually required. Documenting such information could improve future decision making and enhance accountability for the effective and efficient use of USPS resources. USPS acknowledged that the procedures contained gaps when they were initially established. Reviewing and updating the standard procedures to include documentation requirements and clearly defined management responsibilities would provide additional assurance that USPS's Parcel Select NSAs are effectively managed. USPS's costing method for Parcel Select NSAs does not account for package size or weight or use contract-specific cost estimates. Each Parcel Select NSA is required to earn sufficient revenues to cover USPS's costs-referred to as "attributable costs" in the postal context. The Postal Regulatory Commission (PRC), which annually reviews compliance, determined that each contract met this requirement. However, USPS's analysis of attributable costs for Parcel Select NSAs is limited, because USPS has not studied the impact of package size or weight on specific contracts or developed contract-specific cost estimates. Package size and weight information: USPS does not collect information on the size of NSA packages and has not studied the impact of package size and weight on USPS's delivery costs for specific contracts. However, larger and heavier packages can increase USPS's costs. For example, carriers must walk packages that are too large for a centralized mailbox to the customer's door, which increases costs. Moreover, mailers use size and weight to inform their own business decisions. For example, one mailer continually assesses package size and weight to make cost-effective decisions about the delivery method it chooses. If mailers route larger or heavier packages via Parcel Select, collecting and studying such information could improve USPS's analysis of attributable costs for Parcel Select NSAs. Contract-specific cost estimates: USPS's method to determine attributable costs uses average cost estimates for the Parcel Select product instead of contract-specific cost estimates. USPS's use of averages further limits its analysis of the extent to which each Parcel Select NSA covers its attributable costs, because USPS had not studied the extent to which the size and weight of packages shipped under individual contracts deviated from the characteristics of packages USPS used to estimate the average. USPS officials questioned whether the benefits of developing contract-specific cost estimates would exceed the costs; however, USPS has used less intensive methods, such as sampling, to improve estimates for other products in the past.

gov_report

An imbalance between energy intake and energy expenditure will lead to a change in body weight (mass) and body composition (fat and lean masses). A quantitative understanding of the processes involved, which currently remains lacking, will be useful in determining the etiology and treatment of obesity and other conditions resulting from prolonged energy imbalance. Here, we show that a mathematical model of the macronutrient flux balances can capture the long-term dynamics of human weight change; all previous models are special cases of this model. We show that the generic dynamic behavior of body composition for a clamped diet can be divided into two classes. In the first class, the body composition and mass are determined uniquely. In the second class, the body composition can exist at an infinite number of possible states. Surprisingly, perturbations of dietary energy intake or energy expenditure can give identical responses in both model classes, and existing data are insufficient to distinguish between these two possibilities. Nevertheless, this distinction has important implications for the efficacy of clinical interventions that alter body composition and mass. Obesity, anorexia nervosa, cachexia, and starvation are conditions that have a profound medical, social and economic impact on our lives. For example, the incidence of obesity and its co-morbidities has increased at a rapid rate over the past two decades [1], [2]. These conditions are characterized by changes in body weight (mass) that arise from an imbalance between the energy derived from food and the energy expended to maintain life and perform work. However, the underlying mechanisms of how changes in energy balance lead to changes in body mass and body composition are not well understood. In particular, it is of interest to understand how body composition is apportioned between fat and lean components when the body mass changes and if this energy partitioning can be altered. Such an understanding would be useful for optimizing weight loss treatments in obese subjects to maximize fat loss or weight gain treatments for anorexia nervosa and cachexia patients to maximize lean tissue gain. To address these issues and improve our understanding of human body weight regulation, mathematical and computational modeling has been attempted many times over the past several decades [3]–[19]. Here we show how models of body composition and mass change can be understood and analyzed within the realm of dynamical systems theory and can be classified according to their geometric structure in the two dimensional phase plane. We begin by considering a general class of macronutrient flux balance equations and progressively introduce assumptions that constrain the model dynamics. We show that two compartment models of fat and lean masses can be categorized into two generic classes. In the first class, there is a unique body composition and mass (i. e. a stable fixed point) that is specified by the diet and energy expenditure. In the second class, there is a continuous curve of fixed points (i. e. an invariant manifold) with an infinite number of possible body compositions and masses at steady state for the same diet and energy expenditure rate. We show that almost all of the models in the literature are in the second class. Surprisingly, the existing data are insufficient to determine which of the two classes pertains to humans. For models with an invariant manifold, we show that an equivalent one dimensional equation for body composition change can be derived. We give numerical examples and discuss possible experimental approaches that may distinguish between the classes. The human body obeys the law of energy conservation [20], which can be expressed as (1) where ΔU is the change in stored energy in the body, ΔQ is a change in energy input or intake, and ΔW is a change in energy output or expenditure. The intake is provided by the energy content of the food consumed. Combustion of dietary macronutrients yields chemical energy and Hess' s law states that the energy released is the same regardless of whether the process takes place inside a bomb calorimeter or via the complex process of oxidative phosphorylation in the mitochondria. Thus, the energy released from oxidation of food in the body can be precisely measured in the laboratory. However, there is an important caveat. Not all macronutrients in food are completely absorbed by the body. Furthermore, the dietary protein that is absorbed does not undergo complete combustion in the body, but rather produces urea and ammonia. In accounting for these effects, we refer to the metabolizable energy content of dietary carbohydrate, fat, and protein, which is slightly less than the values obtained by bomb calorimetry. The energy expenditure rate includes the work to maintain basic metabolic function (resting metabolic rate), to digest, absorb and transport the nutrients in food (thermic effect of feeding), to synthesize or break down tissue, and to perform physical activity, together with the heat generated. The energy is stored in the form of fat as well as in lean body tissue such as glycogen and protein. The body need not be in equilibrium for Equation 1 to hold. While we are primarily concerned with adult weight change, Equation 1 is also valid for childhood growth. In order to express a change of stored energy ΔU in terms of body mass M we must determine the energy content per unit body mass change, i. e. the energy density ρM. We can then set ΔU = Δ (ρMM). To model the dynamics of body mass change, we divide Equation 1 by some interval of time and take the limit of infinitesimal change to obtain a one dimensional energy flux balance equation: (2) where I = dQ/dt is the rate of metabolizable energy intake and E = dW/dt is the rate of energy expenditure. It is important to note that ρM is the energy density of body mass change, which need not be a constant but could be a function of body composition and time. Thus, in order to use Equation 2, the dynamics of ρM must also be established. When the body changes mass, that change will be composed of water, protein, carbohydrates (in the form of glycogen), fat, bone, and trace amounts of micronutrients, all having their own energy densities. Hence, a means of determining the dynamics of ρM is to track the dynamics of the components. The extracellular water and bone mineral mass have no metabolizable energy content and change little when body mass changes in adults under normal conditions [21]. The change in intracellular water can be specified by changes in the tissue protein and glycogen. Thus the main components contributing to the dynamics of ρM are the macronutrients - protein, carbohydrates, and fat, where we distinguish body fat (e. g. free fatty acids and triglycerides) from adipose tissue, which includes water and protein in addition to triglycerides. We then represent Equation 2 in terms of macronutrient flux balance equations for body fat F, glycogen G, and protein P: (3) (4) (5) where ρF = 39. 5 MJ/kg, ρG = 17. 6 MJ/kg, ρP = 19. 7 MJ/kg are the energy densities [3], IF, IC, IP are the intake rates, and fF, fC, 1−fF−fC are the fractions of the energy expenditure rate obtained from the combustion of fat, carbohydrates (glycogen) and protein respectively. The fractions and energy expenditure rate are functions of body composition and intake rates. They can be estimated from indirect calorimetry, which measures the oxygen consumed and carbon dioxide produced by a subject [22]. The intake rates are determined by the macronutrient composition of the consumed food, and the efficiency of the conversion of the food into a utilizable form. Transfer between compartments such as de novo lipogenesis where carbohydrates are converted to fat or gluconeogenesis where amino acids are converted into carbohydrates can be accounted for in the forms of fF and fC. The sum of Equations 3,4, and 5 recovers the energy flux balance Equation 2, where the body mass M is the sum of the macronutrients F, G, P, with the associated intracellular water, and the inert mass that does not change such as the extracellular water, bones, and minerals, and ρM = (ρFF+ρGG+ρPP) /M. The intake and energy expenditure rates are explicit functions of time with fast fluctuations on a time scale of hours to days [23]. However, we are interested in the long-term dynamics over weeks, months and years. Hence, to simplify the equations, we can use the method of averaging to remove the fast motion and derive a system of equations for the slow time dynamics. We do this explicitly in the Methods section and show that the form of the averaged equations to lowest order are identical to Equations 3–5 except that the three components are to be interpreted as the slowly varying part and the intake and energy expenditure rates are moving time averages over a time scale of a day. The three-compartment flux balance model was used by Hall [3] to numerically simulate data from the classic Minnesota human starvation experiment [21]. In Hall' s model, the forms of the energy expenditure and fractions were chosen for physiological considerations. For clamped food intake, the body composition approached a unique steady state. The model also showed that apart from transient changes lasting only a few days, carbohydrate balance is precisely maintained as a result of the limited storage capacity for glycogen. We will exploit this property to reduce the three dimensional system to an approximately equivalent two dimensional system where dynamical systems techniques can be employed to analyze the dynamics. The various flux balance models can be analyzed using the methods of dynamical systems theory, which aims to understand dynamics in terms of the geometric structure of possible trajectories (time courses of the body components). If the models are smooth and continuous then the global dynamics can be inferred from the local dynamics of the model near fixed points (i. e. where the time derivatives of the variables are zero). To simplify the analysis, we consider the intake rates to be clamped to constant values or set to predetermined functions of time. We do not consider the control and variation of food intake rate that may arise due to feedback from the body composition or from exogenous influences. We focus only on what happens to the food once it is ingested, which is a problem independent of the control of intake. We also assume that the averaged energy expenditure rate does not depend on time explicitly. Hence, we do not account for the effects of development, aging or gradual changes in lifestyle, which could lead to an explicit slow time dependence of energy expenditure rate. Thus, our ensuing analysis is mainly applicable to understanding the slow dynamics of body mass and composition for clamped food intake and physical activity over a time course of months to a few years. Dynamics in two dimensions are particularly simple to analyze and can be easily visualized geometrically [34], [35]. The one dimensional models are a subclass of two dimensional dynamics. Three dimensional dynamical systems are generally more difficult to analyze but Hall [3] found in simulations that the glycogen levels varied over a small interval and averaged to an approximate constant for time periods longer than a few days, implying that the slow dynamics could be effectively captured by a two dimensional model. Reduction to fewer dimensions is an oft-used strategy in dynamical systems theory. Hence, we focus our analysis on two dimensional dynamics. In two dimensions, changes of body composition and mass are represented by trajectories in the L–F phase plane. For IF and IL constant, the flux balance model is a two dimensional autonomous system of ordinary differential equations and trajectories will flow to attractors. The only possible attractors are infinity, stable fixed points or stable limit cycles [34], [35]. We note that fixed points within the context of the model correspond to states of flux balance. The two compartment macronutrient partition model is completely general in that all possible autonomous dynamics in the two dimensional phase plane are realizable. Any two or one dimensional autonomous model of body composition change can be expressed in terms of the two dimensional macronutrient partition model. Physical viability constrains L and F to be positive and finite. For differentiable f and E, the possible trajectories for fixed intake rates are completely specified by the dynamics near fixed points of the system. Geometrically, the fixed points are given by the intersections of the nullclines in the L–F plane, which are given by the solutions of IF−fE = 0 and IL = (1−f) E = 0. Example nullclines and phase plane portraits of the macronutrient model are shown in Figure 1. If the nullclines intersect once then there will be a single fixed point and if it is stable then the steady state body composition and mass are uniquely determined. Multiple intersections can yield multiple stable fixed points implying that body composition is not unique [4]. If the nullclines are collinear then there can be an attracting one dimensional invariant manifold (continuous curve of fixed points) in the L–F plane. In this case, there are an infinite number of possible body compositions for a fixed diet. As we will show, the energy partition model implicitly assumes an invariant manifold. If a single fixed point exists but is unstable then a stable limit cycle may exist around it. The fixed point conditions of Equations 8 and 9 can be expressed in terms of the solutions of (26) (27) where I = IF+IL, and we have suppressed the functional dependence on intake rates. These fixed point conditions correspond to a state of flux balance of the lean and fat components. Equation 26 indicates a state of energy balance while Equation 27 indicates that the fraction of fat utilized must equal the fraction of fat in the diet. Stability of a fixed point is determined by the dynamics of small perturbations of body composition away from the fixed point. If the perturbed body composition returns to the original fixed point then the fixed point is deemed stable. We give the stability conditions in Methods. The functional dependence of E and f on F and L determine the existence and stability of fixed points. As shown in Methods, an isolated stable fixed point is guaranteed if f is a monotonic increasing function of F and a monotonic decreasing function of L. If one of the fixed point conditions automatically satisfies the other, then instead of a fixed point there will be a continuous curve of fixed points or an invariant manifold. For example, if the energy balance condition 26 automatically satisfies the fat fraction condition 27, then there is an invariant manifold defined by I = E (F, L). The energy partition model has this property and thus has an invariant manifold rather than an isolated fixed point. This can be seen by observing that for f given by Equation 15, Equation 26 automatically satisfies condition 27. An attracting invariant manifold implies that the body can exist at any of the infinite number of body compositions specified by the curve I = E (F, L) for clamped intake and energy expenditure rates (see Figure 1C). Each of these infinite possible body compositions will result in a different body mass M = F+L (except for the unlikely case that E is a function of the sum F+L). The body composition is marginally stable along the direction of the invariant manifold. This means that in flux balance, the body composition will remain at rest at any point on the invariant manifold. A transient perturbation along the invariant manifold will simply cause the body composition to move to a new position on the invariant manifold. The one dimensional models have a stable fixed point if the invariant manifold is attracting. We also show in Methods that for multiple stable fixed points or a limit cycle to exist, f must be nonmonotonic in L and be finely tuned. The required fine-tuning makes these latter two possibilities much less plausible than a single fixed point or an invariant manifold. Data suggest that E is a monotonically increasing function of F and L [36]. The dependence of f on F and L is not well established and the form of f depends on multiple interrelated factors. In general, the sensitivity of various tissues to the changing hormonal milieu will have an overall effect on both the supply of macronutrients as well as the substrate preferences of various metabolically active tissues. On the supply side, we know that free fatty acids derived from adipose tissue lipolysis increase with increasing body fat mass which thereby increase the daily fat oxidation fraction, f, as F increases [37]. Furthermore, reduction of F with weight loss has been demonstrated to decrease f [38]. Similarly, whole-body proteolysis and protein oxidation increases with lean body mass [39], [40] implying that f should be a decreasing function of L. In further support of this relationship, body builders with significantly increased L have a decreased daily fat oxidation fraction versus control subjects with similar F [41]. Thus a stable isolated fixed point is consistent with this set of data. We have shown that all two dimensional autonomous models of body composition change generically fall into two classes - those with fixed points and those with invariant manifolds. In the case of a stable fixed point, any temporary perturbation of body weight or composition will be corrected over time (i. e., for all things equal, the body will return to its original state). An invariant manifold allows the possibility that a transient perturbation could lead to a permanent change of body composition and mass. At first glance, these differing properties would appear to point to a simple way of distinguishing between the two classes. However, the traditional means of inducing weight change namely diet or altering energy expenditure through aerobic exercise, turn out to be incapable of revealing the distinction. For an invariant manifold, any change of intake or expenditure rate will only elicit movement along one of the prescribed F vs. L trajectories obeying Equation 12, an example being Forbes' s law (14). As shown in Figure 2, a change of intake or energy expenditure rate will change the position of the invariant manifold. The body composition that is initially at one point on the invariant manifold will then flow to a new point on the perturbed invariant manifold along the trajectory prescribed by (12). If the intake rate or energy expenditure is then restored to the original value then the body composition will return along the same trajectory to the original steady state just as it would in a fixed point model (see Figure 2 solid curves). Only a perturbation that moves the body composition off of the fixed trajectory could distinguish between the two classes. In the fixed point case (Figure 2A dashed-dot curve), the body composition would go to the same steady state following the perturbation to body composition but for the invariant manifold case (Figure 2B dashed-dot curve), it would go to another steady state. Perturbations that move the body composition off the fixed trajectory can be done by altering body composition directly or by altering the fat utilization fraction f. For example, body composition could be altered directly through liposuction and f could be altered by administering compounds such as growth hormone. Resistance exercise may cause an increase in lean muscle tissue at the expense of fat. Exogenous hormones, compounds, or infectious agents that change the propensity for fat versus carbohydrate oxidation (for example, by increasing adipocyte proliferation and acting as a sink for fat that is not available for oxidation [42]–[44]), would also perturb the body composition off of a fixed F vs. L curve by altering f. If the body composition returned to its original state after such a perturbation then there is a unique fixed point. If it does not then there could be an invariant manifold although multiple fixed points are also possible. We found an example of one clinical study that bears on the question of whether humans have a fixed point or an invariant manifold. Biller et al. investigated changes of body composition pre- and post-growth hormone therapy in forty male subjects with growth hormone deficiency [45]. Despite significant changes of body composition induced by 18 months of growth hormone administration, the subjects returned very closely to their original body composition 18 months following the removal of therapy. However, there was a slight (2%) but significant increase in their lean body mass compared with the original value. Perhaps not enough time had elapsed for the lean mass to return to the original level. Alternatively, the increased lean mass may possibly have been the result of increased bone mineral mass and extracellular fluid expansion, both of which are known effects of growth hormone, but were assumed to be constant in the body composition models. Therefore, this clinical study provides some evidence in support of a fixed point, but it has not been repeated and the result was not conclusive. Using data from the Minnesota experiment [21] and the underlying physiology, Hall [3] proposed a form for f that predicts a fixed point. On the other hand, Hall, Bain, and Chow [10] showed that an invariant manifold model is consistent with existing data of longitudinal weight change but these experiments only altered weight through changes in caloric intake so this cannot rule out the possibility of a fixed point. Thus it appears that existing data is insufficient to decide the issue. We now consider some numerical examples using the macronutrient partition model in the form given by Equations 18 and 19, with a p-ratio consistent with Forbes' s law (13) (i. e. p = 2/ (2+F), where F is in units of kg). Consider two cases of the model. If ψ = 0 then the model has an invariant manifold and body composition moves along a fixed trajectory in the L–F plane. If ψ is nonzero, then there can be an isolated fixed point. We will show an example where if the intake energy is perturbed, the approach of the body composition to the steady state will be identical for both cases but if body composition is perturbed, the body will arrive at different steady states. For every model with an invariant manifold, a model with a fixed point can be found such that trajectories in the L–F plane resulting from energy intake perturbations will be identical. All that is required is that ψ in the fixed point model is chosen such that the solution of ψ (F, L) = 0 defines the fixed trajectory of the invariant manifold model. Using Forbes' s law (14), we choose ψ = 0. 05 (F−0. 4 exp (L/10. 4) ) /F. We then take a plausible energy expenditure rate of E = 0. 14L+0. 05F+1. 55, where energy rate has units of MJ/day and mass has units of kg. This expression is based on combining cross-sectional data [36] for resting energy with a contribution of physical activity of a fairly sedentary person [3]. Previous models propose similar forms for the energy expenditure [5], [7], [13], [18]. Figure 3 shows the time dependence of body mass and the F vs. L trajectories of the two model examples given a reduction in energy intake rate from 12 MJ/day to 10 MJ/day starting at the same initial condition. The time courses are identical for body composition and mass. The mass first decreases linearly in time but then saturates to a new stable fixed point. The dashed line represents the same intake rate reduction but with 10 kg of fat removed at day 100. For the invariant manifold model, the fat perturbation permanently alters the final body composition and body mass, whereas in the fixed point model it only has a transient effect. In the fixed point model, the body composition can ultimately exist only at one point given by the intersection of the nullclines (i. e., solution of I = E and ψ = 0). For the invariant manifold, the body composition can exist at any point on the I = E curve (dotted line in Figure 2D). Since a ψ can always be found so that a fixed point model and an invariant manifold model have identical time courses for body composition and mass, a perturbation in energy intake can never discriminate between the two possibilities. The time constant to reach the new fixed point in the numerical simulations is very long. This slow approach to steady state (on the order of several years for humans) has been pointed out many times previously [3], [5], [7], [13], [18]. A long time constant will make experiments to distinguish between a fixed point and an invariant manifold difficult to conduct. Experimentally reproducing this example would be demanding but if the time variation of the intake rates and physical activity levels were small compared to the induced change then the same result should arise qualitatively. Additionally, the time constant depends on the form of the energy expenditure. There is evidence that the dependence of energy expenditure on F and L for an individual is steeper than for the population due to an effect called adaptive thermogenesis [46], thus making the time constant shorter. In this paper we have shown that all possible two dimensional autonomous models for lean and fat mass are variants of the macronutrient partition model. The models can be divided into two general classes - models with isolated fixed points (most likely a single stable fixed point) and models with an invariant manifold. There is the possibility of more exotic behavior such as multi-stability and limit cycles but these require fine-tuning and thus are less plausible. Surprisingly, experimentally determining if the body exhibits a fixed point or an invariant manifold is nontrivial. Only perturbations of the body composition itself apart from dietary or energy expenditure interventions or alterations of the fraction of energy utilized as fat can discriminate between the two possibilities. The distinction between the classes is not merely an academic concern since this has direct clinical implications for potential permanence of transient changes of body composition via such procedures as liposuction or temporary administration of therapeutic compounds. Our analysis considers the slow dynamics of the body mass and composition where the fast time dependent hourly or daily fluctuations are averaged out for a clamped average food intake rate. We also do not consider a slow explicit time dependence of the energy expenditure. Such time dependence could arise during development, aging or gradual changes in lifestyle where activity levels differ. Thus our analysis is best suited to modeling changes over time scales of months to a few years in adults. We do not consider any feedback of body composition on food intake, which is an extremely important topic but beyond the scope of this paper. Previous efforts to model body weight change have predominantly used energy partition models that implicitly contain an invariant manifold and thus body composition and mass are not fully specified by the diet. If the body does have an invariant manifold then this fact puts a very strong constraint on the fat utilization fraction f. Hall [3] considered the effects of carbohydrate intake on lipolysis and other physiological factors to conjecture a form of f that does not lead to an invariant manifold. However, our analysis and numerical examples show that the body composition could have an invariant manifold but behave indistinguishably from having a fixed point. Also, the decay to the fixed point could take a very long time, possibly as long as a decade giving the appearance of an invariant manifold. Only experiments that perturb the fat or lean compartments independently can tell. The three compartment macronutrient flux balance Equations 3–5 are a system of nonautonomous differential equations since the energy intake and expenditure are explicitly time dependent. Food is ingested over discrete time intervals and physical activity will vary greatly within a day. However, this fast time dependence can be viewed as oscillations or fluctuations on top of a slowly varying background. It is this slower time dependence that governs long-term body mass and composition changes that we are interested in. For example, if an individual had the exact same schedule with the same energy intake and expenditure each day, then averaged over a day, the body composition would be constant. If the daily averaged intake and expenditure were to gradually change on longer time scales of say weeks or months then there would be a corresponding change in the body composition and mass. Given that we are only interested in these slower changes, we remove the short time scale fluctuations by using the method of averaging to produce an autonomous system of averaged equations valid on longer time scales. We do so by introducing a second “fast” time variable τ = t/ε, where ε is a small parameter that is associated with the slow changes in body composition and let all time dependent quantities be a function of both t and τ. For example, if t is measured in units of days and τ is measured in units of hours then ε∼1/24. Inserting into Equations 3–5 and using the chain rule yields (28) (29) (30) We then consider the three body compartments to have expansions of the form (31) (32) (33) where 〈F1〉 = 〈P1〉 = 〈G1〉 = 0 for a time average defined by and T represents an averaging time scale of a day. The fast time dependence can be either periodic or stochastic. The important thing is that the time average over the fast quantities is of order ε or higher. We then expand the energy expenditure rate and expenditure fractions to first order in ε: (34) (35) where E0 (t, τ) ≡E (F0, G0, P0, t, τ) +O (ε2) and i∈{F, G, P}. We assume that the expenditure fractions depend on time only through the body compartments. Substituting these expansions into Equations 28–30 and taking lowest order in ε gives (36) (37) (38) Taking the moving time average of Equations 36–38 and requiring that 〈∂F1/∂τ〉, 〈∂G1/∂τ〉, and 〈∂P1/∂τ〉 are of order ε or higher leads to the averaged equations: (39) (40) (41) In the main text we only consider the slow time scale dynamics so we drop the superscript and bracket notation for simplicity. Hence, the system (3–5) can be thought of as representing the lowest order time averaged macronutrient flux balance equations. We note that in addition to the daily fluctuations of meals and physical activity, there can also be fluctuations in food intake from day to day [23]. Our averaging scheme can be used to average over these fluctuations as well by extending the averaging time T. A difference in the choice of T will only result in a different interpretation of the averaged quantities. The dynamics near a fixed point (F0, L0) are determined by expanding fE and (1−f) E to linear order in δF = F−F0 and δL = L−L0 [34], [35]. Assuming solutions of the form exp (λt) yields an eigenvalue problem with two eigenvalues given by where (42) and (43) A fixed point is stable if and only if Tr J<0 and det J>0. In the case of an invariant manifold, detJ = 0, so the eigenvalues are Tr J and 0. The zero eigenvalue reflects the marginal stability along the invariant manifold, which is an attractor if Tr J<0. An attracting invariant manifold implies a stable fixed point in the corresponding one dimensional model. Unstable fixed points are either unstable nodes, saddle points or unstable spirals. In the case of unstable spirals, a possibility is a limit cycle surrounding the spiral arising from a Hopf bifurcation, where Tr J = 0 and det J>0. In this case, body composition and mass would oscillate even if the intake rates were held constant. The frequency and amplitude of the oscillations may be estimated near a supercritical Hopf bifurcation by transforming the equations to normal form. Stability of a fixed point puts constraints on the form of f. Physiological considerations and data imply that ∂E/∂L>∂E/∂F>0 [3], [36]. Thus we can set ∂E/∂F = δ∂E/∂L where δ <1 (the derivatives are evaluated at the fixed point). Then detJ>0 implies that (44) and Tr J<0 implies (45) where K = [δf+γ (1−f) ] (∂E/∂L) /E>0 and γ = ρF/ρL≈5. 2. Hence ∂f/∂F>0 and ∂f/∂L<0 guarantees stability of a fixed point. In other words, if f increases monotonically with F and decreases monotonically with L then there will be a unique stable fixed point. For an invariant manifold, f is given by Equation 15, which immediately satisfies detJ = 0; TrJ<0 is guaranteed if E is monotonically increasing in F and L. For a Hopf bifurcation, we require ∂f/∂F = γ∂f/∂L−K and Equation 44, implying (γ−δ) ∂f/∂L−K>0. Since γ>δ, f must increase with L for the possibility of a limit cycle. However, to ensure that trajectories remain bounded f must decrease with L for very small and large values of L. Hence, f must be nonmonotonic in L for a limit cycle to exist. This can also be seen from an application of Bendixson' s criterion [35], which states that a limit cycle cannot exist in a given region of the L–F plane if (46) does not change sign in that region. In addition, the other parameters must be fine tuned for a limit cycle (see Figure 1D). Similarly, as seen in Figure 1C), for multi-stability to exist, nonmonotonicity and fine tuning are also required.

Understanding the dynamics of human body weight change has important consequences for conditions such as obesity, starvation, and wasting syndromes. Changes of body weight are known to result from imbalances between the energy derived from food and the energy expended to maintain life and perform physical work. However, quantifying this relationship has proved difficult, in part because the body is composed of multiple components and weight change results from alterations of body composition (i. e., fat versus lean mass). Here, we show that mathematical modeling can provide a general description of how body weight will change over time by tracking the flux balances of the macronutrients fat, protein, and carbohydrates. For a fixed food intake rate and physical activity level, the body weight and composition will approach steady state. However, the steady state can correspond to a unique body weight or a continuum of body weights that are all consistent with the same food intake and energy expenditure rates. Interestingly, existing experimental data on human body weight dynamics cannot distinguish between these two possibilities. We propose experiments that could resolve this issue and use computer simulations to demonstrate how such experiments could be performed.

lay_plos

Aedes aegypti, the primary vector of dengue, yellow fever and Zika flaviviruses, consists of at least two subspecies. Aedes aegypti (Aaa) is light in color, has pale scales on the first abdominal tergite, oviposits in artificial containers, and preferentially feeds on humans. Aedes aegypti formosus (Aaf), has a dark cuticle, is restricted to sub-Saharan Africa, has no pale scales on the first abdominal tergite and frequently oviposits in natural containers. Scale patterns correlate with cuticle color in East Africa but not in Senegal, West Africa where black cuticle mosquitoes display a continuum of scaling patterns and breed domestically indoors. An earlier laboratory study did not indicate any pre- or postzygotic barriers to gene flow between Aaa and Aaf in East Africa. However, similar attempts to construct F1 intercross families between Aaa laboratory strains and Senegal Ae. aegypti (SenAae) failed due to poor F1 oviposition and low F2 egg-to-adult survival. Insemination and assortative mating experiments failed to identify prezygotic mating barriers. Backcrosses were performed to test for postzygotic isolation patterns consistent with Haldane’s rule modified for species, like Aedes, that have an autosomal sex determining locus (SDL). Egg-pupal survival was predicted to be low in females mated to hybrid F1 males but average when a male mates with a hybrid F1 female. Survival was in fact significantly reduced when females mated to hybrid males but egg-pupal survival was significantly increased when males were mated to hybrid F1 females. These observations are therefore inconclusive with regards to Haldane’s rule. Basic cytogenetic analyses and Fluorescent In Situ Hybridization (FISH) experiments were performed to compare SenAae strains with the IB12 strain of Aaa that was used for genome sequencing and physical mapping. Some SenAae strains had longer chromosomes than IB12 and significantly different centromeric indices on chromosomes 1 and 3. DAPI staining was used to identify AT-rich regions, chromomycin A3 following pretreatment with barium hydroxide stained for GC-rich regions and stained the ribosomal RNA locus and YOYO-1 was used to test for differential staining. Chromosome patterns in SenAae strains revealed by these three stains differed from those in IB12. For FISH, 40 BAC clones previously physically mapped on Aaa chromosomes were used to test for chromosome rearrangements in SenAae relative to IB12. Differences in the order of markers identified two chromosomal rearrangements between IB12 and SenAae strains. The first rearrangement involves two overlapping pericentric (containing the centromere) inversions in chromosome 3 or an insertion of a large fragment into the 3q arm. The second rearrangement is close to the centromere on the p arm of chromosome 2. Linkage analysis of the SDL and the white-eye locus identified a likely chromosomal rearrangement on chromosome 1. The reproductive incompatibility observed within SenAae and between SenAae and Aaa may be generally associated with chromosome rearrangements on all three chromosomes and specifically caused by pericentric inversions on chromosomes 2 and 3. The mosquito, Aedes aegypti (L), is the principal vector of dengue (DENV1-4) [1,2], Yellow Fever (YF) [3,4] and Zika [5,6] flaviviruses in tropical and subtropical regions world-wide. The ecology and population biology of the species have been studied since the mid-1950s [7–10]. At that time, Ae. aegypti in East Africa was known to have a high frequency of pale cuticle forms that preferred peridomestic sites and readily fed on humans and dark cuticular forms that predominated in the nearby bush and more readily fed upon wild animals [9,10]. This correlation between body color and behavior prompted Mattingly to revisit the biology and taxonomy of Ae. aegypti [11] wherein he defined the type form, Ae. aegypti (Aaa), as having a global tropical and subtropical distribution, a light-tan cuticle, pale scales on the first abdominal tergite, a feeding preference for humans and which laid its eggs in artificial containers (e. g. tires, discarded jars). The dark form was described as a new subspecies, Ae. aegypti formosus (Walker) (Aaf) [11] that was restricted to sub-Saharan Africa, laid its eggs in natural containers (e. g. tree holes), had adults without pale scales on the first abdominal tergite, and only rarely fed upon humans [8]. Later, mark-release-recapture studies in Kenya [12] demonstrated that immature mosquitoes collected from sylvan, peridomestic or domestic breeding containers showed an overwhelming preference for their respective habitats as adults. A 1979 study of hybridization and mating behavior between Aaa and Aaf in East Africa found no evidence of hybrid breakdown or of assortative mating between the two subspecies and concluded that they are part of a single, albeit highly polytypic species[13]. A recent study [14] in East Africa nicely illustrates the differences in color between Aaa and Aaf and reaffirms the correlation between feeding preference for humans, lighter cuticle and the quantity of pale scales on the first abdominal tergite. Furthermore preference for humans in Aaa was tightly linked to increases in the expression and sensitivity of an odorant receptor. Population genetic studies in the early 1970’s and continuing to the present [15–23] have consistently indicated that Aaa and Aaf subspecies are genetically distinct [21], that both originated in Africa but that Aaa is the form that has spread globally whereas Aaf has remained an African endemic taxon [22]. In early population genetic studies it was assumed that Ae. aegypti from West Africa were also Aaf until McClelland performed a comprehensive study of the differences in scaling pattern in 69 different worldwide collections [18]. He found collections of almost pure Aaf in Pensacola, Key West and Miami, Florida. Conversely, collections from Kenya, Nigeria, Tanzania, Senegal, Ghana, Burkina Faso, Sri Lanka, Calcutta, Jamaica, and the Miami Airport contained diverse mixtures of Aaf and Aaa mosquitoes. He concluded that distinctions between the subspecies based on body color, scaling and behavior were not definitive, even within Africa, the only region in the world where both forms are found. While the presence or absence of scales correlates with genetic differences in East Africa [21] this is not the case in West Africa [19,24–27] where cuticle color is predominantly black but mosquitoes exhibit a continuum of scaling patterns. In addition, Aaf were found breeding domestically indoors in Nigeria [28] and Gabon [29]. Collections from West Africa that varied in scaling patterns were sampled in both the dry and rainy seasons from different vegetation zones and from domestic versus sylvan habitats. They were compared using allozyme markers [24], microsatellites [16,25,26], nuclear SNP loci [15,17,27] and mitochondrial DNA [19,25]. Regardless of collection site or marker type, there was little variance in marker frequencies between mosquitoes with or without scales. Instead most variation was associated with geographic distance, vegetative zones, ecological habitats and season [15,16,24,25,27]. Collectively these studies suggest a more complex and unresolved genetic structure in West African Ae. aegypti. With the goal of performing Quantitative Trait Locus (QTL) mapping of gene regions associated with DENV2 susceptibility, Aaa P1 laboratory strains that are known to be highly susceptible to DENV2 infection were crossed with Senegal Ae. aegypti (SenAae) that are refractory to DENV2 infection [30] to breed F1 intercross families. During this process reproductive incompatibilities were observed between Aaa collected outside Africa and SenAae. Furthermore reproductive incompatibilities were observed within and among SenAae collections. These observations differ markedly from the 1979 study of hybridization and mating behavior between Aaa and Aaf in East Africa which found no evidence of hybrid breakdown or of assortative mating [11]. These leads to the hypothesis that, unlike Aaa and Aaf, Aaa and SenAae are reproductively incompatible. Herein possible biological and genetic causes for the observed incompatibilities within SenAae and between Aaa and SenAae are explored. Throughout this study the lab strains of Ae. aegypti (D2S3 [31], ROCK [32], IB12 [33]) are designated as Aaa because they fit Mattingly’s original description [11]. SenAae that had any scales on the first abdominal tergite are designated as “G” (following McClelland’s nomenclature [18]) rather than Aaa because while they have scales they also have a black cuticle which does not fit the original Aaa description [11]. To date light or tan cuticle Ae. aegypti have not been detected in Senegal (and these differences are very obvious (see Fig 1d in [14]). SenAae without scales on the first abdominal tergite are designated as “F” [18] rather than Aaf because earlier studies showed no reproductive isolation between Aaa and Aaf in East Africa [13,21]. These studies, by necessity collectively involved eight different field-collected SenAae strains. Use of different strains was necessary because of the continuous loss of strains due to poor initial oviposition rates, high larval mortality and low F1 adult fecundity. This prohibited the consistent use of the same SenAae strains in all experiments. The D2S3 lab strain of Ae. aegypti [31] originated from crosses between Puerto Rico and Ibo, Nigeria parents followed by selection for mosquitoes with high DENV2 disseminated infection rates and ROCK [32] which originated from Cuba [34]. SenAae populations were collected as larvae near domestic sites in urban and village environments, as well as from sylvatic habitats throughout Senegal [27] (Table 1 and Fig 1). A complete description of mosquito species found in PK10 during the dry and wet seasons has been published [35]. In each collection, larvae were removed mostly from artificial containers (e. g. tires, water storage containers, discarded trash), reared to adults, blood fed on the senior author’s arm and collected eggs were returned to Fort Collins where they were established as laboratory colonies. The PK10 colonies were established from larvae taken from treeholes at the forest-savannah margin [35]. In each collection, these were raised to adults, transferred to half liter cages, anesthetized with Triethylamine (FlyNap Carolina Biological Supply Company, Burlington, NC) and classified according to the presence/absence of scales on the first abdominal tergite [18,27,35]. At least 50 adults of each sex were individually identified and stored in Purell Advanced Hand Sanitizer for eventual extraction of DNA [36]. All colonized adults were maintained at 28°C, 70–80% relative humidity and for a 12: 12 hour photoperiod. For the D2S3 (Aaa) by PK10 (SenAae) crosses, 10 pairs of virgin P1 parents were each given the opportunity to mate in half liter cartons, blood fed on the senior author’s arm on days 3,5 and 8 post-eclosion, and provided on day 5 with moist paper towels on which to oviposit. Cartons were checked every day for eggs, and if present these were collected and counted until three ovipositions had occurred or 10 days had passed since the day 8 blood feeding. Lack of normality was visually evident (black dots in Fig 2) and so egg counts were log transformed (Log10 (eggs +1) ). Transformed counts were compared using a General Linear Model (glm () ) and contrasts in R 3. 1. 0 [37]. The same statistical procedures were followed for the Sedhiou (F) and Ziguinchor (F) families, except eggs were only collected once, 3–4 days after blood-feeding, before adults were collected and frozen for anticipated eventual use in QTL mapping. Since our goal was to generate large F2 families for QTL mapping and Senegal females generally produce far fewer eggs than the D2S3 or ROCK females, F1 intercrosses were only made in one direction. The data from the Sedhiou (F) and Ziguinchor (F) families (black dots in Fig 3) were normally distributed (as determined by shapiro. test in R) and so Fisher’s least significant difference with Bonferroni’s correction for multiple comparisons were calculated in R 3. 1. 0. using pairwise t-tests on untransformed egg counts among crosses and a Bonferroni corrected probability (R command: pairwise. t. test (Eggs, Cross, p. adj =" bonferroni" ). Individual pairs of newly emerged virgin adults were placed together in half liter cartons and maintained with sugar water for 10 days. Females were blood fed on days 3 and 5. Following oviposition or no later than day 10, females were knocked down with trimethylamine and the spermathecae were dissected in phosphate buffered saline (PBS) from the tip of the abdomen, transferred to a glass slide, a coverslip was gently placed over them and the three spermathecae were inspected at 40X under a compound microscope to check for motile sperm. At the same time, the ovaries were inspected for abnormal development (e. g. no vitellogenin accumulation or hypotrophic ovarioles). Many possible reasons exist for the inability of SenAae to survive and reproduce in the laboratory relative to long established ROCK and D2S3 lab strains. Cursory comparisons were made of ROCK, D2S3 and SenAae larvae and pupae grown alongside one another and fed the same larval food. While mortality occurred in larvae in all containers, there was no obvious differential mortality in SenAae. Different types of food were offered to the larvae and this did not differentially affect survival in SenAae larvae and pupae. Different oviposition container sizes, shapes and colors were tried. Coconut milk (5% (v/v) ) was tried in place of water in the oviposition containers because of the abundance of Ae. aegypti larvae in Saba senegalensis husks [35] in forested regions. None of these conditions affected oviposition rates or larval survival. Therefore the possibility of post-zygotic reproductive incompatibility within and among SenAae was considered. More specifically the possibility was considered that SenAae collections consist of mixtures of cryptic taxa that are reproductively isolated from one another and from Aaa. Haldane’s rule predicts that in fully or partially reproductively isolated species with heterogametic sex chromosomes, the heterogametic sex will be preferentially sterile and inviable in interspecific crosses. Backcrosses were performed to test for postzygotic isolation patterns consistent with Haldane’s rule [38] modified for species, like Aedes, that have an autosomal sex determining locus (SDL) [39]. More specifically, in crosses between the ROCK Aaa strain and SenAae strains, crosses involving hybrid F1 males were expected to have higher sterility but crosses involving hybrid F1 females to be unaffected. Egg hatch was predicted to be low when a female mates to a hybrid F1 male but hatch was predicted to be average when a male mates to a hybrid F1 female. F1 mosquitoes were generated by placing 50 virgin ROCK females in a 30 cm x 30 cm x 30 cm cage with 50 PK10 (F) males, these were blood fed once. Reciprocal crosses were made in a second cage and blood fed once. Control crosses were generated by placing a) 50 virgin ROCK females with 50 virgin ROCK males and f) 50 virgin PK10 (F) females with 50 virgin PK10 (F) males. Eight backcrosses were then made with the F1 progeny. These were: b) ROCK x (ROCK x PK10 F1), c) ROCK x (PK10 x ROCK F1), d) (ROCK x PK10 F1) x ROCK, e) (PK10 x ROCK F1) x ROCK, g) ROCK x (ROCK x PK10 F1), h) ROCK x (PK10 x ROCK F1), i) (ROCK x PK10 F1) x PK10, j) (PK10 x ROCK F1) x PK10. For each of the ten crossing types, 40 families were set up in half liter cartons. Eggs and then pupae arising from each of these crossing types were counted in each of the ten crossing types. Egg counts in most crosses were zero-inflated (see S1 Table), so zero-inflated negative binomial (ZINB) regression in R 3. 1. 0 was used with the zeroinfl function in the pscl package. In addition glm () in R 3. 1. 0 was used to compare log10 (eggs) including only those females that laid eggs. The proportions of eggs surviving to pupae were compared using glm () in R 3. 1. 0. Eggs that failed to hatch were checked under a dissecting scope to determine if they were desiccated (collapsed). If not they were then soaked in fresh Trpis bleach [40] for 4 hours and then observed at 20–40X under a compound microscope. No evidence of embryo formation (e. g. segmentation) was observed in these unhatched eggs. To evaluate the possibility that chromosome rearrangements occur within SenAae and between SenAae and Aaa, chromosomes were measured, stained to identify heterochromatic regions and analysed with Fluorescent In Situ Hybridization (FISH) with markers of known position in the Aaa genome to test for chromosome rearrangements. In addition, linkage distances in cM were estimated between the sex determining locus and white eye on chromosome 1 and compared among collections. Chromosomal preparations were made from 4th instar larvae [41]. Chromosomes were stained with 1 μM YOYO-1 iodide solution (Invitrogen Corporation, Carlsbad, CA, USA) in 1x PBS and enclosed under anti-fade Prolong Gold reagent (Invitrogen Corporation, Carlsbad, CA, USA). Chromosome lengths were measured using Zen 2009 light edition software (http: //www. zeiss. de). Based on these measurements, chromosome indexes (the percentage of each arm in the total chromosome length) and centromeric indices (length of the short arm p relative to the total length of the chromosome) were calculated and P-values were determined [42]. Chromosomes were stained with a DAPI Prolog Gold Reagent (Invitrogen Corporation, Carlsbad, CA, USA) and chromomycin A3 (Sigma-Aldrich Corporation, St. Louis, MO, USA) to identify differences in heterochromatic regions between strains. Fluorescent staining was performed with DAPI/Chromomycin A3 [43]. FISH was performed as described previously [44]. BAC clones with known locations on chromosomes of the IB12 strain [45] were utilized. Unspecific hybridization was blocked by using unlabeled repetitive DNA (C0t3) fractions. The linkage distance of the white-eye marker [46] at 20 cM and the sex determining locus (SDL) at 34 cM [47] on chromosome 1 was estimated as a quick and inexpensive way to test for rearrangements on chromosome 1. Forty P1 families were set up in each of four SenAae collections (Mont Rolland, Bignona, PK10 and Goudiry (Fig 1) and in 20 families from an Aaa collection from Merida, Mexico (Fig 1). To assess survival and fecundity within SenAae and between SenAae and Aaa collections, a series of crosses were set up that involved the laboratory strain D2S3 and PK-10 SenAae (Fig 1) that either had scales on the first abdominal tergite, PK10 (G), or had no scales, PK10 (F). There were 7 parental combinations (Fig 2): a) female x male D2S3, b) female x male PK10 (F), c) female x male PK10 (G), d) D2S3 x PK10 (F), e) PK10 (F) x D2S3, f) PK10 (G) x PK10 (F), and g) PK10 (F) x PK10 (G). Table 2 lists the hypothesis being addressed by each contrast in the General Linear Model. The numbers of eggs produced in three ovipositions ranged from very high in the D2S3 family to nearly zero in crosses between PK10 (F) and PK10 (G) parents (Fig 2). D2S3 families produced twice the number of eggs as PK10 (F) and ~7x more than PK10 (G) families and these differences were significant (Table 2—Contrast 1). The numbers of eggs produced in PK10 (F) and PK10 (G) families were not significantly different (Table 2—Contrast 2). Reciprocal crosses between D2S3 and PK10 (F) produced equal numbers of eggs (Table 2—Contrast 3) but half the number produced by D2S3 (Table 2—Contrast 4). There was a large and significant drop in fecundity in the PK10 (F) family as compared to PK10 (F) x PK10 (G) crosses (Table 2—Contrast 5) and in the PK10 (G) family as compared to PK10 (G) x PK10 (F) crosses (Table 2—Contrast 6) is undocumented. PK10 (F) x PK10 (G) reciprocal crosses had similarly low fecundity (Table 2—Contrast 7). These experiments were terminated prior to intercrossing F1 siblings because most crosses yielded too few eggs to continue onto the F2. To assess fecundity in F1 siblings, another series of F1 intercrosses were established with SenAae from Sedhiou and Ziguinchor from the Casamance area of southern Senegal (Fig 1) to determine whether the results seen in the first experiment were unique to PK10. These collections also had a large proportion of SenAae without scales on the first abdominal tergite. Ziguinchor (F) and Sedhiou (F) were crossed with one another and to D2S3. Egg to pupal survival in P1 and F1 eggs were recorded as was F2 pupal-to-adult survival. Fig 3A shows the number of eggs produced by a) D2S3, b) Sedhiou (F), and c) Ziguinchor (F) families, as well as d) D2S3 females crossed to Sedhiou (F) males and e) D2S3 females crossed to Ziguinchor (F). All but the Sedhiou families produced similar numbers of eggs. There was wide variation in pupal survival (pupae/eggs) within all five crossing types but all were statistically homogeneous (Fig 3B). The number of eggs produced when intercrossing the F1 siblings to generate F2 families in each of the five crossing types was also recorded (Fig 4A). The number of eggs produced was statistically homogeneous among the five crossing types. Pupal and adult survival in each of the five crossing types was also measured (Fig 4B and 4C). Pupal survival was homogeneous among D2S3 families, D2S3 x Sedhiou (F) and D2S3 x Ziguinchor (F). However pupal and adult survival in Sedhiou (F) and Ziguinchor (F) families were significantly lower. Thus while all five types of crosses produced similar numbers of F1 eggs (Fig 3A) and had similar F1 pupal survival rates (Fig 3B) survival of these eggs to F2 adults was only 0. 02–0. 16 in Sedhiou (F) and Ziguinchor (F) families and in the D2S3 x Sedhiou (F) and D2S3 x Ziguinchor (F) hybrids. SenAae exhibit poor egg-adult survival in the laboratory as do hybrids of these strains when mated to long established lab colonies. To determine whether the results in Figs 2–4 were specific to D2S3, the experiment was repeated with the ROCK strain and Sedhiou (F). Similar results were obtained and no further attempts were made to construct F1 intercross families for QTL mapping. A series of crosses were performed next to test for prezygotic barriers to gene flow among SenAae. Eggs from the February 2010 collected SenAae PK10 (F), Sedhiou (F), and Ziguinchor (F) experienced large-scale hatch failures even after only 2–3 weeks of storage following oviposition. A newly collected July 2010 SenAae from Bignona (Fig 1) from which separate G and F strains could be selected was used instead. In none of the 9 parental crosses were females uninseminated and on average 77% of spermathecae contained motile sperm (Fig 5A). Furthermore there were very few females in which sperm were immotile. Ovarian development in all cases appeared to be normal. F1 egg-to-pupal survival was monitored in all of these females and was uniform among all crosses except for the D2S3 families, Bignona (F) intercrosses and the Bignona (G) x Bignona (F) cross (Fig 5B). A second set of experiments were set up to test for evidence of assortative mating using the recessive white eye allele [46] as a marker for Aaa. Fifty Higgs White Eye (HWE) females were located in a cage with 25 HWE males and 25 Richard Toll (F) males to test for assortative mating by Aaa females. Of 44 families generated, 24 were white eye, not significantly more than the 22 expected if there was no discrimination (χ2[1 d. f] = 0. 36. P = 0. 55). To test for assortative mating by Richard Toll (F) females, again 50 females were located in a cage with 25 HWE males and 25 Richard Toll (F) males. All F1 offspring were wild type and offspring adults were backcrossed to HWE adults to assess their genotypes. Of 31 backcross (BC) families, 17 had white eye offspring, not significantly more than the 15 expected if there was no discrimination (χ2[1 d. f] = 0. 29. P = 0. 59). Mating between Richard Toll (F) and HWE did not appear to be assortative. This experiment was repeated but using PK10 (F) (2011) choosing between 25 HWE and 25 Richard Toll (F) males. Of 36 families generated, 24 were white eye, slightly more than the 18 expected if there was no discrimination (χ2[1 d. f] = 4. 0. P = 0. 046). All surviving F1 families were wild type and offspring were backcrossed to HWE adults to assess their genotypes but this only produced 2 BC families. This experiment was repeated with Goudiry (F). Of 20 HWE families, 6 were white eye, not significantly fewer than the expected 10 if there was no discrimination (χ2[1 d. f] = 3. 20. P = 0. 074). When Goudiry (F) females were located in a cage with equal numbers of HWE and Goudiry males, only 6 BC families survived. These experiments did not reveal any obvious prezygotic barriers to mating among Aaa and SenAae. Haldane’s rule predicts that in fully or partially reproductively isolated species with heterogametic sex chromosomes, the heterogametic sex will be preferentially sterile and inviable in interspecific crosses. Presgraves and Orr [39] tested Haldane’s rule in Aedes mosquitoes, which contain homomorphic sex chromosomes that contain a sex determining locus and a dominant male allele, and determined that mosquitoes in the genus Aedes follow Haldane’s rule for sterility, but not for inviability. To test for patterns of sterility consistent with Presgraves and Orr [39] caveat to Haldane' s rule, Ten crosses were made: A. 50 virgin ROCK females mated with 50 virgin ROCK males, B. ROCK x (ROCK x PK10 F1), C. ROCK x (PK10 x ROCK F1), D. (ROCK x PK10 F1) x ROCK, E. (PK10 x ROCK F1) x ROCK, F. 50 virgin PK10 (F) females mated with 50 virgin PK10 (F) males, G. PK10 x (ROCK x PK10 F1), H. PK10 x (PK10 x ROCK F1), I. (ROCK x PK10 F1) x PK10, J. (PK10 x ROCK F1) x PK10. Haldane’s rule could not be tested in the strictest sense because no markers exist to distinguish reproductively isolated taxa in SenAae. Instead trends consistent with Haldane’s rule in SenAae were tested for assuming collections contain both reproductively compatible and incompatible individuals. More specifically, in crosses between the ROCK Aaa strain and SenAae strains, crosses involving hybrid F1 males were expected to have higher sterility but crosses involving hybrid F1 females were expected to be unaffected. Egg hatch is predicted to be low when a female mates to a hybrid F1 male but hatch is predicted to be average when a male mates to a hybrid F1 female. The number of eggs/female (Fig 6A) and egg-pupal survival (Fig 6B) in each of the ten crossing types (a–j) were analyzed. Statistical analyses were performed of the proportion of females ovipositing (S1 Table), numbers of eggs produced by all females (S2 Table), numbers of eggs produced by ovipositing females (S3 Table) and egg-pupal survival (S4 Table). The qualitative results with respect to Haldane’s rule are summarized in Tables 3 and 4. The first four contrasts in Table 3A indicate that offspring of crosses involving ROCK/PK10 hybrid F1 males had lower egg-pupal survival than offspring of ROCK families as predicted by Haldane’s rule if Aaa and SenAae are separate taxa. The next four contrasts show that offspring of crosses involving ROCK/PK10 hybrid F1 males had the same low egg-pupal survival as offspring of PK10 families. This pattern suggests that SenAae consists of a single taxon. The first four contrasts in Table 3B show that offspring of crosses involving ROCK/PK10 hybrid F1 females have greater egg-pupal survival than offspring of ROCK families. This pattern is inconsistent with Haldane’s predictions that egg-pupal survival should be unaffected in crosses involving hybrid females. The next four contrasts show that offspring of crosses involving ROCK/PK10 hybrid F1 females had the same egg-pupal survival as offspring of PK10 families. This pattern also suggests that SenAae consists of a single taxon. The first four contrasts in Table 4A are equivocal. The fecundity of crosses involving hybrid F1 males was reduced in the first two contrasts and unaffected in the next two. This pattern is therefore inconclusive with respect to Haldane’s predictions. The next four contrasts are similarly inconclusive with crosses involving ROCK/PK10 hybrid F1 males having the same fecundity as offspring of PK10 families in three of the four contrasts. Table 4B is also inconclusive. In summary, patterns of egg-pupal survival are only partially consistent with Haldane’s predictions if Aaa and SenAae were fully or partially reproductively isolated species. But patterns are consistent with SenAae consisting of a single taxon. In contrast, fecundity patterns are inconclusive with Haldane’s predictions because most of the reduced fecundity was caused by a lack of oviposition by mated females rather than reduced survival in her offspring. A reliable protocol to prepare somatic chromosomes of Ae. aegypti from leg imaginal discs in fourth instar larvae has been developed [41] and has allowed the physical mapping of scaffolds onto Ae. aegypti chromosomes using FISH [48]. In this study this approach was utilized for cytogenetic analysis of four of the SenAae strains: Kedougou, Sedhiou, PK10, and Goudiry (Fig 1). As a control the IB12 strain of Aaa was used because it was the genome that had been sequenced [33] and physically mapped [48]. To determine the presence of chromosomal rearrangements in SenAae strains, basic cytogenetic analyses (chromosome measurements and differential staining) and FISH experiments were performed. Although all SenAae strains have the typical Ae. aegypti karyotype of three pairs of metacentric chromosomes [49], basic chromosome analysis revealed differences between them. Sedhiou and Goudiry mosquitoes have significantly longer chromosomes (P = 0. 0065 and <0. 0001 for Sedhiou and Goudiry strains, respectively) as compared to IB12 strain of Aaa (Fig 7A). This is probably due to differences in repetitive element content. Relative chromosome lengths were identical suggesting the absence of inter-chromosomal rearrangements such as translocations (Fig 7B). The Goudiry strain had significant differences from other strains in centromeric indexes of chromosomes 1 and 3 suggesting the possibility of pericentric inversions (Fig 7C). However, the differences were only 1. 5% and 1%, and P-values were 0. 0051 and 0. 0122 for chromosomes 1 and 3, respectively Polymorphisms in the amount and positions of C-positive heterochromatic bands have been previously described in natural population of Ae. aegypti [50–52]. Two fluorescent dyes were used for identification of heterochromatic regions: DAPI for staining AT-rich regions and chromomycin A3 with pretreatment with barium hydroxide for staining of GC-rich regions [53] and YOYO-1 was used to test for differential staining. These experiments indicated that chromosome patterns of SenAae strains are different from the patterns in Aaa (Fig 8). In addition to chromomycin stained (GC-rich) band, which represents the ribosomal RNA locus of all strains (Fig 8D), the Goudiry strain had DAPI stained (AT-rich) band (Fig 8E) in the centromere of chromosome 1. A FISH experiment with 18S rDNA probe has indicated the presence of an additional ribosomal locus on one of homologous chromosomes in this region (Fig 8F). PK10 and Kedougou strains had polymorphic bright YOYO-1 bands in centromeres of chromosome 1 (Fig 8B) and 3. Thus, all strains had differences in the structure of heterochromatic regions of chromosomes and suggest that the strains are genetically diverged. In addition to the basic cytogenetic analyses, 40 BAC clones previously physically mapped on Aaa chromosomes were used as markers for the identification of chromosome rearrangements between the two subspecies. BAC clones were hybridized to the chromosomes of Sedhiou strain using the standard FISH protocol[44]. Although the resolution of this mapping was relatively low ~30 Mb, the differences in the order of markers revealed the presence of two chromosomal rearrangements between Aaa and SenAae strains. The first rearrangement was found on chromosome 3 (Fig 9A). Markers LF417 and LF92 dramatically changed their positions from the 3p arm in Aaa to the 3q arm in all SenAae strains. Importantly, non-specific hybridization of all repetitive sequences including transposable elements was blocked in the FISH experiments by adding unlabeled C0t3 DNA fractions to the probe before hybridization to the chromosomes. The positions of 14 markers on chromosome 3 in the Goudiry strain (Fig 9B) were analyzed using the program Genome Rearrangements In Man and Mouse program (grimm. ucsd. edu/GRIMM/). The difference in the patterns between Aaa and SenAae strains suggests the presence of two overlapping pericentric inversions in chromosome 3 or an insertion of a large fragment into 3q arm (Fig 9C). The second rearrangement was found closely located to the centromere on the 2p arm. Positions of markers LF158 and a14 were found only in the standard arrangement in IB12 chromosomes (Fig 10A and 10B) and in the standard or standard/inverted arrangements (Fig 10C and 10D) in the Sedhiou and Goudiry strains. Prior studies document inversion polymorphisms on chromosome 1 in Aaa [54,55]. The linkage distance of the white-eye marker [46] and the sex determining locus (SDL) were determined to test for chromosome rearrangements on chromosome 1. These markers are at 20 cM and 34 cM on the standard genetic map of chromosome 1 in Ae. aegypti [47]. This was done as a quick and inexpensive test for altered recombination rates, as would be expected if rearrangements exist on chromosome 1. Using the mating scheme outlined in Fig 11,40 P1 families were set up in each of four West African collections and 20 families from a Mexican collection. In each family the observed numbers of parental and recombinant backcross (BC) progeny were compared to expected numbers under one of three alternative genetic models (r = 14 cM; r = 0 cM (complete linkage); r ≥ 50 cM (unlinked) ) using a χ2-goodness of fit test. Fig 1 show that r = 0. 14 in 74% of 27 PK-10 (F) families and r ≥ 0. 5 in the remaining families. Amongst 27 Goudiry families r = 0. 14 in 41% of families and r ≥ 0. 5 in 59% of families. In Mont Rolland, in northwestern Senegal, r = 0. 14 in 69% of 35 families, r ≥ 0. 5 in 20% of families and r = 0. 0 in 11% of families. Only 5 Bignona BC families survived and r = 0. 0 in 4 of these and r ≥ 0. 5 in one family. In the Vergel collection, 11 BC families produced > 20 offspring. Amongst these r = 0. 14 in 9 families and r = 0. 0 in two families. This study suggested widespread and abundant polymorphisms in chromosome 1 among SenAae populations however it does not suggest mechanisms to explain why r ≥ 0. 5 between white-eye and SDL in many families and r = 0. 0 in some families. Over the last 15 years many F1 intercross families with various Aedes species have been constructed for use in QTL mapping experiments [56–65]. The same degree of sterility and low viability as seen in the present experiments was not observed in these earlier studies. An interesting and possibly unique aspect of this study concerns the ways that sterility became manifest as low oviposition rates in F1 and F2 females and poor egg-pupal-adult survival. Some of the fecundity results shown in Fig 2 are consistent with already well documented genetic differences in fecundity among Ae. aegypti strains [66]. That study also demonstrated that fecundity behaved as an additive quantitative genetic trait with hybrids (e. g. crosses d and e in Fig 2) having intermediate fecundities between the parents (e. g. crosses a and b in Fig 2). However the main reason for this drop in fecundity involved a failure to oviposit (Fig 2). Only one D2S3 mother failed to oviposit, while eight and nine females from PK10 (G) x PK10 (F) and the reciprocal cross failed to oviposit and five females failed to oviposit in both PK10 (F) and PK10 (G) families (Fig 2). The key observation is that PK10 females laid fewer eggs when crossed to a PK10 male but with a different abdominal scale pattern. Thus a large component of fecundity loss appears to be associated with a reduction in the tendency of SenAae females to oviposit under laboratory conditions and depended on the male with which they mated. Haldane' s rule [38] is one of the few empirical generalizations about speciation that holds true across different groups of animals and plants. Specifically, Haldane’s rule predicts that in fully or partially reproductively isolated species with heterogametic sex chromosomes, the heterogametic sex will be preferentially sterile and inviable in interspecific crosses. Dominance and rapid male evolution are often cited as the primary forces causing Haldane’s rule. Dominance is based upon the observation that alleles causing hybrid problems are most often deleterious and/or lethal recessives [67–70] and if they are sex-linked then heterogametic individuals, because they are hemizygous, will suffer their full effects. But deleterious and lethal recessives will be masked in the homogametic sex if one allele is wild-type. Amongst Drosophila species dominance appears to largely explain Haldane’s rule [71–73] for hybrid inviability. It has also been proposed that sexual selection may drive the more rapid evolution of male-expressed genes [74–76] as well as variation in expression of male-expressed genes [77]. More rapid male evolution would also explain Haldane’s rule because hybrid male sterility would occur before hybrid female sterility. Experiments in Drosophila suggest that faster male evolution causes Haldane’s rule for sterility [72,78]. Presgraves and Orr [39] tested Haldane’s rule in mosquitoes, a family in which members of the subfamily Anophelinae have heterogametic sex chromosomes while species in the subfamily Culicinae (Aedes) have a sex determining locus (SDL) on an autosome. Male (“M”) alleles at the SDL are dominant so that males are “Mm” heterozygotes and females are homozygous recessive “mm. ” Presgraves and Orr showed that for the most part Anopheline mosquitoes follow Haldane’s rule for sterility and inviability while Aedes mosquitoes only follow Haldane’s rule for sterility. The authors speculated that faster male evolution is driven by sexual selection which occurs irrespective of the sex determination mechanism. Alternatively, dominance is unlikely to act in Aedes because it lacks heterochromatic sex chromosomes and should exhibit little or no sex-limited inviability. Considering only reproductively isolated species and tabulating hybrid sterility and inviability separately, Presgraves and Orr classified the outcome of each interspecific cross as “male-affected”, “female affected”, or “both-sexes–affected. ” Anopheles species obeyed Haldane’s rule for heterogametic sex while Aedes obeyed Haldane’s rule for hybrid sterility supporting the hypothesis that Haldane’s rule occurs even in species with autosomal sex determination. In all comparisons male-only hybrid sterility was noted, whereas female-only sterility was not seen. This also provided support for faster male evolution in Aedes. Sexual selection, which clearly operates in the genus Aedes [79,80], seems a likely candidate that could drive more rapid male evolution. However, most of the sterility in the present study arose from the absence of egg-laying, especially when females were crossed to hybrid F1 males. It was documented in 1958 that uninseminated Ae. aegypti females tend not to oviposit even when fully gravid [81]. In 1965, it was shown that male accessory gland substances, rather than sperm, acted as oviposition stimulants [82] in Ae. aegypti. Similarly in Drosophila melanogaster, the male accessory gland protein Acp26Aa stimulates oviposition [83–85]. It is possible that females in the present study failed to detect the male accessory gland proteins of a different species or subspecies and therefore failed to oviposit. This would be consistent with Presgraves and Orr suggestion that faster evolution occurs in male-specific genes. The Ae. aegypti semen proteome has been recently characterized [86–90] and reveals that while seminal fluid protein classes are conserved, many of the proteins evolve rapidly, necessitating identification of male reproductive gland proteins in each individual species. Applying study designs similar to those used by McLain and Rai [79,80] no evidence was found for prezygotic factors associated with the observed loss in fertility and survival. If the results of the insemination experiment with Bignona (G) and Bignona (F) (Fig 5) can be extended to the other SenAae, then there is no evidence for assortative mating or sperm inviability. But strong inferences cannot be made concerning assortative mating under laboratory conditions. Chromosomal rearrangements have frequently been associated with speciation in taxa including mosquitoes [91]. Pericentric inversions are only rarely detected within animal species but can be abundant among species [92,93]. Acentric fragments and dicentric “bridge” chromosomes arise when recombination occurs in parents that are heterozygous for pericentric inversions. These in turn yield aneuploid gametes and inviable zygotes. Therefore most F1 offspring arising from a cross between parents with (Goudiry Fig 9, Sedhiou Fig 10) and without pericentric (IB10) inversions are predicted to produce fewer viable larvae which will be homozygous for either of the two parental inversions. The rearrangement on chromosome 2 was close to the centromere on the 2p arm. Positions of markers LF158 and a14 were found only in the standard arrangement in IB12 chromosomes (Fig 10A and 10B) and in the standard or standard/inverted arrangements (Fig 10C and 10D) in the Sedhiou and Goudiry strains. This result suggests that we found this inversion is only detectable in the heterozygote arrangement. Interestingly, the genetic marker LF158 is located at 36. 7 cM, which is close to the position of the Black-Tergite genetic locus responsible for scaling patterns on the first abdominal tergite (34 cM) [47]. A major impediment to further cytogenetic studies was our inability to maintain SenAae isofemales lines. Although evidence of inversions and ribosomal gene duplications have been presented, maintenance of the strains with these rearrangements is difficult. Thus to date these rearrangements have not been validated nor have their geographic distributions been determined. The large variation documented in recombination distances along chromosome 1 among SenAae collections (Fig 1) could occur through insertions, inversions or translocations. The variation could also occur because other genes elsewhere in the genome have come to control sex determination as has been noted for example in Musca domestica (L) populations [94]. Crosses between genotypes with different inversion types will have suppressed recombination rates, so if this was the cause then the original genetic map of white-eye could presumably have had an inversion polymorphism on chromosome 1 while crosses where r ≥ 0. 5 would be the same inversion type. Further cytogenetic analyses and examination of additional collections will be needed to sort out these alternative explanations. A critical question is whether all or some of our SenAae collections contain more than one species? Is hybrid sterility arising between two (or more) discrete subspecies? Alternatively are they occurring between individuals from the same collection? The critical test for cryptic species would be to identify a priori two or more types of mosquitoes based on genetic markers, cytogenetics or phenotype and then show that reproductive incompatibilities exist between these types and not within them. This has not been accomplished far for the many reasons listed above. Good candidates may be mtDNA clades [19,25], chromosome rearrangements, independent linkage of white-eye and SDL (Fig 1) and still possibly scaling patterns on the first abdominal tergites (Figs 2 and 3). An obvious question that remains is how SenAae are related to the Aaf and Aaa in East Africa. The earlier study of hybridization and mating behavior between Aaa and Aaf in East Africa concluded that these two forms are part of a single, albeit highly polytypic species [15]. The results of this study suggest that the taxa found in West Africa differ from the much more intensely studied Aaa and Aaf subspecies in East Africa. Assuming no pre-zygotic barriers and heterozygote breakdown (underdominance), collections that consist primarily of one taxon (few heterozygotes) are predicted to survive better than collections with more-or-less equal numbers of different taxa (many heterozygotes). Crosses within a cryptic species will produce offspring which survive to fully fertile adults. Even though individuals belonging to cryptic taxa can mate with one another, they produce few or no eggs (Fig 2) and if eggs are produced, they have lower survival to adults. The suggestion that one or more new cryptic subspecies exist in Senegal in no way contradicts the phylogenetic patterns among worldwide Ae. aegypti s. l. populations that have been derived over the last 35 years using allozyme markers [20–22], microsatellites [16,25,26], nuclear SNP loci [15,17,27], mitochondrial DNA [19,25] and most recently a SNP-Chip [17]. Instead our results suggest that taxa in some of the clades resolved in these phylogenetic studies may be partially or wholly reproductively isolated from one another and furthermore that some clades identified in these earlier studies may contain reproductively isolated taxa. The results presented here therefore serve as a reminder of the importance of evaluating reproductive isolation among closely related taxa rather than assuming that they belong to a single reproductively continuous population.

Aedes aegypti is one of the best studied mosquito species and it is the principal vector of dengue, Zika, and yellow fever flaviviruses and the Chikungunya alphavirus. Aedes aegypti occurs throughout all tropical and subtropical regions of the world, and previous population genetic studies have shown that the highest genetic diversity occurs in Africa. Aedes aegypti from Senegal, West Africa (SenAae) have a low oviposition rate; those that do oviposit have a low fecundity and poor egg-to-adult survival. Furthermore rearrangements were detected on all three chromosomes in SenAae. These observations are consistent with the presence of at least two cryptic subspecies of Ae. aegypti in Senegal arising from reproductive isolation due to chromosome rearrangements. Genetic control strategies are being considered for the suppression of Ae. aegypti populations worldwide. Barriers to gene flow in African Ae. aegypti populations could compromise these future control efforts.

lay_plos

Natively unstructured regions are a common feature of eukaryotic proteomes. Between 30% and 60% of proteins are predicted to contain long stretches of disordered residues, and not only have many of these regions been confirmed experimentally, but they have also been found to be essential for protein function. In this study, we directly address the potential contribution of protein disorder in predicting protein function using standard Gene Ontology (GO) categories. Initially we analyse the occurrence of protein disorder in the human proteome and report ontology categories that are enriched in disordered proteins. Pattern analysis of the distributions of disordered regions in human sequences demonstrated that the functions of intrinsically disordered proteins are both length- and position-dependent. These dependencies were then encoded in feature vectors to quantify the contribution of disorder in human protein function prediction using Support Vector Machine classifiers. The prediction accuracies of 26 GO categories relating to signalling and molecular recognition are improved using the disorder features. The most significant improvements were observed for kinase, phosphorylation, growth factor, and helicase categories. Furthermore, we provide predicted GO term assignments using these classifiers for a set of unannotated and orphan human proteins. In this study, the importance of capturing protein disorder information and its value in function prediction is demonstrated. The GO category classifiers generated can be used to provide more reliable predictions and further insights into the behaviour of orphan and unannotated proteins. One of the challenges of the post-genomic era is to predict the function of a protein given its amino acid sequence. Most automated function prediction methods rely upon identifying well-annotated sequence and structural homologues to transfer annotations to uncharacterised proteins (see [1,2] for a comprehensive review). Sequence similarity–based methods are relatively successful at annotating homologous proteins; however, they are not applicable to annotating orphan proteins or proteins whose relatives are not themselves functionally annotated. Currently, around 35% of proteins cannot be accurately annotated by homology-based transfer methods [3], highlighting the need for function prediction methods that are independent of sequence similarity. ProtFun [4,5] is an ab initio feature based protein function prediction method that addresses the annotation of orphan proteins and is applicable to any protein whose sequence is known. The method makes use of sequence-based feature descriptors encoded from localisation, secondary structure, and post-translational modification predictions. Function category predictions were made using individual ensembles of neural networks trained to recognise feature patterns associated with particular functions. Similar approaches have been reported using structural properties and sequence information for prediction of enzyme classes [6,7]. One advantage of this type of approach is that features that are important in recognition of different function classes can be easily identified and quantified. Over the past few years, there has been a growing awareness of the fundamental importance of disordered proteins in many biological functions and processes. Disordered regions of proteins can be predicted from amino acid sequence [8,9], allowing for rapid surveying of the occurrence of disorder in entire proteomes. The prevalence of disordered proteins in higher eukaryotes is thought to reflect the complexity of signalling and regulatory process within these organisms [10–12]. Disordered regions in proteins are defined as those which lack a stable well-defined 3-D structure in their native states [13,14]. Intrinsically disordered proteins may be either entirely disordered or partially disordered, characterised by long stretches of contiguously disordered residues. The presence of protein disorder is thought to confer dynamic flexibility to proteins, allowing transitions between different structural states [15]. This increased flexibility is advantageous to proteins that recognise multiple target molecules with high specificity and low affinity [13,15]. The functions of numerous disordered proteins have been characterised experimentally and include DNA and protein recognition, transcription and translation regulation, and targeted protein degradation [16–18]. The disordered regions of these proteins have been shown to be essential for their function, forcing a re-examination of the classical sequence–structure–function paradigm central to the field of structural biology and at the core of most automated function prediction algorithms [19,20]. The Protein Trinity hypothesis [19] states that protein function can arise from any of three states: ordered, molten globule, random coil, or from transitions between any or all of these states. Wright [17] defined a continuum of protein structures ranging from an unstructured conformational ensemble to mostly structured proteins containing only locally disordered regions. The functions of disordered proteins along the continuum are influenced by the presence and type of the unstructured regions. For example, disordered stretches can be flexible linker regions that allow movement between domains or can be sites of molecular attachment that become ordered on binding and give rise to functional specificity. In other proteins, disordered regions are associated with sites of post-translational modification that regulate protein–target interactions. It is clear that protein disorder is an important determinant of some protein functions; however, the value of this information remains unquantified and unexploited in current protein function prediction methods. To investigate the correlation of disorder with function, we considered the human complement of disordered proteins as predicted by DISOPRED2 [9,21]. Based on pattern analysis between the distributions of protein disorder and different function annotations, an encoding scheme for representing the occurrence of disorder in proteins is proposed. We then assess the direct influence of protein disorder in function prediction using single class Support Vector Machines [22] (SVMs) to predict individual Gene Ontology [23] (GO) categories. In this analysis, a protein was considered disordered if it contained a contiguous stretch of predicted disordered residues of ≥30 amino acids. GO categories were identified that were over-represented with disordered proteins as a positive control set of categories likely to be associated with protein disorder features. 31 MF categories and 33 BP categories (Figure 1) were significantly enriched in disordered proteins at corrected p-values of <0. 001. The cutoff was purposefully stringent to ensure virtually no false positive terms were selected. “Transcription factor”, “DNA and protein binding”, “kinase signaling”, and “phosphorylation” molecular function (MF) categories were amongst those enriched in disordered proteins indicated by the highest log ratios of observed/expected occurrence of disordered proteins (Figure 1A). Transcription factor categories were most enriched in disordered proteins, followed by Ion channel and phosphorylation related functions. Metal-ion and nucleotide binding functions exhibited smaller yet significant enrichment in disordered proteins. “Transcription regulation”, “kinase signalling”, “RNA metabolism”, and “phosphorylation” featured in the BP categories (Figure 1B) that were enriched in disordered proteins. These categories were consistent with those functions reported both experimentally [24–26] and those reported in similar analyses of other organisms [10]. We examined the distributions of protein disorder within different GO categories to ensure that the disorder features we used captured the trends and patterns relevant for function prediction. We used location descriptors to encode the position of disordered regions in proteins and length-based descriptors to distinguish short from long contiguous stretches of disordered residues. Correlations between location descriptors and GO categories were demonstrated by calculating the average frequency of disordered residues within different location windows for protein sequences annotated by a GO term (see Methods for more detail, and Figure 2). These averaged values were converted to Z-scores individually for each location window. This procedure normalised for the fact that the false positive rates for prediction of disordered residues are higher at the N and C termini of proteins than in the interior regions [10]. The Z-scores emphasized trends and sampling bias of frequencies of disordered residues directly attributable to the annotation categories. Clustering of annotation categories was performed using Ward' s hierarchical method [27], which minimizes within-cluster variance measured by sums of squares error. The location descriptors showed several trends associated with GO categories. “Transcription regulator”, “DNA binding”, and “RNA pol II Transcription factor” functions were associated with disordered residues in the protein interior, rather than at N and C termini (Figure 2A). “Transcription factor activator”, “Transcription factor repressor”, and “Transcription factor” categories showed significant associations with disordered residues toward the C terminus. Disordered residues were over-represented at the N terminus within the set of Ion Channel and more specifically potassium channel annotated proteins. A further weak association was observed between disorder at the C terminus and the ion channel categories. These observations can be confirmed by crystal structure information. For example, it has been reported that the majority of voltage-gated potassium channel proteins contain intrinsically disordered residues at their N and C terminus [28]. At the N terminus, the residues are responsible for channel inactivation [29]. The disordered residues at the C terminus are adjacent to a PDZ motif mediating binding to scaffold proteins that support the assembly of multiple ion channel subunits into a fully functioning complex [28]. Descriptors for the occurrence of different lengths of disordered regions were also constructed. The link between the length of disordered regions and sequence composition has already been described [30]. To investigate whether this observation also corresponded with functional influences, a similar clustering was performed using descriptors derived from the length distributions of disordered regions within each GO category. The region ranges were selected to reflect the shape of the entire distribution of disordered regions in the human proteome and to avoid sparse descriptors at the upper tail of the distribution (see Figure S1). Clustering the GO categories by the lengths of their disordered regions (Figure 3) revealed a greater degree of function association (more significant Z-scores associated with GO categories) than for the location descriptors. Long regions of more than 500 contiguous disordered residues were over-represented in transcription-related function categories. Shorter regions (50 residues or less) were over-represented in proteins performing metal ion binding, ion channel, and GTPase regulatory functions. Proteins annotated with serine/threonine kinase and phosphatase categories were also over-represented with contiguous stretches of 300–500 disordered residue regions. Again these findings can be supported by structural evidence. Short disordered regions at the mid- to N-terminal regions in small GTPase regulatory proteins mediate a switching mechanism, enabling the protein to interact with multiple binding partners [31,32]. We demonstrate that these correlations are not simply a function of correlations between protein length and GO categories by considering “Ion Channel” and “Transcription factor binding” categories (Figure 3A). We observed a statistically significant association between shorter disordered regions and the Ion Channel GO category, yet the average length of protein within this annotation category is more than 900 amino acids. In contrast, for “Transcription factor binding”, the opposite trend is observed. The average protein length for this class is closer to 700 amino acids, and we have reported an association with long (more than 500 residue) stretches of disorder. The correlations between function category and disorder region length may be symptomatic of the nature of the structurally disordered region. Tompa [33] described a general set of six functional classes for Intrinsically Unstructured Proteins (IUPs) that reflect their capacity to fluctuate freely in conformational space or their ability to partner molecules either permanently or transiently. It may be that the correlations displayed here between disordered region length and GO class represent the degree of structural malleability required by the protein to perform its function. For example, longer disordered regions observed in transcription regulator categories (Figure 3B) predominantly act as assemblers that are entirely unstructured and require great flexibility to function. GO categories that contain proteins whose disordered regions are predominantly display sites, for example those that are phosphorylated or involved in ubiquitination (Ubiquitin cycle in Figure 3B), require only shorter disordered regions conferring local flexibility within the protein. The cluster groupings (Figures 2 and 3) were symptomatic of the relationships between annotation terms in the GO graph structure. Specific terms inherit annotations from general parent terms and thus share protein sequences in common. The fact that inherited terms occupied the same or similar clusters provided evidence for the robustness of the observed trends between different annotation categories. Our systematic analysis of disordered regions in the human proteome revealed significant associations between both lengths and locations of disordered regions within proteins and their different GO categories. Many of the observations can be verified by available experimental structure information, highlighting the potential value in using these attributes of disordered proteins as feature descriptors in a method to predict protein function. Including highly correlated features as inputs to machine learning algorithms often results in little increase in performance, and can sometimes result in decreased performance. To investigate relationships between the disorder features and other features to be used in function prediction, a large set of general feature descriptors was assembled (see Table S1). These were grouped into biological concepts: glycosylation or secondary structure, for example. Redundancy between feature pairs was evaluated using a feature distance matrix (1-Pearson correlation). To represent the important information in the matrix in fewer dimensions, classical Multi-Dimensional Scaling (MDS) was performed. Visualisation of the matrix using the first three dimensions as orthogonal axes (Figure 4) showed three clearly defined groupings. Amino acid composition, phosphorylation, and glycosylation features formed the first group, followed by secondary structure and transmembrane features. Disorder descriptors form a third group less extended from the origin of the plot. The shorter disorder axis reflects the fact that disordered residues are not predicted for all proteins, and, therefore, the information content within these features is comparably less than for amino acid or secondary structure features, which are generic to all proteins. The feature relationships agreed with biological knowledge. For example, sequence features such as hydrophobicity and charge were related to the frequencies of particular amino acids within proteins. The correlations between predicted phosphorylation sites and frequency of Ser, Thr, and Tyr residues (Pearson correlation ∼0. 2) were due to the fact that high frequencies of phosphorylated residues can only be observed when the relevant amino acid types occurred with a high frequency in the protein. Similarly, the frequencies of predicted O and N glycosylation sites displayed correlations with the occurrence of Asn and Ser/Thr residues. The features most closely related to disorder were random coils, PEST, and low-complexity descriptors with correlation values of 0. 472,0. 211, and 0. 307, respectively, at the residue frequency level. These correlations, although relatively weak, indicated that some of the information within the disorder features is also encoded by these related feature descriptors. Disordered regions in proteins frequently contain residues that are also recognised as low sequence complexity [34]; however, a region of low complexity does not always imply structural disorder. For example, fibrous proteins such as collagens and silks are rigidly structured in their native state yet contain repetitive regions of low complexity [16]. PEST motifs are degradation motifs present in proteins involved in protein phosphorylation, protein–protein interactions, and cell adhesion [35]. These motifs have been shown to be enriched in an experimentally characterised database of disordered proteins [36], and the residues that characterise the motifs represent a subset of those amino acids known to be disorder-promoting [18,37]. However, the correlations observed here between predicted occurrences of these features were small. The general spatial isolation of disorder descriptors in feature space suggested that they contain unique biological information not represented by the other features previously used in function prediction. Feature importance estimates for all features were collated across all GO categories using a leave-one-out elimination strategy. The histogram columns (Figure 5) represent the average percentage loss in classifier accuracy for all GO categories belonging to MF and BP ontologies, regardless of their individual category performance. Secondary structure features contributed the most to classifier performance for the majority of MF and BP categories. Disorder features were the second most important feature for BP category recognition. Amino acid composition and secondary structure contributions were higher on average for MF categories than for BPs. For all other features, the importance estimates were higher for BP categories. Our results suggest that disorder patterns are more indicative of the biological process than the molecular activity of the protein. This is striking considering that only one-third of the proteins in the human proteome are predicted to contain significant disordered regions and the information content of the disorder feature set is comparably lower than that for secondary structure or amino acid composition. One possible reason for this observed difference lies in the respective ontology definitions. BP categories describe modules of functions that make up parts of a multi-step process [23], whereas MFs describe a protein' s biochemical activity. For example, the receptor tyrosine kinase signalling BP category annotation describes the series of molecular signals generated as a consequence of a transmembrane receptor tyrosine kinase binding to its physiological ligand. Three example proteins annotated by this term are neurterin precursor a neurotrophic growth factor, Rap guanine nucleotide exchange factor, and erb-B2 receptor tyrosine-protein kinase. These proteins are all unrelated at the primary amino acid sequence and secondary structure level, yet each sequence is predicted to contain at least one 30–50 disordered residue stretch (exemplified in Figure 3B). The role of disordered regions in molecular recognition and in hub proteins in protein–protein interaction networks is well-defined [38–40]. Biologically, it would make sense that proteins that are part of the same multi-step process are more likely to co-localise and possess a common interaction surface such as a disordered region without sharing any similar sequence composition or secondary structure. To evaluate the contribution of disorder features in classification accuracy for individual categories, the performance loss was measured when disorder features were removed from each classifier using the Matthews Correlation Coefficient (MCC). This measure represents the additional value of disorder features in function prediction, accounting for both interaction and compensatory effects between features. Classifier performances were reported for 26 GO categories (Table 1) whose sensitivity at a false positive rate of 10% exceeded 50%. The significance of the improvements in correlation coefficients for individual categories were evaluated using Fisher' s Z test, which considers both the magnitude of the performance increase and the strength of the correlation. The improvements that were significant at the 5% level (p < 0. 05) were marked in bold (Table 1, column MCC+diso). Eleven BP categories and 12 MF categories that were identified as enriched in disordered proteins (Figure 1) showed improvements resulting from the addition of disorder features. Several additional GO classes were identified during feature selection that required disorder features for optimal performance. Seven categories: “UDP-glycosyl transferase”, “hormone”, “growth factors”, “transferase”, “hydrolase”, and “carboxylic acid transporters” were added to the MF set of categories, and “G protein signaling” was added to the BP category set of classifiers. The most notable performance gains were observed for “protein tyrosine kinase signaling, ” “G protein signaling”, “ubiquitin specific protease”, “transcription”, “protein kinase”, and “helicase” categories. For some categories (“cation-channel”, “ion channel”, “metal ion transport”, “purine-nucleotide binding”, “nucleotide binding”, and “DNA binding”), little or no performance increase resulted from the addition of disorder features. Particularly for Ion channel, Metal Ion transport, and Nucleotide binding categories, other features such as transmembrane regions or secondary structure better characterised the relationship between the primary amino acid sequence of the protein and its function. The MCC diso–only values (Table 1) showed the correlation observed when classifiers were trained with only disorder features. Some of the BP categories relating to transcription and the Transcription factor MF category could be recognised with sensitivities of >50% at false positive rates of less than 10%, yielding Matthews correlations of ≥0. 3. For these categories, the increased performance resulting from the addition of disorder features (difference between MCC+diso and MCC–diso columns in Table 1) was much lower than the correlation obtained from disorder features alone. This result can be explained by the representation of mutual information between random coil, low complexity, or PEST features reducing the magnitude of the effect of the disorder features. Conversely, for “G protein signaling” and “Receptor tyrosine kinase” BP categories and “Growth factor”, “Helicase”, “Hydrolase”, and “Ubiquitin specific protease” MF categories, the improvement resulting from the addition of disorder features was greater than the correlation obtained using disorder features alone. This finding indicates that disorder features interacted cooperatively with other features in the dataset to achieve a greater performance increase. Throughout this study, classification performance for GO categories has been reported using the MCC. This measure accounts for the imbalanced class frequencies encountered in the GO term classifiers. For completeness, the classification sensitivities obtained at 10%, 5%, and 1% false positives were reported (Table S2 and Figure 6). The number of positive class labels is also included to stress that different error rates are required for comparable performance between these classifiers. This fact is exemplified by the Receiver Operating Characteristic (ROC) curves (Figure 6 and Table S3) which vary according to class size. The curves have been zoomed in to show the sensitivities at false positive rates of below 50%. The majority of reported classifiers were capable of achieving more than 50% sensitivity at false positive rates of less than 10%. Some categories were not recognised as enriched in disordered proteins using statistical tests due to small class frequencies and low occurrences of proteins containing disordered residues. This finding highlights the advantage in using a machine learning–based approach to assess patterns of disordered features over a simple statistical approach using frequency of occurrence in recognising GO categories for which disorder is an important determinant. In contrast to the finding that disorder features contributed more to BP category recognition, the improvements for MF and BP categories in Table 1 were slightly greater for MF than BP categories. However, these data reflect a subset of the categories for which we were able to produce accurate classifiers. This result highlights the fact that overall more BP categories utilised information from disorder features for classification than MF categories, resulting in a higher feature importance estimate overall. However, for most of these categories, we were not able to produce sufficiently accurate classifiers to be of practical use. Our method differed from the original ProtFun method [4] in several important ways. Firstly, our predictions for structure, disorder, and transmembrane regions utilised PSI-BLAST profiles rather than single sequence predictions as feature inputs. Encoding information from sequence profiles in this manner increased the accuracy of feature predictions for those proteins that belonged to unannotated families. Second, additional secondary structure features were encoded that recorded the frequencies of helices and sheets of particular length ranges within each protein. Despite these differences, we felt it was important to provide a benchmark comparison between our method and an independent method that did not utilise disorder information. To assess the performance of the ProtFun method, the ProtFun server GO category assignments used the 14,055 annotated proteins used in this study. Classifier accuracy was reported for eight common categories (Figure 7 and Table S4). The results indicated that our method outperformed the ProtFun server for all tested categories assessed using the MCC. All of these improvements were significant at the 95% level using Fisher' s Z test for significance of correlation difference, except for the ion channel category. The performance of our method without disorder features (Table 1) was also reported so that the improvements in accuracy could be attributed to the use of disorder features or to the use of different training datasets and machine learning algorithms. Four of the compared function categories; “Ion Channel”, “Voltage gated ion channel”, “Cation channel”, and “Metal ion transport” did not utilise information from disorder features; therefore, improvements resulted from other methodological differences. For the remaining categories, “transcription”, “regulation of transcription”, “hormone”, and “growth factor”, the source of performance improvements were a mixture of these effects and the addition of disorder features. The greatest accuracy increase resulting directly from the addition of disorder features was observed for the “growth factor” category. For the “hormone” category, the increased accuracy resulted equally from the addition of disorder features and the algorithm and encoding differences. “Transcription” and “Regulation of transcription” accuracies were improved more by the feature encoding and more recent training datasets used than the addition of disorder features. This result was not surprising considering that the ProtFun features included low complexity, PEST regions, and random coils that overlap considerably with disorder features within these categories. In this benchmark study, it was difficult to provide an unbiased performance measure that was comparable between the two methods. For ProtFun we were restricted to using the server output alone rather than individual neural network output scores, and any testing dataset was likely to have been used at least partially in the training of this method. However, these results indicate that our method represents a significant improvement in predicting protein function from sequence. The molecular recognition process and function classifiers reported have been used to classify a dataset of unannotated and orphan IPI proteins. A majority rule approach was applied to the annotations such that three of the five classifiers for each GO term must report a positive term assignment. At a confidence cutoff of 0. 6 (see Figure S2 for confidence distributions), we were able to assign putative functions to 317 proteins. The majority of high confidence predictions (>0. 9) were made by “transcription” and “DNA binding” MF classifiers (Table 2). Additionally, the hierarchical nature of the relationships between the GO classes can be exploited to distinguish more confident predictions. For example, many of the proteins predicted to be “regulators of transcription” also receive independent positive assignments from parent terms “transcription” and “regulation of cellular process”. The annotations have been made publicly available at http: //bioinf. cs. ucl. ac. uk/anno/IPI. html. The aim of this study was to investigate the contribution of protein disorder features in protein function prediction. This work extended numerous survey studies that report the occurrence of protein disorder within entire proteomes by identifying relevant trends and patterns of disordered regions that can be used to predict the function of proteins. Additionally, we have extended and enhanced the repertoire of GO categories that can be recognised in prediction methods by incorporating disorder features. Disorder features contributed greater overall improvements in recognition of BP categories than MF categories. In fact, the disorder features were the second most informative feature set in BP category recognition whilst amino acid composition features were the least informative. The differences in feature importance were attributed to the differences in the descriptive nature of the two Ontologies. The anticorrelation observed between the importance of disorder features and amino acid composition for BP categories suggested that associations between disordered region length and location and BP category were not a function of similar amino acid compositions of proteins within BP categories. This finding is particularly relevant for methods that attempt to predict function or possibly protein interactions from amino acid sequence without the use of homologous sequence relationships. The performance of 26 GO category classifiers could be improved using disorder features. Using the disorder features alone, sensitivities above 50% at false positive rates of less than 10% were obtained for some transcription-related BP categories. The results for all other categories were significantly better than random using disorder features as the sole input. These findings were impressive considering that in this study disordered residues were predicted rather than experimentally confirmed. Consequently, the estimates of feature importance were conservative and restricted by the accuracy of the disorder prediction algorithm. DISOPRED2 currently predicts 57% of residues correctly at a false positive rate of 5%. Additionally, whilst structural and compositional subtypes of disordered region have been suggested in the literature [33,41], such classifications have not yet been exploited in a method that predicts disorder from sequence. The potential value of encoding subtypes of disordered region in our function prediction method is indicated by the fact that in most cases the mutual information contained within PEST and low-complexity features was important for recognition of many of our reported GO categories. Finally, we have demonstrated the practical application of our classifiers in predicting function for orphan and unannotated human proteins. The classifiers are applicable to any protein sequence and are well-suited to predicting putative molecular recognition functions that can then be assayed in vivo for activity, or for the purpose of target prioritisation. For the better performing classifiers, such as DNA binding and transcription related categories, identification of function from sequence can be performed. Overall, our findings reflect the importance of capturing protein disorder information and demonstrate the value of disorder features in human protein function prediction. We used the International Protein Index (IPI) [42] as a comprehensive human protein dataset and the Gene Ontology Annotation (GOA) [43] for human. 28,057 proteins were annotated with one or more GO categories. The Cd-Hit [44,45] algorithm was used with a threshold of 60% identity to reduce overall sequence redundancy. The remaining 14,055 sequences were partitioned into five equally sized groups for cross-validation and testing. For rigorous cross-validation, the partitioning algorithm ensured that those sequences with significant homology relationships, defined as having a BLAST E-value ≤ 1e-6, were allocated to either the same training set or the same test dataset but never both. This resulted in five equally sized training and testing sets for each GO term where the maximum sequence identity between pairs of training and testing proteins did not exceed 40% sequence identity or a BLAST E-value of 10−6. Positive and negative training sets for each GO term with at least 50 representative proteins were generated. Positive training examples included those proteins annotated with a particular GO term or any of its child terms in the GO hierarchy. Negative training examples included those proteins not annotated with the particular GO term or any of its children. To avoid potential class labelling errors, proteins annotated with any of the parent or less specific terms in the GO hierarchy were subsequently removed from the negative training sets. These proteins represent incomplete annotations with respect to the GO category under consideration and may belong to either the positive or negative training set for the given term. Fisher' s exact test was performed under the null hypothesis that the occurrence of the GO term annotation and presence of disorder in a protein were independent. The hypothesis was rejected at p-values of <0. 001 after applying Bonferroni multiple testing correction. The calculations were performed using the R package for statistical computing [46]. The degree of over-representation for each GO category was compared using the log odds ratio of observed over expected numbers of disordered proteins. The expected number of disordered proteins represents the background frequency, or occurrence, of disordered proteins by random chance within a sample size equivalent to the size of the GO category. This calculation yields a scale whereby values of zero indicate equality between observed and expected numbers of disordered proteins and higher values indicate a larger difference between the observed and expected values. For a particular GO term, the set of proteins annotated by the term or any of its child terms was considered. For location-based measures, each protein was split into ten segments; N terminus, equally proportioned segments 1 through 8, and C terminus. The frequency of disordered residues within each segment of each protein was calculated. Disordered residues were defined as those residues predicted to be disordered by DISOPRED2 at a threshold of 5% per residue false discovery rate. The set of frequencies of disordered residues within each location window for proteins annotated by each GO term was then averaged. This resulted in a set of ten average frequencies, one for each location region within each GO category. The average frequencies were Z-score normalised independently within each location window to account for the fact that the false positive rate for prediction of disordered residues is greater at the N and C termini than in the protein interior [10]. A similar approach was adopted to assess correlations between disordered region length in proteins and GO term annotations. Disordered regions in proteins were defined as contiguous stretches of ≥30 residues predicted to be disordered. The average frequency of regions that corresponded to each length range across all proteins annotated by the GO term was then calculated and converted to an independent Z-score for each length range. The Support Vector Machine [47] (SVM) is an efficient classification algorithm suitable for solving binary classification problems in high-dimensional spaces. The algorithm separates positive from negative class data by positioning a linear hyper-plane though the class examples. Often, the input data is not linearly separable, and a kernel function is required to map the data into a higher dimensional space to find the optimal separating hyperplane. The SVM was chosen over other machine learning methods of choice due to its capacity and ability to control error without causing overfitting to the data. The SVMlight [48] SVM package was used to train binary classifiers for individual BP and MF GO terms using the radial basis function kernel. Kernel parameters C and γ were selected by exhaustive grid searches performed on a 272 processor Linux cluster that maximised the MCC for each classifier. MCC was chosen as a more informative measure of classifier performance than percent accuracy or error as it avoids bias resulting from unbalanced class frequencies. For example, each of the five testing sets for “GO: 0045449 regulation of transcription” comprised 356 positive class examples and 1,726 negative class examples. A classification accuracy of more than 82% can be obtained by setting all predicted outcomes to be negative, whereas the MCC balances and controls for the bias in class frequencies. The MCC is similar to the Pearson correlation coefficient where 0 represents random classification and 1 implies perfect classification. Feature selection was carried out using a recursive elimination strategy. Initially each classifier was trained and tested using all feature inputs. Optimisation of C and γ kernel parameters was performed at this stage. A single feature set was iteratively removed from the input data and the performance measured in terms of MCC. Feature attributes that did not contribute to classification performance or indeed caused improvements to performance when removed were permanently eliminated from the input data. When no further improvements were observed, a second round of parameter optimisation was performed on the final feature sets to produce final classification performance statistics. The results from feature elimination can be found in Table S2. The features were divided into global (single values per protein) and spatial (multiple descriptors describing feature location within the protein). Global features comprised amino acid composition, sequence features, signal peptides (SignalP 3. 0 [49]), and localisation information (psortII [50]). The sequence features described general protein characteristics calculated directly from the protein sequence such as molecular weight, average hydrophobicity, iso-electric point, charge, and atom counts. Local features Disorder, PEST [51] (motifs rich in proline, glutamate, serine, and threonine), coiled coils, and low-complexity residues were predicted using DISOPRED2 [52], epestfind, coils [53], and pfilt [54] algorithms with default parameter settings. Transmembrane and secondary structure content was predicted using Memsat3 [55] and PSI-Pred [56] algorithms. Post-translational modification features phosphorylation and glycosylation were predicted by NetPhos3. 0, Net-N-Glyc, and Net-O-Glyc software [57]. A detailed list of descriptors for these features can be found in Table S1. All feature descriptors were scaled to between 0 and 1 before use in classification. Frequency-based descriptors such as the number of transmembrane regions were log-transformed prior to scaling. DISOPRED2 was used to predict disordered residues for the representative protein sequence set using three iterations of PSI-BLAST [58] against the UNIPROT database release 6. 0. Residues were predicted as being disordered at a false positive rate of 5%. Residue predictions were post-filtered for the presence of transmembrane regions predicted using MEMSAT 3. 0 [55] set to default parameters. Predicted disordered regions were further filtered for stretches of at least 30 contiguous residues. A dataset comprising 2,157 orphan and unannotated IPI human proteins was compiled. These proteins contained one or more predicted disordered regions and represent a mixture of proteins that are either members of unannotated families or have no detectable sequence homologues by BLAST similarity searches. To calibrate comparable prediction accuracies between classifiers, the SVM outputs (distances from the separating hyperplane) were converted to posterior probabilities [59]. The probabilities were estimated from the testing datasets so that they reflect the performance of the classifiers on unannotated proteins. The predictions for the unnanotated disordered proteins have been made publicly available at http: //bioinf. cs. ucl. ac. uk/anno/IPI. html.

As a result of high throughput sequencing technologies, there is a growing need to provide fast and accurate computational tools to predict the function of proteins from amino acid sequence. Most methods that attempt to do this rely on transferring function annotations between closely related proteins; however, a large proportion of unannotated proteins are orphans and do not share sufficient similarity to other proteins to be annotated in this way. Methods that target the annotation of these difficult proteins are feature-based methods and utilise relationships between the physical characteristics of proteins and function to make predictions. One important characteristic of proteins that remains unexploited in these feature-based methods is native structural disorder. Disordered regions of proteins are thought to adopt little or no regular structure and have been experimentally linked with the correct functioning of many proteins. Additionally, disordered regions of proteins can be successfully predicted from amino acid sequence. To address the requirement for protein function prediction methods that target the annotation of orphan proteins and explore the use of information describing protein disorder, a machine learning method for predicting protein function from sequence has been implemented. The inclusion of disorder features significantly improves prediction accuracies for many function categories relating to molecular recognition. The practical utility of the method is also demonstrated by providing annotations for a set of orphan and unannotated human proteins.

lay_plos

Background To implement a changing and highly technical tax code, IRS publishes approximately 400 tax forms and accompanying instructions each year. More detailed guidance is provided in approximately 100 different publications. Frequent changes to the tax laws necessitate revisions to both forms and publications. The development and revision of tax forms and their accompanying instructions and publications are the responsibility of IRS’ Tax Forms and Publications Division. This division is made up of two branches, the Forms Branch and the Publications Branch. Tax forms and instructions are written by tax law specialists in the Forms Branch. Proposed new forms are reviewed by IRS’ Tax Forms Coordinating Committee (TFCC), which represents all major organizational units in IRS. TFCC provides a forum for all key IRS functions to help ensure that forms meet the overall needs of the agency without placing excessive burden on taxpayers. All new forms and major revisions to existing forms must be approved by TFCC. IRS’ Chief Counsel must also approve the forms to ensure their technical accuracy and consistency with laws and regulations. The Office of Management and Budget (OMB) is responsible for reviewing each tax form once every 3 years. The purpose of OMB’s review is to assess IRS’ compliance with the Paperwork Reduction Act. Among other things, this law compels OMB to evaluate the extent of burden imposed by IRS forms and to ensure that the federal paperwork burden for individuals is minimized. As a consequence, all new forms and major revisions to existing forms must also be reviewed by OMB. Tax law specialists in the Publications Branch write and revise publications. Some publications are designed to accompany specific forms, while others provide general information relating to a variety of forms. Because publications supplement information on forms and instructions, they are not subject to TFCC review or OMB approval. Publications are also periodically revised to reflect changes to the tax laws or forms. Each year, some publications are substantively revised through an intensive review by a team that includes tax law specialists, writer-editors, and technical reviewers associated with the publication’s subject matter. These publications are subjectively selected on the basis of circulation and the need for text revision. The objective of this revision is to make a major improvement in the quality and usefulness of the publication. In 1978, we issued a report citing the tension between IRS’ conflicting responsibilities to develop forms that are technically accurate, yet clear and readable. In that report, we noted that writer-editors and graphic experts played a limited role in the development of only a few forms and that, as a result, technical accuracy was favored at the expense of clarity. While acknowledging the importance of being accurate, we reported that the readability of these documents needed improvement. Our report recommended that IRS institutionalize a broader mix of writing and graphics design experts in a continuous review of forms and instructions. Since then, IRS has made greater use of its writer-editors and included more graphics in its forms and publications. Objectives, Scope, and Methodology Our objectives were to (1) evaluate IRS’ forms and publications development and revision process, (2) identify IRS’ efforts to improve this process, and (3) identify IRS’ initiatives to increase involvement of taxpayers in this process. To assess IRS’ process for developing and revising forms and publications, we examined the agency’s written procedures and discussed them with representatives from IRS’ Tax Forms and Publications Division, the unit with primary responsibility for issuing these documents. We also selected four forms and two publications and tracked their progress through the development and revision phase. We included forms and publications used by individuals and businesses as well as newly created forms and existing forms undergoing revision. To verify IRS’ written procedures and information obtained from IRS officials, we identified the actual steps taken in producing these documents and compared these activities to established procedures. To evaluate the reasonableness of IRS’ process for developing and revising forms and publications, we considered whether its procedures provided for clear lines of responsibility and accountability, specific timeframes, adequate management oversight, sufficient opportunities to evaluate suggestions from internal and external sources, and appropriate strategies for coping with sudden tax law changes. We also interviewed IRS officials involved in preparing these documents and asked them to discuss the development and revision process. To identify IRS’ efforts to improve the process and to increase taxpayers’ involvement in it, we interviewed IRS officials. In addition, we met with OMB officials to discuss OMB’s role in reviewing tax forms for compliance with the Paperwork Reduction Act. We also met with representatives from external professional organizations, which are concerned with the clarity and accuracy of IRS forms and publications. Our discussions with these groups focused on their perceptions of IRS’ willingness to hear their views and to maintain a professional dialogue on matters affecting IRS forms and publications. Appendix I lists these organizations. We also discussed agency initiatives to improve the clarity of tax forms and publications with various IRS officials involved in managing these activities. We did our work at IRS’ National Office in Washington, D.C., from August 1993 to April 1994 in accordance with generally accepted auditing standards. We presented a draft version of this report to appropriate IRS officials including the Chief of Strategic Planning and Communications and the Director of the Tax Forms and Publications Division and obtained oral comments from them on October 25, 1994. IRS’ comments and our evaluation are on page 11 of this report. IRS Faces Continuous Challenge in Keeping Forms and Publications Accurate and Readable IRS’ forms and publications are based on a complex and frequently changing tax code. Since 1980, Internal Revenue Code sections have been amended more than 100 times, resulting in numerous modifications to existing forms and the development of nearly 100 new ones. This combination presents a great challenge to IRS, which is expected to issue documents that are not only technically accurate but readable. IRS is responsible for responding to changes in the tax law by modifying forms and publications and issuing forms on a timely basis. In addition to revisions caused by changes in tax law, IRS reviews all forms for possible revision on a periodic basis. Many forms, such as the Form 1040, “U.S. Individual Income Tax Return,” are for use during a particular tax year and must be updated annually. Other forms, such as the Form 709, “United States Gift (and Generation Skipping Transfer) Tax Return,” may have an extended period of use. IRS automatically considers all forms for revision every 3 years before sending them to OMB for review under the Paperwork Reduction Act. According to IRS officials, the agency currently publishes approximately 400 forms of which about 80 percent are revised each year. Factors beyond IRS’ control may complicate its ability to provide clear forms and publications. Its role as a data gatherer for research purposes may have a direct bearing on form clarity. Some forms may include lines requesting information unrelated to computation of the tax. This information may be gathered for valid statistical or research purposes, but the information may have no bearing on tax liability. These lines may also be intended to aid an IRS examiner conducting an audit. Although these data collection efforts may bear no relationship to tax liability and may actually increase taxpayer burden, they may also serve valuable purposes justifying their presence. In addition, the varying literacy levels of taxpayers, the many forms and publications and their relationships to each other, and the sheer number of annual changes, even those adopted for the sake of clarity, can further contribute to general confusion among taxpayers. Components of IRS’ Process for Developing and Revising Forms and Publications Appear Reasonable We found that IRS’ process for developing new forms and publications and revising existing ones is composed of reasonable components. For example, its procedures (1) establish well-defined roles and responsibilities for staff, clear lines of accountability, and specific timeframes for drafting these documents and (2) ensure sufficient management oversight. In addition, the procedures provide steps to accommodate the passage of legislation late in the calendar year. IRS officials involved in this process identified no major problems with agency procedures that would hamper their ability to create and revise forms and publications, and the officials were generally satisfied with the procedures. Representatives from the professional organizations we interviewed also were generally satisfied with IRS’ process. Our tracking of four forms and two publications also indicated that IRS’ procedures were followed and proved reasonable. IRS has taken steps to build greater readability into its development and revision process since we issued our prior report in 1978. Greater use is now being made of writer-editors. While writer-editors were rarely used at the time of our previous report, they now play an integral role in simplifying language and assisting in the development of suitable graphic presentations intended to enhance readers’ understanding. As part of its annual revisions process, IRS obtains suggestions for improving both the accuracy and clarity of its forms and publications from a variety of internal and external sources such as organizations representing tax professionals and tax preparers, taxpayers, and employees. All suggestions are to be referred to an appropriate tax law specialist for consideration during a document’s next scheduled revision. IRS has established an ongoing dialogue with some of these organizations and addresses many of their concerns during meetings or through written responses. Some of these organizations, such as the American Institute of Certified Public Accountants, annually submit written suggestions to IRS. Others, such as the National Association of Enrolled Agents, prefer to provide occasional suggestions on an as-needed basis. These groups sometimes hold conflicting opinions, and IRS must balance their views along with many other factors in making revisions. Although no organization has all of its suggestions implemented, most of the ones we spoke with expressed satisfaction with IRS’ accessibility and the process itself. IRS Efforts to Improve Forms and Publications Continue IRS recognizes that more efforts need to be devoted to enhancing readability, and the agency is taking steps consistent with its mission of improving customer service and informing and educating taxpayers. The agency’s new Business Master Plan for 1995 through 2001 has identified maximizing customer satisfaction and reducing taxpayer burden as one of its objectives. One of the steps IRS plans to take to achieve this objective is the establishment of form simplification teams. Comprised of IRS field personnel, these teams will be charged with performing in-depth reviews of selected forms and simplifying at least four each year. This effort is to be modelled after an earlier forms improvement project that IRS officials considered successful. Simplified versions of approximately 25 high-volume forms were drafted by field personnel during that previous project. IRS officials expect that these new teams will meet with similar success. A major effort stems from IRS’ realization that taxpayers have difficulty understanding its forms and publications, even when the reading level of these documents is relatively low. IRS’ Compliance Research Division is studying factors influencing taxpayer’s comprehension of tax documents. IRS has concentrated its efforts on how the following 10 factors affect reader comprehension: readability, if-then statements, references to other documents, tax vocabulary, abbreviations and acronyms, arithmetic complexity, headings, text, negative terminology, and graphic usage. On the basis of these factors, selected passages of the Form 1040 and Form 1040EZ instructions were rewritten and tested to see if comprehension improved. IRS is now validating its methodology for this study. Ultimately, IRS hopes to improve taxpayer comprehension by using the study’s findings to develop training for staff involved in writing forms and publications. Another development that may improve the quality of the forms and publications is the possible reorganization of the Tax Forms and Publications Division. Currently under consideration, this reorganization would result in tax law specialists’ becoming responsible for preparing related forms and publications. Presently, Forms Branch staff are not involved in the development of publications. Similarly, Publications Branch staff do not prepare forms. According to IRS officials, if implemented, the reorganization would merge staff into two new branches, one addressing individual tax issues and the other addressing business tax issues. Tax law specialists would be assigned one or more forms and accompanying publications. IRS is considering whether such a reorganization would make forms and publications more readable for taxpayers by improving staff expertise and achieving greater consistency in language between the forms and publications among other things. According to IRS officials, the decision on this reorganization has been postponed pending completion of IRS’ broader internal review of its processes. IRS Needs Data to Better Identify Concerns of Individual Taxpayers While IRS gets information on the clarity of forms and publications for individual taxpayers from several sources, IRS officials acknowledge that there is not a systematic way for obtaining input from the many individual taxpayers not represented by particular interest groups or associations. These officials told us that IRS has not met with the same success in obtaining the views of individual taxpayers as it has attained among the business community and organizations of tax professionals, but the agency has several efforts underway that may help to improve this situation. To obtain the views of individual taxpayers on such matters as the clarity of forms and publications, IRS solicits written comments from taxpayers, holds periodic town meetings where taxpayers can discuss their concerns, distributes customer satisfaction surveys, and conducts focus group sessions on selected forms revisions. However, IRS officials said that these sources generally do not yield many substantive insights as to what specifically confuses individual taxpayers. IRS officials have told us that the concerns expressed by individuals in these forums have not presented a precise view as to what individuals find wrong with the forms. According to these officials, they also do not generally result in useful suggestions for revising the forms. However, the officials said that while focus groups generally provide better results, the groups are costly to conduct. Because of this cost factor, only a limited number of focus groups are held. Also, focus groups do not cover a broad base of taxpayers and only address one or two forms each year. Nonetheless, officials told us that IRS hopes to find new ways of identifying the concerns of individuals. In an effort to better capture these concerns, the Publications Branch will resume its annual interviews with the agency’s telephone assistors, who respond to questions from taxpayers on the toll-free telephone system. Conducted at the end of the filing season to discuss commonly asked questions and points of confusion, these interviews were previously used to identify and clarify passages in forms and publications for future revisions. Publications Branch staff claim that these interviews yielded hundreds of specific suggestions that were ultimately adopted. These interviews were discontinued several years ago due to budgetary constraints. IRS recently decided to resume these interviews in the spring of 1995, after the next filing season. This approach may be one way to tap into existing sources of information. IRS plans to introduce an additional new feature to its toll-free telephone system in January 1995. A new line will be dedicated to taxpayers who wish to leave recorded messages with the agency. Also, the annual “Message from the Commissioner,” which will be included in the 1994 tax packages, will invite taxpayers to call IRS with their ideas for making the forms simpler. IRS hopes these actions will make it easier for taxpayers to comment on forms and publications and will generate more suggestions. IRS may already possess critical information that could provide insights into what areas taxpayers are specifically having difficulty in understanding. For example, IRS’ existing toll-free telephone system may be a source of information as to what taxpayers find confusing. Tracking specific questions at routine intervals may identify specific sections of forms and publications that taxpayers have difficulty understanding. IRS does limited monitoring of the nature of these calls but information captured is too broad to provide specific guidance for form revision. At least one state relies on call-tracking as an indicator that certain passages in its state tax forms are confusing and uses this information to make appropriate changes to these documents. Taxpayers could also benefit from improved use of observations from IRS employees. One potential source of information may be errors made by taxpayers and discovered by IRS during audits. While some errors are inevitable and others may be indicators of intentional noncompliance, some frequently made errors may point to ambiguities in forms and publications, leaving the taxpayer open to an honest mistake. Another opportunity to simplify forms and publications frequently used by individual taxpayers may reside in the agency’s annual process for proposing legislative changes to Congress. Each year the Department of the Treasury asks IRS to offer suggestions to improve tax administration that need legislative approval to be implemented. The Treasury Department’s ultimate objective is to provide Congress with a list of such proposals for its consideration. Simplification of tax forms is one area of tax administration that generates suggestions requiring legislative action. Tax law specialists told us that some forms contain line items or instructions required by law but no longer serving a useful purpose. Removing such line items and instructions could result in some simplified forms, but only could be accomplished by an act of Congress. However, this process has been perceived by some staff in various units, including the Tax Forms and Publications Division, as not productive. As suggestions are forwarded to officials at increasingly higher levels within IRS, then the Treasury Department, and finally at OMB (which must approve the document before it is submitted to Congress), suggested proposals are eliminated through this process. In recent years, few, if any, suggestions have been forwarded to Congress. IRS managers are presently studying ways of revising the process to make it more useful to the Treasury Department and Congress. If improved, this process may encourage greater staff participation and provide a vehicle for conveying simplification ideas, among others, to Congress. Conclusion Although it faces many challenges in developing and revising forms and publications, IRS has instituted a process for doing so that includes reasonable components and is seeking opportunities for improvement. While IRS has established a dialogue with organizations representing tax professionals, it has had difficulty in identifying and responding to the needs of individual taxpayers who are not represented by any particular organization. Because clear and understandable forms and publications help to promote voluntary compliance, readability is a continuous concern. Thus, IRS should continue to seek new ways of identifying what individual taxpayers find most difficult to understand. IRS should also explore the use of potentially helpful, but untapped, sources of information that may reveal points of taxpayer confusion and use existing information in new ways. Recommendation to the Commissioner of Internal Revenue We recommend that the Commissioner of Internal Revenue direct agency staff to make additional efforts to identify the specific concerns of individual taxpayers. Identifying these concerns may be accomplished in a variety of ways, including gathering information concerning the nature of taxpayer questions received through its toll-free telephone system and soliciting information from IRS field personnel including auditors, examiners, and customer-service representatives for the sole purpose of identifying common errors made by taxpayers that may be related to confusing passages in forms and publications. Agency Comments and Our Evaluation We provided a draft copy of this report to, and obtained oral comments from, appropriate IRS officials, including the Chief of Strategic Planning and Communications and the Director of the Tax Forms and Publications Division. These officials suggested several technical modifications that we incorporated in our final report. While they generally agreed with the facts contained in our report and the importance of identifying the specific needs of individual taxpayers, IRS officials did not think the recommendation we proposed was necessary. The officials stated that IRS is continuously seeking the views of individual taxpayers, receiving many employee suggestions to clarify forms and publications, and using available data to improve these documents. The officials noted that IRS has additional plans to enhance customer satisfaction with regard to forms and publications. For example, in response to a 1993 customer satisfaction survey, IRS intends to develop a learning, business, and communication strategy for making forms and instructions more readable. While acknowledging that improvement is always possible, the officials stated that its many current and planned efforts will meet taxpayer needs. We agree that IRS has made efforts to improve its forms and publications. For example, IRS’ efforts to identify factors influencing taxpayers’ comprehension of tax documents was a positive step. We also consider its future plans to dedicate a telephone line to messages from taxpayers and to resume interviewing telephone assistors to be positive steps. However, we continue to support the need for IRS to obtain more specific information about what individual taxpayers find most confusing about forms and publications. We believe that implementing our recommendation will further clarify forms and publications, thus benefitting individual taxpayers. We are sending copies of this report to other congressional committees, the Secretary of the Treasury, the Commissioner of Internal Revenue, and other interested parties. Major contributors to this report are listed in appendix II. If you or your staff have any questions concerning the report, please call me on (202) 512-9110. Organizations Contacted American Institute of Certified Public Accountants, Washington, D.C. American Payroll Association, New York American Society of Payroll Management, New York National Association of Enrolled Agents, Rockville, MD National Taxpayers Union, Washington, D.C. Tax Executives Institute, Washington, D.C. Major Contributors to This Report General Government Division, Washington, D.C. Chicago Regional Office Linda Schmeer, Evaluator Donald R. White, Evaluator The first copy of each GAO report and testimony is free. Additional copies are $2 each. Orders should be sent to the following address, accompanied by a check or money order made out to the Superintendent of Documents, when necessary. Orders for 100 or more copies to be mailed to a single address are discounted 25 percent. U.S. General Accounting Office P.O. Box 6015 Gaithersburg, MD 20884-6015 Room 1100 700 4th St. NW (corner of 4th and G Sts. NW) U.S. General Accounting Office Washington, DC Orders may also be placed by calling (202) 512-6000 or by using fax number (301) 258-4066, or TDD (301) 413-0006. Each day, GAO issues a list of newly available reports and testimony. To receive facsimile copies of the daily list or any list from the past 30 days, please call (301) 258-4097 using a touchtone phone. A recorded menu will provide information on how to obtain these lists.

Pursuant to a congressional request, GAO provided information on the accuracy and clarity of the Internal Revenue Service's (IRS) forms and publications, focusing on: (1) the adequacy of the process IRS uses to revise its tax forms; (2) IRS efforts to improve this process; and (3) IRS efforts to increase taxpayer involvement in the process. GAO found that: (1) IRS efforts to provide taxpayers with accurate and easy-to-read tax forms have been hampered by increasingly complex tax codes, frequent tax code revisions, and taxpayers' reading ability; (2) the IRS process for developing and revising its tax forms and publications appears reasonable and provides clear lines of responsibility and accountability, specific timeframes, adequate management oversight, sufficient opportunities to evaluate suggestions from internal and external sources, and appropriate strategies for coping with sudden tax law changes; (3) IRS periodically reviews and revises its publications and tax forms so that accurate forms and publications are available to taxpayers for each filing season; (4) IRS considers comments from taxpayers and tax preparers, payroll professionals, accountants, and lawyers regarding clarity improvements; (5) IRS has initiated several special projects to study taxpayer comprehension problems and to further improve its forms and publications; (6) despite its ongoing commitment to improvement, IRS does not have a systematic way to identify the specific areas that cause individual taxpayer confusion; and (7) IRS needs to find ways to readily identify the specific concerns of individual taxpayers.

gov_report

Background The Forest Service is responsible for managing over 192 million acres of public lands—nearly 9 percent of the nation’s total surface area and about 30 percent of all federal lands in the United States. In carrying out its responsibilities, the Forest Service traditionally has administered its programs through nine regional offices, 155 national forests, 20 grasslands, and over 600 ranger districts (each forest has several districts). The Forest Service’s implementation, management, and oversight of fuel reduction activities tend to be decentralized and vary by region, although all activities must be carried out under applicable laws. Figure 1 shows a map of the national forests and Forest Service regions. Forest Service projects intended to reduce fuels and restore or maintain desired vegetation conditions generally use prescribed burning, in which fires are deliberately set by land managers, and/or mechanical treatments, in which equipment such as chain saws, chippers, bulldozers, or mowers is used to cut vegetation. Such mechanical treatment may include logging to remove commercial timber. Other approaches include applying chemical herbicides, using grazing animals such as cattle and goats, and allowing the public to remove firewood by hand. To carry out its fuel reduction work, the Forest Service may use agency staff but more commonly contracts it out. The agency generally uses three types of contracts— timber sale contracts, service contracts, and stewardship contracts—to accomplish fuel reduction work. Timber sale contracts are awarded to individuals or companies to harvest and remove trees from federal lands under its jurisdiction. Service contracts are awarded to contractors to perform specific tasks, such as thinning trees or clearing underbrush. Stewardship contracts are generally awarded to contractors who perform both timber harvesting and service activities, and include contracts under which the agency uses the value of commercial products, such as timber, to offset the cost of services received, such as thinning, stream improvement, and other activities. Controversy has surrounded the issue of fuel reduction for some time, particularly in areas where federal lands surround or are adjacent to human development and communities—the wildland-urban interface—and in inventoried roadless areas. Roadless areas have received special attention for decades, as some argue that these areas should be available for appropriate development and timber harvesting, while others believe that the areas should remain roadless to preserve the special values that their condition provides, such as clean water and undeveloped wildlife habitats. Forest Service hazardous fuel reduction activities are typically subject to one of two different internal administrative review processes, each of which has a specific procedure through which the public can challenge the agency’s decisions or proposed decisions to conduct the activities. Specifically: Postdecisional administrative appeals process. The Forest Service has provided an administrative appeals system for review of agency decisions, under certain circumstances, for over 100 years. Although the specific requirements of the appeals system have changed over the years, the Appeals Reform Act of 1993 established the appeals process pertinent to fiscal years 2006 through 2008––the time period covered by our review. When the Forest Service issues a public notice in a newspaper of record of a proposed action, the public has either 30 or 45 days to comment, depending on the type of NEPA analysis document prepared. Once the agency issues a decision, the public has 45 days to file appeals; however, only those individuals who were involved in the public comment process through submission of written or oral comments or by otherwise notifying the Forest Service of their interest in the proposed action may file an appeal. Once the 45-day time frame for filing appeals has expired, the Forest Service must review all appeals and issue a response to each within an additional 45 days. Appeals can result in decisions being affirmed, in which case the Forest Service can proceed with the project as planned, or in decisions being reversed in whole or in part, in which case the agency may revise or even cancel the affected activities. The official (known as the Appeal Deciding Officer) who determines the outcome of the appeal must be, at least, the next higher level supervisor of the individual who made the original decision. There is no further administrative review of the Appeal Deciding Officer’s decision by any other Forest Service or Department of Agriculture official. The types of decisions that can be appealed have changed since GAO last reported on this issue in 2003. In 2003, the Forest Service added several new categorical exclusions related to vegetation management (including one specific to hazardous fuel reduction) that it exempted from appeal. However, as the result of subsequent litigation challenging these exemptions, the Forest Service ultimately was required to allow the public to appeal many (though not all) of these decisions during fiscal years 2006 through 2008, the time period covered by our current review. Predecisional administrative objection process. In 2003, HFRA required the Forest Service to establish an alternative process for authorizing certain hazardous fuel reduction projects, including an alternative predecisional objection process in lieu of the appeals process for certain projects. HFRA authorizes the public to file objections to a proposed project before the agency issues a final decision on the project, instead of the traditional appeals process where the administrative review occurs after the agency’s final decision has been made. According to the Forest Service, this objection process was intended to expedite the implementation of fuel reduction projects and to encourage early public input during the planning process. Only those parties who have previously submitted written comments specific to the proposed project may file objections. (The public has an opportunity to provide these written comments during scoping or other public comment periods.) The public must file objections with the reviewing officer—the next higher level supervisor of the person responsible for the proposed action—within 30 days following the publication date of the legal notice of the proposed environmental assessment or environmental impact statement. (Decisions that are subject to objection cannot use categorical exclusions as the basis for the decision.) If no objection is filed within the 30-day time period, the decision may be finalized on, but not before, the fifth business day following the end of the objection-filing period. If an objection is filed, the Forest Service must issue a written response to the objector addressing the objection within 30 days following the end of the objection-filing period. The reviewing officer may hold a meeting to discuss issues raised in the objection and any potential resolution. There are several ways the Forest Service addresses an objection. The objection can (1) be set aside from review, (2) be reviewed by the Forest Service resulting in a change to the final decision, (3) be reviewed by the Forest Service resulting in no change to the final decision, or (4) result in the reviewing officer directing the appropriate Forest Service official to complete additional analysis prior to issuing a final decision. An objection may be set aside from review for procedural reasons—if, for example, the objection is not received within the allowed 30-day time period, or the objecting individual or organization did not submit written comments during scoping or other public comment opportunities. There is no further administrative review by any other Forest Service or Department of Agriculture official of the reviewing officer’s written response to an objection. Table 1 compares the appeals and objection processes. Some decisions, however, were subject to neither the appeal nor the objection process during the time of our review. As noted, the Forest Service was required to allow appeals of many fuel reduction decisions based on categorical exclusions, but was not required to allow appeals on all such decisions—meaning that certain decisions based on categorical exclusions remained exempt from appeal. These decisions were also exempt from the objection process because HFRA requires that fuel reduction decisions subject to objection use environmental assessments or environmental impact statements rather than categorical exclusions. Number and Type of Decisions Involving Hazardous Fuel Reduction Activities in Fiscal Years 2006 through 2008 and the Associated Acreage For fiscal years 2006 through 2008, national forest managers reported 1,415 decisions involving hazardous fuel reduction activities, affecting 10.5 million acres of national forest land. Most of these decisions were based on categorical exclusions, although decisions based on environmental assessments represented the most acreage of all decision types. Table 2 shows the number of decisions and associated acreage, by decision type. Appendix II provides greater detail on the number of decisions and associated acreage for each Forest Service region. Number of Decisions Involving Hazardous Fuel Reduction Activities That Were Subject to Appeal or Objection; Number of Decisions Appealed, Objected to, or Litigated; and Associated Acreage Of the 1,415 decisions in our review, 1,191—about 84 percent—were subject to the appeals process. In contrast, only 121 decisions—8.5 percent—were subject to the objection process. However, the rate at which decisions subject to the objection process were challenged was higher than for decisions under the appeals process. Specifically, 40 percent of decisions subject to objection were objected to, compared with the 18 percent appeal rate for decisions subject to appeal. Table 3 shows, for all decisions covered by our review for fiscal years 2006 through 2008, the number of appeals, objections, and litigation associated with each decision type. Appendix III provides greater detail on the number of appeals, objections, and litigation for each Forest Service region. In addition to the introduction of the objection process, our survey data reflect two important changes that have occurred since our 2003 report: (1) the extent to which activities associated with categorical exclusions are subject to the appeals process and (2) the decrease in the use of the categorical exclusion for hazardous fuel reduction to authorize hazardous fuel reduction activities. Specifically: Extent to which categorical exclusions were subject to appeal. At the time of our 2003 report, decisions using categorical exclusions were generally not subject to appeal, and in that report we noted that 99 percent of fuel reduction decisions using categorical exclusions in fiscal years 2001 and 2002 were exempt from appeal. Also in 2003, the Forest Service introduced several new categorical exclusions that were exempt from appeal, including one categorical exclusion specific to fuel reduction activities. Beginning later that year, however, the agency’s ability to exempt decisions using categorical exclusions from appeal was challenged in court. As a result of this litigation, the Forest Service was required to allow the public to appeal decisions containing any of 11 types of categorically excluded activities, including fuel reduction—and thus, most fuel reduction decisions in our survey that were made using categorical exclusions were appealable by the public. Specifically, 89 percent of the categorical exclusions identified in our survey were subject to appeal in fiscal years 2006 through 2008, in contrast to the 1 percent that were subject to appeal during our 2003 review. The remaining 11 percent of categorical exclusions in our current review—a total of 103 decisions— were identified by survey respondents as exempt from appeal because they did not contain the activities covered by the litigation. Subsequently, in 2009 the U.S. Supreme Court overturned the lower court’s ruling on procedural grounds, allowing the Forest Service to utilize the provisions of its regulations that exempt categorically excluded decisions from appeal. Appendix X contains data on the type and frequency of the categorical exclusions represented in our survey. Decrease in the use of the categorical exclusion for hazardous fuel reduction. Although Forest Service regulations contain a specific categorical exclusion under which hazardous fuel reduction activities can be authorized, this was not the most commonly reported categorical exclusion in our survey of decisions involving hazardous fuel reduction activities. Instead, the most commonly reported categorical exclusion was one intended for timber stand and/or wildlife habitat improvement. Our survey data show that the total number of decisions authorized under the categorical exclusion for hazardous fuel reduction decreased greatly over the period covered by our survey, while at the same time, the use of the categorical exclusion for timber stand and/or wildlife habitat improvement increased. Specifically, use of the categorical exclusion for hazardous fuel reduction decreased from 214 in fiscal year 2006 to 28 in fiscal year 2008, while the use of the categorical exclusion for timber stand and/or wildlife habitat improvement increased from 145 in fiscal year 2006 to 167 in fiscal year 2008. This decrease in the use of the categorical exclusion for hazardous fuel reduction may have resulted in large part from the chief of the Forest Service’s response to a court order in 2007. In this response the chief directed that no new decisions should be made under the categorical exclusion for hazardous fuel reduction after December 2007. Furthermore, he directed that no additional contracts be initiated to implement projects authorized under this authority—meaning that projects that were not under way did not start, even if a final decision had already been issued. Under the chief’s direction, projects that were near completion could proceed. Of the 379 decisions in our survey originally authorized under the categorical exclusion for hazardous fuel reduction, respondents reported that 207—or about 55 percent—were affected by the chief’s directive. Although we did not systematically gather information on what happened to projects subject to the court decision, respondents indicated that they took a variety of approaches, including the following: using a different categorical exclusion, such as the categorical exclusion for timber stand and/or wildlife habitat improvement, to authorize the project; preparing an environmental assessment subject to the appeals process; stopping or slowing project implementation; and preparing an environmental assessment subject to the predecisional objection process, under HFRA. Additionally, the rate at which decisions were litigated was about the same—2 percent—for decisions that were subject to the Forest Service’s traditional appeals process as for decisions authorized under HFRA—even though the agency’s expectation was that HFRA would reduce the likelihood of litigation. Of the 29 litigated decisions in our study, 26 had been subject to appeal, representing 2 percent of the 1,191 decisions subject to appeal; the remaining 3 litigated decisions had been subject to objection, likewise representing 2 percent of the 121 decisions subject to objection. Outcomes of Appeals, Objections, and Litigation of Decisions with Hazardous Fuel Reduction Activities; Associated Time Frames; and Identities of Appellants, Objectors, and Plaintiffs In fiscal years 2006 through 2008, of the 298 appeals filed, the Forest Service upheld its earlier decision in the majority of the cases without requiring any changes to the decision. Of the 101 objections submitted, the outcome was more evenly divided between those objections resulting in a change to the decision and those that did not. According to time frame information provided by survey respondents, all appeals and objections were processed within the prescribed time frames. For litigated decisions resolved at the time of our review, the Forest Service prevailed slightly more often than the plaintiffs. Outcomes of Appeals, Time Frames, and Identities of Appellants Of the 298 appeals filed on appealable decisions from fiscal years 2006 through 2008, For 160 appeals, the decisions were affirmed—that is, allowed to proceed—with no changes. For 22 appeals, the decisions were affirmed with specified changes. For 24 appeals, the decisions were reversed —that is, not allowed to proceed—based on issues raised by the appellants. A total of 91 appeals were dismissed for various reasons, including 38 appeals that were resolved informally, of which 30 appeals were withdrawn by the appellant and 8 decisions were withdrawn by the agency (when an appeal is resolved informally, changes may or may not be made to the decision); 53 appeals that were dismissed without review, mostly for failing to meet procedural requirements, such as timeliness—however, 23 of these appeals were dismissed without review because, subsequent to receiving the appeal, the agency official who made the decision decided to withdraw the decision; For 1 appeal, the outcome could not be determined based on documentation in the agency’s regional files, according to an agency official. According to time frame information provided by Forest Service officials, all appeals of fiscal year 2006 through 2008 decisions were processed within the time frames prescribed in applicable laws and regulations. See appendix IV for detailed information on appeal outcomes for each Forest Service region. The 298 appeals were filed by 217 appellants. This total includes appeals by 88 different interest groups, mostly environmental groups, and 129 individuals. Of the 88 interest groups, 10—Alliance for the Wild Rockies, Biodiversity Conservation Alliance, John Muir Project of the Earth Island Institute, Native Ecosystems Council, Oregon Wild, Ouachita Watch League, Sierra Club, The Lands Council, Utah Environmental Congress, and the WildWest Institute—each appealed 10 or more decisions. Appendix VI lists each interest group that appeared as an appellant in fiscal years 2006 through 2008 and the number of decisions for which each appellant filed appeals in each region. To protect the privacy of individual appellants, we do not list their names, but in appendix VI we provide information on the number of decisions appealed by individuals in each region. Outcomes of Objections, Time Frames, and Identities of Objectors Of the 101 objections filed for 49 decisions from fiscal years 2006 through 2008, 38 objections resulted in no change to the final decision. 31 objections resulted in a change to the final decision. 4 objections resulted in the Forest Service having to conduct additional analysis. 15 objections were set aside from review. 13 objections were addressed some other way; for example, several agency respondents explained that they addressed objector’s concerns by both agreeing to make a change to the final decision and by setting the objection aside from review. Rather than setting it aside from review for procedural reasons, however, the decisions were set aside because the objector withdrew the objection after the Forest Service agreed to make changes to the final decisions. For objections that the Forest Service does not set aside, the Forest Service reviewing officer is required to respond in writing. Prior to issuing a written response, the objector or reviewing officer may request a meeting to discuss the issues that were raised in the objection and a possible resolution. According to some Forest Service officials we spoke with, these meetings have been used to further satisfy public concerns; however, because meetings are at the discretion of the reviewer, objectors with whom the reviewer decides not to meet may feel that their concerns were not adequately addressed, regardless of the outcome. For example, the Forest Service received 22 objections to the Middle East Fork Hazardous Fuel Reduction decision on the Bitterroot National Forest in west central Montana and east central Idaho, one of the first and, according to Forest Service officials, most contentious decisions authorized under HFRA authority in the Northern Region. One objector requested a meeting with the Forest Service and others expressed a willingness to meet, but the reviewing officer chose not to hold meetings, stating that their objections did not require additional clarification and that a private consultant with whom the forest contracted had determined that additional discussions would not resolve the objector’s concerns. The decision was ultimately litigated. In other cases, however, respondents reported that such meetings successfully addressed objectors’ concerns, sometimes resulting in objectors withdrawing their objections. However, we also determined that different regions follow different approaches in addressing objectors’ concerns. For example, an official in the Pacific Southwest Region told us that officials generally meet with the objectors associated with valid objections (those that are not set aside for procedural reasons), with the goal of informally resolving the objections and having them subsequently withdrawn by the objectors. In contrast, an official in the Northern Region told us that while the region seeks to resolve objections informally, unlike the Pacific Southwest Region, it does not seek to have objectors subsequently withdraw their objections, and none have done so. Seeking to have objectors withdraw their objections, as the Pacific Southwest Region has done, may have important implications for subsequent litigation because, according to Forest Service officials, under HFRA and its implementing regulations, an objector that withdraws an objection has no standing to obtain judicial review of the Forest Service’s final decision. According to time frame information provided by survey respondents, the final decisions for all proposals subject to the objection process from fiscal year 2006 through 2008 were signed in accordance with the time frames set forth by applicable laws and regulations. However, while officials are required to respond to objections within certain time frames, there is no limitation on the amount of time allowed to make a final decision. Of the 49 decisions for which objections were filed, 25 were signed between 35 days and 3 months of legal publication date of the proposed action. The remaining 24 were signed more than 3 months after the legal publication date, including 3 cases in which the final decision was signed more than a year after the legal publication date of the proposed action. The 101 objections were filed by 37 organizations and 41 individuals. Of the 37 organizations, 3—the Center for Biological Diversity, the Idaho Conservation League, and the WildWest Institute— each objected to 5 or more decisions. Appendix VI lists each group that filed objections in fiscal years 2006 through 2008 and the number of decisions for which objections were filed in each region. As with appeals, in appendix VI we do not list the names of individual objectors, but do show the number of proposed decisions objected to by individuals in each region. Outcomes of Litigation and Identities of Plaintiffs Of the 29 decisions that were litigated from fiscal years 2006 through 2008, we are able to report the outcome for 21 of the lawsuits because they had been resolved at the time of our review. According to regional officials, lawsuits for 3 of these 21 decisions were dismissed because the plaintiffs and the Forest Service agreed to settle their claims. District courts reached an outcome on the 18 additional decisions, with 8 decided favorably to the plaintiffs and 10 decided favorably to the Forest Service. Lawsuits on the remaining 8 decisions were continuing at the time of our review. In the 29 litigated decisions, 24 interest groups and 11 individuals were plaintiffs. The interest groups were primarily environmental groups, with three groups—Alliance for the Wild Rockies, Native Ecosystems Council, and the WildWest Institute—each acting as plaintiff in 5 or more decisions. Of the 29 litigated decisions, plaintiff groups and individuals had previously submitted appeals on 24 of the decisions and objections on 3 of the decisions during the administrative process. The remaining 2 litigated decisions were subject to appeal, but the plaintiffs did not submit an appeal during the administrative process. Appendix VI lists each group that acted as a plaintiff in fiscal years 2006 through 2008 and the number of decisions for which lawsuits were filed by each group within each Forest Service region. To protect the privacy of individual plaintiffs, we do not list their names, but in appendix VI provide information on the number of decisions litigated by individuals in each region. Treatment Methods and Contract Types Associated with Fuel Reduction Decisions; the Number of Appeals, Objections, and Lawsuits by Treatment Method and Contract Type; and the Associated Acreage Prescribed burning was the most frequently used treatment method associated with the fuel reduction decisions included in our study, followed by mechanical treatment and commercial logging. Of these three methods, prescribed burning was the method most often challenged through appeals and objections; however, commercial logging was challenged at the highest rate, considering both appeals and objections. Table 4 shows, for all treatment methods in our study, the number and percentage of, and acreage associated with, appeals, objections, and litigation. Appendix VII provides additional information on fuel reduction methods used and the number of appeals, objections and lawsuits by treatment method, for each Forest Service region. Commercial timber sale contracts were the most frequent contract type used to implement the decisions included in our study, and were the type most often challenged through appeals and objections. Decisions using stewardship contracting, however, were challenged at a higher rate than the other contract types, considering both appeals and objections. Table 5 shows, for all the decisions included in our study, the number and percentage of contract types, and acreage associated with, appeals, objections, and litigation. Appendix VIII provides additional information on the contracting methods used for decisions included in our study and the appeal, objection, and litigation rates for each Forest Service region. Decisions Involving Fuel Reduction Activities in the Wildland-Urban Interface and Inventoried Roadless Areas; the Number of Appeals, Objections, and Lawsuits on Those Decisions; and the Associated Acreage Of the 1,415 decisions in our review, respondents identified 954 decisions that included activities in the wildland-urban interface and 169 decisions that included activities in inventoried roadless areas. Both types of decision were appealed at about the same rate, while decisions involving inventoried roadless areas were objected to at a slightly higher rate than those involving the wildland-urban interface. Table 6 shows, for both wildland-urban interface and inventoried roadless areas, the number and percentage of, and acreage associated with, appeals, objections, and litigation. Regarding fuel reduction activities in inventoried roadless areas, the majority of decisions in our study involved no road construction in the roadless area––which is a primary concern related to hazardous fuel reduction activities in roadless areas. About 10 percent included temporary road construction or other road construction activity, with one decision involving the construction of a permanent road in an inventoried roadless area. Appendix IX provides information on the number of decisions with fuel reduction activities in inventoried roadless areas and the number of appeals, objections, and lawsuits for such decisions in each Forest Service region. Concluding Observations Much has changed since we last reported on appeals and litigation of fuel reduction activities 7 years ago. One of the most significant changes to the process has been the passage of HFRA, which has provided a new approach for public challenges of fuel reduction projects by allowing the opportunity to formally object to decisions before they become final, rather than waiting to file appeals until after the decisions are made. Although the passage of HFRA was seen as an important new tool for streamlining fuel reduction decisions, our review indicates that the impact of the act appears to be limited. Most notably, fuel reduction decisions that used HFRA authority represented less than 10 percent of decisions signed during fiscal years 2006 through 2008. As a result, despite the opportunities HFRA introduced for a new approach to the administrative review process, in practice most decisions remained subject to the Forest Service’s traditional postdecisional appeals process. In addition, although the agency’s expectation was that HFRA would reduce litigation of fuel reduction decisions, our review shows that HFRA and non-HFRA decisions were litigated at about the same rate of 2 percent. Another area of ongoing change is the dispute over the Forest Service’s ability to exempt categorically excluded decisions from appeal. Although most of these decisions were subject to appeal during the years we examined, the Supreme Court’s 2009 ruling means that the regulation exempting categorically excluded decisions from appeal is once again in effect. However, two factors suggest ongoing uncertainty about this issue. First, the Supreme Court’s ruling was made on procedural grounds rather than on the merits of the case—meaning that the court did not rule on whether the regulation is consistent with the Appeals Reform Act, allowing for the possibility of future challenges to the regulation. Second, even though the regulation survived the recent lawsuit, the Forest Service is considering changes to it in light of, among other things, the litigation it has engendered. Thus, the ultimate fate of the regulation—and the public’s ability to appeal categorically excluded decisions—remains uncertain. Agency Comments and Our Evaluation We provided a draft of this report to the Forest Service for comment. The Forest Service did not provide comments, although it did provide technical corrections which we incorporated as appropriate. We are sending copies of this report to the Secretary of Agriculture; the Chief of the Forest Service; appropriate congressional committees; and other interested parties. The report also will be available at no charge on the GAO Web site at http://www.gao.gov. If you or your staff members have questions about this report, please contact me at (202) 512-3841 or mittala@gao.gov. Contact points for our Offices of Congressional Relations and Public Affairs may be found on the last page of this report. Key contributors to this report are listed in appendix XIII. Appendix I: Objectives, Scope, and Methodology We examined (1) the number and type of Forest Service decisions involving hazardous fuel reduction activities signed in fiscal years 2006 through 2008; (2) the number of these decisions that were objected to, appealed, or litigated, and the acreage associated with those decisions; (3) the outcomes of these objections, appeals, and lawsuits, including whether they were processed within prescribed time frames, and the identities of the objectors, appellants, and plaintiffs; (4) the treatment methods and contract types associated with fuel reduction decisions, and how frequently the different methods and types were objected to, appealed, and litigated; and (5) the number of decisions involving hazardous fuel reduction activities in the wildland-urban interface (WUI) and inventoried roadless areas (IRA), and how frequently these decisions were objected to, appealed, and litigated. To address our objectives, we implemented a nationwide, Web-based survey of Forest Service officials, to collect information about all fuel reduction decisions signed in fiscal years 2006 through 2008 (See appendix XII for a copy of the survey). We supplemented the survey with a semistructured interview of officials in all nine Forest Service regions to gather additional details about time frames, outcomes and identities related to appeals and litigation of fuel reduction decisions. Details about this process are described below. To identify Forest Service decisions involving hazardous fuel reduction activities signed in fiscal years 2006 through 2008, we asked the agency’s Ecosystem Management Coordinator to query a Forest Service database designed to track decision planning, appeals, and litigation for all Forest Service decisions—-the Planning, Appeals, and Litigation System (PALS). This official queried the PALS database using the following criteria: (1) decisions signed in fiscal years 2006 through 2008, and (2) decisions that included fuels management as a purpose and/or one or more fuel treatment activities. This initial query identified 1,437 decisions in 108 national forest system units. Because PALS was not designed to include all information we sought as part of our review—including information on the number of acres treated, treatment methods and contract types used, and decisions involving activities in the wildland-urban interface or in inventoried roadless areas— we determined that a nationwide survey would be necessary. We began our survey effort by ensuring that we had identified the correct universe of fuel reduction decisions. After reviewing the list of fuel reduction decisions from PALS and correcting for any obvious duplication and other errors, we sent a list of each national forest’s fuel reduction decisions to the corresponding forest supervisor’s office. We asked the supervisor or cognizant official to verify the accuracy of our list, removing any decisions that did not meet our criteria (i.e, that were not signed in fiscal years 2006 through 2008, or that did not involve any hazardous fuel reduction activities), and adding decisions that met our criteria but did not appear in PALS. At this time, we also asked the supervisor or cognizant official to identify Forest Service employees most knowledgeable about these decisions. A total of 1,415 decisions, issued by 108 national forests, were determined to fit our criteria. We gave recipients 3 weeks to respond to our request for information and granted extensions as needed. We obtained a 100 percent response rate from the national forests. To determine the characteristics of each fuel reduction decision, we subsequently administered a Web-based survey to those Forest Service employees identified by each forest supervisor or cognizant official as most knowledgeable about the decisions at all 108 national forests that issued decisions with hazardous fuel reduction activities in fiscal years 2006 through 2008. Appendix XII contains a copy of the survey used to gather these data. The survey asked respondents to provide information about each of the decisions, including the type of environmental analysis used, acres involved, treatment methods and contract types used, the extent to which the decisions included activities in the wildland-urban interface and inventoried roadless areas, and detailed information about the outcomes of those decisions subject to the predecisional objection process. The Forest Service does not have a uniform definition of a hazardous fuel reduction activity, a fact that could affect the information that forest managers reported to us. Many activities have the practical effect of reducing fuels, but their stated purpose may be for something other than, or in addition to, fuel reduction. For example, the cutting and gathering of firewood or forest products to provide a product to the public may have the additional benefit of reducing hazardous fuels. Some forest managers may have included such projects among the decisions they reported in their responses to our survey, while other forest managers with similar decisions may not have included them. Similarly, there are a number of limitations to the acreage data. The data reported by forest managers include a mixture of planned, estimated, and actual treatment acres for decisions included in our review. In our survey, we did not limit responses to acres actually treated because once a decision is made and documented, there are many reasons that activities covered by the decision may be delayed or not implemented, including availability of funding or personnel, weather conditions, and administrative appeals or litigation. In addition, national forests may have submitted more than one decision with activities on the same area of land, or may have planned to use a series of different treatments on the same land. Therefore, the 10.5 million acres covered by decisions in our review may include overlapping acreage. Because this was not a sample survey, there are no sampling errors. However, the practical difficulties of conducting any survey may introduce errors, commonly referred to as nonsampling errors. For example, differences in how a particular question is interpreted, in the sources of information that are available to respondents, or in how the data are entered into a database or were analyzed can introduce unwanted variability into the survey results. We took steps in the development of the survey, the data collection, and data analysis to minimize these nonsampling errors. For example, prior to developing the data collection instruments, we met with Forest Service personnel at the headquarters, regional, and national forest levels to discuss the Forest Service decision- making, appeal, objection, and litigation processes. We also reviewed current policies, legislation, and court cases that are relevant to our questions and the analysis of the survey responses. Survey specialists designed the questionnaire in conjunction with GAO staff with subject matter expertise. The draft survey was then pretested with officials from four national forests in four different regions to ensure that the questions were relevant, clearly stated, and easy to comprehend. Upon receiving survey responses, we verified the accuracy of 5 percent of the surveys by comparing the responses to three survey questions against the decision documents used to complete the surveys, which were provided by respondents at our request. Using this approach, we verified 70 randomly selected decisions. Discrepancies between the survey responses and our data verification were discussed and resolved with the responsible forest official. In addition, we conducted follow-up to clarify ambiguous or incomplete responses that were identified through an internal logic test of all submitted responses. Through our data verification process, we determined that the data submitted were generally reliable. To gather specific details about the outcomes of appeals and litigation, we conducted semistructured interviews with regional appeals and litigation officials in each of the Forest Service’s nine regions. The semistructured interviews were used to gather information about each of the decisions that were appealed or litigated, including related dates, status and outcomes of administrative and court decisions, and the identities of the appellants and litigants. Information collected through these semistructured interviews was also verified for a randomly selected sample of decisions. We verified the accuracy of about 10 percent of the appealed decisions and about 50 percent of the litigated decisions by comparing the information provided in response to several interview questions against the administrative and court decision documents provided to us by interviewees at our request. Any discrepancies between the interview responses and the documents provided were discussed and resolved with the responsible regional official. Through our data verification process, we determined that the data gathered during the semistructured interviews were generally reliable. There are some limitations to the data we gathered. As with any survey, the information obtained from the national forests was self-reported, and we were not able to ensure that all decisions meeting our criteria were identified. In particular, we had no way to determine whether forests were fully reporting their hazardous fuel reduction activities. To get some indication of the completeness and accuracy of the data provided by Forest Service, we contacted several interest groups that, according to our data collection efforts, often appealed and objected to decisions or determinations. We asked these groups to verify the data pertaining to their appeals, objections, and litigation of Forest Service fiscal year 2006 through 2008 fuel reduction decisions and to identify any missing data. The groups generally agreed that the data provided by the agency were complete and accurate. In addition, during these interviews, we asked the groups for their perspectives on the administrative process for challenging decisions, including the objection process authorized under the Healthy Forests Restoration Act. The interviewees’ comments and perspectives are incorporated in this report. We conducted our work from October 2008 through February 2010, in accordance with all sections of GAO’s Quality Assurance Framework that are relevant to our objectives. The framework requires that we plan and perform the engagement to obtain sufficient and appropriate evidence to meet our stated objectives and to discuss any limitations in our work. We believe that the information and data obtained, and the analysis conducted, provide a reasonable basis for any findings and conclusions in this product. Appendix II: Number of Fuel Reduction Decisions and Associated Acreage, by Forest Service Region Figure 2 shows, for each of the Forest Service’s nine regions, the number of fuel reduction decisions and the total associated acreage. As shown, the Southern Region (Region 8) had the largest number of decisions and the largest acreage, while the Alaska Region (Region 10) had the fewest decisions and the smallest acreage. Appendix III: Number of Appeals, Objections, and Lawsuits of Fuel Reduction Decisions, by Forest Service Region Figure 3 shows, for each of the Forest Service’s regions, information on appeals, objections, and litigation of fuel reduction decisions, including the total number of appeals, objections, and litigation and the percentage of decisions appealed, objected to, and litigated. The Southern Region (Region 8) had the highest combined total of decisions subject to appeal and objection; however, decisions in the Northern Region (Region 1) were challenged at the highest rate, considering both appeals and objections. Appendix IV: Appeal Outcomes for Fuel Reduction Decisions, by Forest Service Region Figure 4 shows, for each Forest Service region, the outcomes of appeals filed on fuel reduction decisions within the region. While six of the eight regions reporting appeal activity allowed the majority of appealed decisions to proceed without changes, the Southwestern Region (Region 3) had no appealed decisions that were allowed to proceed without changes and the highest rate of reversed decisions. Appendix V: Litigation Outcomes for Fuel Reduction Decisions, by Forest Service Region Figure 5 shows, for each Forest Service region, the outcomes of litigation filed on fuel reduction decisions within the region. Six of the nine regions experienced litigation during the period covered by our survey. The Northern Region (Region 1) had the highest number of decisions judicially challenged as well as the greatest number of ongoing lawsuits. Appendix VI: List of Appellants, Objectors and Litigants, by Forest Service Region Tables 7, 8, and 9 list, by Forest Service region, the appellants, objectors, and litigants of fuel reduction decisions. We list the identities of organizations filing appeals, objections, and litigation, but summarize data on individuals to protect their privacy. As shown, organizations were most active in the Northern Region (Region 1) for appeals, objections, and litigation. Individuals were likewise most active in the Northern Region for objections, but were most active in the Eastern Region (Region 9) for appeals and litigation. Appendix VII: Fuel Reduction Treatment Methods and Number of Appeals, Objections, and Lawsuits, by Forest Service Region Figure 6 shows, for each Forest Service region, the number of decisions using various fuel reduction treatment methods and the number and frequency of appeals, objections, and litigation by fuel reduction method. The rate at which treatment methods were used varied by region. For example, the Southern Region (Region 8) and the Eastern Region (Region 9) used prescribed burning more than any other treatment method, whereas the remaining regions used mechanical treatment the most. In addition, the Northern Region (Region 1) used commercial logging at a higher rate than any other region. Appendix VIII: Contract Types and Number of Appeals, Objections, and Lawsuits, by Forest Service Region Figure 7 shows, for each Forest Service region, the number of decisions using various contract types and the number and frequency of appeals, objections, and litigation by contract type. The use of different contract types varies among regions. The Eastern Region (Region 9) has the highest rate of commercial timber sale contract use compared with other regions, while the Rocky Mountain Region (Region 2) has the highest rate of stewardship contracting use. Appendix IX: Fuel Reduction Decisions in the Wildland-Urban Interface and Inventoried Roadless Areas, by Forest Service Region In this appendix, Figures 8 and 9 provide information about appeals, objections, and litigation of fuel reduction activities in the wildland-urban interface and in inventoried roadless areas. Figure 8 shows, for each Forest Service region, the number of decisions with fuel reduction activities in the wildland-urban interface and the number and frequency of appeals, objections, and litigation of such decisions by region. The Southern Region (Region 8) had the most decisions in the wildland-urban interface, while the Northern Region (Region 1) had the highest number of appeals and objections of such decisions, and the highest rate at which decisions were challenged, considering both appeals and objections. According to survey respondents, over half of these decisions (696) contained definitions of wildland-urban interface that were based on the definition provided in the January 4, 2001, Federal Register as refined by HFRA. HFRA Section 101 (16) defines wildland-urban interface as an area within or adjacent to a community that is identified as at risk in a community wildfire protection plan. In addition, for areas for which a community wildfire protection plan is not in effect, the definition in HFRA includes areas (1) extending 1/2 mile from the boundary of an at-risk community, or (2) within 1 1/2 miles of the boundary of an at-risk community, including any land that has for example, a sustained steep slope, a geographic feature that could help when creating an effective firebreak, or Condition Class 3 land, or (3) is adjacent to an evacuation route. Further, while many additional survey respondents who did not select this definition provided their own definition of wildland-urban interface, we found that 36 such respondents had definitions very similar to that contained in HFRA. Other respondents said they defined wildland-urban interface as it is referenced in their forests’ National Forest Land Management Plans. Others said they used a combination of definitions from multiple sources. For example, in the Pacific Southwest Region, several wildland-urban interface definitions were based on both the Federal Register and their forests’ National Forest Land Management Plans. Still others defined wildland-urban interface as an area within some distance from private land, or private lands with structures. The remaining respondents either said they did not have a definition for wildland-urban interface (14) or did not know the definition they used to identify the wildland-urban interface (49). Figure 9 shows, for each Forest Service region, the number of decisions with fuel reduction activities in inventoried roadless areas and the number and frequency of appeals, objections, and litigation of such decisions by region. The Intermountain Region (Region 4) had the most decisions with activities occurring in inventoried roadless areas and also the highest number of appeals, objections, and cases litigated. However, the Pacific Northwest Region (Region 6) had the highest rate at which decisions were challenged, considering both appeals and objections. Appendix X: Types of Categorical Exclusions Authorized by the Forest Service and Represented in the GAO Survey A categorical exclusion (CE) is a category of actions for which neither an environmental assessment nor an environmental impact statement is required because the agency has determined that it does not individually or cumulatively have a significant effect on the quality of the human environment. Agencies develop a list of categorical exclusions specific to their operations when they develop or revise their implementing procedures for the National Environmental Policy Act (NEPA), in accordance with the Council on Environmental Quality’s NEPA regulations. When the Forest Service determines that activities of a proposed decision fall within a category of activities the agency has already determined have no significant environmental impact, it approves it using one of the predetermined categorical exclusions established by the Secretary of Agriculture or the Chief of the Forest Service. Table 10 shows the types and frequency of categorical exclusions reported in our survey. They are divided into two types: those that require the agency to prepare a decision memo for each action approved using a categorical exclusion, and those that do not require such documentation. Appendix XI: Major Litigation Affecting Appeal Procedures for Categorical Exclusions A summary of the major litigation that affected the exemption of categorical exclusions from the requirements of the National Environmental Policy Act process is shown in table 11. Starting in late 2003, these exemptions were challenged in court and were the subject of a Supreme Court ruling. Table 12 summarizes the litigation centered specifically on the validity of the Hazardous Fuel Reduction categorical exclusion, or Fuels CE, also known as CE #10. Appendix XII: Survey Questions to National Forests Appendix XIII: GAO Contact and Staff Acknowledgments Staff Acknowledgments In addition to the individual named above, Steve Gaty (Assistant Director), Ulana Bihun, Sandra Davis, Justin Fisher, Cathy Hurley, Richard P. Johnson, Stuart Kaufman, Armetha Liles, Diane Lund, Robin Nazzaro, Alison O’Neill, and Shana Wallace made key contributions to this report.

Increases in the number and intensity of wildland fires have led the Department of Agriculture's Forest Service to place greater emphasis on thinning forests and rangelands to reduce the buildup of potentially hazardous vegetation that can fuel wildland fires. The public generally has an opportunity to challenge agency hazardous fuel reduction decisions with which it disagrees. Depending on the type of project being undertaken, the public can file a formal objection to a proposed decision, or can appeal a decision the agency has already made. Appeals and objections must be reviewed by the Forest Service within prescribed time frames. Final decisions may also generally be challenged in federal court. GAO was asked, among other things, to determine, for fiscal years 2006-2008, (1) the number of Forest Service fuel reduction decisions and the associated acreage; (2) the number of decisions subject to appeal and objection, the number appealed, objected to, and litigated, and the associated acreage; and (3) the outcomes of appeals, objections, and litigation, and the extent to which appeals and objections were processed within prescribed time frames. In doing so, GAO conducted a nationwide survey of forest managers and staff, interviewed officials in the Forest Service's regional offices, and reviewed documentation to corroborate agency responses. GAO requested, but did not receive, comments from the Forest Service on a draft of this report. Through a GAO-administered survey and interviews, Forest Service officials reported the following information: (1) In fiscal years 2006 through 2008, the Forest Service issued 1,415 decisions involving fuel reduction activities, covering 10.5 million acres. (2) Of this total, 1,191 decisions, covering about 9 million acres, were subject to appeal and 217--about 18 percent--were appealed. Another 121 decisions, covering about 1.2 million acres, were subject to objection and 49--about 40 percent--were objected to. The remaining 103 decisions were exempt from both objection and appeal. Finally, 29 decisions--about 2 percent of all decisions--were litigated, involving about 124,000 acres. (3) For 54 percent of the appeals filed, the Forest Service allowed the project to proceed without changes; 7 percent required some changes before being implemented; and 8 percent were not allowed to be implemented. The remaining appeals were generally dismissed for procedural reasons or withdrawn before they could be resolved. Regarding objections, 37 percent of objections resulted in no change to a final decision; 35 percent resulted in a change to a final decision or additional analysis on the part of the Forest Service; and the remaining 28 percent were set aside from review for procedural reasons or addressed in some other way. And finally, of the 29 decisions that were litigated, lawsuits on 21 decisions have been resolved, and 8 are ongoing. Of the lawsuits that have been resolved, the parties settled 3 decisions, 8 were decided in favor of the plaintiffs, and 10 were decided in favor of the Forest Service. All appeals and objections were processed within prescribed time frames--generally, within 90 days of a decision (for appeals), or within 60 days of the legal notice of a proposed decision (for objections).

gov_report

Fumarase is distributed between two compartments of the eukaryotic cell. The enzyme catalyses the reversible conversion of fumaric to L-malic acid in mitochondria as part of the tricarboxylic acid (TCA) cycle, and in the cytosol/nucleus as part of the DNA damage response (DDR). Here, we show that fumarase of the model prokaryote Bacillus subtilis (Fum-bc) is induced upon DNA damage, co-localized with the bacterial DNA and is required for the DDR. Fum-bc can substitute for both eukaryotic functions in yeast. Furthermore, we found that the fumarase-dependent intracellular signaling of the B. subtilis DDR is achieved via production of L-malic acid, which affects the translation of RecN, the first protein recruited to DNA damage sites. This study provides a different evolutionary scenario in which the dual function of the ancient prokaryotic fumarase, led to its subsequent distribution into different cellular compartments in eukaryotes. The enzyme fumarase (classII, fumarate hydratase in higher eukaryotes) is a conserved protein in all organisms from bacteria to human with respect to its sequence, structure, and enzymatic activity (Akiba et al., 1984). Fumarase is a dual targeted protein in eukaryotes; its echoforms are distributed between mitochondria and the cytosol/nucleus (Yogev et al., 2011; Yogev et al., 2010). Moonlighting of proteins is also a well-known phenomenon, which is defined by single proteins that can perform different functions in the cell (Gancedo et al., 2016; Espinosa-Cantú et al., 2015). Fumarase is also a moonlighting protein since it performs functions in the tricarboxylic acid (TCA) cycle in mitochondria and it participates in the DNA damage response (DDR) in the nucleus (Boukouris et al., 2016). In the TCA cycle, fumarase converts fumaric acid to L-malic acid while in the nucleus it catalyses the opposite reaction, thereby supplying fumaric acid as a signaling molecule for the DDR (Yogev et al., 2010). In human cells, fumaric acid has been shown to inhibit certain histone dimethylases and prolyl hydroxylases (Jiang et al., 2015; Gottlieb and Tomlinson, 2005; Isaacs et al., 2005). Worth mentioning is that fumarase associates with different protein partners in the two compartments (in the mitochondria where it is part of the TCA cycle, it interacts with other TCA cycle enzymes such as malate dehydrogenase, while in the cytosol/nucleus, it interacts with components of the DDR such as kinases and histones). Two questions with regard to the evolution of fumarase come to mind; (1) how and when did dual targeting of the protein evolve? And (2) how and when did dual function of the protein evolve? With regard to the first question, fumarase has been shown to distribute between the mitochondria and the cytosol/nucleus by different mechanisms in different eukaryotes (Yogev et al., 2011). In Arabidopsis, there are two fumarase-encoding genes, which are highly homologous, besides the fact that one encodes a mitochondrial targeting signal (MTS), while the other lacks it (Pracharoenwattana et al., 2010). In human, there is a single gene, however, it makes multiple mRNAs, which either encode or lack a MTS. In these cases, described above, two different types of mRNA are made which determine dual localization of fumarase echoforms (Dik et al., 2016). In yeast (S. cerevisiae), mitochondrial and cytosolic echoforms of fumarase, are encoded by a single nuclear gene (FUM1) and follow an intriguing mechanism of protein subcellular localization and distribution (Yogev et al., 2011). Translation of all FUM1 messages initiates only from the 5' -proximal AUG codon and results in a single translation product that contains the MTS (Yogev et al., 2007). The precursor of yeast fumarase is first partially translocated into mitochondria, so that the N-terminal signal is cleaved. While a subset of these molecules continues to be fully translocated into the organelle, the rest are folded into an import-incompetent conformation and are released by the retrograde movement back into the cytosol. Thus, protein folding is the driving force for fumarase dual targeting in yeast, a mechanism termed reverse translocation (Karniely and Pines, 2005; Kalderon and Pines, 2014). Dual localization is a very abundant phenomenon in eukaryotes, and in fact, we estimate that a third of the yeast mitochondrial proteome is dual targeted (Ben-Menachem et al., 2011). Therefore, dual targeting or dual localization of proteins is a major outcome of gene expression and as such, the evolutionary pressures governing this phenomenon are of fundamental importance. In a major discovery, we have shown that dual-targeted proteins are significantly more evolutionary conserved than exclusive mitochondrial proteins. We reached this conclusion by employing codon usage bias, propensity for gene loss, phylogenetic relationships, conservation analysis at the DNA level, and gene expression (Dinur-Mills et al., 2008; Kisslov et al., 2014). This has changed the way we think about dual targeting since we now assume that the majority of dual targeted proteins have discrete functions in the different subcellular compartments, regardless of their dual-targeting mechanism. Thus we hypothesize that dual targeting is maintained due to separate selective pressures administered by the different compartments to maintain the functions associated with the protein sequences (Kisslov et al., 2014). Here, we use the enzyme fumarase as a paradigm of this evolution and show for the first time that the single fumarase gene of B. subtilis is involved in the bacterial DDR, in addition to its role in the TCA cycle. This finding suggests that dual function of fumarase, in the bacterial progenitor, preceded the dual targeting that we find in eukaryotes. Intriguingly, fumarase in bacteria, similarly to eukaryotes, appears to be linked to the DDR by metabolite signaling, however the active molecule in this case is L-malic acid and not fumaric acid. The conservation of fumarase dual function in eukaryotes without conservation of the mechanism of dual targeting has brought us to examine the following hypothesis: Dual function of fumarase in bacteria preceded dual targeting in eukaryotes; in other words, it occurred in the prokaryotic ancestor prior to evolving of eukaryotes. Thus, dual function was the driving force for fumarase dual localization in the eukaryotic cell. To examine this question we employed the Gram positive bacterium Bacillus subtilis, which contains a single class II fumarase gene (fum-bc). As shown in Figure 1A (compare the right and left panels), B. subtilis deleted for the citG gene (fum-bc) which encodes fumarase, exhibits very poor growth on defined medium (S7) containing a low level of glucose (0. 1%) that requires respiration and the TCA cycle for efficient growth. Thus, as expected, Fum-bc has a crucial function within the TCA cycle. Intriguingly, fum-bc deleted strains are sensitive to DNA damaging agents such as ionized radiation (IR) (Figure 1A, middle panel) or methyl methanesulfonate (MMS) (Figure 1B). We expressed Fum-bc from the ectopic amyE locus (Δfum+Fum-bc, Figure 1—figure supplement 1) and this strain exhibits similar resistance to MMS treatment as the wild type (Figure 1B, compare the first and third rows). The slight difference in resistance may be since fumarase is expressed from the amyE locus and not from its native promoter. Quantitative experiments following colony forming units (CFU) are shown in Figure 1Ci and 1Cii. The difference between wild type and Δfum strains is insignificant (compare the two left bars of Figure 1Ci and Figure 1Cii) while the difference between these strains following DNA damage is highly significant (bars 4 and 5, p<0. 01). These results suggest, as we hypothesized, that dual function of fumarase can already be found in prokaryotes. To examine the ability of Fum-bc to perform its two functions (TCA and DDR) in a eukaryotic model, we took advantage of our experience with the yeast Saccharomyces cerevisiae. fum-bc was cloned into a yeast expression vector under the control of the GAL promoter. The levels of fumarase in cultures grown in galactose medium of wild type yeast, yeast deleted for the chromosomal FUM1 and such a deletion strain expressing bacterial fum-bc, are shown in Figure 1—figure supplement 2. The fum-bc gene was expressed in a yeast strain deleted for the endogenous FUM1 gene (Δfum1). This strain was grown on glucose (dextrose) as a control medium that does not require respiration for growth, and on ethanol which does require respiration and the function of the TCA cycle. As shown in Figure 1D, yeast strains deleted for the endogenous FUM1 and expressing fum-bc can partially complement the lack of yeast fumarase for growth on ethanol (compare row 4 [fum-bc expression] to row 2 [no fumarase expression]). Fum-bc does not contain a mitochondrial targeting sequence and it is not targeted to, or imported into mitochondria. We have performed subcellular fractionation of yeast cells expressing fum-bc. The bacterial protein is located only in the ‘cytosol’ and not in mitochondria (Figure 1—figure supplement 3). From previous studies, it turns out that fumarase, located outside mitochondria, can nevertheless function in the TCA cycle (Sass et al., 2003; Stein et al., 1994). The explanation for this is that the metabolites fumarate and malate can enter and exit the organelle (via specific inner membrane transporters) thereby completing the TCA cycle. To examine the activity of Fum-bc in the DNA damage response, the yeast FUMm strain was employed. The FUMm strain harbors a chromosomal FUM1 deletion (Δfum1) and a FUM1 ORF (open reading frame) insertion in the mitochondrial DNA, thereby allowing exclusive mitochondrial fumarase (Fum1) expression, but lacking extra-mitochondrial (cytosolic/nuclear) fumarase (Yogev et al., 2010). Accordingly, the FUMm strain exhibits a functional TCA cycle and the ability to respire; however, it displays sensitivity to DNA damaging agents such as ionized radiation. When fum-bc is expressed in the yeast FUMm strain, it can complement the lack of extra-mitochondrial fumarase with respect to sensitivity to ionized radiation (Figure 1E, compare row 3 [cytosolic fum-bc expression] to row 2 [no cytosolic fumarase expression]). These results support our hypothesis that bacterial fumarase has the capacity to function both in the TCA cycle and the DNA damage response. To examine the role of fumarase in the DDR, Bacillus subtilis cells were grown to log phase and then incubated in the presence or absence of MMS. Cell extracts were analyzed with time by western blot using a mixture of anti yeast fumarase (anti yFum) and anti human fumarase-FH (anti hFum) antibodies. As shown in Figure 2A the level of Fum-bc gradually increases (compare the top panel to the SigmaA loading control in the lower panel. After 60 min of MMS treatment, the amount of fumarase rises two fold (Figure 2B). This finding is consistent with the proposed function of Fum-bc in the DDR. As shown In Figure 2—figure supplement 1, the levels of other TCA cycle enzymes, citrate synthase 2 (citZ) and isocitrate dehydrogenase (ICDH), do not change significantly upon treatment with MMS. We next asked whether the appearance and localization of Fum-bc changes upon induction of the DNA damage response. For this, we monitored fluorescence in B. subtilis strains harboring genomic Fum-bc-GFP fusions, in combination with DAPI for DNA staining and FM4-64 for membrane staining. Fum-bc-GFP retains full fumarase activity in cell extracts (Figure 2—figure supplement 2). As shown in Figure 2C, in untreated cells (top panels) Fum-bc does not generally colocalize with the bacterial DNA (top right panel); only 13% of Fum-bc-GFP foci showed colocalization with the DNA (Figure 2D). Following treatment with MMS, we detected full colocalization of Fum-bc-GFP fluorescence with the DNA DAPI stain (bottom right panel); more than 95% of the foci showed colocalization with the DNA (Figure 2D). Upon DNA damaging treatment, fumarase clearly coincides with the condensed DNA and in fact as shown in the two lower right panels of Figure 2C, the Fum-bc-GFP fluorescence and the DNA DAPI stain perfectly coincide during this process. Without DNA damaging treatment the fum-bc-GFP foci and DAPI stained DNA do not regularly superimpose. Nevertheless one does see some co-staining which can be explained by simple coincidence or naturally occurring low random DNA damage for example at DNA replication sites. Furthermore, we found that without induction of double strand breaks (DSBs) (-MMS) most of the cells show between three to four Fum-bc-GFP foci, while after induction of DSB (+MMS) most of the cells show one to two extensive foci that overlap with the DAPI stained DNA (Figure 2E). Thus, Fum-bc appears to be recruited to the DNA during the DNA damage response supporting a role for the bacterial fumarase in this response. To examine whether the role of Fum-bc in the DDR requires its enzymatic activity, we first identified mutations within the fumarase active site that may abolish its enzymatic activity. According to the literature (Weaver et al., 1997; Alam et al., 2005; Kokko et al., 2006) fumarase has a known active site in eukaryotes (S. cerevisiae) and in prokaryotes (Escherichia coli). Based on sequence similarity between eukaryotic and prokaryotic fumarases, we created two separate substitution mutations within the suspected Fum-bc active site; H186N and H127R. H186N has been suggested to be a residue of the active site of E. coli fumarase while H127R has been suggested to be a residue of the active site of the eukaryotic S. cerevisiae fumarase. We created fum-bc point mutations corresponding to the above single amino acid changes in the respective active sites, and examined expression of the proteins by western blot (Figure 3—figure supplement 1). While extracts of cells expressing only the H186N mutated fumarase, were essentially devoid of enzymatic activity, those expressing H127R displayed 40% of the wild type activity (Figure 3—figure supplement 2). Accordingly, cells expressing H186N exhibit sensitivity to MMS and defective growth on S7 medium (low glucose, Figure 3A, right panel), while H127R grows normally on S7 plates and is not sensitive to MMS (Figure 3—figure supplement 3). This indicates that enzymatic activity is required for both DDR and respiration-related functions of fumarase. Fumarase catalyses the reversible conversion of fumaric acid to L-malic acid as part of the TCA cycle in mitochondria. In yeast and human cells, defective for extra mitochondrial fumarase, the sensitivity to DNA damage can be complemented by fumaric acid, added to the growth medium in the form of an ester (monoethyl fumarate, which is cleaved in the cells to form the free acid) (Yogev et al., 2010; Jiang et al., 2015). To examine if products or substrates of the fumarase reaction in B. subtilis may complement the lack of fumarase in the DDR, bacteria were grown in the presence of organic acids added to the medium. As shown in Figure 3B, B. subtilis cells deleted for the fum-bc gene are protected from the DNA damaging treatment with MMS, by L-malic acid (compare the third and fourth rows of the right and left panels). In contrast, succinic and citric acids have no protective effect (two middle panels respectively). Since, fumaric acid is not soluble; we also examined the capacity of esters of the other organic acids - to protect the bacterial cells against MMS. While diethylmalate protects the cells from MMS the other organic acids (monoethyl fumarate and monoethyl succinate), had a much weaker effect (Figure 3—figure supplement 4). Organic acids, and in particular L-malic acid, appear to play a role in the DNA damage response in B. subtilis. To correlate changes in the intracellular levels of these organic acids we employed GC-MS of cell lysates. As shown in Figure 3C, following induction of DNA damage with MMS, the relative levels of L-malic acid increase while those of succinic and fumaric acids decrease (compare the two left sets of bars). This clearly fits the role of L-malic as a DDR signaling molecule and that succinic and fumaric acids do not have such a role. In B. subtilis, strains deleted for fum-bc, the single fumarase gene, accumulate higher levels of fumaric and succinic acid (Figure 3C). This is expected from a block in the TCA cycle at the conversion step of succinic to fumaric acid and subsequently to L-malic acid by fumarase (succinic - > fumaric - > L malic). Interestingly, upon treatment with MMS the levels of fumaric and succinic acids are even higher (compare the fourth and third sets of bars), indicating an induced flow of metabolites through TCA cycle upon DNA damage. To further implicate Fum-bc expression and its function in the DNA damage response, we decided to examine certain B. subtilis DDR components in conjunction with fumarase. RecN appears to be one of the first proteins recruited to DNA damage sites in live cells (Cardenas et al., 2014; Alonso et al., 2013). B. subtilis cells deleted for the fumarase gene exhibit an alteration in the localization and appearance of RecN (Figure 4A, see description below). RecN appears to be the first protein detected as discrete foci (repair centers) in live cells in response to DNA double strand breaks (Cardenas et al., 2014). It is cytoplasmically located in untreated cells and upon treatment with DNA damaging agents is recruited to damage sites followed by RecO and then RecF (Alonso et al., 2013). As shown in Figure 4B, a strain deleted for RecN shows weak DNA damage sensitivity to MMS (Sanchez et al., 2007), while a double knock out of RecN and fumarase exhibits an additive effect with the cells exhibiting much higher sensitivity (Figure 4B). This effect can be reversed by addition of L-malic acid to the medium (Figure 4B, right panel, compare rows 4 and 2 to row 1). Thus, according to the results with RecN, we conclude that fumarase is involved in the resistance to DNA damage and its absence can be complemented by L-malic acid. To examine whether the appearance and localization of RecN-GFP changes upon knockout of the fumarase gene and/or induction of the DNA damage response, we created B. subtilis strains harboring genomic RecN-GFP fusions. Following treatment with MMS of wild type cells, there appears to be only a small increase in the proportion of cells containing RecN foci (less than 5%), nevertheless, this increase is statistically significant (Figure 4—figure supplement 1), and the appearance of these foci remained unchanged under MMS treatment (Figure 4A, compare the RecN-GFP untreated control to RecN-GFP MMS). In strains deleted for the fumarase gene, we observed a similar number of RecN associated foci (Figure 4A, third row, RecN-GFP Δfum) when compared to the RecN-GFP control (fourth row). In the presence of MMS (DNA damage) strains deleted for the fumarase gene displayed a drastic change in the number of cells which contain foci (a two fold increase) and in their appearance (Figure 4A and Figure 4—figure supplement 1, compare RecN-GFP Δfum + MMS to untreated RecN-GFP Δfum). Upon DNA damage the RecN-GFP fluorescence, in the Δfum strain, does not appear as discrete foci but rather this fluorescence coincides with the DNA DAPI stain of the condensed B. subtilis chromosomes. Thus, in the absence of fumarase, RecN appears to be recruited differently to the DNA during the DNA damage response. In addition to the DNA damage sensitivity and subcellular appearance, we wished to examine the effect of fumarase on RecN levels in the cell. Cells deleted for the fumarase gene were grown to early logarithmic phase (OD = 0. 4), MMS was added for 30 min and then cell lysates were subjected to western blot analysis (we could not detect RecN without induction of DSB- data not shown). As shown in Figure 5A, RecN is expressed about three fold higher in the strain deleted for the fumarase gene when compared to the wild type (Figure 5A, compare lane 2 to lane 1 of the top panel, quantification, Figure 5B). In contrast, when L-malic acid is added to the medium the levels of RecN are essentially the same in wild type and strain deleted for the fumarase gene (Figure 5A, upper panel, compare the two right lanes, quantitation Figure 5B). Worth mentioning is the fact that we have tried to coimmunoprecipitate fumarase and RecN with no positive indications (see the Supplementary methods). Together these data support the notion that fumarase affects RecN function by producing the metabolite L-malic acid and not by direct interaction of the proteins. The primary goal of this research was to implicate prokaryotic fumarase in the DNA damage response. The finding that fumarase and L-malic acid affect the expression and subcellular appearance of RecN fully supports this hypothesis. Nevertheless, we decided to also examine at which level of gene expression, do fumarase and L-malic acid affect RecN cellular levels. Cells deleted for the fumarase gene and the corresponding wild type were grown to early logarithmic phase (OD = 0. 4), MMS was added for 30 min and then the levels of RecN mRNA were determined. We employed quantitative RT-PCR (see materials and methods) and observed no difference between the mRNA levels (data not shown). To further make the point that transcription does not play a role in RecN higher levels in Δfum strains, we grew B. subtilis strains in the presence of rifampicin. Rifampicin inhibits bacterial RNA polymerase and blocks transcription initiation, thus, in its presence; changes in protein synthesis do not result from changes in transcription. As shown in Figure 5C and D, cells treated with MMS in the presence of rifampicin for 10 or 20 min, revealed significantly higher levels of RecN in the Δfum strain than in the wild type strain. These results indicate that the increased synthesis of RecN, in the absence of fumarase, is due to translation and not transcription. To rule out the possibility that the different RecN levels in strains result from differences in protein stability, we assessed RecN-protein turnover in the presence of chloramphenicol following induction of DNA damage. Chloramphenicol prevents protein chain elongation by inhibiting the peptidyl transferase activity of the bacterial ribosome. As presented in Figure 5E the decrease in RecN levels in this experiment shows that both Δfum strain and wild type exhibit similar RecN turnover kinetics (Figure 5F). How did moonlighting and dual targeting of proteins evolve in eukaryotes? The notion is that a protein with a single function, activity or location, acquired new traits through evolution. With regard to fumarase the notion was that the enzyme acquired its second function in the DNA damage response after endosymbiosis and the creation of mitochondria. An example of the acquisition of novel functions after endosymbiosis are roles assumed by yeast Hsp60 and aconitase in mitochondrial genome stability through binding of mitochondrial DNA (Chen et al., 2005; Kaufman et al., 2000). Fumarase has been shown to distribute between the mitochondria and the cytosol/nucleus by different mechanisms in different eukaryotes (Yogev et al., 2011). It seems unlikely that the dual targeting/dual function arose independently in different eukaryotic ancestors. A different possibility is that dual function arose prior to dual localization and actually it was the function that was the driving force for the evolution of fumarase dual targeting. Our results support a second function for fumarase (in addition to its function in the TCA cycle) in the DDR of bacteria: We have shown that dual-targeted proteins are significantly more evolutionary conserved than exclusive mitochondrial proteins, strongly suggesting that dual function drives the evolution of dual targeting or at least its maintenance (Ben-Menachem et al., 2011; Kisslov et al., 2014). Our model depicts fumarase as a protein with dual function in the bacterial ancestor of mitochondria. Upon transfer of the endosymbiont gene into the eukaryotic nucleus, the selective pressure due to the two functions that are ‘needed to be carried out in different subcellular compartments’, resulted in dual targeting of this enzyme. A similar model can be assigned to the dual-targeted yeast aconitase, which is a component of the TCA cycle in mitochondria and the glyoxylate shunt in the cytosol (Chen et al., 2005; Rouault et al., 1991). This is a simpler example, compared to fumarase, in which aconitase functions in two parallel metabolic pathways that coexist in the prokaryotic cytosol. The main goal of this study was to determine that B. subtilis Fum-bc has both TCA cycle and DDR-associated functions. Nevertheless, we have also gained insight into some mechanistic features of fumarase function within the DDR. The most exciting finding is that intracellular signaling of the DDR is achieved via L-malic acid, the product of the reaction catalyzed by fumarase. This conclusion was reached not only due to the fact that L-malic acid, added to the medium, can complement Δfum strains upon DNA damage, but it is also consistent with other data in this study. B. subtilis fumarase enzymatic activity is required for its DNA damage protective function. In addition, upon DNA damage B. subtilis accumulates L-malic acid versus lower levels of fumaric and succinic acids. Furthermore, upon DNA damage Δfum cells accumulate extremely high levels of fumaric (and succinic) acids as though the cells are ‘trying to make more L-malic acid’ in order to signal DNA damage. This finding is intriguing since in yeast and human cells, the signaling molecule associated with fumarase, with respect to the DDR, is fumaric not L-malic acid as we find for B. subtilis (Yogev et al., 2011; Jiang et al., 2015). Thus, although the fumarase protein sequence and the dual function/targeting of the enzyme are conserved, the signaling metabolite is different. In other words, when we talk about conservation of function of fumarase in the DDR we do not mean that all aspects of dual function are conserved but rather that the metabolic pathway with specific organic acid intermediates are recruited. This raises questions on how intermediates of primary metabolism were chosen during evolution as signaling molecules in different organisms. Our results indicate that fumarase does not interact directly with RecN, but rather, the effect is via L-malic acid. How does L-malic acid precisely exert its effect on the DDR? A number of possibilities come to mind; the organic acid binds components of the DDR directly, thereby, modulating their activity. A good example of such a scenario are succinate and fumarate that can inhibit prolyl hydroxylases (PHDs), resulting in the stabilization of HIF1-α and activation of downstream hypoxic pathways in human cells (Gottlieb and Tomlinson, 2005; Isaacs et al., 2005; Pollard et al., 2005; Sudarshan et al., 2007). Another example are local concentrations of fumarate produced by phosphorylated fumarase (bound to histone H2A. Z) which inhibits KDM2B, histone dimethylase, which in turn results in enhancement of histone H3 dimethylation and downstream activation of the DDR (Jiang et al., 2015). Another possibility is that L-malic acid affects components of the DDR by affecting their expression. RecN fits the profile of an L-malic acid target since its expression is affected by the acid; the level and appearance of RecN is altered in Δfum cells following DNA damage which is correlated with lower levels of L-malic acid in the cells. RecN does not appear to be regulated at the mRNA level, transcription or protein stability. We claim that L-malic acid affects the translation of RecN mRNA, since upon DNA damage, B. subtilis cells lacking fumarase, have three fold higher amounts RecN protein in cell extracts. Importantly, this higher level of RecN can be reversed by growth of the cells in the presence of L-malic acid. To summarize these notions, the activity of fumarase and L-malic acid are required for an efficient DNA damage response and their effect on RecN has two consequences: (1) A change in the localization of RecN, (upon DNA damage induction), from foci throughout the cell to co-localization with the condensed DNA and (2) A two fold over-expression of RecN at the protein level. While we do not know how lack of fumarase and accumulation of L-malic acid affect the localization of RecN we do know that the change in expression of RecN occurs at the level of translation. How could L-malic acid affect RecN translation? There are three different ways to temporally regulate gene expression at the translational level: through trans-acting proteins, through cis-acting mRNA elements, acting as riboswitches (Kirchner and Schneider, 2017; Perez-Gonzalez et al., 2016) and through transacting RNAs (small RNA) (Kim et al., 2009). There are no known riboswitches in the RecN gene yet riboswitches have been detected in the upstream gene, ahrC, of the RecN operon, which appears to be highly regulated in Bacillus (Dar et al., 2016). The ahrC gene participates in the metabolism of arginine and the riboswitches were identified by term-seq which is quantitative mapping of all exposed RNA 3′ ends in bacteria. This allowed unbiased, genome-wide identification of genes that are regulated by premature transcription termination. Small untranslated RNA SR1, from the Bacillus subtilis genome, is a regulatory RNA involved in fine-tuning of arginine catabolism (Gimpel et al., 2012). SR1 is an sRNA that acts as a base-pairing regulatory RNA on the ahrC mRNA. The interaction of SR1 and ahrC mRNA does not lead to degradation of ahrC mRNA, but inhibited translation at a post-initiation stage (Heidrich et al., 2006). Overexpression of RecN has been shown to be lethal for B. subtilis cells. Thus, one of the roles of L-malic acid may be to maintain appropriate RecN levels. B. subtilis strains are listed in Supplementary file 1, S. cerevisiae strains are listed in Supplementary file 2 of the supplemental material. The plasmids and primers referred to in this study are described in Plasmid construction. All general methods were carried out as described previously (Harwood and Cutting, 1990). Molecular biological methods for Bacillus. Wiley, Chichester, United Kingdom). Cultures were inoculated at an optical density at 600 nm (OD600) of 0. 05 from an overnight culture, and growth was carried out at 37°C in LB medium. During logarithmic phase (OD600 of 0. 4 to 0. 6), 0. 5% xylose or 0. 1% IPTG was added to induce citG (fumarase) expression, as indicated. Pfum-GFP (fum-GFP-kan), containing the 3’ region of fum-bc fused to gfp, was constructed by amplifying the 3’ region by PCR using primers: F GAATTC TTC CAT GAT AAA TGT GCT GT R CTCGAG CGC CTT TGG TTT TAC CAT G, which replaced the stop codon with a XhoI site. The PCR-amplified DNA was digested with EcoRI and XhoI and was cloned into the EcoRI and XhoI sites of pKL168 (kan) (Lemon and Grossman, 1998), which contains the in frame gfp coding sequence. Pfum-bc (amyE: : fum-spc), containing flanking amyE sequences and the spc gene, was constructed by amplifying the citG gene by PCR using primers: F GTCGAC ATG GAA TAC AGA ATT GAA CGA R GCTAGC G CAG CCG TTC TTC CTA TTA. The PCR-amplified DNA was digested with SalI and NheI and cloned into the SalI and NheI sites of pDR111 (amyE: : spc). PH127R, pDR150 [amyE: : fum-bc (spec) ] is an ectopic integration containing the xylose-inducible promoter. pDR150 was generated by site-directed mutagenesis using the KAPAHiFiTM kit, using primers: F CGT CCA AAT GAT GAC GTG AAC P R A ATC GTT TGA TCA GAG TTC TTC P PH186N, pDR150 [amyE: : fum-bc (spec) ] is an ectopic integration containing the xylose-inducible promoter. pDR150 was generated by site-directed mutagenesis using the KAPAHiFiTM kit, using primers: F GAT CTT CAG GAT GCT ACG R CGT GCG TCC GAT TTT GAC Pyfum yeast expression vector yep51, was constructed by amplifying the citG gene by PCR using primers: F GTCGAC ATG GAA TAC AGA ATT GAA CGA R GGATCC CGC CTT TGG TTT TAC CAT G. The PCR-amplified DNA was digested with SalI and BamHI and cloned into the SalI and BamHI sites of YEp51. Samples (0. 5 mL) of a given culture were removed, centrifuged briefly, and resuspended in 10 µL of PBS × 1 (Phosphate-Buffered Saline) supplemented with 1 µg/mL FM4–64 (Molecular Probes, Invitrogen). Cells were visualized and photographed using an Axioplan2 microscope (Zeiss) equipped with CoolSnap HQ camera (Photometrics, Roper Scientific) or an Axioobserver Z1 microscope (Zeiss) equipped with a CoolSnap HQII camera (Photometrics, Roper Scientific). System control and image processing were performed using MetaMorph 7. 2r4 software (Molecular Devices). GC-MS analysis of three organic acids was performed using gas chromatograph (Agilent 7890A) coupled to the mass selective (Agilent 5975C MSD). The gas chromatograph was equipped with the CTC COMBI PAL autosampler. Mass spectrometer was operated in SIM mode (single ion monitoring). Plasma samples were dissolved in water following the addition of isotopically labeled succinic acid – D6. The samples were cleaned by SPE (Phenomenex Strata X-AW) and dried over a stream of nitrogen. Acids were chemically derivatized by trimethyl silylation before GC-MS analysis. For RecN half-life determination, strains RecN-GFP and RecN-GFP, Δfum were grown to an OD600 = 0. 4 at 37°C in LB. MMS (0. 07 v/v) was added to an aliquot, and the cells were incubated for 30 min. Then, rifampicin (100 μg/ml) or chloramphenicol (20 μg/ml) were added. Aliquots were then collected at variable times, and cell growth was halted by addition of NaN3 (10 μM). Cells were harvested in lysis buffer containing: 10 mM Tris pH 8,10 mM MgCl2,0. 2 mg/ml AEBSF (MegaPharm-101500), 0. 5 mg/ml Lysozyme (USBiological-L9200), 5 µg/ml DnaseI (Sigma DN25). Protein concentrations were determined using Bradford analyses. Samples were separated on 10% SDS-PAGE gels, and then transferred onto PVDF membranes (Millipore). The following primary antibodies were used: Polyclonal anti-yeast fumarase and anti-human FH were generated in rabbits injected with the purified proteins. Monoclonal anti GFP was a product of Roche). Monoclonal anti SigmaA was kindly provided by M. Fujitas lab,. Polyclonal anti ICDH and anti citZ were product of kerafast. Blots were incubated with the appropriate IgG-HRP-conjugated secondary antibody. Protein bands were visualized using the ECL immunoblotting detection system (GE Healthcare) and developed on an ImageQuant LAS4000 mini Fuji luminescence imagining system. For the analysis of protein expression, bands from at least three independent experiments were quantified by densitometry using Image J analysis software. Total RNA was extracted by using a FastRNA blue kit (MP) according to manufacturer' s instructions. For integrity assessment and purification level of extracted RNA, 2% agarose gel electrophoresis as well as spectrophotometric assays were performed. The extracted RNA was reverse-transcribed into cDNA by using a Maxima First Strand cDNA Synthesis Kit with dsDNase (Thermo scientific). To reveal the modification in the expression of RecN, real-time reverse transcriptase PCR (real-time RT PCR) was exploited using SYRB Green dye (Thermo scientific) and the Mic system (Bio Molecular Systems). The raw data were further normalization to the yoxA gene. Three independent experiments were conducted. Sequences of the primers used in the current study were as follows: yoxA: F ATACAATGCGGACGGAAAAC R GGCTCCAGCACTTGTAAACC RecN F CAGGCTCCTTGAACTGCTG R CGT CAG TTC CTC AAT AAT GGC RecN (set2) F TGC ATT ACA CAC CTG CCT CA R CGC TAC CTT TTC CTG CTT AG When more than two groups were compared, statistical analysis was performed by one-way repeated measure analysis of variance with Duncan’s test. When only two groups were compared, significance was analyzed by the paired t test.

Living cells make an enzyme called fumarase. It converts a chemical called fumaric acid into L-malic acid. This is a crucial step in primary metabolism and aerobic respiration, the process of using oxygen to release energy for life. Yet it is not the only role that fumarase plays. In the cells of eukaryotes such as plants, animals and even baker's yeast, aerobic respiration happens inside compartments called mitochondria. Yet fumarase is also found in the nucleus, which contains the cell's genetic material. Inside the nucleus, this enzyme takes part in the DNA damage response that senses and repairs damage to the genetic code. Simpler organisms, like bacteria, do not have mitochondria or a nucleus. Instead, all their reactions take place inside the main space within the cell. The current model for the evolution of fumarase is that the enzyme evolved in an ancient bacterium for the production of energy. Then, in more complex organisms, becoming split between the mitochondria and the nucleus allowed it to take on a second role in the DNA damage response. Singer et al. now challenge that model, and show that fumarase takes part in DNA damage repair in bacteria too. Bacillus subtilis has one fumarase gene, known as fum-bc. Singer et al. showed that, without this gene, the bacteria do not grow well under conditions where they need to use aerobic respiration. But, the bacteria also became sensitive to DNA-damaging agents such as ionizing radiation or a chemical called methyl methanesulfonate. Singer et al. then expressed the bacterial fum-bc gene in baker's yeast, Saccharomyces cerevisiae. This organism has mitochondria and a cell nucleus. With the yeast' s own fumarase gene switched off, the bacterial fumarase was able to take on both roles - aerobic respiration and the DNA damage response. In bacteria grown with the DNA-damaging chemical, the level of fumarase started to rise. A fluorescent tag revealed that it also changed location, moving close to the bacteria's DNA. As such, even in bacteria, fumarase has two roles. Further experiments showed that the L-malic acid made by fumarase affects the production of a protein called RecN, and it is this protein that triggers DNA repair. These findings shed new light on the evolution of fumarase, and suggest that its dual role evolved before its dual location in eukaryotes. The next step is to find out exactly how L-malic acid affects the production of RecN.

lay_elife

SECTION 1. SHORT TITLE. This Act may be cited as the ``Tobacco Use Prevention and Public Health Act of 1999''. SEC. 2. WAIVER OF RECOUPMENT OF MEDICAID TOBACCO-RELATED RECOVERIES. (a) In General.--Section 1903(d)(3) of the Social Security Act (42 U.S.C. 1396b(d)(3)) is amended-- (1) by inserting ``(A)'' before ``The''; and (2) by adding at the end the following: ``(B) Subparagraph (A) and paragraph (2)(B) shall not apply to any amount recovered or paid to a State as part of a settlement or judgment reached in litigation initiated or pursued by a State against one or more manufacturers of tobacco products, as defined in section 5702(d) of the Internal Revenue Code of 1986, if (and to the extent that), with respect to amounts recovered or paid to a State in a fiscal year, the Secretary finds that following conditions are met: ``(i) The State will spend at least 25 percent of the amounts so recovered or paid in any fiscal year on tobacco control activities in accordance with subparagraph (C). ``(ii) The State will spend at least 25 percent of the amounts so recovered or paid in any fiscal year for health activities, including public health activities and expenditures described in subparagraph (D), but excluding amounts expended to meet the requirement of clause (i). ``(iii)(I) Subject to subclause (II), the amounts described in clauses (i) and (ii) will be spent only in a manner that supplements (and does not supplant) funds previously being spent by the State (or local governments in the State) for such or similar activities. ``(II) At the request of a State, the Secretary may waive the requirement of subclause (I), with respect to not supplanting State and local government expenditures for tobacco control activities, to the extent that the total level of such expenditures (taking into account expenditures required under clause (i)) is sufficient to provide for a well-funded, effective program of tobacco control in the State. ``(C) For purposes of subparagraph (B)(i), expenditures for tobacco control activities are made in accordance with this subparagraph if-- ``(i) the expenditures are made for any (or all) of the following activities: ``(I) Activities to reduce the use of tobacco products using methods that have been shown to be effective, such as tobacco use cessation programs, State or local counter-marketing programs, enforcement of laws relating to tobacco products, community-based programs to discourage the use of tobacco products, and school-based and child-oriented education programs to discourage the use of tobacco products, and for ongoing evaluations of these programs. ``(II) Activities to fund research related to nicotine addiction, tobacco use cessation, and prevention of the use of tobacco products, including surveillance and epidemiology research related to tobacco. ``(III) Activities to assist in economic development efforts designed to aid tobacco farmers and workers and communities as they transition to a more broadly diversified economy; and ``(ii) the expenditures include an annual contribution by the State of its pro rata share for the National Tobacco Use Prevention Program (under section 3 of the Tobacco Use Prevention and Public Health Act of 1999). ``(D) For purposes of subparagraph (B)(ii), the expenditures described in this subparagraph are State expenditures for outreach activities in enrolling individuals under this title and State expenditures for medical assistance with respect to individuals who are eligible for such assistance only on the basis of a State plan amendment approved on or after January 1, 1999. ``(E)(i) For purposes of subparagraph (C)(ii), a State's `pro rata share' for a fiscal year is equal to $605,000,000 (or, if less, the sum of the amounts provided under subsections (a), (b), and (c) of section 3 of the Tobacco Use Prevention and Public Health Act of 1999 for the fiscal year) multiplied by the ratio of (I) the amount of the State's aggregate recoveries or payments described in subparagraph (B) for the fiscal year, to (II) the total of such aggregate recoveries or payments for all the States for the fiscal year. ``(ii) Amounts contributed under subparagraph (C)(ii) shall be deposited in a separate account in the Treasury to be known as the `National Tobacco Use Prevention Account'. Amounts in such Account shall remain available until expended for obligation under section 3 of the Tobacco Use Prevention and Public Health Act of 1999. ``(F) For purposes of applying subparagraph (B) for amounts described in such subparagraph recovered or paid in a fiscal year before fiscal year 2000, such amounts shall be treated as being recovered or paid in fiscal year 2000.''. (b) Effective Date.--The amendments made by subsection (a) apply to amounts recovered or paid to a State before, on, or after the date of enactment of this Act. SEC. 3. NATIONAL TOBACCO USE PREVENTION PROGRAM. (a) National Public Awareness Campaign.--There shall be made available from the National Tobacco Use Prevention Account (provided for under section 1903(d)(3)(B)(E)(ii) of the Social Security Act) to the Secretary of Health and Human Services, without fiscal year limitation, $500,000,000 for each fiscal year (beginning with fiscal year 2000) to implement a national public awareness campaign to discourage the use of tobacco products. (b) National Program To Reduce Tobacco Use by Minority Groups.-- There shall be made available from such National Tobacco Use Prevention Account to such Secretary, without fiscal year limitation, $100,000,000 for each fiscal year (beginning with fiscal year 2000) to implement the recommendations contained in the 1998 report by the Surgeon General entitled ``Tobacco Use Among U.S. Racial/Ethnic Minority Groups''. The Secretary shall implement such recommendations through the Deputy Assistant Secretary for Minority Health and in consultation with an advisory committee composed of individuals from the private sector who are experienced with respect to minority health concerns. (c) National Economic Stabilization Program for Tobacco Farmers.-- There shall be made available from such National Tobacco Use Prevention Account to the Secretary of Agriculture, without fiscal year limitation, $5,000,000 for each fiscal year (beginning with fiscal year 2000 and ending with fiscal year 2005) to promulgate and enforce regulations to provide economic stabilization for tobacco farmers. Such regulations-- (1) shall provide that any manufacturer of tobacco products in the United States which purchases (or which controls a person who purchases) tobacco grown in a foreign country in any year, beginning with 2000 and ending with 2004, may not reduce in such year its level of purchases of tobacco from farmers in the United States below the level of such purchases in 1997; (2) may be enforced through an action brought by such Secretary in an appropriate district court of the United States to enjoin any failure to comply with such regulations or to impose a civil penalty for such failure to comply of not more than $25,000 per day of violation; and (3) shall not apply to purchases made on or after January 1, 2005. (d) Adjustment.--If the aggregate amount deposited into such National Tobacco Use Prevention Account for a fiscal year is less than the sum of the amounts specified under subsections (a), (b), and (c) for that fiscal year, the amounts so specified shall be reduced in a pro-rata manner so that the total of such amounts for the fiscal year is equal to the aggregate amount so deposited for the fiscal year.

Tobacco Use Prevention and Public Health Act of 1999 - Amends title XIX (Medicaid) of the Social Security Act to prohibit as the treatment of an overpayment for Medicaid-related purposes any amount recovered or paid to a State as part of a settlement or judgment reached in litigation initiated or pursued by the State against one or more manufacturers of tobacco products (recovered amounts), if (and to the extent that), with respect to such recovered amounts in a fiscal year, the Secretary of Health and Human Services (HHS) finds that specified conditions will be met, including that the State will use at least 25 percent of recovered amounts for a fiscal year on: (1) specified tobacco control activities, including certain tobacco use reduction activities such as tobacco use cessation programs and school-based and child-oriented education programs to discourage tobacco use, as well as the enforcement of laws relating to tobacco products; and (2) health activities, including public health activities, which include State expenditures for outreach activities. Includes further among such activities: (1) tobacco-related research concerning nicotine addiction; and (2) activities to assist tobacco farmers and workers and communities as they transition to a more broadly diversified economy. Creates the National Tobacco Use Prevention Program (NTUPP), composed of various specified HHS and Department of Agriculture (DOA) subprogram and public awareness campaign components. Includes subprograms to reduce tobacco use by minorities (HHS) and to provide economic stabilization for tobacco farmers (DOA) within NTUPP. Establishes in the Treasury the National Tobacco Use Prevention Account, subject to a specified fiscal year adjustment.

billsum

Monoclonal antibodies targeting the Epidermal Growth Factor Receptor (EGFR), such as cetuximab and panitumumab, have evolved to important therapeutic options in metastatic colorectal cancer (CRC). However, almost all patients with clinical response to anti-EGFR therapies show disease progression within a few months and little is known about mechanism and timing of resistance evolution. Here we analyzed plasma DNA from ten patients treated with anti-EGFR therapy by whole genome sequencing (plasma-Seq) and ultra-sensitive deep sequencing of genes associated with resistance to anti-EGFR treatment such as KRAS, BRAF, PIK3CA, and EGFR. Surprisingly, we observed that the development of resistance to anti-EGFR therapies was associated with acquired gains of KRAS in four patients (40%), which occurred either as novel focal amplifications (n = 3) or as high level polysomy of 12p (n = 1). In addition, we observed focal amplifications of other genes recently shown to be involved in acquired resistance to anti-EGFR therapies, such as MET (n = 2) and ERBB2 (n = 1). Overrepresentation of the EGFR gene was associated with a good initial anti-EGFR efficacy. Overall, we identified predictive biomarkers associated with anti-EGFR efficacy in seven patients (70%), which correlated well with treatment response. In contrast, ultra-sensitive deep sequencing of KRAS, BRAF, PIK3CA, and EGFR did not reveal the occurrence of novel, acquired mutations. Thus, plasma-Seq enables the identification of novel mutant clones and may therefore facilitate early adjustments of therapies that may delay or prevent disease progression. Colorectal cancer (CRC) is an important and highly prevalent health problem and improvements in outcomes associated with novel targeted therapies could have important health impacts. To this end, molecular markers are increasingly being used for predictive and prognostic applications in CRC. For example, mutant KRAS is a predictor of resistance to treatment with monoclonal antibodies targeting the Epidermal Growth Factor Receptor (EGFR), such as cetuximab (Erbitux) [1], [2] or panitumumab (Vectibix) [3]. However, almost all patients with wild type KRAS and clinical response to anti-EGFR therapies develop acquired resistance within a few months of starting therapy [4], [5]. Other factors than KRAS mutation status likely affect response to anti-EGFR therapy, because the response rates among patients with wild-type KRAS are less than 20% [1], [6], [7]. Recent investigations have identified genes and proteins downstream of KRAS in the mitogen-activated protein kinase signaling pathway, which affect unresponsiveness to anti-EGFR therapy, including the BRAF V600E mutation, mutations in NRAS or PIK3CA (exons 9 and 20), or loss of PTEN or AKT expression [8]–[10]. Furthermore, several mechanisms of acquired (secondary) resistance to anti-EGFR therapies, such as expression of EGFR ligands [11], deregulation of the EGFR recycling process [12], amplifications of the genes ERBB2 (also called HER2) [13], [14], KRAS [15], [16], and MET [17], have been identified. In addition, the EGFR ectodomain mutation S492R has recently been found to confer resistance to cetuximab [18]. On the other hand, several studies reported evidence that an increased EGFR copy number enhances response rates to anti-EGFR therapy [10], [19]–[21]. Hence, there is a growing number of markers predictive of response and survival in patients treated with anti-EGFR therapy. However, the evolution of these markers during disease course is unknown at present due to a lack of follow-up genetic data. To this end investigations are now increasingly employing blood-based assays that characterize cell-free DNA (cfDNA) in the plasma of patients with cancer [22]–[32]. Cancer cells can release tumor DNA into the circulation, which is frequently referred to as circulating tumor DNA (ctDNA) and ctDNA is a component of cfDNA [33], [34]. ctDNA can be used to deduce characteristics from the tumor genome non-invasively from a blood sample [33], [34]. For example, using the ctDNA in plasma the emergence of secondary KRAS mutations, which are responsible for acquired resistance in patients with CRC who had initially responded to cetuximab or panitumumab, has recently been reported [16], [35]. Using plasma-Seq we investigated whether genetic alterations associated with acquired resistance to anti-EGFR therapy can be identified by analysis of cfDNA. Plasma-Seq employs a benchtop high-throughput platform, i. e. Illuminas MiSeq instrument, and performs whole-genome sequencing from plasma at a shallow sequencing depth (i. e. 0. 1–0. 2×) to establish a genome-wide copy number profile of the tumor at low costs (<300€) within 2 days [32]. Thus, plasma-Seq allows an easy assessment about clonal evolution of the tumor genome. Furthermore, we performed highly sensitive deep sequencing for mutations in KRAS (exon 2), PIK3CA (exons 9 and 20), BRAF (V600E), and EGFR (S492R mutation in patients who received cetuximab). We conducted targeted deep sequencing for the 7 most common KRAS mutations in codons 12 and 13 (i. e. G12R, G12D, G12C, G12A, G12S, G12V, G13D), the BRAF V600E mutation, exon 9 and 20 PIK3CA mutations, and for the EGFR S492R mutation in patients who received cetuximab (Table S1). In addition, we included plasma samples from our previous study [26] with known percentage of ctDNA reflected in KRAS mutations as positive controls. Plasma-Seq allows an estimation of tumor DNA fraction in the plasma [32], which was above 10% of total cfDNA in all samples. Hence, the detection limit of deep sequencing, which is in the range of 1% [26], was sufficient for mutation detection in our plasma samples. However, sequencing of the aforementioned genes revealed mutations only in exon 9 of the PIK3CA gene in the plasma of 3 patients (#2, #5, and #8) (Table S1). Such exon 9 PIK3CA mutations were discussed not to have an independent effect on anti-EGFR efficacy [8] and in all three patients we found the same mutation also in pretreatment samples, i. e. primary tumor (#2 and #5) or metastasis (#8) (Table S1) suggesting that targeted deep sequencing has not contributed to the identification of therapy related changes in the tumor genomes of our patient cohort. Altogether we analyzed 18 plasma samples from the 10 patients, the mean coverage for the entire genomes was 0. 16× (Table S2). For comparison one representative plasma copy number profile from each patient and an example of a control, i. e. plasma-Seq from a male person without cancer, are shown in Figure S1. All plasma samples from patients showed CRC associated copy number changes (www. progenetix. org; [36]), such as loss of the chromosomal 5q22 region harboring the APC (adenomatous polyposis coli) gene (n = 1; P3_1 in Figure S1), and loss of chromosome arms 17p (n = 5; P1_2, P2_1, P4_1, P7_1, and P9_1 in Figure S1), and 18q (n = 7; P2_1, P3_1, P4_1, P6_1, P7_1, P8_1, and P9_1 in Figure S1). Interestingly, we observed loss of 8p and gains of 8q and 20q, which are among the most commonly observed copy number changes in CRC (www. progenetix. org; [36]), in all patient derived plasma samples. In order to determine whether the number of sequenced reads for an individual patient sample deviates from patterns in normal samples, we calculated z-scores. We and others [22], [32] had defined z-scores of <−3 and >3 as significantly under- and overrepresented, respectively. To this end we first calculated log2-ratios, which we used for segmentation to achieve regions with similar copy-number values [32]. These segments were then used for calculation of so-called “segmental z-scores” by comparing the respective log2-ratios with those from a cohort of individuals of the same sex but without cancer [32]. Using these z-score calculation criteria we determined genetic alterations at chromosomal levels, such as focal amplifications and chromosomal polysomies. Focal amplifications refer to high-level genomic gains of circumscribed genomic regions, often encompassing just one or a few genes. In contrast, chromosomal polysomies represent variable degrees of chromosomal gains and often affect larger chromosomal regions, i. e. chromosome arms or entire chromosomes. Losses of chromosomal regions were also determined based on segmental z-score calculations. Focal amplifications of KRAS on chromosome 12p12. 1 [15], [16], MET (7q31. 2) [17], and ERBB2 (17q12) [13], [14] have been shown to be associated with acquired resistance in tumors that do not develop KRAS mutations during anti-EGFR therapy. Furthermore, several studies reported evidence of a relationship between polysomies involving the EGFR gene (7p11. 2) and anti-EGFR efficacy. Mean EGFR copy numbers in the range of 2. 5–2. 9/nucleus in ≥40% of analyzed cells were suggested as relevant cutoff points to discriminate between responders and non-responders to anti-EGFR therapy [19]–[21]. Hence, the main focus of our study was on the regions known to affect anti-EGFR treatment response, i. e. KRAS, MET, ERBB2, and EGFR. Details on read-counts, log2-ratios, z-scores, and relative copy numbers for the segments harboring the respective genes are listed in Table S3; a summary of findings is in Table S4. Plasma-Seq indeed allowed us to observe the emergence of novel copy number changes, which were closely associated with the development of clinical anti-EGFR resistance. When patient #1 was diagnosed he already had liver metastases and only biopsies from the primary tumor were obtained. Whole-genome sequencing of primary tumor DNA (PT1) revealed multiple of the aforementioned copy number changes frequently observed in colorectal cancer (Figure 1a). When we performed our 1st plasma-Seq analysis one month later (P1_1) we observed, as expected, an almost identical pattern of copy number changes (Figure 1a). For the following 16 months the patient received various palliative treatments and was then switched to panitumumab monotherapy. Initially the patient responded with a marked decrease of tumor markers carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9) (Figure 1b). However, after 7 months of treatment radiological progression was noted (new liver metastases). At this time we obtained our 2nd blood sample (P1_2) and plasma-Seq revealed a similar pattern of copy number changes as in the previous analyses (Figure 1a). However, a novel focal amplification of the chromosomal region 12p12. 1, harboring the KRAS gene was identified (Figure 1a, b). The segmental z-score for the 12p12. 1 region was 23. 9 corresponding to a high-level gain. For the next 4 months the patient was treated with FOLFOX (FOL-Folinic acid [leucovorin]; F-Fluorouracil [5-FU]; OX-Oxaliplatin [Eloxatin]) in addition to panitumumab, which resulted in stable disease (Figure 1b). During this time, we obtained our 3rd blood sample (P1_3), which confirmed the presence of the KRAS amplification (Figure 1a, b). When a maintenance treatment with panitumumab monotherapy was tried the patient did not respond, but showed radiological progression and increase of tumor markers CEA and CA19-9 (Figure 1b). Another focal amplification on 17q11. 2 (chr17: 26,205,340–29,704,695) had already been present in both pretreatment samples (PT1) and (P1_1) and did not contain the ERBB2 gene (exact position of ERBB2: chr17q12: 37,844,167–37,886,679) (Figure S2), and is therefore likely unrelated to the development of anti-EGFR resistance. For patient #2 we analyzed a first plasma sample after a disease course of 2 years and 3 months, immediately before his therapy was switched to panitumumab because of progressive disease. Despite the long time interval between initial diagnosis and our blood collection the copy number changes in the plasma sample showed a marked resemblance to those of the primary tumor (compare PT2 with P2_1 in Figure 2a). With respect to the above listed anti-EGFR therapy relevant regions we noted gains of 7p (EGFR z-scores: PT2: 7. 79; P2_1: 10. 9) and 17q (ERBB2 z-scores: PT2: 6. 69; P2_1: 5. 6) in both samples (Figure 2a). The EGFR gain correlated excellently with an initial good response to anti-EGFR therapy as previously reported [19]–[21], because tumor markers CEA and CA19-9 decreased (Figure 2b) and when we repeated our plasma-Seq analysis two months later we observed an almost balanced copy number profile, indicating a very low ctDNA percentage (P2_2 in Figure 2a). However, another 4 months later, employing plasma-Seq, we identified a KRAS amplification (z-score KRAS: 10. 59; P2_3 in Figure 2a) for the first time. This plasma-Seq result prompted a re-staging of the patient, which indeed revealed progressive disease. Patient #3 received two cycles of cetuximab each for a 4 months period of time, which were administered 22 and 30 months, respectively, after diagnosis of the primary tumor (Figure 3). After the 2nd cycle she developed resistance as documented by increasing CEA levels and radiographic progress (increasing size of abdominal metastases). As pre-treatment samples material from the primary tumor and of a liver metastasis, which had been resected 9 months after initial diagnosis, were available. Both primary tumor (PT3) and liver metastasis (LM3) shared many copy number changes as revealed by array-CGH (Figure S3); however, chromosome 12 was balanced in the primary tumor whereas it was lost in the liver metastasis (Figure 3; Figure S3). Our plasma-Seq analysis (P3_1), performed 35 months after the initial diagnosis, identified a novel high-level gain of the entire short arm of chromosome 12, which included the KRAS gene. A chromosome 12p z-score of 28. 99 suggested that this chromosome arm was not only duplicated but present in multiple copies (Figure 3a). We also observed the co-occurrence of a KRAS focal amplification with another focal amplification of an anti-EGFR therapy relevant gene, i. e. the MET gene. For patient #4 we tried to analyze the primary tumor (PT4) as a pre-treatment sample. However, as the primary tumor was inoperable, it was not resected but only biopsied so that only very little material was available. Hence, in this case it was not possible to tell whether the analyzed material was indeed representative for the primary tumor (Figure 4a). Patient #4 was treated with panitumumab and initially responded very well. However, after 5 months with anti-EGFR treatment increases of CEA and CA 19-9 were noted (Figure 4b). In a post-treatment plasma sample (P4_1) we observed two focal amplifications, again of 12p12. 1 including KRAS and of 7q31. 2 harboring the MET gene (Figure 4a). The respective z-scores were 13. 63 for KRAS and 28. 13 for MET. Furthermore, plasma-Seq revealed gains close to the centromeres of chromosomes 16 and 17. The focal amplicon on chromosome 16p11. 2 (chr16: 32,163,432–33,818,739) did not contain any gene previously implicated in anti-EGFR response, whereas the gain on chromosome 17 did not include the ERBB2 gene (z-score: 2. 54). Although patient #5 received panitumumab and irinotecan for a period of six months, his liver metastases continued to progress. Copy number profiles of both the primary tumor (PT5) and a plasma sample (P5_1) obtained after panitumumab treatment had marked similarities (Figure 5a), although the time interval between the samples was 2 years and 11 months. In both samples we observed copy number changes in three regions which can affect anti-EGFR therapy: a focal amplification of the MET gene (z-scores PT5: 20. 6; P5_1: 19. 0), and polysomies of 7p (EGFR z-scores PT5: 12. 0; P5_1: 10. 5), and the 17q12 region (ERBB2 z-scores: PT5: 5. 7; P5_1: 5. 0). Additional amplifications on other chromosomes, e. g. on chromosome 12q13. 13-12q13. 3 (chr12: 51,639,133–56,882,181), which did not contain the KRAS gene on 12p12. 1, were also present in both pre- and post-treatment samples. These amplicons did not contain genes which have yet been discussed within the context of affecting anti-EGFR therapies. As amplification of the MET gene has recently been shown to drive resistance to anti-EGFR therapies [17], this copy number change is the best candidate to explain the poor treatment response. In patient #6 plasma-Seq (P6_1) revealed gains of 7p (EGFR z-score: 11. 7) and of 17q (ERBB2 z-score: 10. 4) (Figure 5b) prior to cetuximab therapy, i. e. a similar copy number constellation as patient #2 prior to his therapy. This patient responded also very well to the treatment with remission of intrahepatic metastases and retroperitoneal lymph nodes and low CEA and CA 19-9 levels (Figure 5b) after treatment with cetuximab for 8 months. However, treatment had to be terminated because of cutaneous side effects and as several weeks later an increase of CEA and CA 19-9 levels was observed, and treatment had to be continued with chemotherapy (Figure 5b). We obtained a second blood sample during this time, but the ctDNA fraction was very low so that we did not obtain novel information. In patients #2 and #6 we had observed increased z-scores for both EGFR and ERBB2. In both cases there was an initial good response confirming previous reports that increased EGFR copy numbers enhance response rates to anti-EGFR therapy [19]–[21]. However, at the same time amplifications of ERBB2 were reported to be associated with resistance [13], [14]. Hence, in these two cases the EGFR and not the ERBB2 copy number appeared to have determined treatment outcome. However, another patient, i. e. #7, may contribute to the elucidation of the role of ERBB2 in anti-EGFR therapy. This patient was treated with a combination of cetuximab and irinotecan after a disease course of 10 months (Figure 6). However, after only 3 months massive radiographic progress (increasing size of intrahepatic metastases and retroperitoneal lymph nodes) was noted. Plasma-Seq (P7_1) performed at this time revealed a focal high-level amplification of ERBB2 with a z-score of 196. 4. Furthermore, the short arm of chromosome 12 with the KRAS gene (z-score: 7. 3) and the entire chromosome 7 (z-scores for EGFR: 17. 5, and MET: 19. 3) were also overrepresented (Figure 6a). The only available pre-treatment sample was a biopsy of the primary tumor and immunohistochemistry and SISH (silver in situ hybridization) revealed an extensive ERBB2 immunoreactivity (immunoreactive score: 3+) and increased ERBB2 signals with a highly increased Her2/CEP17 ratio (12. 6) suggesting that the amplification had already been present at the time of initial diagnosis (Figure 6b). Based on previous reports [13], [14] the high-level ERBB2 amplification may have been the main driver for the primary resistance to cetuximab although additional contributions by KRAS and MET are possible. In 3 cases plasma-Seq did not reveal candidate regions as possible explanations for treatment response. In patient #8 we had as pretreatment samples a metastasis (M8) resected 5 months and a plasma sample (P8_1) obtained 45 months after initial diagnosis. Despite this long time interval copy number profiles of both samples were very similar (Figure S4). He had only a brief response to panitumumab treatment and relapsed within 4 months of treatment (radiological progression and increasing tumor markers). A post-treatment blood sample (P8_2) obtained 10 months later demonstrated again an almost identical copy number profile despite the progressive disease [P8_1 (z-scores: EGFR: 8. 71; MET: 7. 65), P8_2 (z-scores: EGFR: 4. 73; MET: 4. 73) ] (Figure S4). As deep sequencing had also not revealed a novel mutation, we could not find a good explanation for the resistance towards panitumumab in this case. We observed a focal amplification on 16q12. 1 (Chr16: 51,265,518–52,958,468; z-score: 19. 299), which was not present in the metastasis, in both plasma samples. As this amplicon had already been present in one of the two pre-treatment samples, i. e. P8_1, it was not acquired during anti-EGFR therapy. However, this amplicon represents an example that plasma-Seq identifies novel changes during tumor evolution, which may warrant further investigation. Furthermore, we observed low ctDNA allele fractions which resulted in relatively unremarkable copy number plots in two patients. Patient #9 responded to cetuximab for more than 2 ½ years (Figure S5). After this long period of time a mild progress and mild increase of CEA and CA 19-9 were noted. Patient #10 received treatment with panitumumab after a disease course of 10 years, yet she did not respond well. In both cases plasma-Seq analysis showed only few copy number changes reflecting a low ctDNA fraction, in patient #10 even in three serial analyses (an exemplary profile is shown as P10_3 in Figure S1). As plasma-Seq is a new technique we verified the results for the genes KRAS, MET, and ERBB2 by quantitative real-time PCR (RT-PCR). The quantitative RT-PCR results reflect relative but not real copy numbers because the fraction of ctDNA within the cfDNA varies and thus modulates the copy number. Nevertheless, the relative copy numbers of KRAS, MET, and ERBB2 showed a close correlation to the respective log2-ratios (r2 = 0. 686) (Figure S6). Furthermore, we also observed a close correlation between the quantitative RT-PCR relative copy numbers and the segmental z-scores (r2 = 0. 557) (Figure S6) demonstrating that plasma-Seq reliably detects copy number changes. Colorectal carcinomas that are wild type for KRAS are often sensitive to EGFR blockade [1], [3] and therefore KRAS testing can prevent both ineffective treatment and treatment-associated toxicity. However, to define CRC as KRAS mutant versus KRAS wild-type underestimates additional heterogeneity and calls for the identification of novel biomarkers for truly personalized medicine. Indeed, additional factors affecting anti-EGFR treatment have recently been identified, many of which are genes and proteins downstream of KRAS in the mitogen-activated protein kinase signaling pathway, such as BRAF, NRAS, PIK3CA, PTEN and AKT [8]–[12]. Furthermore, other RAS mutations than KRAS exon 2 mutations have recently been discussed as predictive markers [37]. However, despite this progress, the EGAPP (Evaluation of Genomic Applications in Practice and Prevention) Working Group (www. egappreviews. org) found only adequate evidence for an association of KRAS genotype at codons 12 and 13 with diminished treatment response to anti-EGFR therapy, but not for BRAF V600E, mutations in NRAS or PIK3CA, or loss of PTEN or AKT expression [6]. One reason for this uncertainty is the lack of genetic follow-up data in clinical studies. Often therapies are administered months or years after initial diagnosis and due to the instability of tumor genomes the status of predictive biomarkers obtained from the primary tumor may have changed over time. Recent progress in plasma DNA and CTC analyses now allows the monitoring of tumor genomes by non-invasive means [34], [38], [39]. Here we used plasma-Seq [32] and demonstrated that genetic follow-up data may include novel, acquired copy number changes, such as focal amplifications and chromosomal polysomies, which likely affect response to anti-EGFR therapy. Regarding KRAS we made several interesting observations. Recently, it has been shown that the presence of KRAS amplification directly affects response to EGFR targeted agents and that KRAS amplification is a mechanism of resistance to EGFR targeted therapies in CRC [15], [16]. Currently, the frequency of KRAS amplifications is unknown. In colorectal cancer specimen KRAS amplifications were observed in only 0. 67% (7/1039) [15] or 2. 1% (2/96) [40] of tumors. In plasma samples from patients who progressed on cetuximab acquisition of KRAS mutations were a frequent finding [16], [35], whereas KRAS amplification was only observed in one patient [16]. However, these studies used 454 deep sequencing [16] or a digital ligation assay [35], i. e. methods with a high sensitivity for the detection of mutations but unsuitable to establish copy number levels. These differences in methods may explain our surprising and unexpected finding of acquired KRAS copy number changes in 4 of 10 (40%) patients. Although it will have to be further validated in larger cohorts an intriguing finding of our study is that KRAS amplifications as response to anti-EGFR therapy may be more prevalent than currently thought. Interestingly, in patient #1 the KRAS amplification was detected in plasma DNA at a time when CEA and CA19-9 levels were still at background levels. Similarly, in patient #2 the detection of the KRAS amplification was the reason for the initiation of a re-staging, which then showed clear evidence that the patient was indeed progressive. These two cases exemplarily demonstrate the potential power of plasma-Seq. However, a potential shortcoming of our study is that we had no access to post-treatment tissues to confirm the plasma-Seq results. At the same time there were several lines of evidence supporting our interpretations: 1. We observed a very close correlation between the emergence of KRAS amplifications and the respective clinical course. 2. As mentioned before, KRAS amplifications are a recently established, novel mechanism of resistance against anti-EGFR therapy [15], [16]. 3. The KRAS amplification was the only new, acquired copy number change in plasma-Seq and we excluded genetic alterations in other oncogenes known to modulate EGFR signaling, such as mutations in BRAF [41], PIK3CA [8], or EGFR [18], [42] and ERBB2 amplification [13], [14], which are established key determinants of resistance to anti-EGFR therapies. 4. We confirmed copy number changes observed with plasma-Seq by another method, i. e. with quantitative RT-PCR. Recently it has been suggested that the development of resistance to EGFR blockade might be caused by rare cells with KRAS mutations that preexist at low levels in tumors with ostensibly wild-type KRAS genes [35]. Given that previous studies [15], [40] did not find KRAS amplifications in primary tumors in relevant numbers, it will be interesting to extend the analyses of primary tumors with more sensitive methods to identify the possible presence of such amplifications at the subclonal level. Furthermore, our data confirm previous suggestions [15] that KRAS mutations and amplifications occur mutually exclusively. However, we also made the novel observation that KRAS amplification may co-occur with other anti-EGFR associated amplifications, such as the MET gene. Although the prevalence of MET amplification in untreated metastatic colorectal cancer was also reported to be low [17], [43]–[45], we identified two of them in 10 patients. It was shown that anti-EGFR therapies may select MET-amplified preexisting clones, which may then limit the efficacy of anti-EGFR therapies [17] and this may have been the mechanism of resistance in patient #4. Activation of ERBB2 signaling, e. g. through ERBB2 amplification, leads to persistent ERK 1/2 signaling, which was shown to be the principle mechanism of both primary and secondary resistance to cetuximab-based therapy in colorectal cancer patients [13], [14]. This may be the reason why patient #7 with a high-level focal amplification did not benefit from treatment with cetuximab. The level of ERBB2 overrepresentation may determine treatment response, as lower gains in other cases did apparently not affect treatment response. Importantly, the high-level ERBB2 amplification was only noted through our plasma-Seq analysis, as from the primary tumor only small samples from bioptic procedures insufficient for a detailed analysis of the tumor genome were available. Plasma-Seq initiated reanalysis of the remaining bioptic material by immunohistochemistry and FISH, which confirmed the ERBB2 amplification. This suggests that in cases where only limited tumor material is available, a “base-line” plasma-Seq profile established at the time of initial diagnosis may help to guide therapeutic decisions. Plasma-Seq may also contribute to the identification of patients who will likely benefit from anti-EGFR therapy. For example, several studies reported evidence of a relationship between increased EGFR copy number and anti-EGFR efficacy [19]–[21]. Indeed, patients with 7p polysomies were initially good responders. In fact, an interesting observation of our study is that copy number analyses alone were very powerful to detect associated mechanisms relevant for anti-EGFR treatment. In contrast, mutation analyses did not identify novel acquired mutations in genes previously associated with anti-EGFR resistance, such as KRAS [16], [35], BRAF [41], PIK3CA [8], or EGFR [18], [42]. A further potential limitation of our approach is that we cannot assess all possible mechanisms, which can affect response to anti-EGFR therapy. For example, decreased PTEN expression can be the result of mutations, allelic loss, and hypermethylation of the PTEN promoter region [10], [46], [47]. Our approach would miss epigenetic alterations, such as hypermethylation of promoters, and furthermore it cannot establish expression levels of genes. In this study we focused mainly on regions known to affect anti-EGFR treatment, i. e. EGFR, MET, KRAS, and ERBB2. However, our genome-wide approach should also allow mapping of novel regions which may be involved in anti-EGFR resistance. However, this will require larger cohorts and could not be achieved with the limited number of samples used in this study. We demonstrated here that the status of predictive anti-EGFR markers may change in tumor genomes of patients with metastatic CRC. Thus, treatment decisions should not depend on the marker status of the primary tumor but on the current status as established by liquid biopsies [33], [34]. Hence, prospective clinical trials need to include evaluation of drug resistance mechanisms at the time of disease progression, which can now be achieved by non-invasive means. At present such trials should analyze post-treatment samples, i. e. tissue samples, in parallel to the liquid biopsies whenever possible to establish which approach yields the more representative result. Plasma-Seq is of particular utility in metastatic disease, i. e. the target population for nearly all early phase clinical trials. To this end, plasma-Seq represents an easy, fast, and affordable tool to provide the urgently needed genetic follow-up data in clinical studies. Hence, plasma-Seq may contribute to the identification of novel determinants of therapeutic response and may enable the early initiation of combination therapies that may delay or prevent disease progression. The study was approved by the Ethics Committee of the Medical University of Graz (approval number 21-229 ex 09/10), conducted according to the Declaration of Helsinki, and written informed consent was obtained from all patients. The clinical data is summarized in Table 1. All patients had metastatic CRC and were seen in the Department of Internal Medicine, Division of Oncology, at the Medical University of Graz. We could isolate DNA from pre-treatment tumor specimens in 5 patients where tumor tissue was available through surgical or bioptic procedures. In one patient immunohistochemistry (4B5 antibody, Ventana) and silver in situ hybridization (SISH, Ventana) were performed on biopsy material to establish the Her2/neu status before treatment (using a Benchmark Ultra platform). Partial data regarding copy number profiles made by array-CGH from patients #1 (P1_1), #7 (P7_1), #3 (P3_1), #6 (P6_1) were described in our previous papers [26], [48]. All plasma-Seq or whole-genome sequencing analyses and all mutation analyses presented here have not been previously published. Array-CGH was carried out as previously described [26] using a genome-wide oligonucleotide microarray platform (Human genome CGH 60K microarray kit, Agilent Technologies, Santa Clara, CA, USA), following the manufacturer' s instructions (protocol version 6. 0). The methods were described in detail previously [26], [32]. In brief, plasma DNA was prepared using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). Shotgun libraries were prepared using the TruSeq DNA LT Sample preparation Kit (Illumina, San Diego, CA, USA) following the manufacturer' s instructions with some exceptions: we used 5–10 ng of input DNA, we omitted the fragmentation step since plasma DNA has an enrichment of fragments in the range of 160 to 340 bp, for selective amplification of the library fragments that have adapter molecules on both ends we used 20–25 PCR cycles. The libraries were sequenced on an Illumina MiSeq (Illumina, San Diego, CA, USA). On the MiSeq instrument the run was initiated for 1×150 bases of SBS sequencing, including on-board clustering. On the completion of the run data were base called, demultiplexed on the instrument (provided as Illumina FASTQ 1. 8 files, Phred+33 encoding), and FASTQ format files in Illumina 1. 8 format were used for downstream analysis. Deep sequencing was performed with the Illumina MiSeq as described [48]. In brief, target specific primers were designed for all mutations as listed in Table S1 and Illumina specific adapters were attached to the 5′ ends in a second PCR run. Obtained sequence reads were base called using the Illumina MiSeq Reporter Software. Subsequently, reads were aligned to the human hg19 genome using Burrows-Wheeler Alignment (BWA, MEM-algorithm) [49] and alignments with mapping quality <15 were filtered. Bases sequenced with Phred-scores lower than 20 were masked in the alignment using an in-house script and mutations were visualized using Integrative Genomics Viewer (IGV) [50]. We set the threshold for reliable detection of a sequence variation at 1%, allelic fractions of <1% were considered as sequencing errors. We masked the pseudoautosomal region (PAR) of the hg19 genome and divided it into 50,000 windows, each containing the same amount of mappable reads. Low-coverage whole-genome sequencing reads were mapped to the PAR-masked hg19 genome and reads in each window were counted and normalized by the median read-count obtained for each sample. We further normalized read counts according to the GC-content of each genomic window using LOWESS-statistics. In order to avoid position effects we normalized the sequencing data with GC-normalized read counts of plasma DNA of controls without malignant disease and calculated log2-ratios as detailed in [32]. Subsequently, we generated segments of similar copy-number values by applying both, circular binary segmentation (CBS) [51] and Gain and Loss Analysis of DNA (GLAD) [52] reflecting a summary of both algorithms as provided by the R-package CGHWeb [53], which generate mean log2-ratios for each identified segment. The log2-ratio threshold for plotting of copy numbers was set to 0. 2 for gains and −0. 2 for losses. Owing to variable ratios of tumor specific and normal alleles in plasma, accurate copy number calculations are not applicable. Furthermore, log2-ratios only indicate the relative copy number changes and are less sensitive at low allele fractions of tumor-specific DNA. For this reasons we applied z-score statistics. Z-scores indicate whether a region is gained or lost at a significant level (i. e. z-score >3 or <−3) compared to controls, i. e. individuals without cancer. Z-Scores for each segment were calculated by subtracting the mean GC-corrected read count of controls and dividing by standard-deviation of controls. In order to check for the copy-number status of genes we calculated gene-specific z-scores (a z-score applied to test whether the copy number of a specific gene, i. e. ERBB2, significantly deviates from the control samples). Therefore, the chromosomal region of a specific gene was defined as a window and z-scores were calculated as described above [32]. However, for genes <100 kb in length gene-specific z-scores are not applicable since the standard deviation might be too large owing to the shallow sequencing depth. Hence, since most analyzed genes (except BRAF and MET) were <100 kb in length we used z-scores of the called segments where the genes are located. We used TaqMan Copy Number Assays from Life Technologies, Carlsbad CA, USA, to validate the copy number status of KRAS, MET and ERBB2. The respective assays are commercially available under the IDs Hs0239788_cn, Hs04993403, and Hs00450668_cn, respectively. We ran the TaqMan Copy Number Assays simultaneously with a TaqMan Copy Number Reference Assay (hTERT) in a duplex real-time polymerase chain reaction (PCR). The target assay contained two primers and a FAM dye labeled MGB probe and the reference assay contained two primers and a VIC dye-labeled TAMRA probe. PCR setup was performed according to the manufacturer' s recommendations with the following exceptions: we increased the number of PCR cycles to 45 and decreased the amount of input DNA to 1–1. 5 ng. The number of copies of the target sequence was determined by relative quantitation (RQ) using the comparative CT (ΔΔCT) method. Post-PCR data analysis of copy number quantitation experiments was done with Applied Biosystems CopyCaller Software. All sequencing raw data were deposited at the European Genome-phenome Archive (EGA, http: //www. ebi. ac. uk/ega/), which is hosted by the EBI, under the accession number EGAS00001000582.

Targeted therapies based on characteristics of the tumor genome are increasingly being offered to patients with cancer. For example, colorectal carcinomas that are wild type for KRAS are frequently treated with monoclonal antibodies targeting the Epidermal Growth Factor Receptor (EGFR). However, almost all patients with clinical response to anti-EGFR therapies develop resistance and underlying mechanisms are poorly understood. Because of the instability of tumor genomes the status of predictive biomarkers, such as the KRAS gene, can change during the course of disease. So-called "liquid biopsies", e. g. analyses of circulating tumor DNA, provide genetic follow-up data non-invasively from peripheral blood. When using whole genome sequencing of plasma DNA (plasma-Seq) we observed that specific copy number changes of genes, such as KRAS, MET, or ERBB2, can be acquired under therapy and determine responsiveness to therapy. In fact, our data suggest that non-invasive genome profiling is capable of predicting responsiveness or emerging resistance to anti-EGFR therapy in the majority of cases. Hence, non-invasive testing of the current status of the tumor genome can help reduce of harm from erroneous therapeutic decisions and optimize treatment for maximal efficacy and minimal side effects, which is important for decreasing metastasized CRC-related morbidity and mortality.

lay_plos

SECTION 1. SHORT TITLE AND TABLE OF CONTENTS. (a) Short Title.--This Act may be cited as the ``Small Business Opportunity Preservation Act of 1996''. (b) Table of Contents.-- Sec. 1. Short title and table of contents. Sec. 2. Statement of policy. Sec. 3. Definition of contract bundling. Sec. 4. Assessing proposed contract bundling. Sec. 5. Fostering contractor teaming. Sec. 6. Reporting of bundled contract opportunities. Sec. 7. Evaluating subcontract participation in awarding contracts. Sec. 8. Improved notice of subcontracting opportunities. Sec. 9. Deadlines for issuance of regulations. SEC. 2. STATEMENT OF POLICY. (a) Sustaining Small Business Participation in Government Contracting Despite Contract Bundling.--Section 2 of the Small Business Act (15 U.S.C. 631) is amended by adding at the end the following new subsection: ``(j) In complying with the statement of congressional policy expressed in subsection (a)(2)(B), relating to fostering the participation of small business concerns in the contracting opportunities of the Government, each Federal agency, to the maximum practicable extent, shall-- ``(1) comply with the provisions of this Act expressing congressional intent to foster the participation of small business concerns as prime contractors, especially section 15; ``(2) structure its contracting requirements to facilitate competition by and among small business concerns, taking all reasonable steps to eliminate obstacles to their participation; ``(3) avoid the bundling of contract requirements that preclude small business participation as prime contractors; and ``(4) comply with the provisions of this Act expressing congressional intent to foster the participation of small business concerns as subcontractors (including suppliers), especially section 8(d).''. (b) Conforming Amendment.--Section 2(a) of the Small Business Act (15 U.S.C. 631(a)) is amended-- (1) in the first sentence, by striking ``The essence'' and inserting ``(1) The essence''; and (2) by striking the fifth sentence and inserting the following: ``(2) It is the declared policy of the Congress that the government should aid, counsel, assist, and protect, in so far as is possible, the interests of small business concerns in order to-- ``(A) preserve free competitive enterprise; ``(B) insure that a fair proportion of the total purchases for property or services (including construction) be placed with small business concerns as prime contractors or subcontractors (including suppliers); ``(C) insure that a fair proportion of the total sales of Government property be made to small business concerns; and ``(D) maintain and strengthen the overall economy of the Nation.''. SEC. 3. DEFINITION OF CONTRACT BUNDLING. Section 3 of the Small Business Act (15 U.S.C. 632) is amended by adding at the end the following new subsection: ``(o) For the purposes of this Act, the terms `contract bundling', `bundled contract', and `bundling of contract requirements' mean the practice of consolidating two or more procurement requirements of a type that were previously solicited and awarded as separate smaller contracts into a single contract solicitation likely to be unsuitable for award to a small business concern because of-- ``(1) the diversity and size of the elements of performance specified; ``(2) the aggregate dollar value of the anticipated award; ``(3) the geographical dispersion of the contract performance sites; or ``(4) any combination of the factors described in paragraphs (1), (2), and (3).''. SEC. 4. ASSESSING PROPOSED CONTRACT BUNDLING. (a) In General.--Section 15(b) of the Small Business Act (15 U.S.C. 644(b)) is amended to read as follows: ``(b)(1)(A) To the maximum extent practicable, procurement strategies used by the various agencies having contracting authority shall facilitate the maximum participation of small business concerns as prime contractors. ``(B) Whenever a proposed procurement strategy reflects a bundling of contract requirements, such proposed procurement strategy shall-- ``(i) identify specifically the benefits anticipated from bundling the contract requirements; ``(ii) assess the specific impediments to participation by small business concerns as prime contractors and specify actions designed to maximize small business participation as subcontractors (including suppliers) at various tiers; and ``(iii) include a specific determination that the anticipated benefits of the proposed bundled contract justify its use. ``(2)(A) The Administration, acting through one of its Procurement Center Representatives (or such other employee of the Administration as may be designated), is empowered to review for a period of 30 days a proposed solicitation for compliance with the requirements of this subsection and subsection (a). The 30-day review shall occur concurrently with other reviews required prior to the issuance of the solicitation. ``(B) Within 15 days after receipt from a procurement activity of a Federal agency of any proposed contract solicitation that in the opinion of the representative would constitute a bundling of contract requirements, the representative (or other designee of the Administration) shall-- ``(i) request the head of the procurement activity to furnish recommendations to modify the procurement strategy and the proposed solicitation for the purpose of increasing the probability of participation by small businesses as prime contractors; or ``(ii) recommend to the procurement activity an alternative procurement strategy that would increase the probability of participation by small businesses as prime contractors. ``(C) Whenever the Procurement Center Representative and the head of the procurement activity fail to agree to a revision of the procurement strategy (or the proposed solicitation) under subparagraph (B), the matter may be submitted by the Administrator to the head of the agency in which the procurement activity is located for determination. ``(D) Any determination by an agency head to issue a contract solicitation with no revision of the procurement strategy (or the proposed solicitation) shall be supported by findings and an assessment addressing the matters described in subparagraph (E). Such determination and findings shall be submitted to the Administrator. ``(E) The findings accompanying a determination made pursuant to subparagraph (D) shall include-- ``(i) the estimated benefits of the proposed bundling of contract requirements, including improved performance of programmatic objectives to be met by the contract, savings in terms of acquisition costs and contract administration costs, and how such estimated benefits were calculated; ``(ii) specific adverse impacts on the participation of small business concerns as prime contractors, especially small business concerns that are performing (or have previously performed) contracts of the type that are proposed for inclusion in the solicitation for the bundled contract; ``(iii) specific actions to foster the participation of small businesses in the performance of the bundled contract as subcontractors (including suppliers) at various tiers; and ``(iv) such other matters as the agency head considers appropriate. ``(F) Unless otherwise authorized by the head of the agency for urgent and compelling reasons, the solicitation shall not be issued until the determination under subparagraph (D) has been made by such agency head and submitted to the Administrator.''. (b) Conforming Amendment.--Section 15(a) of the Small Business Act (15 U.S.C. 644(a)) is amended by striking the third, fourth, fifth, and sixth sentences. (c) Responsibilities of Agency Small Business Advocates.--Section 15(k) of the Small Business Act (15 U.S.C. 644(k)) is amended-- (1) by redesignating paragraphs (5), (6), (7), (8), and (9) as paragraphs (6), (7), (8), (9), and (10), respectively; and (2) by adding after paragraph (4) the following new paragraph (5): ``(5) identify and report on proposed solicitations that represent bundling of contract requirements, and work with the agency acquisition officials and the Administration to revise the procurement strategies for such proposed solicitations to increase the probability of participation by small businesses as prime contractors, or to facilitate small business participation as subcontractors and suppliers, if a solicitation for a bundled contract is to be issued,''. SEC. 5. FOSTERING CONTRACTOR TEAMING. Section 15(b) of the Small Business Act (15 U.S.C. 644(b)), as amended by section 2, is further amended by adding at the end the following new paragraph: ``(3)(A) A small business concern intending to submit an offer for an anticipated bundled contract may propose to the Administration for approval a team of subcontractors meeting the requirements of subparagraph (B) without regard to the requirements of subsection (o) or the regulations of the Administration regarding findings of affiliation or control, either direct or indirect. ``(B) A subcontracting team proposed under subparagraph (A) may include-- ``(i) other small business concerns; and ``(ii) business concerns other than small business concerns, whose aggregate participation may not represent more than 25 percent of the anticipated total value of the contract. ``(C) Any subcontracting team proposed under subparagraph (A) and approved by the Administrator shall be subject to such alternative requirements regarding subcontracting and affiliation or control as may be specified by the Administrator.''. SEC. 6. REPORTING OF BUNDLED CONTRACT OPPORTUNITIES. (a) Data Collection Required.--The Federal Procurement Data System described in section 6(d)(4)(A) of the Office of Federal Procurement Policy Act (41 U.S.C. 405(d)(4)(A)) shall be modified to collect data regarding contract bundling. The data shall reflect the determination made by the employee of the Small Business Administration exercising the responsibilities of section 15(b) of the Small Business Act (15 U.S.C. 644(b)) (as amended by section 3) regarding whether a particular solicitation constitutes contract bundling. (b) Definitions.--For purposes of this section, the term ``contract bundling'' has the meaning given such term in section 3(o) of the Small Business Act (15 U.S.C. 632(o)) (as added by section 2). SEC. 7. EVALUATING SUBCONTRACT PARTICIPATION IN AWARDING CONTRACTS. (a) In General.--Section 8(d)(4) of the Small Business Act (15 U.S.C. 637(d)(4)) is amended by striking ``(4)(A)'' and all that follows through the end of subparagraph (D) and inserting the following: ``(4)(A) Each solicitation for the award of a contract (or subcontract) with an anticipated value of $1,000,000, in the case of a contract for construction (including repair, alteration, or demolition of existing construction) or $500,000, in the case of a contract for all other types of services or supplies, that can reasonably be expected to offer opportunities for subcontracting in the business judgment of the contracting officer, shall-- ``(i) in the case of a contract to be awarded using competitive procedures, include solicitation provisions described in subparagraph (B); ``(ii) in the case of a contract to be awarded using procedures other than competitive procedures, require submission and acceptance of a subcontracting plan pursuant to subparagraph (C); and ``(iii) in the case of a subcontract award, require submission and acceptance of a subcontracting plan pursuant to subparagraph (D). ``(B) With respect to subcontract participation by the various types of small business concerns listed in paragraph (1), the solicitation shall-- ``(i) specify, whenever practicable, minimum percentages for subcontract participation by the various types of small business concerns listed in paragraph (1), determined in the exercise of business judgment by the contracting officer considering the matters described in subparagraph (F)(iii), that must be met for an offer to be considered responsive; ``(ii) assign a weight of not less than the numerical equivalent of 5 percent of the total of all evaluation factors to a contract award evaluation factor that recognizes incrementally higher subcontract participation rates in excess of the minimum percentages, if any; ``(iii) require the successful offeror to submit a subcontracting plan that incorporates the information prescribed in paragraph (6); and ``(iv) assign a significant weight in the evaluation of past performance by offerors in attaining subcontract participation goals. ``(C)(i) The apparent successful offeror for a contract to be awarded using procedures other than competitive procedures shall negotiate with the contracting officer-- ``(I) separate goals for subcontract participation by the various types of small business concerns listed in paragraph (1); and ``(II) a plan for the attainment of the goals that incorporates the information prescribed in paragraph (6). ``(ii) The goals and plan shall reflect the maximum practicable opportunity for participation of small business concerns in the performance of the contract, considering the matters described in subparagraph (F)(iii). If, within the time limits prescribed in the Federal Acquisition Regulation, the apparent successful offeror fails to negotiate such goals and subcontracting plan, such offeror shall be ineligible for award of the contract. ``(D) An apparent subcontract awardee shall negotiate with the prime contractor (or higher-tier subcontractor) a goal for the participation of the various types of small business concerns listed in paragraph (1), and a plan for the attainment of those goals which incorporates the information prescribed in paragraph (6). Such goals and plan shall reflect the maximum practicable opportunity for the participation of such small business concerns in the performance of the contract, considering the matters described in subparagraph (F)(iii).''. (b) Conforming Amendments.--Section 8(d) of the Small Business Act (15 U.S.C. 637(d)) is amended as follows: (1) Paragraph (5) is amended to read as follows: ``(5) [Reserved.]''. (2) Paragraph (6) is amended-- (A) in the matter preceding subparagraph (A), by striking ``or (5)''; and (B) in subparagraph (D), by striking ``or (5)''. (3) Paragraph (7) is amended by striking ``(4), (5),'' and inserting ``(4)''. (4) Paragraph (10) is amended-- (A) in the matter preceding subparagraph (A), by striking ``(4), (5),'' and inserting ``(4)''; and (B) in subparagraph (B), by striking ``paragraphs (4) and (5)'' and inserting ``paragraph (4)''. SEC. 8. IMPROVED NOTICE OF SUBCONTRACTING OPPORTUNITIES. (a) Use of the Commerce Business Daily Authorized.--Section 8 of the Small Business Act (15 U.S.C. 637) is amended by adding at the end the following new subsection: ``(k) Notices of Subcontracting Opportunities.-- ``(1) In general.--Notices of subcontracting opportunities may be submitted for publication in the Commerce Business Daily by-- ``(A) a business concern awarded a contract by an executive agency subject to subsection (e)(1)(C); and ``(B) a business concern which is a subcontractor or supplier (at any tier) to such contractor having a subcontracting opportunity in excess of $10,000. ``(2) Contents of notice.--The notice of a subcontracting opportunity shall include-- ``(A) a description of the business opportunity that is comparable to the description specified in paragraphs (1), (2), (3), and (4) of subsection (f); and ``(B) the due date for receipt of offers.''. (b) Regulations Required.--The Federal Acquisition Regulation shall be amended to provide uniform implementation of the amendments made by this section. (c) Conforming Amendment.--Section 8(e)(1)(C) of the Small Business Act (15 U.S.C. 637(e)(1)(C)) is amended by striking ``$25,000'' each place it appears and inserting ``$100,000''. SEC. 9. DEADLINES FOR ISSUANCE OF REGULATIONS. (a) Proposed Regulations.--Proposed amendments to the Federal Acquisition Regulation or proposed Small Business Administration regulations shall be published not later than 120 days after the date of enactment of this Act for the purpose of obtaining public comment pursuant to section 22 of the Office of Federal Procurement Policy Act (41 U.S.C. 418b) or chapter 5 of title 5, United States Code, as appropriate. The public shall be afforded not less than 60 days to submit comments. (b) Final Regulations.--Final regulations shall be published not later than 270 days after the date of enactment of this Act. The effective date for such regulations shall be at least 30 days after the date of publication.

Small Business Opportunity Preservation Act of 1996 - Amends the Small Business Act to state as a policy under such Act that each Federal agency: (1) foster the participation of small businesses as prime contractors; (2) structure its contracting requirements to facilitate competition by and among small businesses; (3) avoid contract bundling (the practice of consolidating two or more procurement requirements into a single contract likely to be unsuitable for award to a small business); and (4) comply with requirements intended to foster the participation of small businesses as subcontractors. (Sec. 4) Requires procurement strategies used by Federal agencies to facilitate the maximum participation of small businesses as prime contractors. Requires specific information to be included in any proposed procurement strategy that reflects a bundling of contract requirements, including impediments caused to small businesses as prime contractors. Authorizes the Small Business Administration (SBA) to review proposed contract solicitations for compliance with such requirements and to act within 15 days toward the modification of procurement strategies to increase the probability of participation by small businesses as prime contractors. Requires a determination not to modify a procurement strategy to be supported by specified findings and an assessment which addresses matters concerning contract bundling and its impacts on small businesses. (Sec. 5) Authorizes a small business intending to submit an offer for an anticipated bundled contract to propose to the SBA for approval a team of small business subcontractors (or a team of small businesses and other businesses whose participation may not represent more than 25 percent of the contract value) to perform the contract. (Sec. 6) Requires the Federal Procurement Data System to be modified to collect data regarding contract bundling. (Sec. 7) Requires, in a solicitation for the award of construction contracts of $1 million or other types of contracts for $500,000, the inclusion of provisions which specify minimum percentages of participation by various types of small businesses in subcontracting under such contracts. Requires the successful offeror for such a contract to negotiate with the contracting officer in order to meet specified goals for subcontract participation by small businesses. (Sec. 8) Authorizes notice of subcontracting opportunities to be submitted for publication in the Commerce Business Daily by the appropriate prime contractors. (Sec. 9) Provides deadlines for the publication of proposed amendments made to the Federal Acquisition Regulation or to SBA regulations.

billsum

Series Finale (Part 1 of 2) [Scene: A set dressed up like Dawson's Bedroom. Actors playing Colby and Sam are acting out a scene from Dawson's New TV show "The Creek."] Colby: Feelings and emotions have an inexplicable way of manifesting themselves in subconscious and not always se-aware behavior. Sam: Verbal deconstruction of teen angst is really outdated, Colby. There's nothing going on between me and Petey. He's just a friend. Your best friend, I might add. I have no subconscious subliminal intentions towards him. Colby: Just a friend, huh? Sam: Yes. That's all. Period. End of sentence, dissertation, and postmodern diatribe. Now can we go to sleep? Colby: So, is Petey a friend the same way you and I are just friends? Sam: Yeah... of course. Colby: That's what I was afraid of. [They lie back down on the bed together, and lie together awkwardly. The camera pulls back from the TV and we see that Joey was watching the show on the couch with a tear in her eye. Another man comes over to her.] Christopher: Thank god that's over. Joey: Tread lightly, book boy. Christopher: Watch this. This'll be fun. [He grabs the remote and turns off the TV] Joey: Hey! Christopher: Ahh... back to intelligent life as we know it. Yeah, you know that show? It's like bad airplane food. You know, the teen hyperbole, it's hard on the stomach. And the writers must sit around with a thesaurus just seeking out 4-syllable ways to abuse the English language. Joey: Well, I only watch it to torture you. Christopher: Yeah? Well, it works. I mean, who talks like that Colby guy? He's like some mutant English professor, psychotherapist freak. Joey: We can't all be brilliant literary snobs like yourself. You should read some of the crap I have to edit. Christopher: You edit my stuff. Joey: [Whispering] Except your stuff. [They begin kissing and starting to make out on the couch, when Joey stops him.] Joey: Ok, ok, ok, ok. I'm sorry. I'm sorry. I have to finish this, and I have 2 more after that. Go write a book or something. Christopher: I can't. That show has officially destroyed some very necessary brain cells. Joey: [Sighs] Get over it. What is the big deal? So I like a teen soap. So what? Christopher: The way it possesses you is what frightens me, honestly. Every Wednesday at 8:00, you enter this supernatural portal of teen angst. Joey: I have an emotional connection to it you wouldn't understand. Christopher: Will Sam and Colby ever get together? Will Sam choose Petey? Will Sam choose Colby? Find out next week as we continue to beat a dead dog all the way into syndication! Joey: You know, I think subconsciously, you like the show just as much as I do. Christopher: Spoken very much like the indecisive, noncommittal character of Sam. Joey: How dare you? [They begin kissing again when Joey stops again] Joey: Do you really think I sound like her? [Opening Credits] [SCENE_BREAK] [Scene: The Production Office for The Creek. Dawson and his assistant are walking quickly down one of the hallways, while his assistant is going through her notebook and reading things off for him.] Dawson: We'll have to loop Colby's virgin speech in the third act. His mumbling out of control. Assistant: And don't forget about the network notes. They did not clear "m*st*rb*t*" as acceptable dialogue. Dawson: It's a clinical term. Assistant: They suggested "walking your dog." Dawson: Walking your dog? Assistant: He has no idea that his character's coming out of the closet. He's going to lose it. Dawson: After lunch. I've gotta step in with the writers. [Dawson goes into a meeting room where the writers are brainstorming.] Man: They're soul mates. They have to end up together. Second man: Ah, but not in the first season. You got nowhere to go after that. Dawson: What are we talkin' about? Man: The perpetual dilemma, Dawson. Does Sam hook up with feisty Petey or soul mate Colby in the season cliffhanger? Woman: If she chooses Petey, it will break convention and surprise the audience. Man: Surprise, not satisfy. Colby is Sam's soul mate. It's destiny. Second man: This show is about twisting the convention, right? Let's break this notion of destiny and fate. Dawson, what do you think? Dawson: I think you guys are onto something. [Dawson grabs his stuff and leaves them to continue bickering. He joins his Assistant who is finishing a phone call.] Assistant: Look, I've gotta go. Here he comes. I picked up your suit for the wedding. It's hanging on the back of your door. And don't forget that the car is going to come and pick you up for the airport tomorrow at noon. Dawson: Right. Assistant: And Rebecca called to confirm dinner. Dawson: Postpone it. Again. And don't give me that look. Assistant: What look? Dawson: For the record, I'm a nice guy. I'm just... late for editing. Assistant: [Sighs] [Scene: A road on the outskirts of Capeside. Jack is driving down the road listening to the radio, when her hears a siren and looks back to see a cop car behind him. HE pulls over and the cop car stops behind him. Doug gets out of the car and walks up to Jack's door.] Doug: Looks like somebody's in quite the hurry this morning. Jack: Sheriff Doug, hey, look, I'm sorry. I'm late and I'm trying to get to class. Doug: Well, I have no choice but to issue you a citation for doing 40 in a 25. Jack: Look, I already have all these points against my license. You think maybe there's something I could do to...get you to look the other way. Doug: May I remind you, Mr. McPhee, that attempting to bribe a public official is a serious offense? Jack: Uh, you know, I was just hoping that maybe, um... I could appeal to your greater sense of compassion? [Doug leans in and kisses him] Doug: All right. Just this once, I'll let you go with a warning. Jack: Thanks, honey. Doug: No, no, never, never call me honey. Jack: Dude, it's a deserted road. Chill. Doug: Don't call me dude, either. Jack: All right, tonight? Dinner later? Doug: Yeah. I'll cook. My place. Jack: Great. I'll bring the handcuffs. Kidding. Doug: [Clears throat] Jack: I'll see you. Doug: Yeah, I'll see you. Jack... slow down. Jack: You got it. Doug: All right. [Engine starts] [Scene: Outside the new Icehouse Restaurant. One of the bus boys is cleaning up when Pacey comes over to give him a hand.] Cory: I got that, Mr. Witter. You're the boss, remember? Pacey: I'm just trying to lend a hand. The rush today was crazy. Cory: Ka-ching. That's a good thing. Pacey: Yeah, 6 months and going strong. No complaints, right? [Pacey turns to see a woman standing by the bar, and she turns and sees him.] Woman: Oh, Pacey, there you are. I've got your new menu designs along with all your revisions. Pacey: Excellent. I've been meaning to take a look at those. Hey, Cory, I'll be in my office if anybody's looking for me, ok? Cory: Yeah. Pacey: Shall we? [Cut to inside Pacey's office and the two of them are inside, when Pacey closes the door and they begins making out.] Pacey: We're gonna have to find a new pretense to meet. This is...the third time I've had to review the menu design. Woman: I'm sure no one suspects a thing. Pacey: I'm not exactly the most invisible guy in Capeside these days. I'm sure somebody's starting to suspect. Woman: As long as it's not my husband. [Scene: Jack's High School English Class. A student is reading some poetry from a book uncomfortable in front of the entire class while Jack is sitting in one of the desks towards the back.] Hampton: "We two boys together clinging... "one the other, never leaving... "up and down, the roads going north and south excursions making..." Jack: Mr. Hampton... is there a problem? Hampton: Do I have to keep reading? Jack: Well, the poem's not finished. Hampton: No, offense, Mr. McPhee, but this is a poem by a guy about another guy. It's, like, a gay poem. [Students laugh] Jack: I wasn't aware that poems had sexual orientation. [Students laugh] Jack: See, this gay poem and others like it actually got Whitman fired from his job. See, he was an outcast most of his life. But he didn't care. He loved his country. He loved the freedom that it stood for, and he celebrated the American spirit every chance that he got. And here we are 150 years later, and we're still laughing at him. [The bell rings and Jack looks to the door and sees Jen standing in the door way waiting for the class to leave.] Jack: Your assignment for this weekend is to find a way to say, through the conventions of poetry, what you are afraid to say. I want you guys to write about something that you're scared of. We're gonna read these things out loud on Monday to each other, so I would hope that you give the same courtesy to each other that you did not give to Mr. Whitman today. Have a good weekend. [Everyone leaves, and Jen walks in pushing a baby carriage.] Jack: Hey, hey, hey. Jen: Hi. Jack: Oh, let me see my goddaughter. Hey, gorgeous. [Jack leans down and looks into the carriage.] Jack: Oh, my god. [Laughs] She's beautiful. So are you. [They hug] Jen: Hi, sweetheart. Jack: God, it's good to see you. Jen: You, too. Mmm... Jack: ah, too long. Jen: So I just caught some of your act. Looks like Capeside finally hired a good teacher. Jack: Good teachers are just traumatized students trying to erase whatever went wrong with their own high school experience. Jen: Do you think that you could erase my memories while you're at it? Jack: That would be a lifetime in a nunnery. Jen: So, what's on your agenda tonight, huh? Jack: Well, I gotta have dinner with the sheriff, but if you want to catch a drink later? Jen: Yeah, sure. Gosh, 6 months in, you two are already an old married couple, huh? Jack: Yes, but with the added element of pretending we don't know each other every time we pass on the street. Jen: Oh, still? Jack: Yeah, what can I say? He's a paranoid, closeted freak... but he's my paranoid, closeted freak. You staying at the Potter B&B? Jen: Mm-hmm. I'll be there, awaiting your phone call. Jack: All right, let's get out of here before you make me late for my next class. Jen: Ok, sorry. Jack: It's good to see you. [Scene: Dawson's House. Dawson walks into the house carrying his bags, and looks around remembering a lot. When Gale comes running and gives him a huge hug.] Gale: Ah! You're here! Yay! Ha ha ha! Oh, look at you. You get handsome every time I see you. Dawson: Mom, I look terrible. I've aged 10 years in the past 9 months. Gale: Well, you wear it well. So, how was your flight? Dawson: Oh, it was ok. I've got 5 scenes to write by tomorrow, and the season finale shoots in a week, I still don't have an ending. But...other than that... Gale: I'm just so glad you're here. Dawson: And miss my mom's wedding? Not a chance. Not a chance. [Lilly comes running down the stairs and gives Dawson another hug] Lily: Dawson! Dawson: Lily. Hey, hey, hey, you got tall! Dawson, I just got Annie Hall on DVD. Wanna watch it with me? Dawson: Sure. Go set it up. [Lilly runs off] Dawson: Annie hall? [Cut to upstairs in Dawson's room. Dawson looks around and sees a Director's chair with his name on it and a poster of his show on the wall] Dawson: Hmm. [He puts down his bags and looks around and picks up a picture of him and Joey happy together.] [Scene: The Ice House. Pacey walks over to one of the tables and stops to talk to the people sitting there.] Pacey: Greg. Hey, I'm sorry to interrupt you while you guys are eating, but thanks for coming back. It's good to see you. [Pacey looks up to see Joey come walking up to the Place. She doesn't see him and she stops one of the waiter.] Joey: Excuse me. I'm looking for Pacey Witter. [Pacey runs up from behind Joey and grabs her and picks her up in a bear hug] Pacey: Joey potter! As I live and breathe! Hi! Joey: Oh! Pacey: Good god, woman, you're heavy! Joey: Watch it, Witter. Pacey: I'm just kidding. They told me you weren't gonna be able to make it. Joey: Uh...long story. Pacey: Everybody! This is Josephine Potter, official talent alumni and quality Capeside stock, so what do you say we give her a nice warm welcome home, huh? [Cheering] Joey: Pacey, is this all yours? Pacey: Mine, the bank's, several family members. You hungry? Joey: I'm starving. Pacey: Look at you. I gotta give you another hug. God, it's good to see you! Joey: Good to see you. Pacey: It has been way too long, Jo. [They hug, and as they pull apart we see that Dawson is just walking up and sees them.] Joey: Hey. [There is some Love triangle feelings again] [SCENE_BREAK] [Scene: The Ice House. Later that evening. All of the gang have met up at the Ice house and are all sitting around a large table joking and reminiscing together] [Jack and Jen come walking up to join] Jack: Hey, hey, hey, hey! Pacey: Hey! Hey, girl! [Jen and Pacey hug] Pacey: Hey, pretty mama! Jack: Ah, come here! Dawson: What's up?! Pacey: God, it's good to see you, man! Jen: How are you?! Jack: Good. I'm great. Joey: Handsome! Pacey: Ok, in honor of this momentous occasion, open bar for everyone! [Pacey goes quickly into the bar and Jen and Joey follow.] Jack: Give it up! What's up, buddy? [Dawson and Jack hug and then join them] [Cut to later around the table] Joey: Do you remember that time I painted you in the nude? Jen: Oh, you got excited and you sprouted-- Jack: Oh, whoa, hey, hey! Joey: A little bit more than I bargained for. Jack: Whatever. It's all good. It wasn't meant to be. Besides, Dawson probably would eventually kill me for taking his only soul mate, right? [All of the members of ht triangle have a small uncomfortable giggle at this] Jack: Hey, what's up with Audrey, anyway? Anybody talk to her lately? Joey: Audrey's singing backup for John Mayer. She's touring Europe, and she's got some boyfriend she calls the anti-Pacey. He's totally boring and... really sweet or something. Pacey: And "really sweet," as opposed to the actual Pacey? And that from my ex-girlfriend, no less. Joey: Pacey...thank you so much for reopening this place. I did not know how much I missed it. Pacey: Maybe if your daddy hadn't burned it down in the first place, it'd still be yours Joey: Ohh, nice, Pacey. Nice. Dawson: I couldn't write this stuff if I tried. Joey: How long has it been? Pacey: Not long enough, apparently. Jen: Oh Dawson... remember when I de-virginized you? Pacey: What?! Jack: Ok. Uh, yeah, on that note, maybe we should, uh, depart. Pacey: What do you mean? You can't leave now. We're just getting to the good stuff. Jack: Yeah, I'm afraid she's gonna divulge a little too much info. Jen: Oh, my goodness. Jack: Here, I'll give you a ride. [He picks Jen up and flings her onto his shoulder] Jen: I'm sorry. Oh, no! Jack: We're out. Jen: Hey, guys, remember the time when my boyfriend knocked me up and left me to raise a baby on my own? [Awkward silence] Jack: Ok, see you at the wedding. Jen: Wait, stop. One more thing. Oh, god, you guys are the best friends I've ever had in my life. I love you so much. That is all. Good night. Jack: We'll see you. Pacey: Good night. Joey: Good night. Pacey: And on that note, get the hell out of my restaurant. I gotta clean up. [Joey and Dawson get up to leave.] Pacey: You guys need a ride? Joey: I'm gonna walk. Dawson: I can drive you. Joey: That's ok. It's right down the block. It'll sober me up. Dawson: All right. Joey: Good night, Dawson. [They hug] Joey: Pace, thank you. [Joey hugs Pacey] Pacey: A pleasure, as always, darlin'. Walk safe. Joey: I will. Bye, boys. [Joey leaves, and Dawson gives Pacey a hand cleaning up all the beer bottles on the table] Pacey: My god, that woman's amazing. Dawson: Yeah. Pacey: Man, that girl you cast on your show cannot hold a candle to her. Dawson: I know. I did nail Petey, though. That's perfect casting. Pacey: Yeah, well, he does have the certain requisite roguish charm, I guess. Dawson: Whew. Man... a lot's changed. You've changed. Pacey: Yeah, well, life happens. So, are you happy? You know, with everything that's happened, you happy? Dawson: The stock answer's a resounding yes. Anything else, I'd sound like a whiny Hollywood brat, right? Pacey: Yeah, but right now you're talking to me. Dawson: To be honest with you, I haven't given it much thought. You? Pacey: Heh. Uh... oh, you know me. I'd be miserable if I was happy. You don't have to help me do this. It's my job, remember? It's good to see you, Dawson [They hug]. Dawson: You, too. Pacey: I'll see you tomorrow at the wedding. Dawson: All right. Welcome home. [Scene: Potter B&B. Jack is changing Amy's diaper when Jen comes into the room and is taking a couple of pills. ] Jack: The last time I checked, this was not on the list of godparent job requirements. Amy: [Crying] Jack: That's ok, that's ok. Jen: You're doing just fine. Jack: Is she always this sad naked? [Jack puts the wipes away and finds a pill bottle] Jack: What's this? Jen: Oh, they're grams'. I just keep it on hand in case she forgets it. Jack: I didn't know she was still on painkillers. She looks like she's in good health. Jen: Looks can be deceiving that way. Jack: Oh, it's ok, it's ok. There you go. [He hands Amy to Jen] Jen: Mmm... Jack: you ever wonder if you could survive without her? Think you could really do this alone? Jen: I used to think I couldn't, but, uh, really, I don't feel that way anymore. It's really wonderful-- parenting. I mean, I can't program my Tivo, but I can take care of a child. Jack: You've changed the most. Out of all of us. Jen: If I have, it's not because of me. Amy changed me. I mean, I'm just along for the ride. Jack: [Sighs] What am I doing here? I mean, what sane gay man of this era goes back to a suburb? Jen: Jack... I don't think this is about Capeside. I think you're... you're frustrated with Doug. Jack: I know. I mean, I feel like I'm back in the closet, Jen. Although this time it's not in my closet. Jen: Well, you have to give him some time. Jack: Why should I? I mean, come on, it's not my fault my boyfriend lives back in the stone age. Jen: I know but he's so scared. Jack: What's there to be scared of? Jen: Of what's real. It terrifies us. All of us. And you say that I've changed. And you're right. But, I mean, I went kicking and screaming. And if you think that anything of any value in this world comes at an easier price, you're wrong. Jack: Your wise, sage superpowers have increased with motherhood. I think I might have to buy you a cape pretty soon. Jen: You can be my sidekick, hmm? My boy wonder. Jack: Anytime. [Scene: Dawson's Room. Dawson is sitting at his desk, and staring at his laptop computer screen, but cannot figure out what he wants to type. He is startled by a sound coming from outside the window and he grabs his laptop ready to use it to hit whatever comes through the window and it is Joey who falls to the floor while trying to climb through the window.] Joey: Oh! Ow! Ow. Dawson: You scared the hell out of me. Joey: That's not as easy as it once was. I think I might have broken something. Dawson: What are you doing here? Joey: Well, I was, um... walking to the B&B, and I just decided to keep walking. I haven't been home for so long and... did you know they put up a McDonald's on Hudson? We've officially been invaded, Dawson. We are one Old Navy away from being destroyed. Dawson: Are you ok? Joey: Oh, yeah. I'm fine. So, I, um... I kept walking, and then... it was too late to ring the bell, and I saw the light on, so I thought, why not? Were you sleeping? Because I can leave. Dawson: No, no. I'm glad you're here. We didn't really get much alone time tonight. There's so much I want to know about you and New York and... everything. Joey: Ow. That did hurt. [Laughing] [Scene: Outside the Ice House. Pacey is locking up as the rest of the Employees have just left the building. ] Guy: See you later, boss. Pacey: Good night, guys. Woman: Good night. [He locks the door and turns to see a guy standing there] Pacey: I'm sorry, we're all closed up for the night. Man: Oh. I'm guessing you know why I'm here. Pacey: [Sighs] Well, it's a long shot, but... sleeping with your wife, maybe? Ok, well, I'll make it easy on you. I deserve it. Do your worst. [Pacey turns to see two other guys walk up, and they are both rather large men.] Pacey: Heh heh. Oh, now, look. I've been drinking a bit tonight, so you could probably take me out all by yourself. Man: Yeah, what's the fun in that? Pacey: [Sighs] [Pacey hits the first guy, and tries to run when the other two grab him, and the three of them begin beating him up.] [Scene: Dawson's Bedroom. Dawson and Joey are still there sitting down on his bed and talking to one another about how they have been spending the past few years.] Joey: Well, I met Christopher when I was assigned to proof his book. This was before I got promoted to junior editor. Dawson: Ok. Joey: And we bickered and argued so much, so we naturally started dating, and we've been fighting ever since. Dawson: Is it serious? Joey: Um... well, we're-- we're definitely... at that moment when it either is or isn't. I don't know what's gonna happen. What about you? Anything going on in L.A.? Dawson: [Sighs] I'm absolutely, 100%, intensely committed to my show... and nothing else. Joey: Well... that's today. It's not always gonna be like that, Dawson. Dawson: I... I'm not complaining. But... sometimes I feel like... what am I doing? You know, I wanted to be Spielberg. Joey: Dreams aren't perfect, Dawson. They come true, not free. Dawson: Nicely put. Joey: Someone famous said it. Dawson: Hmm. I'm just so tired. Joey: I'll go. I'll let you get some sleep. Dawson: No, no. I meant just tired...in general. Joey: Well, I should go anyway. It's late. We have a wedding tomorrow. Dawson: All right. I'll drive you. [He stands up and lets out a huge yawn] Dawson: [Yawning] On second thought, no, I won't. Go to sleep. Joey: What? Dawson: You know the drill. That's your side, that's mine. Lie down and go to sleep. Joey: Are you kidding me? Dawson, I can't. Dawson: Hey, you already climbed through the window. It's only fitting, wouldn't you say? No one's pining away for each other or masking their feelings. So, just sleep for sleep's sake. Joey: Works for me. We are adults. [They get in bed and try to get to their positions] Joey: Good night, Dawson. Dawson: Good night, Joey. [The camera pulls away and we see them both laying there awkwardly together] [SCENE_BREAK] [Scene: A wedding outside. Dawson and Joey are standing as bride and groom in front of the preacher giving their vows.] Joey: We've been through so much, Dawson. So many good times and bad. When I loved you, you loved Jen. And when you loved me, I needed to be on my own. So I left you for Jack, and then he realized he was gay. Dawson: And then I convinced you to turn your dad in for trafficking cocaine, and...you said you'd never speak to me again. Joey: But I did. I offered myself to you at that party after you crashed your dad's boat. Dawson: And I refused... for some reason. And so you fell for Pacey. Joey: And years passed... until finally here we are... saying, "I do." The way it should be... the only way it can be for star-crossed, ill-fated soul mates. So, I do. Dawson: I do, too. [They leaning and kiss and everyone applauds. The camera pulls back from the kiss and we wee Sam and Colby there instead of Joey and Dawson.] Dawson: Cut! [Dawson sits up in his bed waking up form the dream. He looks over at the pillow next to him, and there is a note from Joey. "See You Later, Joey"] [Scene: Outside along the waterfront. Doug and Jack are jogging together and are coming up to their place, and they begins to walk together to cool off before going inside] Doug: Hey, I booked us a room for next weekend at Stonybrook lodge. You know, I figured we could do a little hiking, some mountain biking, and, you know, other various activities. Don't look so excited. Jack: I--I mean, you know, do you think that's what we need right now, another weekend away from Capeside? Doug: Oh, I'm sorry I made the mistake of planning something nice for us. Jack: No, I appreciate it. I do. You know, it's just, I'd like it a little bit better if we were hanging out here. Doug: Look, you knew when we started seeing each other that I wasn't ready for all of Capeside to know my business. Jack: Doug, that was 6 months ago. You know, we do live in a post Will & Grace world. I mean, do you really think people still care who you sleep with? Doug: Yeah. Yeah, I do. Jack: Why do you even care what anybody thinks, anyway? I mean, your family knows. They have no problem with it. Pacey, he couldn't be happier. Doug: That's because Pacey gets the last laugh. Jack: No, it's because Pacey loves you, and so do I. Doug, this really sucks that we have to go through this. I mean, how many weekends away are we gonna have to go on before we can be together like a normal couple? It's like we're having some kind of affair, yet neither one of us are even married. Doug: Not all of us were fags at 15, Jack. It's not so easy for some of us. Jack: I--I can't even believe you just said that. Doug: I'm--I'm sorry. I didn't-- Jack: No, you're not. You know what the difference is between you and me, Doug? You were a fag at 15. You just haven't stopped hating yourself for it. [Scene: The reception after Gale's wedding. Gale and her new husband are cutting the wedding cake. Dawson goes up to her and gives her a hug after she cuts the first slice. ] Dawson: I'm so happy for you. And dad would be, too. Gale: Thanks, honey. [Cut to later. Pacey walking along, and sees the married woman, and she turns and sees him and mouths "You and me later" to him. He walks on, and sees Jen and goes over to her, and she is taking another couple of pills.] Pacey: Hey... Jen: Oop! Pacey: What you taking? Jen: Oh, you caught me. I'm medicating. I'm an anxiety-ridden mother. They help. [He hands her his wine so she can wash down the pill] Jen: Thank you. Pacey: You, uh...mind sharing? 'Cause I could really use it today. [He takes off his sunglasses to show the nice shiner he has] Jen: Oh, no. Pacey: Oh, yes. [HE looks over and sees the woman who is looking over at him still] Pacey: Oh... [Cut to later and Pacey and Joey are dancing together.] Joey: So, are you gonna tell me how you got that shiner, or am I gonna have to assume that you beat up the priest before the ceremony? Pacey: Let's just say that mistakes were made. Joey: What's going on, Pace? Pacey: What's going on? What's going on is that I forget how much fun I have when I'm with you, and it's really, really nice to be reminded. [Jen and Dawson who are dancing together come over to join them] Jen: Hey. Let's switch. Dawson won't let me lead. Pacey: Ok. [The switch partners] Dawson: Hey. Joey: Hey. Dawson: You left in a hurry this morning. Joey: Yeah. Well, I had to get ready. Plus you were snoring, plus... the whole deja vu thing was kind of hard to take. I mean, it was kind of like an acid flashback without all the colors and stuff. I mean, I guess. Not like I've actually ever done acid. Dawson: I'm glad you're nervous, too. Joey: What do you mean? Dawson: You ramble when you're nervous. Joey: Is this "rag on Joey Potter day"? Dawson: I like that you ramble when you're nervous. I like that I know that you ramble when you're nervous. I like that I still make you nervous. [Jen and Pacey come over again] Jen: [Laughing] Pacey's stepping on my toes. Will you take him back, please? [They trade partners again] Pacey: It's a conspiracy. New shoes, I think. Joey: New shoes. Pacey: Must be the new shoes. [Joey and Pacey dance together and Joey notices the woman staring at Pacey.] Joey: Ahem. Pacey, I think we may have an audience. Pacey: Oh, boy... Joey: Nice to see some things never change. Still breaking hearts? Pacey: Yeah. Her heart, my jaw. Joey: It's all starting to make sense. Pacey: Do you want to help me out here? Joey: How? [He dips her and gives her a deep kiss.] Pacey: Thanks. [The woman storms off. Jen and Dawson are dancing together when Jen collapses to the ground, out cold.] Dawson: Whoa, whoa, Jen! We were dancing. She just collapsed. Jack: Jen! Grams: My god. Somebody get her bag. She needs her pills. Pacey: I just saw her take one. Grams: Oh. Dear lord, this should not be happening. Jack: What's going on? Grams: Hurry up, get an ambulance. Dawson: Ok. What's wrong with her? Grams: For god's sake, hurry up. She needs to go to the hospital now! Jack: Grams, what the hell is going on? Grams: She is sick. Jack: What do you mean, sick? She was fine. Grams: She's not fine. She's very sick. It's her heart. Oh, dear god. My baby Jen. Oh, Jen. Ooh. [SCENE_BREAK] [Scene: The hospital waiting room. Dawson is pacing worriedly as the rest are sitting in their chairs uncomfortably. While the doctors are in looking at Jen, and she is still unconscious.] Dawson: It's been hours. Why won't they talk to us? [He finally takes a seat with the rest of them] Dawson: What's going on? Grams: Well, they're monitoring her. Her vitals have been compromised. And, uh, they need to confer with her New York doctors before they can tell us anything. Joey: What's wrong with her? You know, don't you? Grams: It's a small problem, really. It's--it's been there all along. We just didn't notice it until the pregnancy. There's an abnormality in her heart, which is why she fainted. It's nothing serious, really. I mean, she'll be more embarrassed than anything else when she wakes up. Joey: Really? Grams: Yes, really. [The doctor comes over] Doctor: Excuse me Mrs. Ryan. [Grams goes over to talk to the doctor] Joey: Jack, did you know anything about this? Jack: No. Pacey: What is a heart abnormality? Is that like a murmur or something? 'Cause I don't mean to be an alarmist, but that sounds serious to me. [Grams comes back to talk to them.] Grams: All right. She's stable. She fainted from a palpitation caused by her medication. We won't know anything more for now. Um, so there's nothing much else to be done... at the moment. Look, I really think you should all just go home and rest. It's been...dramatic, and I--I... please, just-- just go now. Dawson: Is she gonna be ok? Grams: Of course she's gonna be all right. Her mother's in Europe. I, uh... I really need to-- to call and... excuse me. [Scene: Pacey's Car. Pacey is driving Dawson and Joey back to their places. Dawson is in the front seat and Joey is in the back.] Joey: I'm worried. This isn't good. Dawson: She's gonna be fine. Right? I mean, we don't know anything. Let's not jump to conclusions. Pacey: Yeah. And she's young. She's healthy. Dawson: Best thing we can do is just be ourselves-- carry on in our typical, usual, distracting... Pacey: Sordid love triangle ways. Dawson: [Laughs] Leave it up to you to say the most inappropriate thing possible. Pacey: Aw, I'm always dependable, my friend. Joey: So very not funny. [Cell phone rings] Joey: Hi, Christopher. Dawson: And the triangle becomes a square. Pacey: Well put. [Scene: The hospital. Jack is pacing in the hallway outside Jen's room. He notices, Grams and the doctor come out of the room, and begins to make his way over to them. She is crying, and he gives her a hug to comfort her. We cut to inside Jen's room. She is now conscious and Jack comes into her room to join her] Jen: Hey, you. Jack: Hey. Jen: What are you doing here? It's late. Doesn't this hospital have visitation hours? Jack: I, uh... flirted with a nurse. I can be quite charming when I want to be. Jen: What happens when she finds out you're gay? Jack: It's a male nurse. Jen: Cute? Jack: Look, Jen... I would love nothing more than to engage in our patented, meaningless, good-humored Jack-Jen fag-hag banter, but... first I was kinda wondering, I mean, since you're lying here in this hospital bed and you're hooked up to all these machines... how come? How come you didn't tell me, 'cause I thought I was your best friend? Jen: Because I didn't want you to be worried. I was already worrying enough. Because I thought that if I pretended it didn't exist, it would just go away. Because I like it. I like our... patented, meaningless fag-hag banter, and 'cause I was scared if I said it out loud, it would be true, because I was just-- I was just scared. 'Cause I was an idiot. Jack: You are an idiot. I--I could've handled it. I could've helped you handle it. Jen: Well, you will help me handle it. I need you. Jack: Jen, straight up. How bad is it? Jen: Decreased left ventricular-systolic function. It's a whole blood-not-pumping-right to my heart. And it's causing a lot of problems in my lungs. It's called pulmonary congestion. Jack: All right, so what do we do about it? I mean, what, surgery? Treatment? What? Jen: Nothing. I have been doing everything. And at first, the odds were good. But... you know me and odds. Jack: Jen, there's gotta be something we can do. I mean, what about a specialist? Jen: I've been there, repeatedly. I'm so sorry laying this all on you like this. I really thought that I'd make it to Capeside and back in one piece. Yeah. Yeah. Been trying to get ok with this, but I can't do it alone anymore. 'Cause I am gonna die, Jack. And like everything else in my life, I don't really know how to do that. But I'd like to not screw up. I'd like it to be something that I get right for once. Jack: I'm here. I will do anything that you want me to do. Jen: Right now... I want you to get in bed with me and tell me all about this cute nurse and make me forget everything that I just told you. Jack: Yeah. Jen: Come here. Yeah. Come here. It's ok. Now tell. Jack: [Crying] His name's Max. Jen: Mm... I like max. Jack: He has a goatee. Jen: Hmm. We could work on that. [Scene: A montage of scenes before the commercial. First Pacey sitting along drinking a beer outside his restaurant, when he sees Jack come walking up, and they hug as Jack tells Pacey. Cut to the Potter B&B. Joey is sitting with her head on Jack's shoulder and Bessie is sitting with them on the couch as he tells them about Jen. Cut to Dawson hanging up the phone after Jack has told him. Gale and Lily are standing in the next room looking reassuringly at him. Cut to a Jen, Grams and Amy sitting together in the hospital bed. Cut to Dawson opening his door to find Joey outside. They silently grab one another in a deep heart felt hug, and never release one another. Camera fade to black.]

Five years have passed and everyone is reunited in Capeside for a special wedding. Dawson has been living in Los Angeles producing his autobiographical television series The Creek and has never forgotten the love of his life, Joey. She is now a successful book editor living in New York City with her boyfriend, Chris, but comes home to realize she still loves her childhood sweethearts. Pacey is the owner of the new Ice House and still carries a torch for Joey, Jen is a single mom living with Grams in New York and managing an art gallery in SoHo, and Jack is a teacher at Capeside High and has fallen in love with Doug who is now the Sheriff of Capeside. Together all the secrets come out as tragedy and love becomes clear. With Jen in the hospital dying from a terminal heart condition, the gang tries not to be too sad about it. Dawson helps Jen make a video for her daughter, asking her to never stop loving and dreaming. Jen also asks Joey as her last wish to end the chasing and running and to finally pick between Pacey and Dawson. Joey tells Jen that she has always known who she is supposed to be with. Jack tells Jen that she's his soul mate and asks to raise baby Amy for her. Jen passes away and while mourning the death of their close friend, the gang has to move on. Doug tells Jack he's willing to spend his life with him and wants to help raise Amy. Joey breaks up with Chris over the phone while she tries to decide whom she will choose: Dawson or Pacey. After Jen's funeral, Pacey privately tells Joey that she's "off the hook"; despite his endless love for her, he can't hold her back any more despite them growing up together and relating to each other over their unhappy childhood and adolescence; Joey dealing with her mother's death from cancer and her father's imprisonment for drug trafficking, plus Pacey dealing with his outrageously immature and neglectful parents. But as much as Joey wanted, all her life, was to leave Capeside and travel the world, Pacey seems forever destined to be stuck there just like his alcoholic loser father and grandfather, etc. Before returning to New York, Joey sits with Dawson and confesses that even though they may never be together, he and she are soul mates and nothing should ever change that, but, in the end, she decides it is Pacey she wants to be with. In the final moments of the series, Dawson answers a phone call from Pacey and Joey who have just watched an episode of The Creek. Dawson tells them that he's meeting someone tomorrow and Pacey and Joey figure out it is Steven Spielberg, Dawson's idol. The episode ends when the camera lingers on a picture on Dawson's desk of him and his friends before cutting to a scene of them all walking down the beach from season one. In 2011, the whole two-part finale was ranked #16 on the TV Guide Network special, TV's Most Unforgettable Finales.[2]

summ_screen_fd

[PREVIOUSLY_ON] Art: You're hit! Alison: [panting] No, i-it's not me. It's you. Tim: He lost a lot of blood. He went out in the ambulance. Hasn't opened his eyes since. Dewey: That's my heroin! Boyd: Dewey ... Dewey: Or you don't think I'll shoot?! I will shoot ... the way I shot Wade Messer! Raylan: We got you on tape, Dewey, talking heroin, big dreams. Dewey: You heard that? Raylan: About Messer, too. Raylan: You're saying you're going after Boyd Crowder? Vasquez: We're going after him under the rico statute. We're gonna bury him. Raylan: And you want me to help? Rachel: Before you go? Yeah. Katherine: You were lousy at running heroin, Boyd Crowder. But from what I'm told, you are really good at robbing banks. Raylan: I came here to go over the rules. How you make a call. How you record a conversation. Ava: How I send an emergency signal. What to do if I'm in danger. Yeah. Raylan: And you're clear? Ava: Mm-hmm. Raylan: Then we're good. Winona: Are you serious? This is ... is really happening? Raylan: Paperwork's been filed. Just as matter of routine approvals. I'll be there, all moved in, in a few weeks. God damn, she's beautiful. Oh, little lady, you're wide awake, aren't you? [baby cooing] Want mister turtle? [sighs] I'll tell you a secret. Your daddy is pretty tough, but he is no match for the graveyard shift. [cooing continues] [chuckles] Oh, Raylan, what in the world is worth missing this for? [loud music] [horns honking] [all conversing in Spanish, laughter] [scoffs] Raylan: Bourbon. Tequila. Aguilar: Hey, yanqui. If you're looking for the whorehouse, it's just around the corner, just past the Starbucks. [laughter] Raylan: I'm looking for a Federale named Aguilar. Don't suppose that's you? Aguilar: It depends. Raylan: On? Aguilar: What it is you want. Raylan: [exhales sharply] Never really developed a taste for tequila. Kind of hard to understand how you make a drink out of something like that ... sharp, inhospitable. Same reason I never understood the fascination with the artichoke. Aguilar: Who are you? D.E.A.? Raylan: Now, bourbon is easy to understand. Tastes like a warm summer day. Aguilar: Texas Ranger? Raylan: U.S. Marshals service. Just got a couple questions, I'll be on my way. Four weeks ago, you reported finding an abandoned truck in the desert with the bodies of some drug-running Americans. Aguilar: You read the report. I have nothing more to say. Raylan: Not looking to jam you up. Makes no difference to me if you hijacked the truck hoping to find some drugs, instead got a wagonload of dead gringos. Don't even care if a little money was exchanged. How you boys do things down here is none of my business. I just want to know about the other men in that desert, the ones that walked out alive. [billiard balls clack] Aguilar: [chuckles] You walk through that door, putting your hat on like you mean business, like god gave you a big set of swinging cojones. Look around you. This is Mexico, cabron. And that star you wear don't mean sh1t. Now, a ranger badge ... ah, a ranger badge ... that means something. They bang it out of a 1948 Mexican silver coin. And [scoffs] you don't even have a gun, which is a good thing, or else I'd stick your yanqui ass in a Mexican prison where you can eat sh1t for all I care. You take that worthless star and get out of my city. Raylan: Thank you for the drink. I'll be in touch. Aguilar: [speaking Spanish] This is Mexico, cabron! [speaks Spanish, knocks on table] [drunkenly singing in Spanish] [chuckles] [all conversing in Spanish] [laughs] [drunkenly singing in Spanish] [keys jangling] [dog barking in distance] [insects chirping] [grunting] [engine turns over] [tires squeal] [barking continues] [groaning, breathing heavily] Raylan: I told you I'd be in touch. [music] [car door closes] Welcome to the United States of America, chief. Around these parts, this star means something. [groans, spits] Raylan: Feel like talking? [dogs barking] [faucet squeaks] [tires screech] [gearshift clicks] Boyd: You want to smoke that sh1t, do it outside my truck. Joyce: And one more. [stamper clicks] Boyd: "I believe that banking institutions are a greater threat to our liberties than standing armies." Thomas Jefferson. [stamper clicks] Joyce: Right this way, Mr. Crowder. Would you like a private room, Mr. Crowder? Boyd: No, ma'am. I believe this will be a quick one. [door closes, lock engages] [keys jingle] The Pig: Excuse me! Can I please get some help? Boyd: I'll be fine. Joyce: Thank you, Mr. Crowder. The Pig: Thank you. Joyce: You're welcome. [briefcase clicks] The Pig: [in distance] I appreciate your help. [indistinct conversation in distance] [bottle spraying] [camera shutter clicking] [title theme] On this lonely road, trying to make it home doing it by my lonesome, pissed off, who wants some? I'm fighting for my soul, god, get at your boy you try to bogard, fall back, I go hard on this lonely road, trying to make it home doing it by my lonesome, pissed off, who wants some? I see them long, hard times to come [birds chirping] [bang in distance] [bed creaks] Ava: [sighs] [vehicle door closes] [sighs] [whirring] Boyd! [whirring stops] Where's Hollis? Boyd: [sighs] I let him go. Ava: Yet I hired him to do the job. Boyd: Well, Ava, I figure what's mine is yours, what's yours is mine, so I... thought I'd just do it myself. Besides, all that's left is to paint. I can get to that tomorrow. Ava: Then what? Boyd: You tell me. Ava: [sniffs] I got to wake up. Do you want a coke or a coffee? Boyd: Well, you got a cold beer? Ava: I do. Boyd: You got two? Ava: I just got out of bed. Boyd: I'm just trying to make you smile. Look, Ava, I know what you're going through. Being in prison ain't easy on anybody. [i]I mean, I have been there ... more than once. If you want to talk about it... I mean, hell, girl, if you want to talk about anything, really. Ava: Let me get you that beer. [bottles clinking] [cap clatters] [bottles clink] [cellphone vibrates] [SCENE_BREAK] [sighs] [telephone ringing] [knock on door] Mexico went well. Raylan: How'd you know? Rachel: You seem happy. You never seem happy. Raylan: What are you talking about? I'm always happy. Rachel: You found the federale? Raylan: Found him, had a very fruitful conversation, and in return, I set him up with some nice accommodations in the El Paso lockup so he doesn't disappear. He gave me a witness who was with Boyd when Johnny Crowder and the rest were killed. We get his testimony, Boyd's looking at murder one. Make that the centerpiece of your RICO case. Rachel: Who's the witness? Dewey Crowe. Vasquez: Dewey Crowe? Dewey Crowe. Rachel: Should I tell him, or you? Vasquez: Dewey Crowe is being released. Raylan: That's unfortunate. Vasquez: Charges didn't stick. Rachel: His confession to killing Messer ... he said he was just joking. He also says that he took the heroin just to keep it off the streets. Vasquez: You know, doing his civic duty. Raylan: Call Reardon. Get him to block the release. Rachel: It's not that simple. Raylan: Why not? Vasquez: Because of your history with Mr. Crowe, Raylan. I'm sure you remember the federal government awarding him 300 grand a couple months back, tacitly admitting harassment by the Marshals service, in the person of Raylan Givens. Rachel: To which his lawyer believes his current detention bears more than a passing resemblance. Vasquez: Bottom line is, Dewey Crowe's getting out in about three hours. Raylan: Well, maybe that's not such a bad thing. Yeah. Screw it. Let's not even fight it. We'll give him some rope. Dewey's gonna screw up again. When he does, I'll be there. Vasquez: Except lawyers being lawyers, she's petitioned that you stay 1,000 feet away from him. Raylan: Now I got to keep my distance? Rachel: Pretty much. Raylan: But we get Dewey, we get Boyd. That is why I'm here. Vasquez: Yes. Officially, my boss is not gonna let me let you violate Dewey Crowe's civil rights. So... what are you gonna do? [indistinct speaking over intercom] [door buzzes] Man: You're off the chain, man. Good luck. Guard: Keep moving. [gate buzzes] Dewey: [sighs] Thank you, Jesus. Raylan: ?como esta, Dewey? [bird squawking] Dewey: Christ... Raylan? Raylan: How's it going, amigo? Dewey: We ain't amigos. Raylan: Oh, so, you speak a little Spanish. That must have come in handy down in Mexico. I got no idea what you're talking about. Raylan: Federale named Aguilar who says different. Dewey: Hey, my lawyer told me you come within 1,000 feet, that's harassment. Raylan: You best back up. Dewey: They got cameras all over. His description fits you to a "T" ... Nazi tattoo, funny hair, stupid-looking. Dewey: That could be 10,000 people. How am I supposed to read that? It's in Mexican. Raylan: Oh, then you just have to take my word for it. Mexican government wants to extradite you back to Nuevo Laredo, put you on trial for the murder of Johnny Crowder and a truckload of others. Well, I ain't never been to Mexico in my life. Raylan: That's not what's in question, Dewey. I didn't kill Johnny Crowder. Well, then tell me who did. Maybe I can help you out. Yeah, I ain't a rat. And like I said, I wasn't even there. Raylan: Then you got yourself a problem. I can give you maybe a week to jog your memory, and then it's off to a Mexican jail. And we both know that's a world away from titties and tequila. Yeah, I'm done talking. I got a bus to catch. Raylan: You're a card in fate's right hand. Don't you see how it's gonna play out? What the hell does that mean? Raylan: It means you need to be smart. What I need... is a $6 blow job. A smarter move, I cannot imagine. [sighs] [bird squawking] [insects chirping] [music] [dog barking] Dewey: Oh, you're kidding me, man. Goddamn government seizure? [bird squawks] [indistinct conversations] Mina: Get you something? Dewey: Teena? Mina: Mina, Dewey. And it's, uh, back to Abigail. Dewey: What the hell happened? Mina: When Audry's closed down, put me at the crossroads. I didn't know which way to go. The next day, God sent me a sign ... "now hiring." Dewey: What happened to Mina? I mean Teena ... or whatever the hell. Mina: I don't know. She wasn't my sister, exactly. Dewey: It was nice when you played like you were, though. Huh? [chuckles] Mina: You want something Dewey? On me. Dewey: Ooh, hey. [chuckles] What you got in mind? Mina: Um, how about the twofer plus two? It's two pancakes and two eggs with either two slices of bacon or a couple of links. Dewey: W... [chuckles] Any chance I can maybe jam one last bone in you? Reggie: He bothering you? [sighs] I'm okay, baby. Dewey: Didn't I knock him out? Well, look at you. Got your freedom, and then ... bam! ... New job, new man, whole new deal. Mina: Not like I thought about it too hard. Just kind of happened, like fate or something. Dewey: Nah, you saw a sign. Well, I'll be damned. Don't you get it? Don't you see? You lost it, I found it, and then I found you. It's my sign. Mina: Okay. Well, I got to get back to it. How about that twofer? Dewey: No, no. I'm good. But thank you. Thank you! Mina: Anytime! [birds squawking] [wind chimes jingle] Boyd: You look pretty. Early day. Where you off to? Ava: Tammy Lee took me back at the beauty salon, gave me the morning slot. - I didn't know you were working. Ava: First day back. Needed a reason to get dressed before 4:00 in the afternoon. Boyd: [chuckles] Ava, there's something we need to talk about. Tammy Lee's waiting. Boyd: It won't take but a minute. [clears throat] While you were locked up, I-I-I had some time to think, time to look around. Harlan's dying. Mines mostly shut down, stores closed or closing, no money, no work ... not ... not worth having ... no ... no offense. People are giving up, selling what they got, and moving on. But if there's a chance for us, Ava, it's not here. Now, i-i-if I were to come into a good sum... soon... [sighs] would you come away with me? Ava: What are you talking about? Come away where? Boyd: Costa Rica, Brazil, maybe ... someplace they got white sand and blue water. Ava: No. I don't want to hear this. How ... How much money you comin' into, Boyd? I don't want to hear this. Boyd: I-I ain't talking about running from trouble, Ava. Ain't gonna be no trouble. I'm talking about leaving Harlan, escaping. Ava: This is our home, Boyd, the home that we're fixing up. And you want to leave? Boyd: If we stay in this ghost town, Ava, together or otherwise, how long you think it's gonna be before we turn into ghosts ourselves? Ava: Oh, you saying that like we ain't dead already. I gotta go to work. All right. I will be back in a jiff. [hair dryer whirs] [sighs] [birds squawking] Raylan: [sniffs] Ava: What the hell, Raylan? What are you doing here? Raylan: Come to see you. What are you thinking? I mean, are you sh1tting me? I work here! Yeah. Few days now. That's good. Good place to meet. You can't do this. You can't sneak up on me like this. Raylan: First off, I didn't sneak up. Second, yes, I can. Ava: What if Boyd came? What would you do then? Raylan: Boyd would not be caught dead near a beauty parlor, though I bet he's thrilled you get 10% off his hair spray. Ava: Funny. Raylan: I use a paste. Ava: [chuckles] I don't think I can do this. Raylan: What you need to do is contact me... particularly when I reach out again and again and I don't hear from you. That way, there are no surprises. Ava: Fine. Raylan: [inhales sharply] Sit down. [both sigh] Ava: I'm afraid to look him in the eye, say good morning the wrong way, give him cause to put a hole through my head. Raylan: Why would he have any reason to do such a thing? Ava: I don't know. Do I have to know? I mean, can't I just have a feeling? Raylan: How's he acting? Doing anything different ... making changes, talking about buying a new car, boat, making big plans? He bought a $300 tow truck. Raylan: There you go. How's that for a plan? Raylan: Could be something. Ava: [chuckles] He could fix it up, sell it. Yeah. It'd be a $20 profit. [sighs] Raylan: Okay. The reason I'm here ... Dewey's out of prison. He's gonna come and see Boyd. I want you to keep an eye on him. Ava: Fine. Is that all? Raylan: That's all. That, and be cool. We're setting up a command post at Arlo's. You need anything, I'm that close. Boyd: Hey! Where the hell is everybody? Carl: Yeah. Back here. Boyd: Well, what's going on? Earl and the Pig went to eat. Boyd: They did what?! They said they'd be quick. Boyd: Well, call them and tell them to get back here ... and to bring me something. I'm so hungry, I could eat the ass out of a low-flying duck. Why you still standing there like that? Carl: Found him outside, grabbed him up. Dewey: It's good to see you again, Boyd. I was hoping we could talk. Hey! Boyd: Frisk him. Dewey: Boyd! I come in peace. [Dewey thuds] He's clean. Boyd: How did you get here? Dewey: What do you mean? Boyd: How did you get here? Dewey: I-I walked. Boyd: From prison? Dewey: The pancake house. I left the pen yesterday by bus. I-I came partway in, and then I hot-wired a car. Boyd: What kind of car? Dewey: I don't know. The one with the horse. Toyota? Boyd: Ford Mustang. You lying to me. I ain't lying. Boyd: Cassette player or CD? Dewey: Cassette. Boyd: Any of them lying around? Dewey: ARS live, man, "Champagne jam." Boyd: I love that record. Dewey: Me, too. W-why you asking all these questions, Boyd? I-I know I didn't l-leave you on good terms. Boyd: That is correct. Robbing me at gunpoint was not on good terms. Dewey: Yeah, and I'm sorry about that, Boyd. I'm real sorry. But... I found this. This is the only link I have with family, Boyd. I gave it to someone special, and the bitch lost it. But now it's found. That's a powerful sign, Boyd. My turtle dog come back to me, and I come back to you. See? Now, I gotta talk to you about something. Only thing that matters is what I... want to talk about, Dewey. Now, I want to know exactly why you're not sitting in a prison cell as we speak. I was there, Dewey. I heard you say you killed Wade Messer. Now, all of a sudden, you're here, sitting in my bar. Dewey: They can't touch a hair on Dewey Crowe's head, else it's harassment, on account of the civil suit that I won against the federals, particularly Raylan Givens. Boyd: Well, what do you want? Dewey: I just want back in, Boyd. I just want you to trust me again. Carl. Carl: Come on. [SCENE_BREAK] [birds chirping] [gearshift clicks] Tim: Tow truck? Tow truck. Tim: Wasn't Crowder involved running drugs in a tow truck? Raylan: Like hot rod Dunham before him. Tim: Well, if he's still in the drug-running business, might explain this. That's Cyrus Boone, known dealer in Harlan. I got those about... 36 hours ago. Raylan: Maybe we should pay Cyrus a visit. Tim: This a friend of yours? [music] Raylan: Never seen him before in my life. Walker: Hey! Good morning! Raylan: Where you from? Walker: Maryland. Raylan: Way it works hereabouts, a man comes onto another man's land with no seeming purpose, that man might be taken as a trespasser, might get himself shot. Walker: Just assessing the land, devising a number, unless I misread the sign on the road ... "for sale by owner." You are the owner? Raylan: De facto. Walker: You might want to reconsider before threatening a potential buyer. House has been on the market for some time. Is that because you can't let it go or because it's stuck in a bad market? You don't strike me as the sentimental type. I'm gonna go with bad market. But I am prepared to pay your full asking price in cash... today. Raylan: And by "today," you mean "immediately." Walker: Indeed. [briefcase clicks] Raylan: Forgive me if I ain't the run-of-the-mill tater tot whose eyes go all pinwheels at a stack of stolen money. Walker: Stolen? I am offended, sir. Raylan: You don't even want to come up and see the house. I can see it fine from here. But it does have... uh, curb appeal, my daddy pretty much having let it go to ruin, but it does have history. My kin moved to this land from Miller's Creek in 1903. A lot of them gave their lives to hold on to it, a couple of them buried right up there by the house. Walker: I stand corrected. You are the sentimental type. Alas, all things must pass. What do you say, friend? Isn't it time to sell? Raylan: It is time to sell, but not to you. Walker: You have second thoughts, you'll have no trouble finding me. Raylan: You have no idea. [car door opens and closes] [engine turns over] Tim: What's the call on Cyrus? [knock on door] [dog barking in distance] Cyrus: [groans] Crackpot: Yo, man. You hook me up? Cyrus: Damn, Crackpot. What, you on the fast train? I just saw you yesterday! Crackpot: So? Cyrus: "So?" So, if you're O.D., it's gonna be your own goddamn fault. You hear me? Don't be goose-necking in here. Come on. [door hinges creak] [scoffs] Tim: You understand? Cyrus: sh1t. Yeah. Yeah. I understand. I'm cool! [music] [door closes] [shovel clangs] Raylan: Halt! U.S. Marshals. Boyd: What? No Cyrus. Boyd: What does that mean? He was abducted by aliens? Carl: He wasn't answering his phone. Earl just got back. The house is empty. I don't like it, Boyd. What are we doing? Are we really doing this thing? Boyd: Yeah. Carl: Why? Boyd: Because. Any more questions? Carl: Yeah. How we gonna get away with this with the feds up our ass? We needed Cyrus. Dewey: 8 ball, side pocket. Boyd: You want back in? I got a job that needs doing. Dewey: Anything you say, Boyd... anything. Hell, yeah. Raylan: See that? Good things happen to those who wait for stupid. Tim: I believe that was in the Sermon on the mount. Raylan: We get Dewey transporting, we get our leverage back. Tim: He tells a judge about Crowder-on-Crowder crime. Raylan: You want to follow Crowder or Crowe? Tim: [sighs] Your call. Raylan: Let's go after Dewey. [music] You know, he once told me he worked at Disney World dressed as Goofy ... in a water-skiing show. Tim: Well, some guys just peak too early. [music] [brakes squeal] [engine sputters] Dewey: sh1t, sh1t, sh1t, sh1t. [gearshift clicks] [music] [brakes squeal] Raylan: Want to hang back, let KSP do their thing? Tim: Yeah. Why not? Dewey: How you doing there? Laplante: Do you mind steppin' out of your vehicle? Dewey: I do. Laplante: We need to search your vehicle. Dewey: What, this vehicle? Laplante: License and registration, please. Dewey: Not today, friend. Laplante: What? Dewey: Oh, they didn't tell you who I am? I am Dewey goddamn Crowe, and I'm gonna keep on driving Sso I can save the state of Kentucky and the government of the United States of America another $300,000 for harassment, "nelgigence," and general "mal-fee-essence." So, have a nice day and kiss my ass. Tim: sh1t. [engine revs] [gunshots] [tires screech] [horn blaring] [music] [tires screeching] [crashing] [music stops playing] [birds squawking] Raylan: You all right, Dewey? Dewey: Raylan? Tim: 1,000 feet? Raylan: I'm pretty sure that's just a figure of speech. Are you okay? Dewey: I'm fine. Raylan: Good. Now get your ass out of that truck. Dewey: No. You can't touch me. I'm teflon. [trunk opens] Raylan: I'll only ask once. I'm gonna help you out of your vehicle. You understand? Dewey: What I understand is my constitutional ... Ohh! Ohh! Ohh, you broke my jaw! Goddamn! You broke my jaw! What'd you have to do that for?! Tim: What's in the bag? Dewey: What bag? Tim: That bag, dipshit. Dewey: I've never seen that bag before in my life. Raylan: Dewey, you're in a world of sh1t. First, you're facing extradition to Mexico. Second ... Tim: Second, whatever we find in this bag, that's on you. Raylan: If we find drugs in that bag, what do we call that? Tim: Transportation and distribution of illegal substances. Raylan: That don't look good on a resume. Tim: No it does not. [sirens wailing] Raylan: How many years would he be in for that? Tim: Well, he's not a first-time offender. Raylan: No. Tim: It's got to be at least 7 to 10. Raylan: Depends on the judge. Could be more. Tim: Uh-huh. [tires screech] Maybe just let KSP handle this. Tim: Maybe. Raylan: Dewey, you think Boyd gives a sh1t about you? Dewey: Boyd gives a big sh1t about me. Raylan: Open the bag. Dewey: I heard you the first time. Where is it? Tim: Where is what? Dewey: I don't know, but whatever it is, it ain't here. Raylan: [inhales sharply] Which bank did you say you saw Boyd in? Tim: First River. Boyd: Let's do this. [engine revs] [tires squeal] [music] [brakes squeal] [gun cocks] Carl: Lick the floor! [gun cocks] I said now! [gunshot] [all scream] I said down! That means you, too, Dolly Parton. [gasps] [whimpering] Please... Carl: All right, everybody, keep your head down and your mouth shut! This will all be over in a minute! Nobody gets hurt! The Pig: Got it? Boyd: Got it. Let's do it. [static hisses] [walkie-talkie chirps] [tires screech] [tires squeal] [indistinct conversations] [brakes squeal] [vehicle doors open] [police radio chatter] Tim: Yeah, let's follow Dewey. Yeah. Good call, Gutterson. [insects chirping] Ava: You reached out. Here I am. See? I can listen. Raylan: Why just the security-deposit boxes? Ava: What's that, a riddle? Come on, Raylan. It's late, and I'm cold. Was it Boyd's idea to send me after the truck, or was that you? Ava: I don't ... I don't know what we're talking about. We're talking about the bank job Boyd pulled off this afternoon. Ava: First I heard of it. That's gonna be your line? Ava: It's the truth. If it ain't, you're going back to prison. Worse, you'll be a suspect. Ava: Suspect? Worse, you'll be a suspect. Ava: Suspect? Raylan: Yeah. Criminal co-conspirator. You'll be looking at Boyd's charges, plus a few of your own. If someone gets killed, that's on you, too. Ava: Jesus, Raylan, I'm not lying. I'm prone to believe you. The problem is, if you didn't know about it, then you're of no use to us, in which case, you're going back to prison. You understand your dilemma? Ava: [exhales sharply] Ava, am I being clear? Ava: Yes, Raylan, crystal clear! I'm just having a nervous collapse here, is all. Raylan: You don't have that luxury. You need to pull yourself together and do the job. Ava: So, I should just start asking probing questions like I'm curious, like ... like it ain't about anything particular? Hell, I was in "Brigadoon" in seventh grade. [chuckles] I guess I can act. Ava, I know you. Ava: [sighs] You knew me then. What do you know now? Raylan: Then, now ... it's the same. It's the same. You remember the day I came back to Harlan? Ava: Hardly. I recall it well. You opened your screen door, kissed me, and invited me in, then showed me a bloodstain on your dining-room carpet. Ava: Bowman. Christ, that was a long time ago. Raylan: You recall the days leading up to it? Ava: [sighs] It was clear to me. I knew I would shoot him. I'd had enough. And I just had to see it through. Raylan: You made him dinner, a whole spread ... his favorites, if I recall. Ava: He was delighted. Raylan: He had no sense of your intention. Ava: He never saw it coming... because he believed me... [gasps] because I acted like everything was the way it always was. Raylan: Because he had never suspected otherwise. Ava: No, he did not. Raylan: See? I know you, Ava. I know you can do this. So you damn well better. [tapping on metal] Boyd: [grunts] [crowbar clangs] Carl: What the hell, Boyd? What is this? Paperwork? Goddamn notebook? Boyd: Looks like a ledger, some deeds. The Pig: We got the wrong box. No, we got the right box. Carl: This isn't money. Where's all the money?! [sighs] [scoffs] Art: Well, you're lucky. Leslie's not supposed to let people in here that might upset me. Raylan: Sorry. I should have come a week ago. Art: Me who's sorry. All this sh1t. [glasses thud] Slipping in my old age. Raylan: He didn't get you 'cause you're slipping, Art. I brought you something. Art: [chuckles] I don't suppose that's doctor recommend. Raylan: Civil war doctor, maybe. You want to abstain, I'll drink in your honor. Art: Just waft the fumes in my direction. Raylan: To your speedy recovery. Art: So, how's it going? Willa has been baptized a catholic. [liquid pours] Art: Mm. That grandma's idea? Raylan: She fears for her immortal soul. Art: Well, do you blame her? Kid's got half your DNA. How's work? Raylan: Going after Boyd Crowder ... at a snail's pace, I might add, so not to spook him. Art: That explains why you haven't scooted down to see your papist daughter. Raylan: Big deal now ... RICO case, all eyes watching. Got to be neat and tidy, by the book. Art: Not how you'd like it done. Doubt that's how we'll get him. Art: Your C.I. in the Crowder camp isn't some help? Yeah. I know more than I let on. And, no, I would not have green-lit Ava Crowder as said C.I., nor you as her handler. But I'm not in charge at the moment, may not be again, don't know why I'm even interested, except out of sheer damn boredom. Cheers. Art: Well, I know you didn't come here just to check on my condition and jeopardize my convalescence with some high-class bourbon. Raylan: Remind me why I'm gonna be patient. Art: Not back him into a corner and force him to draw? Plug him face-to-face. Art: Get'r done quick. Plug him face-to-face. Art: Get'r done quick. Save us all a big headache. Make the world a better place for a little while. Yeah, you could kill Boyd, then you'd be headed down to Florida without a star and a gun. And you might be seeing your daughter through the glass on visitation days at the penitentiary. Or, Raylan, there's another way it goes, where you try and you fail and the bullet finds you. Raylan: Unlikely. Art: I know you think so, but if you'll allow me ... you get to be my age, do the job as long as you do... sometimes it just doesn't go your way. Dewey: Y-you tell him I got to see him! Carl: All right. Dewey: You tell him it's Dewey Crowe! Carl: All right! [clears throat] It's Dewey Crowe. Dewey: What the hell, Boyd?! Boyd: What the hell, what? Dewey: You set me up! Boyd: How you figure that? Dewey: The staties and the feds were waiting for me. They knew I was coming before they even saw me. Boyd: You said you wanted back in, so I gave you a job. You told me what was in that bag was important! Boyd: I told you that the job was important. Dewey: What, carrying your underpants? Boyd: Well, it hadn't have been underpants, your sorry ass would be sitting in jail right now. What in the hell happened to your face? Dewey: Raylan broke my jaw. Why? Dewey: I don't know why! But I'm ... I'm tired of it! [breathing heavily] I'm tired of it. You my friend, Boyd? Yeah, I'm your friend. Dewey: I got to talk to you, Boyd... about ... [inhales deeply] Mexico, and I-I... and I-I-I don't know. I just ... I'm tired. [sighs] Boyd: What do you want, Dewey? Dewey: [voice breaking] I want to go back. I want it to be like it used to be at the church, when we was Crowder's commandos. You, me, and Devil. Dewey: Yes! And the Pork brothers, giving me no end of sh1t. [chuckles] The music cranked so loud that we almost blew the roof off that old church. Boyd: [chuckles] Dewey: Bombing around in my Cadillac and making war plans and drinking 'shine. Why can't it be like that again, Boyd? Boyd: Those were simple days, good days. Weren't they? They were good days. They were. [exhales sharply] Boyd: Carl, go out front and pour me and Dewey a couple glasses, would you? Come on over here. Have a seat. Dewey, I'm gonna tell you something in confidence. It's all coming to an end. Dewey: What do you mean? Boyd: Well, look around you. Whatever it was we was hoping for ... those days have long since passed. Now, I ... I ain't saying I'm giving up, but I am saying I never thought that it would be this hard. It seems like maybe those good days are gone forever. Dewey: I don't know, man. I feel like ... I feel like we can make it like it was before. Boyd: Hmm. I want to show you something. You see that man right there? That's my Granddaddy. Fella next to him is my Great Uncle, with all their Union brothers. Now, Harlan county was a boomtown then. Those men saw hard, bitter times, Dewey Crowe, but they also saw a future. Now, you look closely, you can see it in their eyes. Go on. Take a look. Dewey: [sniffles] Can't even see their eyes. [gunshot, body thuds] Carl: Boyd! [footsteps approach] Whew. You, uh, think he was a ... a rat for the federals? Boyd: I think I couldn't trust him anymore. Now, they'll follow me, no doubt, but you be sure. You wait 20 minutes after I leave. Then you roll him up in a carpet, and you make him disappear. [dog barking in distance] [music]

Raylan remains separated from his infant daughter so he can work on the RICO case against Boyd Crowder, abducting crooked Federale Agular from Mexico where he is compelled to say that Dewey Crowe was a witness to Boyd's murder of Johnny and the others. Dewey has just been released from jail on a bad confession and a record of harassment from Raylan, who despite a petition to keep a thousand feet away attempts to coerce Dewey into turning on Boyd. Dewey claims ignorance and wanders Harlan until circumstances cause him to return to Boyd, apologizing and asking Boyd to trust him and bring him back in. When Raylan busts drug dealer Cyrus who had recently met with Boyd, Boyd employs Dewey to drive a truck (apparently using Hot Rod Dunham's old trick of towing a car with a bag of drugs in the trunk). Raylan decides to pull surveillance off Boyd to follow Dewey, who gets scared at a state police roadblock and drives off the road, Raylan realizing too late that Dewey was a decoy. Boyd uses the opportunity to rob the safety deposit boxes of a bank he'd cased earlier, but his crew are frustrated to find that the boxes do not contain cash as promised but a ledger and deeds. Raylan pressures a scared Ava to fulfill her obligations as an informant and produce actionable information on Boyd or be returned to prison. Ty Walker earns Raylan's suspicion by offering a suitcase of cash for Arlo's old house and farmland. At the end of the episode, Dewey reminisces with Boyd about Crowder's Commandos saying it was the happiest time of his life, but Boyd believes there's no future left in Harlan and those days are behind them. Boyd kills Dewey, saying that he couldn't trust Dewey any more, then in the final scene is shown contemplating a sleeping Ava.

summ_screen_fd

EXT. - MOUNTAIN PASS - THE OLD WEST - DAY [A couple of cowboys in dusters hurriedly climb up a precariously rocky path. A woman in a petit coat and hat lags behind, stumbling. Cowboy #1, played by Burr Connors, stops to look in the bullet chamber of his six-shooter. Horses neigh in the distance. Cowboy #2, played by Rod Sebring, stops and turns to him.] Cowboy #1: We're done for now. Cowboy #2: We'd be clear to Barker Pass by now if you hadn't brought that uppity preacher's daughter. Cowboy #1: Sometimes a man's gotta do what a man's gotta do. Cowboy #2: Yeah. [The preacher's daughter walks up and puts her hands on her hips.] Preacher's Daughter: Looking for these, boys? [She has a couple of bullet-laden bandoliers strapped across her chest.] Cowboy #1: What the... [She steps up to Cowboy #1 and grabs him and kisses him.] Preacher's Daughter: For last night. [She slaps him.] Preacher's Daughter: For this morning. Next time, don't undercook the yolks. [Cowboy #1 looks at Cowboy #2, who smirks at him. The director shouts through a megaphone.] Director: (off screen) And cut! [We see the cameras and the crew standing nearby. The woman playing the preacher's daughter rubs Burr's cheek.] Woman: Are you okay? Burr: (to director) Do I get extra pay for that? Director: (megaphone) Back to ones, everybody. [The director gets up and walks off. Several people scramble around the set.] [Title card: On location for with Burr Connor 1985, Lone Pine, California] EXT. - PARKING LOT - BURR CONNOR'S TRAILER - DAY [It's a foggy, rainy day in the mountains, on the set of Burr's movie. He steps out of his trailer, into the parking lot. The trailer next to his, belonging to co-star Rod Sebring, is rocking. Burr looks around, approaches, and goes in.] INT. - ROD SEBRING'S TRAILER - DAY [Rod is on the bed having s*x with a guy named Ben. Burr freaks. He pulls Rod off the guy.] Burr: Get the hell off of him! Ben: Oh, sh1t! Oh! [Burr grabs the naked Ben and drags him toward the door by his neck.] Rod: Burr, c'mon! Burr! [Ben shouts in terror. Rod follows. Burr mutters angrily, then throws Ben out the door.] Burr: Get the f*ck outta here! Ben: Argh! [Ben crashes hard on the wet pavement.] Burr: Go on! [Burr tosses his clothes out.] Burr: Don't come back! [Ben grabs his clothes and runs away.] Burr: (to Rod) What the f*ck are you doing?! Rod: It's none of your business! Burr: It is absolutely my business! What if that kid talks, huh? Did you ever think of that? [Rod rubs his face, frustrated.] Burr: Huh? What if he goes to The Star and he says that Rod Sebring is an ass bandit? Rod: No. No. Ben wouldn't do that. Burr: Ben wouldn't do that. Rod: No. He wouldn't. Burr: You don't know that. And I have too much riding on this picture. [Burr walks out.] Rod: Burr. Burr! [Opening credits.] INT. - ALICE'S APARTMENT - BEDROOM - MORNING [Alice and Dana are cuddling in bed, asleep. The clock radio comes on. Alice rolls over and groggily smacks it. Alice rolls back over on top of Dana. They cuddle. Alice starts to kiss Dana's back, then slides a hand under the covers. Dana's eyes pop open.] Dana: Oh, my God. Oh, my God! [Dana squirms out from under Alice. Alice flops over onto the other side of the bed.] Alice: God, don't freak, it's Sunday, we're allowed to get up late. [Dana scrambles to put some clothes on.] Dana: Alice, it's Monday. [Alice sits up, still groggy.] Alice: No. Mm-mm. No, it's Sunday. Dana: Alice, think about it. Yesterday, we got brunch delivered, and then the night before, we watched "Saturday Night Live". Alice: (gasping) Right! Oh, f*ck! Oh, my God, I have my KCRW thing tomorrow! (sits up) I haven't come up with a single idea! I'm totally f*cking up! Oh, God. [Now Alice scrambles off the bed and gets dressed. Dana sits on the bed.] Alice: Uh, it's - uh! What am I gonna do for my audition?! A once-a-week, three-minute culture spot. Great. I need f*cking ideas! Dana: People who ruin their lives because they can't stop having s*x? Alice: 'Kay. That's really not helping. [Alice puts on her glasses and grabs her laptop.] Dana: I'm sorry. Look, you're not the only one who's blown off important things to stay in bed f*cking for five days, okay? I should be training. Alice: You should always be training. So. Dana: Alright, look. [Dana sits up on her knees and takes Alice's face in her hands.] Dana: I'm gonna go make us some coffee, okay? Alice: Okay. [They kiss.] Dana: Work. [They kiss.] Alice: Okay. [Dana leaves for the kitchen.] Dana: Get to work! Alice: Okay, okay. [Alice plops down on her stomach on the bed, with her laptop.] INT. - THE GARAGE - DAY [Mark is watching Shane on his computer. She stands in the middle of her bedroom, looking at her battered face in a handheld mirror. He copies the clip from the video and puts it into the timeline on his video editing program.] INT. - THE PLANET - DAY [Bette sits at a table by herself. Tina walks in and approaches. They smile at each other. Tina's face lights up when she smiles at her.] Bette: Hey. Tina: Hi. [Tina sits. Bette starts to pour her a cup of tea.] Bette: I got you tea. I wasn't sure what you were eating these days, but... Tina: Thank you, that's nice. [Bette finishes pouring and sets the pot down. She smiles modestly at Tina, trying to look away each time she looks at her. It's no use.] Bette: I spoke to Dan Foxworthy this morning. I was thinking about going back to therapy. Tina: Oh. I don't know about therapy. I think we should just focus on the baby. Bette: Oh, no, not for us - I - I was thinking about... going to see him... myself. (smiles) [Tina seems hugely impressed. She smiles huge, her face lighting up again.] Tina: Oh! That's great, that's really great. [The smile fades a little.] Tina: That'll be good for you. Bette: (smiling) Yeah. [The moments are tense between them. Bette is about to take a sip of her drink, but stops.] Bette: Can I just say one thing? Tina: You can say anything you want. Bette: (smiling) No, I can't. I mean, you've made it clear that you don't exactly wanna hear what I have to say right now and that's fine, but I, um... I never got to say... how happy I am for you. For us. [Bette looks deeply sad and happy at the same time. Tina gives her a genuine smile.] Tina: I'm glad you're happy. [Bette stares at her sadly and takes a deep breath.] Tina: This should be your happiness too. Let's, um... just focus on being really good parents. [They both smile and chuckle.] Tina: It'll be a new kind of partnership for us. [Bette smiles then looks down, sad. Tina watches her. Bette looks up again, smiles, and raises her glass to Tina. Tina raises hers. They toast.] EXT. - SIDEWALK OUTSIDE THE PLANET - DAY [Carmen and Jenny are walking down the sidewalk, toward The Planet.] Jenny: My teacher, Charlotte Birch, is pushing me. And I'm really freaked out because she's intimating there's some kind of mysterious challenge in all of this for me. Carmen: Okay, well can I tell you what I think about Charlotte? [They cross the street.] Carmen: I think - Jenny: What. Carmen: You have a crush on her. Jenny: No, I don't. Carmen: I think you do and I think it's totally okay, and - and I think that you are allowed to have a crush on your teacher. And you know what? I think you should go for it. Totally go for it. Jenny: f*ck you. Carmen: What? [They stop in front of The Planet and face each other. Behind them, Helena pulls up and parks her car on the curb. Jenny stares at Carmen.] Carmen: You want me to be jealous? [Jenny nods. Carmen kisses her.] Carmen: Well, if you think about f*cking her, I will kill you. [Jenny kisses Carmen.] INT. - THE PLANET - BETTE'S TABLE - DAY [Tina is looking in her day planner.] Tina: My next sonogram is Wednesday the 7th at 10 a.m. Bette: Well, I'd love to be there - I mean, if that's okay with you. (smiling) I'd really like to see the baby. Helena: How exciting. [Neither of them have noticed Helena. Helena puts her arm on Tina's shoulder. Bette seems to draw back a little.] Helena: You ready to go? The agent has four listings planned out for us. [Tina gets her things.] Helena: (to Bette) We're real estate hunting. Bette: Real estate hunting. Helena: Mm-hmm. Tina: Oh. Helena wants to rent a house in L.A. [Tina kind of shakes her head, indicating to Bette it's not a house for them both.] Helena: The Chateau Marmont is getting so tedious, I mean, I can't walk from one end of the lobby to the other without being offered to do blow or finance four independent films starring Maggie Gyllenhaal. Bette: (laughing) All totally unsolicited, I'm sure. Helena: (straight faced) Totally. Tina: (to Bette) Um... I'll see you on the 7th. Okay? I'll call you. [Tina gets up and leaves with Helena. Helena puts a hand on Tina's back as they walk off.] INT. - SLEAZY PRODUCER'S OFFICE - DAY [Mark and Gomey sit in the office of a sleazy producer, listening to him talk.] Producer: Look, the thing about reality is it's gonna draw the audience in. Mark: Right. Producer: Basically, I need pussy that we can smell and taste. Gomey: Absolutely. Mark: Right. Of course. I mean... the thing about this film, though, is it's got that, but it's got more. It's um... it's fascinating, I mean... we're giving people access to a world that they've never seen before. You know. It's like journalists who infiltrate mosques, it's witness - Producer: Why don't you show me footage? I mean, usually, I don't write checks sight-unseen. Mark: I completely understand. Um... I just need a little more time. [The producer pinches the bridge of his nose.] Mark: I mean, this isn't scripted, so it's - something's happening, I mean, it's real but we can't force - Gomey: (to producer) We'll cut you something together. And it will be dripping with hot lesbian pussy. (to Mark) Won't it, dude? Mark: Yeah. Yeah. I'll work on cutting something together. Producer: You can, uh... show yourselves out. Mark: Great. Well, thank you. Gomey: Yeah, thanks. INT. - HOUSE FOR RENT #1 - FOYER - DAY [A real estate agent guy is ushering Helena and Tina into a purely massive house full of marble everything, chandeliers, old furniture, and classical sculpture.] Agent: Did you see the movie Hannibal? Uh, Freddie versus Jason? Helena: (scoffs) Please. You don't honestly think that regaling me with a list of the owners' mediocre movies is really going to influence whether I rent this property or not? [Tina walks around the foyer, which is as big as most one-bedroom apartments.] Helena: May I see the specs, please? [The agent hands Helena the folder with the details about the house. Helena smiles at Tina.] Tina: I think I'm gonna go take a look around. Helena: Mkay. But, um... [Helena steps up to Tina.] Helena: Don't go too far. [Helena kisses Tina in front of the agent, who watches. After a couple of seconds, Tina pulls away, blushing but embarrassed. She walks into the expansive parlor, furnished with a chandelier, grand piano, a huge, old couch and a massive fireplace.] Helena: (to agent) So, I'm assuming the tenant will move out of the guest house if I decide to rent this behemoth? [The agent nods and smiles at her.] Helena: Ah. Now, why don't you tell me about the household staff that come along with this property? [They walk into another part of the house.] INT. - ALICE'S APARTMENT - BEDROOM - DAY [Alice sits on the bed with her laptop. Dana sits on the edge of the bed, lifting weights.] Alice: Okay. Listen to this. Um... [Alice starts to talk in a radio commentator voice - soft, soothing, and enunciated.] Alice: (reading) "Walking through Fred Segal this week, I could sense all was not right in the world of consumerism. Something's wrong." Dana: You said that. Alice: What? Dana: Well, you said all was not right, and then you said something's wrong. Just... it seems redundant. Alice: Okay. May I? Dana: Yeah, go ahead. Alice: Okay. (clears throat) (reading) "There's an invasion underway. Our troops are in Iraq, in Afghanistan, and we are here, shopping." Dana: What are you doing to your voice? Alice: It's my radio commentator voice. You have to have one. Can I continue now? Dana: Yeah. Sorry. Alice: Thank you. (clears throat) (reading) "Is the main mission of our troops the protection of our way of life, and is our way of life defined by our consumerism? Are women and men dying in Iraq so that back home we can shop til we drop?" Dana: Is consumerism the right word? [Alice stops and stares flatly at Dana.] INT. - HOUSE FOR RENT #1 - TERRACE - DAY [Tina walks around the terrace and looks out one of the huge floor-to-ceiling windows. Helena walks in.] Helena: Well. It's a little vulgar. But it's amusing. Tina: Could you live like this? [Helena tosses her bag on the nearby desk and walks toward Tina.] Helena: Why don't we find out? [Tina chuckles.] Helena: Hey. [Helena puts her arms around Tina and starts to try and kiss her.] Tina: What are you doing? [Helena kisses her and backs her up against a desk.] Helena: I couldn't be expected - [Helena kisses her. Tina sits up on the desk, smiling. Helena stands between her legs.] Helena: - to have a property like this without seeing if it's conducive - [Helena kisses Tina, then unbuttons her shirt and kisses her chest.] Helena: - to this. [Tina turns around to see if the agent guy is around.] Helena: Hey. [Tina turns back around. Helena kisses her, then kisses her chest. Tina sighs. Helena looks up at her. Tina goes stiff and moans. Helena moves forward a little; Tina closes her eyes and shudders. Helena's face is an inch away. She teases her, almost kissing her.] Helena: Could you live like this? [Tina sighs, her breath shaking.] Tina: Oh yeah... [Helena opens her mouth, gasping a little. Tina kisses her, then grabs her hair and kisses her harder. Behind them in the parlor, the agent walks in. Neither of them notice.] Agent: Helena. You've seen the matching - [Helena kisses Tina, and starts to push her further back on the desk. Tina rocks back in ecstasy and moans - until she realizes the agent is talking to them.] Agent: - his and hers walk-in closets? [Tina hops off the desk and looks at the agent, embarrassed. The agent smiles.] Agent: Or should I say hers and hers? EXT. - JENNY AND SHANE'S HOUSE - BACK PORCH - DAY [Shane walks out the back door, wearing sunglasses. Mark stops her.] Mark: Shane! Hey. Shane: Hey. Mark: (smiling) You're alive. I haven't seen you around, how've you been? [Shane nods.] Mark: How's, uh... (points to eye) Shane: Oh, it's uh... it's better, thank you. [Shane starts to walk away.] Mark: I've been worried about you. Shane: Thanks. Mark: Um. So where you off to? Shane: Work. Mark: Hair job, or Veronica Bloom job? Shane: Hair job. It's a day call. [Shane starts to walk off again.] Mark: Um. Well, have a good one. (smiles) Shane: You too. [Shane walks off. Mark walks into the garage.] INT. - THE GARAGE - DAY [Gomey is sitting at Mark's computer, watching video in fast-forward. Mark takes off his jacket.] Gomey: Most of this stuff is just crap, man. They're not doin' nothin' but talkin'. Sometimes they eat. Mark: (irritated) What do you think, lesbians just f*ck all the time? Gomey: Obviously not, but if they don't start soon... we're not gonna... [Gomey looks closer at the monitor. It's Shane, looking in the mirror at her black eye.] Gomey: What the f*ck is this, man? [Mark walks over and pushes Gomey out of the way. He takes the mouse and starts to mess with the video.] Mark: It's none of your f*cking - why are you so f*cking nosey? God. Gomey: (disgusted) I'll tell you what it is. It's some total lez who's never gonna give you the time of day and you're rock hard for her! Jesus. I bet you just f*cking sit here all night jacking off while you watch her sleep! [Mark turns and shoves Gomey to the ground.] Mark: You shut the f*ck up! [Gomey gets up quickly.] Gomey: Great. This is just f*ckin' perfect. We've been best friends for how long? Mark: Just get out of here. [Mark sits down at the computer and runs a hand through his hair.] Gomey: And now you're gonna f*ck up this gig we've been working our asses off on for some chick? News flash, man! This girl you're crushing on is never gonna be with you! You've got a real live dick! And that disqualifies you from getting up in there! [Mark ignores him. Gomey leaves.] INT. - THE CAC - BETTE'S OFFICE - DAY [Bette sits at her desk, writing in a notebook. There's a knock at her door, then it slides open. It's Franklin and Leo.] Franklin: (smiling) Bette. Leo and I have something we're very excited about and we'd like to share with you. Leo? Leo: We just wanted to let you know that Helena Peabody is going to be joining the CAC's steering committee. [Bette looks dumbfounded.] Bette: What? Franklin: We're going to welcome her at our regular board meeting tomorrow. I'm sure you can appreciate what a coup it is for the CAC to have a Peabody on the Board of Directors. [Bette looks almost let down.] Franklin: Not to put too fine a point on it, but she's bringing Allyn Barnes with her to her first Board of Directors meeting. Bette: Allyn Barnes is coming to our board meeting? Franklin: Mm-hmm. Leo: Allyn and Peggy are old friends. She's known Helena since she was a little girl. Bette: There's a rumor that Allyn Barnes has been contemplating a career retrospective. The Modern's been after her for years. Leo: She's pretty resistant. I mean, everybody knows Allyn hates the art world. But, Helena thinks she might be ready. Now I've asked your department to put together some press clippings of the CAC so we can present it to her tomorrow. Bette: Well, I'd like to go over those materials. Franklin: This is Leo's strength, Bette. Why don't we just let him do his thing? We'll see you tomorrow at six. Hm? (to Leo) Come on. [Franklin and Leo leave. Bette sighs and reaches in her desk drawer. She pulls out a candybar and takes a couple of big bites. She frowns as she chews.] INT. - HOUSE FOR RENT #2 - DAY [The agent leads Helena and Tina through a spacious California beach house. The interior is white, with wood accents, and is sparsely furnished with modern designs. The ceilings are low and the walls are mostly made of windows.] Tina: This is more like it. Helena: Do you think? Tina: Bette saw this house in "Architectural Digest" - she said it was the quintessential California beach house. Helena: Hm. Did she, now? Agent: There are eight bedrooms all together, uh, not including the guesthouse, and there's a nanny's quarters down below. [Helena's cell phone rings. She answers.] Helena: (phone) What, Walter? (listens) Please just tell me what you have to tell me - no digressions. [The agent opens the sliding glass door to a small patio behind them. We hear the sound of the ocean, and seagulls.] Agent: Amazing view. [Tina walks over and checks out the winding stairwell that goes down a few floors.] Helena: (phone) (agitated) No, no, Walter, I do not ex - Walter, what did I charge you with? Don't tell me things I don't want to hear! That's not what I pay you for! Tina: (to agent) Do you wanna show me the kitchen? [The agent leads Tina down the long, winding staircase.] Helena: (phone) Walter, no, here's what's happening. I'm moving to Los Angeles, and I want my children here with me, so just f*cking well make it happen, and call me when it's done! [Helena disconnects the call. She walks over to the stairwell and looks down at Tina and the agent.] Helena: So? Do we like it? [Tina and the agent stop and look up.] Tina: It's spectacular. Helena: (smiling) Okay. (to agent) I'll move in tomorrow. [The agent continues down the stairwell. Tina smiles at Helena, a little floored by the snap decision.] Tina: Okay... (chuckles) INT. - BETTE'S HOUSE - NIGHT [Bette answers the front door. It's Winnie Mann, and the kids, Wilson and Jun Ying.] Winnie: Hi, I'm Winnie Mann. Thanks for seeing me. [They shake hands.] Bette: Really nice to meet you. I, uh, I didn't know that you would be bringing - Winnie: I don't have a nanny, so when the kids are with me, they are with me. I hope it's not - Bette: Oh, no, no. It's fine. It's great. [They walk in. Bette smiles down at the kids.] Bette: Do you remember me? I'm - we met in New York. [Winnie chuckles. The kids stare up at Bette.] Winnie: Don't take it personally, it takes more than one meeting. [Wilson sees a display by a lamp. It's five jars with little items in them.] Winnie: Wilson, wait, um, uh - [Wilson immediately picks up one of the jars, opens it, and crams his hand inside.] Bette: Oh, sweetie, sweetie! [Bette dashes over. And gently takes the jar and replaces the cap.] Bette: No, no, no, no, no. Oh. See, this... is a Richard Prince, and it goes right there. [Bette puts the jar back on the shelf and sighs, relaxing.] Bette: Hey, would you guys like to go swimming? Wilson: Yeah, that'd be great! Jun Ying: Yeah, can we? Winnie: Uh, well, I don't really swim, do you think you might go in with them? [Bette doesn't look like that's what she had in mind. She looks at Wilson. He nods excitedly. Bette chuckles.] Bette: Sure. INT. - ALICE'S APARTMENT - LIVING ROOM - NIGHT [Alice and Dana are sitting around on the couch. Alice is writing on a notepad and Dana is watching a cooking show.] Alice: Okay. Okay, will you listen to this one? [Alice clicks the off button on the remote in Dana's hand.] Alice: Okay, so basically I'm gonna get someone from the left, someone from the right, someone from the center - local, elected officials, whatever. And then I'm gonna interview them about trends. 'Kay? So. (reading in commentator voice) "State Attorney General Wachtel, have you shopped for your new granddaughter at the new "La La Ling" baby store in Las Feliz?" So. Dana: Mm-hmm. Alice: So, I'm trying to get that intersection between culture and politics. [Dana looks lost.] Dana: I don't know. Al, it's good. It's just - it's not funny. Alice: It's not supposed to be funny. Hello? Have you listened to KCRW? Dana: Yeah, but didn't Mimi ask you personally to try out for this? Alice: So? Dana: So, she must want you to be you. Alice: No, I'm going to be a (makes air quotes) funny person on the radio. I don't know why everybody thinks I'm so funny. Dana: Because you are. Alice, you're a funny lady, and it's one of the things I love about you, and I'm not the only one. [Alice pouts and scribbles on her notepad.] Alice: I don't like you very much right now. [Dana takes her pen and notepad and sets them on the coffee table. She gets up on her knees on the couch and pulls Alice's legs around her waist.] Dana: It makes me so hot when you're angry. Alice: Oh yeah? Dana: Oh yeah. Alice: (giggling) You're totally topping me again. Dana: I'm sorry, I couldn't hear you through the feathers, Pillow Queen. [Alice laughs. Dana leans back and forth, keeping Alice's legs wrapped around her.] Alice: Have you always been a top? Dana: I'm not a top. Alice: Yeah, you are. Dana: Uh-huh. Alice: Yeah, you are. [Alice pulls Dana forward.] Alice: You know what I want you to do? Dana: What? [Dana leans close. Alice whispers quietly in her ear.] Alice: (whispering) I want you to f*ck me really hard with a strap-on. [Dana leans up.] Dana: I don't know if I can do that. Alice: But you're doing it right now without even using one. [Dana grinds slowly against Alice. Alice smiles at her. They kiss.] Dana: Okay. Alice: Yeah? Dana: Twisted my arm. EXT. - BETTE'S HOUSE - POOL - NIGHT [Bette is in the pool with the kids. She twirls Jun Ying around in the water. Winnie sits on a chair at the edge.] Winnie: You know, Bette, I - I know you don't know me, but, um, I'm taking a wild stab that maybe we have some interests in common. Bette: What are you asking me to do? Winnie: I'd like you to be a character witness... against... you know who. I know she's coming on to your board of directors. Bette: Yeah, she's everywhere. Can't seem to get away from her. Winnie: That's how she deals. She colonizes. Bette: She f*ckin' plunders and pillages, that's - Winnie: Uh. (clears throat) [Bette looks at Jun Ying in her arms.] Bette: I'm sorry. I'm sorry. That was bad. Winnie: Bette, we've got a court hearing in six weeks. Bette: You want me to appear in court? Winnie: Yes. Bette: Jesus, Winnie. I don't know. I mean, I'm really gonna have to think about it. It's, you know, it's not just that she's on my board of directors, but there's Tina, the... Winnie: Well, f*ck, I understand if you don't want to - Bette: No, it's not that I don't want to. Believe me, I would love nothing more than to stand up in court and say what I think of... you know who. (to Wilson) You wanna play again? Wilson: Yeah. Bette: Okay. One, two, three. [Bette tosses Wilson through the air a couple of feet. He splashes into the water.] Wilson: Ahh! Bette: Woo! INT. - JENNY AND SHANE'S HOUSE - HALLWAY OUTSIDE SHANE'S ROOM - NIGHT [Carmen is walking down the hallway. Shane starts to step out of her room. She sees Carmen, and goes back inside. Carmen hesitates, then opens the door.] INT. - JENNY AND SHANE'S HOUSE - SHANE'S ROOM - NIGHT [Shane is leaning against the door. When Carmen opens it, she backs away. Carmen enters. Shane keeps her back to her; she's wearing her sunglasses.] Carmen: Um. I - I - I just saw you out there, and it - I dunno, it looked like you were hiding. Shane: I'm not hiding. Carmen: I don't believe you. [Shane faces her, then grabs her keys nearby; Carmen grabs the keys and tosses them aside. Carmen tries to remove the sunglasses. Shane turns her head, but Carmen manages to remove them. She sees the bruises.] Carmen: Oh my God. Oh my God. Sh - [Carmen tries to touch Shane's face. Shane grabs her arm.] Carmen: Shane, who did this? Who did this to you? Shane: It doesn't matter. Carmen: No, tell me. I will f*cking kill them. Who did this to you? Shane: You did it. [Carmen is stunned.] Carmen: What? [She stares up at Shane, hurt.] Carmen: f*ck you. [Carmen opens the door. Shane pulls her back in.] Shane: Wait. Wait, wait. Look, I'm sorry. That was a fucked-up thing to say. [Shane closes the door and faces Carmen.] Carmen: Well, maybe you meant something by it. Maybe we should just talk and see what happ - Shane: No. No. No. Can we... can we just forget what I said? Let's go back to being friends. Carmen: Is that what you want? Shane: Really a lot. [They both stare at each other. Shane opens the door. As she pulls the door open, she steps closer to Carmen. Their faces come close. Shane turns away. Carmen leaves.] INT. - BETTE'S HOUSE - KITCHEN - NIGHT [Bette and Winnie sit at the dining room table, having some water. The kids sit at the kitchen table a few feet away, eating chicken nuggets.] Winnie: I'm so angry I can barely control myself. But, then I know the more I act like that, the more this person can "F" me up. But, there I am screaming like a crazy B-I-T-C-H. Bette: That's gotta be hell. Winnie: She made my life a living hell. Bette: What were you doing with her, Winnie? Winnie: Is it so hard to understand? Look at Tina. [Bette looks down.] Winnie: You two were separated when she met? Bette: Yeah. (smiling) And now she has her completely enthralled. Winnie: Yeah. My first play had just debuted at PS 122. The Voice gave it a rave, and this person shows up and underwrites the entire run of the show. The next thing you know, I've got my own theatre company, I'm living in a five million-dollar loft in Tribeca, and... Bette: And starting a family. Winnie: Yeah. Hey, she'd never even thought about kids, but it was a dream of mine, and she's in the business of making people's dreams come true, until she co-opts and makes them her own. Wilson: Mommy, I'm finished with my chicken nuggets. Can I have some ice cream? Winnie: Uh... Bette: (to Winnie) I think I have some if it's okay. Winnie: (to Wilson) Did you and your sister finish your salad? Wilson: Yep. Winnie: Alrighty. Bette: Then you get the big prize. [Bette heads to the freezer and gets the ice cream. Winnie leans over the kitchen bar.] Winnie: Can I give you some advice about it? Bette: I could sure use some. Winnie: Drives her crazy when you don't react. So, she'll bait and bait and bait, and if you just don't take it... (winks) INT. - JENNY AND SHANE'S HOUSE - NIGHT [Jenny and Carmen are making out on the couch. The TV is showing the old Burr Connor movie that was being filmed at the beginning of the episode.] Cowboy #1: (TV) "We're done for now." Cowboy #2: (TV) "We'd be clear of Barker's Pass by now if you hadn't brought that uppity preacher's daughter." [Mark enters and plops down on the couch next to them.] Mark: Hey ladies. Carmen: (annoyed) Oh, hello. It's Mark. Mark: Excuse me. Is that how you greet your roommate? What are we watching? Jenny: We're watching a Burr Connors film festival. [On the TV, Burr kisses the uppity preacher's daughter.] Carmen: Yes, Miss Jenny over here is going to ghostwrite his memoirs. Jenny: (to Carmen) I'm gonna be his stenographer. Mark: No way. Do you know that I had a Burr Connor action figure doll as a child? Jenny: You did? Mark: I did, and I made him beat up my G.I. Joe on a daily basis. Jenny: Are you serious? You actually thought this guy, Burr Connor, is more - is tougher than G.I. Joe? Mark: Oh, pffpt. Look at him. Way tougher. [On the TV, Burr stands in his cowboy hat and duster, looking tough.] Jenny: This is sentimental claptrap. Mark: How could you possibly call these macho men sentimental? Jenny: Um, because it's a pretext for telling stories about, like, the purity of male friendship and devotion. I mean, Mark, okay, look at all these films. They're all f*cking the same. Mark: Chick flicks are the same! Carmen: Okay, okay, you know what? You cannot tell that theory to Burr Connors. No. (to Jenny) Kiss. Now. [Carmen grabs Jenny's face and plants a kiss on her.] Mark: Thelma and Louise Carmen: (kissing) Mwah! (to Mark) No more. Mark: Where's Shane, by the way? Carmen: Um, I - I think she, uh, went out. Mark: Where? Carmen: I dunno. Mark: To a club? Jenny: (giggling) We don't know, Mark. [They all laugh.] Carmen: Yeah. Mark: Sixteen Candles. Jenny: Dukes of Hazzard. Mark: Gone With the Wind, Funny Girl. [Carmen suddenly looks distracted. Jenny strokes her hair.] [SCENE_BREAK] INT. - CHURCH CONFESSIONAL - NIGHT [Shane sits in a confessional with a priest. She's crying.] Shane: Bless me father, for I have sinned. It's been... sixteen years... since my last confession. Priest: And what brought you here tonight? Shane: Everyone... wants something from me, and... I don't feel like I have anything left to give. Priest: What have you been giving up until now? Shane: s*x. That's mainly what people want. Actually, I... I don't even know at this point. I don't - I don't - I don't know. [Shane hangs her head and sniffs.] Priest: Do you feel you have to have s*x with everyone who wants it? Shane: In church I didn't. I used to, uh, live in a church shelter, so... Priest: When was that? Shane: I guess I was 10. And I ran away from my foster family because someone told me my real mom was back in Austin. And she used to go to that shelter when she was trying to get clean. Priest: Your mother was a drug addict? Shane: Yeah. Priest: Have you ever considered joining a church group? Shane: No. No. No, no, I don't like groups. The thing I... I like about confession is... you don't have to see the other person's face. And you don't have to see how - how hurt they are when they realize that you can't be that thing they want you to be. Priest: You might find that there are people who don't want anything from you. [Shane chuckles uneasily.] Priest: They just want to know you. Shane: Yeah, I haven't met anyone like that. Anyway, there's nothing to know. [Shane gets up and walks out.] Priest: My friend, would you just consider it? [The priest sits in the confessional while we hear Shane's bootsteps growing farther away.] INT. - ALICE'S APARTMENT - BATHROOM - DAY [Alice is getting out of the shower. Dana hands her a towel.] Alice: Ah, thank you. Dana: Sure. [Dana goes to the mirror.] Dana: Alice! Alice: What? (looks up) Oh. Yeah. [Alice climbs out of the shower. Dana is pointing to something on her neck. It's a love bite.] Alice: Yeah, I thought that would go away. Dana: You knew you did this to me? Alice: (smiling) Well, I wasn't doing it on purpose. You bruise easy. Dana: You're not the only one. [Dana goes to the toilet. Alice gawks at herself in the mirror. She has a love bite on her neck, too.] Alice: Oh, my god! I look like I've been beaten! (looks at Dana) I have my interview today! Dana: It's radio. Alice: I have to make an impression. Dana: And I don't. You're right. I have a photo shoot with Women's Fitness today. I'm the first out lesbian they've ever put on the cover. [Alice messes with her hair in the mirror.] Alice: Well, you'll just be saying you can be gay and a slut. [Dana pulls her pants up and smiles, and heads for the door.] Alice: Is there any more? Dana: Yeah! Alice: Where. [Dana leaves. Alice turns around and around, trying to see her back.] INT. - THE PLANET - DAY [Jenny, Carmen, Kit, and Bette are sitting at a table. Carmen is telling a joke. Everyone is laughing.] Kit: Oh, really! [Alice and Dana walk to the table, arm in arm, both of them dressed up and also wearing neck scarves. Everyone is excited to see them and they all say hi. Alice and Dana sit.] Jenny: I haven't seen you guys in a couple days. What have you been doing? [Alice and Dana speak over each other.] Alice: Working. Dana: Training. [Dana gawps at Alice. Everybody chuckles. They don't notice when Tina walks up behind them. She pats their heads.] Tina: They've been having s*x all weekend. Alice: Hi! Dana: Hey! Tina: Hi, everyone. Kit: Hi. [Kit looks unsure of Tina's presence. Bette looks sad now. Helena walks up behind Tina.] Bette: Um, why don't you two join us? Tina: Okay... (smiles) Thank you. That's nice. [Helena and Tina go to sit. Alice and Dana look at Bette. Bette smiles and keeps her game face on.] Carmen: Hi. Hello. [Carmen and Jenny hold hands. Helena and Tina sit at the end of the table, near Bette.] Jenny: So, um... Tina was just saying that Dana and Alice are having s*x. [Alice and Dana grin and giggle, along with everyone else.] Helena: (smiling) There's nothing to be ashamed of. They'll be in good company. [Helena puts her hand on Tina's and smiles at her. Bette leans back in her seat and a little shocked, but tries to smile. She and Tina catch each other's glance for just a moment, and both quickly look away.] Alice: Yeah, we haven't, though. I've been busy and she's been busy and... Dana: Working. Kit: Oh, yeah, uh, let me see. [Kit pulls away Dana's scarf.] Dana: No... Kit: Busy! Everyone: Ohh! Carmen: Okay, let's see. [Carmen leans forward and pulls Alice's scarf away a little.] Carmen: Um, working! [Everyone giggles.] Carmen: Oh, my goodness! [Bette is grinning at the pair. She sneaks a glance at Tina. Again, they catch each other's glances, and again, they both look away quickly.] Kit: Working it, working it, uh-huh. Carmen: Let us see, let us see! Open it up. [Alice undoes her scarf.] Alice: Alright. I have... [Alice shows off the love bite. Everyone claps and laughs.] Carmen: Oh! Jenny: Oh, my god! Carmen: Oh, my god! Alice: I have my KCRW audition and I kinda don't know what to do so I was... (smiles) Helena: Look, you know... [Helena stands, untying the scarf from around her waist, and walks over to Alice.] Helena: Why don't you take this. Because passion should never cost you respect. [Helena drapes the expensive silk scarf around Alice's neck.] Alice: No that - (chuckling) Helena, I couldn't accept this. It's Hermes. Helena: It's vintage. And for having such a good eye, you get to keep it. Kit: Ohh! Carmen: Wow. [Helena walks back and sits. Alice, still smiling, politely removes the scarf. Dana is watching Bette.] Bette: You know, I think I'd better be getting to work. Helena: Well, that's a shame. Why don't you take the morning off? Bette: Well, because it's not really an option for me. Helena: Of course it is. You're having breakfast with one of your board members. And we're gonna discuss what we're gonna talk about with Allyn Barnes tonight. Kit: (mumbling to Bette) You've gotta be kidding. Bette: (to everyone) Uh, Helena has just joined the board of directors for the CAC. [Alice and Dana raise the brows. Helena beams. Tina smiles but looks a little embarrassed.] Bette: (to Helena) I'll see you at the meeting. [Bette stands to go.] Helena: Well - Bette, why don't you come over later tonight, after the meeting? (smiling at Tina) Tina and I have found a house. Tina: (smiling) Oh - no - Helena has found a house. [Bette frowns.] Helena: Well, you know what, why don't you all come over tonight, we'll have a, uh - Tina: (shrugs, smiling) Yeah. Helena: - a house warming party! [Dana's brows shoot up. Alice looks at everyone. Kit sits quietly, staring at the table.] Tina: (to Bette) You should see this house. It's amazing. It's the one on Broad Beach. The one in Architectural Digest? It's gorgeous. Bette: (smiling) Of course it is. [Bette glances at Tina, then Helena.] Bette: You know, that sounds really nice, Helena. [Helena smiles and nods.] Bette: Maybe if I'm not too tired. Helena: Good. [Bette starts to walk off.] Alice: Bye, Bette. Jenny: Bye. Bette: Bye, you guys, good luck. Alice: Thanks. [Bette leaves. Kit follows.] INT. - JENNY AND SHANE'S HOUSE - KITCHEN - DAY [Shane stands at the counter, fixing food. Mark walks in the back door.] Mark: Hey. Shane: Hey. [Mark sits on the counter.] Mark: Did I say something to offend you? Shane: No. Look. You saved my ass. And I'm sorry if I didn't thank you adequately. So thank you. Mark: Do you know anything about, uh... about the Samurai code, Ronin? Shane: That one of those Hong Kong action flicks? Mark: No, it's a book actually. I was really into that sh1t when I was like 13, 14, got my first degree black belt in Shotoka. Shane: Lucky for me, huh? Mark: Yeah. Well, according to the Samurai code, if you save someone's ass you're forever indebted to that person. You're - it's the greatest honor and privilege you can ever achieve. Shane: Look, I - I said thank you. I don't know what else you want me to do. Mark: Not you, me. I'm indebted to you for giving me that opportunity. [Shane leans against the counter and looks at him.] Mark: No, so, it's like, basically, I'm your servant, now. Shane: (thinking) No thank you. Mark: It's just the way it's gotta be. You're stuck with me. [Mark pulls out a chair at the table.] Mark: Sit, please. [Shane grabs a bag of food and opens the fridge.] Mark: Oh, oh, oh! Allow me. [Mark takes the food from her hand, and the rest of the stuff on the counter. He puts them in the fridge. Shane stands in the middle of the room, stupefied.] Mark: (fake British accent) Will you be having a beverage, my lord? [Shane stands dumfounded.] Shane: No. EXT. - BURR CONNOR'S DRIVEWAY - DAY [Jenny pulls up to the intercom at the gate and presses the button.] Woman: (intercom) Hello? Jenny: Hi. Um, this is Jenny Schecter for Mr. Burr Connor, please? Woman: (intercom) Yes, Miss Schecter, come on in. Jenny: Thank you. [The gate opens. Jenny drives up the driveway, to a huge mansion.] INT. - BURR CONNOR'S HOUSE - DAY [Jenny and Burr sit in the parlor, talking. Burr is an attractive man in his 40s.] Jenny: It's a hard feeling to describe. But I think that I've known for a really long time, and I fought against it because I knew that my... family... wouldn't approve, but I actually think that I've always known. Burr: Yeah, I know exactly what you mean. Jenny: Yeah? Burr: Mm-hmm. From the time I was a small boy, I... I always knew I wanted to be an actor. Jenny: How cool. Burr: So... like, what - what kinda writers would you say you model yourself after? Judith Krantz, Danielle Steele? Like that? [Jenny's jaw drops.] Jenny: Ew! You're kidding. I'm sorry. Burr: Looking at you, having spent a few minutes with you, um... I'm good at this. [Burr stares at Jenny for a moment.] Burr: Mary Gaitskill. Jenny: (gasps) Wow! Thank you. [Burr smiles.] Jenny: I don't know. I mean, Charlotte's pushing me really hard right now, so my style is just evolving pretty fast. Burr: Well, I won't be pushing you. Okay? I, uh... I basically just need you to correct my grammar... Jenny: Mm-hmm. Burr: And add an adjective or two here and there. Jenny: 'Kay. Have you - have you ever written anything before? Burr: No, not really. I've been tape-recording myself. Jenny: Oh, yeah? Burr: Yeah. Jenny: Oh, okay. Actually, I have this. [Jenny reaches in her bag and pulls out a micro-cassette recorder.] Burr: No. Uh. You can put that away. Jenny: Okay, then I'll put it away. [Jenny puts it back in her bag, then sits up and smiles, then goes back into her bag.] Jenny: Can I - can I just use um, my notebook? [Jenny whips out a notebook and pen.] Burr: No, go ahead. Jenny: Um, okay. I have a couple of questions for you, to begin. Burr: Mm-hmm. Jenny: Rod Sebring. It appears as though you have a very profound connection with him. Burr: Mm-hmm. Jenny: Yeah. Burr: Well, we did three pictures together. Jenny: Yeah. The scene in, uh, in Hard Man To Know, where you carry Mr. Sebring across Texas after he's been shot... it's beautiful and quite romantic. [Burr looks a little uncomfortable.] Burr: Mm-hmm. You like that movie? Jenny: I loved it! I really - I - I thought it was like - I loved it. Burr: Really? Jenny: Yeah. Burr: You don't seem like the type. Jenny: Oh, my god. I've seen all of your films. Burr: I'm sure you have, yeah. Jenny: Yep. Burr: In the last two days, right? Jenny: Yes, but (laughs)... Okay, but - but - but my girlfriend actually has seen all your films. Burr: Your girlfriend. Jenny: Yeah. When she was a little kid, she and her step-dad used to go to all your films so now she can recite all your dialogue. Burr: So she's a tomboy? Jenny: (thinking) Gosh, I've never thought of her like that. I think she's just beautiful. Burr: Well... (standing) Miss Schecter, thank you so much for coming on over. And I'll be in touch with you to let you know if I'll be needing your help. Jenny: Oh. Okay. Burr: Okay. [Jenny puts her notebook in her bag and stands.] Jenny: Um, are you firing me, um... because I'm gay? Burr: Well, I wasn't aware that I'd actually hired you. But I do prefer to have certain things flaunted in my face. [Jenny walks out.] INT. - THE PLANET - DAY [Kit is taking stock behind the bar. Alice sits on a barstool, typing on her laptop.] Alice: My big catch-phrase-ending could be, um, "Go ask Alice 'cause she knows what's goin' on!" Like... (looks at Kit) Kit: No, that's good. That's good. Kit: Um, look, I'm gonna go talk to my new chef. Alice: What? Kit: Yeah! Alice: You got a new chef, that's so cool. Kit: Girl, I stole her from A.O.C. She was training under Suzanne Goins. Alice: Maybe I should write about that. I mean, it - it's gotta be better than what I have. Kit: No, no, yours is really good, Alice! I mean, you just gotta (lowers voice) relax... (raises voice) (smiling) and breathe. Alice: Benjamin. Right. Kit: You bet. Alice: Yeah. Kit: Okay? Alice: Yeah. [Kit walks away. Alice stares at her laptop screen.] Alice: (sighs) (mumbling) This is Alice Pieszecki reminding you to relax and breathe. Relax and... [Gabby Devaux enters, sees Alice, and walks over.] Alice: (mumbling) Reminding you to relax and breathe. Gabby: Alice. [Alice looks up.] Alice: (shocked) Gabby! Gabby: (smiling) I thought you'd have a more interesting demise than muttering to yourself at The Planet. Alice: Yeah - wow. [Gabby smiles and opens her arms.] Gabby: Gimme a hug. Alice: Oh. 'Kay. Oh. Gabby: Hi! Alice: Hi. Gabby: You look good. Alice: Thanks. What's wrong with you? Gabby: Mm, nothing except that I'm in love. [Alice raises her brows.] Gabby: Yeah. Gabby Devaux in love. Alice: (surprised) Wow. Gabby: How are you? What's going on with you? Alice: Um - I'm in love too! Gabby: (smiles) Aw. Alice: Yeah. You can't tell? Um, maybe I'm not showing it or something - I'm very private, so. Gabby: Alice "tell me all your secrets and I'll sell them to the Daily Planet" Pieszecki? [Alice smiles.] Gabby: So, who is tDS

When Alice tries to catch up on her latest journalism assignment, she accidentally runs into her ex-lover Gabby and is more surprised to find out that Gabby is now dating Dana's ex-girlfriend, Lara. Bette and Tina try to reach an agreement over their relationship, as Tina goes house-hunting with Helena who comes on board the CAC's board of directors. Bette meets Winnie who wants to recruit Bette over helping her win her custody battle over Winnie and Helena's kids. Jenny is worried that she may have lost the job of ghost-writing a TV star named Burr Connor's autobiography because she lets it slip out about her lesbianism. Mark and his pal, Gomey, meet with a sleazy producer for their lesbian reality video, while Shane finds some comfort in a church over her recent emotional setbacks.

summ_screen_fd

By. Associated Press and Daily Mail Reporter. PUBLISHED:. 23:00 EST, 17 March 2013. |. UPDATED:. 17:10 EST, 18 March 2013. Prosecutors said Monday that they won't charge a Texas couple in the death of a 3-year-old adopted boy from Russia, a case that has become the latest flashpoint in the debate over whether American families should be allowed to adopt Russian children. Ector County District Attorney Bobby Bland said his office would not charge Alan and Laura Shatto in the Jan. 21 death of Max Alan Shatto, who was born Maxim Kuzmin. 'The grand jury determined there was insufficient evidence to charge them with anything,' Bland said at a news conference. Autopsy: The medical examiner ruled that the death of Max Shatto was an accident. Accused: Kremlin accused Laura Shatto, right, of beating little Max, left, to death in January. Laura Shatto told authorities she. found Max unresponsive outside their Gardendale, Texas, home while he. was playing with his younger brother, Ector County Sheriff Mark. Donaldson has said. The boy was pronounced dead at a hospital a short. time later. Preliminary autopsy results indicated Shatto had bruises on. several parts of his body, though four doctors reviewing the final. autopsy result ruled his death to be accidental. Bland, the top prosecutor in Ector. County, about 350 miles west of Dallas, previously said that the bruises. on Max's body appeared to be self-inflicted, and that no drugs were. found in Max's system. Russian authorities and state-run. media have blamed the Shattos for Max's death and used the case as. justification for a recently enacted ban on all American adoptions of. Russian children. Parents: The boy's adoptive parents, Alan and Laura Shatto, denied knowing about the allegations being made by Russian authorities. No arrests have been made. Tragedy: Max Shatto, 3, died last month after being adopted by a family in Texas. Russian authorities are claiming he was abused. Russian born: Maxim was born the town of Pskov, near Russia's western border with Estonia. The boy lived with a family in Gardendale, Texas with his family before his death. Russia's Investigative Committee has said it has. opened its own investigation. It's unclear whether the committee could. charge the Shatto family or force their prosecution. U.S. State Department officials and adoption agency advocates have called for caution. The Russian government passed the ban. in December in retaliation for a new U.S. law targeting alleged Russian. human-rights violators. The ban also reflects lingering resentment over. the perceived mistreatment of some of the 60,000 children Americans. have adopted over the last two decades. At least 20 of those children. have died, and reports of abuse have garnered attention in Russia. Outrage: Demonstrators hold portraits of adopted Russian children who have died in the U.S., during a massive rally in Moscow. Protests: This woman holds a sign that reads 'Juvenile judiciary. Give us parents back!' Demonstrators walked along Moscow streets to support the new law prohibiting the adoption of Russian children by Americans. Rally: Activists from pro-Kremlin children's advocacy groups march through Moscow, demanding the return of an adopted boy whose brother, Max Shatto, died in Texas. Keeping mum: The driveway to the Shatto family home, rear left, is seen in Gardendale, Texas, with a sign that reads: 'No Comment' Foreign Ministry official Konstantin. Dolgov has called Max's death 'yet another case of inhuman treatment of a. Russian child adopted by American parents.' Texas Child Protective Services. spokesman Patrick Crimmins said the agency was investigating allegations. that Max was subject to physical abuse and neglect but had not. determined whether those allegations were true. The agency that. processed the Shattos' adoption, the Gladney Center for Adoption in Fort. Worth, was cleared in a separate state investigation to find out. whether it followed all guidelines. The Shattos adopted Max and his. biological half-brother, 2-year-old Kristopher, from the same orphanage. in western Russia. Since Max's death, Kristopher has remained with his. adoptive parents. Russian state media have interviewed the. boys' biological mother, Yulia Kuzmina, who lost custody over. negligence and serious drinking problems. Sad: The little boy, pictured right, was pronounced dead in hospital on Jan. 21. In a tightly choreographed Feb. 21. interview on state television, Kuzmina insisted that Russian custody. officials seized her children unfairly and said that she wanted to be. reunited with her other son, born Kirill Kuzmin. She said she had given. up drinking, found a job and pledged to fight to get the boy back. Russian President Vladimir Putin's. spokesman, Dmitry Peskov, has said it is necessary 'to temper emotions' over the case, and U.S. Ambassador to Russia Michael McFaul has called. for'sensational exploitations of human tragedy to end and for. professional work between our two countries to grow, on this issue and. many others.' Russia's Investigative Committee said that it had opened an. investigation into the little boy's death. 'Three-year-old Maxim was beaten,. according to the investigators, by his adoptive mother, who fed him. psychoactive drugs over a long period of time, saying that he had some. psychiatric illness,' said Pavel Astakhov, the Russian Children's Rights. Commissioner alleges, according to The Telegraph. Plea: In a tightly choreographed interview, Max's biological mother, 23-year-old Yulia Kuzmina, tearfully appealed to President Putin to have her surviving son returned to her. Accusations: Pavel Astakho has alleged that Maxim was beaten to death by his American mother. American authorities have not confirmed the allegations are still investigating. Look of love: Alan Shatto embraces his son, Maxim, in this photo. Max's parents wrote in his obituary, 'When we get to Heaven, we know we will hear your sweet voice singing with the angels. We love you and will always miss you' Timing: Max's death comes weeks after Russian authorities announced it was banning all adoptions by Americans

Ector County Medical Examiner's office on Friday revealed that the death of Maxim Shatto, 3, was accidental. Little Max died on Jan. 21 after being adopted by a family in Texas. Russian authorities alleged he was abused by his adoptive mother and given psychiatric drugs. Texas officials believe the bruises found on his body were self-inflicted. Latest in ongoing battle between two counties over adoptions and death comes weeks after Russians banned all adoptions to Americans. American authorities investigating claims but no arrests have been made.

cnn_dailymail

FEMA Has Taken Actions to Address Grant Management Concerns but Needs Better Coordination DHS and FEMA have streamlined application and award processes, enhanced the use of risk management principles in its grant programs, and proposed consolidation of its various grant programs to address grant management concerns. In February 2012, we reported that better coordination and improved data collection could help FEMA identify and mitigate potential unnecessary duplication among four overlapping grant programs—the Homeland Security Grant Program, the Urban Areas Security Initiative, the Port Security Grant Program, and the Transit Security Grant Program. FEMA has proposed changes to enhance preparedness grant management, but these changes may create new challenges. FEMA Has Streamlined Application and Award Processes and Enhanced Use of Risk Management Principles Since its creation in April 2007, FEMA’s Grant Programs Directorate (GPD) has been responsible for the program management of DHS’s preparedness grants. GPD consolidated the grant business operations, systems, training, policy, and oversight of all FEMA grants and the program management of preparedness grants into a single entity. GPD works closely with other DHS entities to manage grants, as needed, through the grant life cycle, shown in figure 1. For example, GPD works with the U.S. Coast Guard for the Port Security Grant Program and the Transportation Security Administration for the Transit Security Grant Program. Since 2006, DHS has taken a number of actions to improve its risk-based grant allocation methodology. Specifically, in March 2008, we reported that DHS had adopted a more sophisticated risk-based grant allocation approach for the Urban Areas Security Initiative to (1) determine both states’ and urban areas’ potential risk relative to other areas that included empirical analytical methods and policy judgments, and (2) assess and score the effectiveness of the proposed investments submitted by the eligible applicants and determine the final amount of funds awarded. We also reported that DHS’s risk model for the Urban Areas Security Initiative could be strengthened by measuring variations in vulnerability. Specifically, we reported that DHS had held vulnerability constant, which limited the model’s overall ability to assess risk and more precisely allocate funds. Accordingly, we recommended that DHS and FEMA formulate a method to measure vulnerability in a way that captures variations in vulnerability, and apply this vulnerability measure in future iterations of this risk-based grant allocation model. DHS concurred with our recommendations and FEMA took actions to enhance its approaches for assessing and incorporating vulnerability into risk assessment methodologies for this program. Specifically, FEMA created a risk assessment that places greater weight on threat and calculates the contribution of vulnerability and consequence separately. In June 2009, we reported that DHS used a risk analysis model to allocate Transit Security Grant Program funding and awarded grants to higher-risk transit agencies using all three elements of risk—threat, vulnerability, and consequence. Accordingly, we recommended that DHS formulate a method to measure vulnerability in a way that captures variations in vulnerability, and apply this vulnerability measure in future iterations of this risk-based grant allocation model. DHS concurred with our recommendations and FEMA took actions to enhance its approach for assessing and incorporating vulnerability into risk assessment methodologies for this program. In November 2011, we reported that DHS had made modifications to enhance the Port Security Grant Program’s risk assessment model’s vulnerability element for fiscal year 2011. Specifically, DHS modified the vulnerability equation to recognize that different ports have different vulnerability levels. We also reported that FEMA had taken actions to streamline the Port Security Grant Program’s management efforts. For example, FEMA shortened application time frames by requiring port areas to submit specific project proposals at the time of grant application. According to FEMA officials, this change was intended to expedite the grant distribution process. Further, we reported that to speed the process, DHS took actions to reduce delays in environmental reviews, increased the number of GPD staff working on the Port Security Grants, revised and streamlined grant application forms, and developed time frames for review of project documentation. FEMA Needs Better Coordination and Improved Data Collection to Reduce Risk of Unnecessary Duplication Despite these continuing efforts to enhance preparedness grant management, we identified multiple factors in our February 2012 report that contributed to the risk of FEMA potentially funding unnecessarily duplicative projects across the four grant programs we reviewed—the Homeland Security Grant Program, the Urban Areas Security Initiative, the Port Security Grant Program, and the Transit Security Grant Program.and geographic locations, combined with differing levels of information that FEMA had available regarding grant projects and recipients. We also reported that FEMA lacked a process to coordinate application reviews across the four grant programs. These factors include overlap among grant recipients, goals, Overlap among grant recipients, goals, and geographic locations exist. The four grant programs we reviewed have similar goals and fund similar activities, such as equipment and training in overlapping jurisdictions, which increases the risk of unnecessary duplication among the programs. For instance, each state and eligible territory receives a legislatively-mandated minimum amount of State Homeland Security Program funding to help ensure that geographic areas develop a basic level of preparedness, while the Urban Areas Security Initiative grants explicitly target urban areas most at risk of terrorist attack. However, many jurisdictions within designated Urban Areas Security Initiative regions also apply for and receive State Homeland Security Program funding. Similarly, port stakeholders in urban areas could receive funding for equipment such as patrol boats through both the Port Security Grant Program and the Urban Areas Security Initiative, and a transit agency could purchase surveillance equipment with Transit Security Grant Program or Urban Areas Security Initiative funding. While we understand that some overlap may be desirable to provide multiple sources of funding, a lack of visibility over grant award details around these programs increases the risk of unintended and unnecessary duplication. FEMA made award decisions for all four grant programs with differing levels of information. In February 2012, we reported that FEMA’s ability to track which projects receive funding among the four grant programs varied because the project information FEMA had available to make award decisions—including grant funding amounts, grant recipients, and grant funding purposes—also varied by program due to differences in the grant programs’ administrative processes. For example, FEMA delegated some administrative duties to stakeholders for the State Homeland Security Program and the Urban Areas Security Initiative, thereby reducing its administrative burden. However, this delegation also contributed to FEMA having less visibility over some grant applications. FEMA recognized the trade-off between decreased visibility over grant funding in exchange for its reduced administrative burden. Differences in information requirements also affected the level of information that FEMA had available for making grant award decisions. For example, for the State Homeland Security Program and Urban Areas Security Initiative, states and eligible urban areas submit investment justifications for each program with up to 15 distinct investment descriptions that describe general proposals in wide-ranging areas such as “critical infrastructure protection.” Each investment justification encompasses multiple specific projects to different jurisdictions or entities, but project-level information, such as a detailed listing of subrecipients or equipment costs, is not required by FEMA. In contrast, Port Security and Transit Security Grant Program applications require specific information on individual projects such as detailed budget summaries. As a result, FEMA has a much clearer understanding of what is being requested and what is being funded by these programs. FEMA has studied the potential utilization of more specific project-level data for making grant award decisions, especially for the State Homeland Security Program and Urban Areas Security Initiative. However, while our analysis of selected grant projects determined that this additional information was sufficient for identifying potentially unnecessary duplication for nearly all of the projects it reviewed, the information did not always provide FEMA with sufficient detail to identify and prevent the risk of unnecessary duplication. While utilizing more specific project-level data would be a step in the right direction, at the time of our February 2012 report, FEMA had not determined the specifics of future data requirements. FEMA lacked a process to coordinate application reviews across the four grant programs. In February 2012, we reported that grant applications were reviewed separately by program and were not compared across each other to determine where possible unnecessary duplication may occur. Specifically, FEMA’s Homeland Security Grant Program branch administered the Urban Areas Security Initiative and State Homeland Security Program while the Transportation Infrastructure Security branch administered the Port Security Grant Program and Transit Security Grant Program. We and the DHS Inspector General concluded that coordinating the review of grant projects internally would give FEMA more complete information about applications across the four grant programs, which could help FEMA identify and mitigate the risk of unnecessary duplication across grant applications. In our February 2012 report, we note that one of FEMA’s section chiefs said that the primary reasons for the current lack of coordination across programs are the sheer volume of grant applications that need to be reviewed and FEMA’s lack of resources to coordinate the grant review process. She added that FEMA reminds grantees not to duplicate grant projects; however, due to volume and the number of activities associated with grant application reviews, FEMA lacks the capabilities to cross-check for unnecessary duplication. We recognize the challenges associated with reviewing a large volume of grant applications, but to help reduce the risk of funding duplicative projects, FEMA could benefit from exploring opportunities to enhance its coordination of project reviews while also taking into account the large volume of grant applications it must process. Thus, we recommended that FEMA take actions to identify and mitigate any unnecessary duplication in these programs, such as collecting more complete project information as well as exploring opportunities to enhance FEMA’s internal coordination and administration of the programs. In commenting on the report, DHS agreed and identified planned actions to improve visibility and coordination across programs and projects. We also suggested that Congress consider requiring DHS to report on the results of its efforts to identify and prevent duplication within and across the four grant programs, and consider these results when making future funding decisions for these programs. FEMA Has Proposed Changes to Enhance Preparedness Grant Management, but these Changes May Create Challenges In the President’s Fiscal Year 2013 budget request to Congress, FEMA has proposed consolidating its various preparedness grant programs— with the exception of the Emergency Management Performance Grants and Assistance to Fire Fighters Grants—into a single, comprehensive preparedness grant program called the National Preparedness Grant Program (NPGP) in fiscal year 2013. FEMA also plans to enhance its preparedness grants management through a variety of proposed initiatives to implement the new consolidated program. According to FEMA, the new NPGP will require grantees to develop and sustain core capabilities outlined in the National Preparedness Goal rather than work to meet mandates within individual, and often disconnected, grant programs. NPGP is intended to focus on creating a robust national response capacity based on cross-jurisdictional and readily deployable state and local assets. According to FEMA’s policy announcement, consolidating the preparedness grant programs will support the recommendations of the Redundancy Elimination and Enhanced Performance for Preparedness Grants Act, and will streamline the grant application process. This will, in turn, enable grantees to focus on how federal funds can add value to their jurisdiction’s unique preparedness needs while contributing to national response capabilities. To further increase the efficiency of the new grant program, FEMA plans to issue multi-year guidelines, enabling the agency to focus its efforts on measuring progress towards building and sustaining national capabilities. The intent of this consolidation is to eliminate administration redundancies and ensure that all preparedness grants are contributing to the National Preparedness Goal. For fiscal year 2013, FEMA believes that the reorganization of preparedness grants will allow for a more targeted grants approach where states build upon the capabilities established with previous grant money and has requested $1.54 billion for the National Preparedness Grant Program. FEMA’s Fiscal Year 2013 Grants Drawdown Budget in Brief also proposes additional measures to enhance preparedness grant management efforts and expedite prior years’ grant expenditures. For example, to support reprioritization of unobligated prior year funds and focus on building core capabilities, FEMA plans to: allow grantees to apply prior years’ grant balances towards more urgent priorities, promising an expedited project approval by FEMA’s Grant Programs Directorate; expand allowable expenses under the Port Security Grant Program and Transit Security Grant Program, for example, by allowing maintenance and sustainment expenses for equipment, training, and critical resources that have previously been purchased with either federal grants or any other source of funding to support existing core capabilities tied to the five mission areas contained within the National Preparedness Goal. The changes FEMA has proposed for its fiscal year 2013 National Preparedness Grants program may create new management challenges. As noted by Chairman Bilirakis in last month’s hearing by the House Homeland Security Committee’s Subcommittee on Emergency Preparedness, Response, and Communications, allocations under the new grant program would rely heavily on a state’s Threat and Hazard Identification and Risk Assessment (THIRA). However, nearly a year after the THIRA concept was first introduced as part of the fiscal year 2011 grant guidance, grantees have yet to receive guidance on how to conduct the THIRA process. As we reported in February 2012, questions also remain as to how local stakeholders would be involved in the THIRA process at the state level. In March 2012, FEMA’s GPD announced that FEMA has established a website to solicit input from stakeholders on how best to implement the new program. According to Chairman Bilirakis, it is essential that the local law enforcement, first responders, and emergency managers who are first on the scene of a terrorist attack, natural disaster, or other emergency be involved in the THIRA process. They know the threats to their local areas and the capabilities needed to address them. Finally, according to FEMA’s plans, the new National Preparedness Grant Program will require grantees to develop and sustain core capabilities; however, the framework for assessing capabilities and prioritizing national preparedness grant investments is still not complete. As we noted in our February 2012 report, FEMA’S efforts to measure the collective effectiveness of its grants programs are recent and ongoing and thus it is too soon to evaluate the extent to which these initiatives will provide FEMA with the information it needs to determine whether these grant programs are effectively improving the nation’s security. FEMA Has Not Yet Completed National Preparedness Assessment Efforts to Address Longstanding Concerns DHS and FEMA have had difficulty in implementing longstanding plans to develop and implement a system for assessing national preparedness capabilities. For example, DHS first developed plans in 2004 to measure preparedness by assessing capabilities, but these efforts have been repeatedly delayed and are not yet complete. FEMA’s proposed revisions to the new NPGP may help the agency overcome these continuing challenges to developing and implementing a national preparedness assessment. DHS and FEMA’s Longstanding Plans to Develop and Implement a National Assessment of Preparedness Have Not Been Fulfilled Since 2004, DHS and FEMA have initiated a variety of efforts to develop a system of measuring preparedness. From 2005 until September 2011, much of FEMA’s efforts focused on developing and operationalizing a list of target capabilities that would define desired capabilities and could be used in a tiered framework to measure their attainment. In July 2005, we reported that DHS had established a draft Target Capabilities List that provides guidance on the specific capabilities and levels of capability at various levels of government that FEMA would expect federal, state, local, and tribal first responders to develop and maintain.to organize classes of jurisdictions that share similar characteristics— such as total population, population density, and critical infrastructure— into tiers to account for reasonable differences in capability levels among groups of jurisdictions and to appropriately apportion responsibility for development and maintenance of capabilities among levels of government and across these jurisdictional tiers. According to DHS’s Assessment and Reporting Implementation Plan, DHS intended to implement a capability assessment and reporting system based on target capabilities that would allow first responders to assess their preparedness by identifying gaps, excesses, or deficiencies in their existing capabilities or capabilities they will be expected to access through mutual aid. In addition, this information could be used to (1) measure the readiness of federal civil response assets, (2) measure the use of federal assistance at the state and local levels, and (3) assess how federal assistance programs are supporting national preparedness. DHS planned DHS’s efforts to implement these plans were interrupted by the 2005 hurricane season. In August 2005, Hurricane Katrina—the worst natural disaster in our nation’s history—made final landfall in coastal Louisiana and Mississippi, and its destructive force extended to the western Alabama coast. Hurricane Katrina and the following Hurricanes Rita and Wilma—also among the most powerful hurricanes in the nation’s history—graphically illustrated the limitations at that time of the nation’s readiness and ability to respond effectively to a catastrophic disaster; that is, a disaster whose effects almost immediately overwhelm the response capabilities of affected state and local first responders and require outside action and support from the federal government and other entities. In June 2006, DHS concluded that target capabilities and associated performance measures should serve as the common reference system for preparedness planning. In September 2006, we reported that numerous reports and our work suggested that the substantial resources and capabilities marshaled by federal, state, and local governments and nongovernmental organizations were insufficient to meet the immediate challenges posed by the unprecedented degree of damage and the resulting number of hurricane victims caused by Hurricanes Katrina and Rita. We also reported that developing the capabilities needed for catastrophic disasters should be part of an overall national preparedness effort that is designed to integrate and define what needs to be done, where it needs to be done, how it should be done, how well it should be done, and based on what standards. FEMA’s National Preparedness Directorate within its Protection and National Preparedness organization was established in April 2007 and is responsible for developing and implementing a system for measuring and assessing national preparedness capabilities. Figure 2 provides an illustration of how federal, state, and local resources provide capabilities for different levels of “incident effect” (i.e., the extent of damage caused by a natural or manmade disaster). In October 2006, Congress passed the Post-Katrina Act that required FEMA, in developing guidelines to define target capabilities, to ensure that such guidelines are specific, flexible, and measurable. In addition, the Post-Katrina Act calls for FEMA to ensure that each component of the national preparedness system, which includes the target capabilities, is developed, revised, and updated with clear and quantifiable performance metrics, measures, and outcomes. We recommended in September 2006, among other things, that DHS apply an all-hazards, risk management approach in deciding whether and how to invest in specific capabilities for a catastrophic disaster. DHS concurred with this recommendation and FEMA said it planned to use the Target Capabilities List to assess capabilities to address all hazards. In September 2007, FEMA issued an updated version of the Target Capabilities List to provide a common perspective in conducting assessments that determine levels of readiness to perform critical tasks and identify and address any gaps or deficiencies. According to FEMA, policymakers need regular reports on the status of capabilities for which they have responsibility to help them make better resource and investment decisions and to establish priorities. In April 2009, we reported that establishing quantifiable metrics for target capabilities was a prerequisite to developing assessment data that can be compared across all levels of government. At the time of our review, FEMA was in the process of refining the target capabilities to make them more measurable and to provide state and local jurisdictions with additional guidance on the levels of capability they need. Specifically, FEMA planned to develop quantifiable metrics—or performance objectives—for each of the 37 target capabilities that are to outline specific capability targets that jurisdictions (such as cities) of varying size should strive to meet, recognizing that there is not a “one size fits all” approach to preparedness. In October 2009, in responding to congressional questions regarding FEMA’s plan and timeline for reviewing and revising the 37 target capabilities, FEMA officials said they planned to conduct extensive coordination through stakeholder workshops in all 10 FEMA regions and with all federal agencies with lead and supporting responsibility for emergency support-function activities associated with each of the 37 target capabilities. The workshops were intended to define the risk factors, critical target outcomes, and resource elements for each capability. The response stated that FEMA planned to create a Task Force comprised of federal, state, local, and tribal stakeholders to examine all aspects of preparedness grants, including benchmarking efforts such as the Target Capabilities List. FEMA officials have described their goals for updating the list to include establishing measurable target outcomes, providing an objective means to justify investments and priorities, and promoting mutual aid and resource sharing. In November 2009, FEMA issued a Target Capabilities List Implementation Guide that described the function of the list as a planning tool and not a set of standards or requirements. Finally, in 2011, FEMA announced that the Target Capabilities List would be replaced by a new set of national Core Capabilities. However, it is not clear how the new approach will help FEMA overcome ongoing challenges to assessing national preparedness capabilities discussed below. FEMA Has Not Yet Fully Addressed Ongoing Challenges to Assessing National Preparedness Capabilities FEMA has not yet fully addressed ongoing challenges in developing and implementing a system for assessing national preparedness capabilities. For example, we reported in July 2005 that DHS had identified potential challenges in gathering the information needed to assess capabilities, including determining how to aggregate data from federal, state, local, and tribal governments and others and integrating self-assessment and external assessment approaches. In analyzing FEMA’s efforts to assess capabilities, we further reported in April 2009 that FEMA faced methodological challenges with regard to (1) differences in data available, (2) variations in reporting structures across states, and (3) variations in the level of detail within data sources requiring subjective interpretation. As noted above, FEMA was in the process of refining the target capabilities at the time of our review to make them more measurable and to provide state and local jurisdictions with additional guidance on the levels of capability they need. We recommended that FEMA enhance its project management plan to include milestone dates, among other things, a recommendation to which DHS concurred. In October 2010, we reported that FEMA had enhanced its project management plan by providing milestone dates and identifying key assessment points throughout the project to determine whether project changes are necessary. Nonetheless, DHS and FEMA have had difficulty overcoming the challenges we reported in July 2005 and April 2009 in establishing a system of metrics to assess national preparedness capabilities. As we reported in October 2010, FEMA officials said that, generally, evaluation efforts they used to collect data on national preparedness capabilities were useful for their respective purposes but that the data collected were limited by data reliability and measurement issues related to the lack of standardization in the collection of data. FEMA officials reported that one of its evaluation efforts, the State Preparedness Report, has enabled FEMA to gather data on the progress, capabilities, and accomplishments of the preparedness program of a state, the District of Columbia, or a territory. However, they also said that these reports included self-reported data that may be subject to interpretation by the reporting organizations in each state and not be readily comparable to other states’ data. The officials also stated that they have taken actions to address these limitations by, for example, creating a Web-based survey tool to provide a more standardized way of collecting state preparedness information that will help FEMA officials validate the information by comparing it across states. We reported in October 2010 that FEMA had an ongoing effort to develop measures for target capabilities that would serve as planning guidance, not requirements, to assist in state and local capability assessments. FEMA officials had not yet determined how they planned to revise the Target Capabilities List and said they were awaiting the completed revision of Homeland Security Presidential Directive 8, which was to address national preparedness. That directive, called Presidential Policy Directive 8 on National Preparedness (PPD-8), was issued on March 30, 2011. In March 2011, we reported that FEMA’s efforts to develop and implement a comprehensive, measurable, national preparedness assessment of capability and gaps were not yet complete and suggested that Congress consider limiting preparedness grant funding until FEMA completes a national preparedness assessment of capability gaps at each level based on tiered, capability-specific performance objectives to enable prioritization of grant funding. In April 2011, Congress passed the fiscal year 2011 appropriations act for DHS, which reduced funding for FEMA preparedness grants by $875 million from the amount requested in the President’s fiscal year 2011 budget. The consolidated appropriations act for fiscal year 2012 appropriated $1.7 billion for FEMA Preparedness grants, $1.28 billion less than requested. The House committee report accompanying the DHS appropriations bill for fiscal year 2012 stated that FEMA could not demonstrate how the use of the grants had enhanced disaster preparedness. According to FEMA’s testimony in a hearing on the President’s Fiscal Year 2013 budget request before the House Committee on Homeland Security’s Subcommittee on Emergency Preparedness, Response, and Communications, FEMA became the federal lead for the implementation of PPD-8 in 2011. The new presidential policy directive calls for the development of both a National Preparedness Goal and a National Preparedness System (both of which were required by the Post-Katrina Act in 2006). FEMA issued the National Preparedness Goal in September 2011, which establishes core capabilities for prevention, protection, response, recovery, and mitigation that are to serve as the basis for preparedness activities within FEMA, throughout the federal government, and at the state and local levels. These new core capabilities are the latest evolution of the Target Capabilities List. According to FEMA officials, they plan to continue to organize the implementation of the National Preparedness System and will be working with partners across the emergency management community to integrate activities into a comprehensive campaign to build and sustain preparedness. According to FEMA, many of the programs and processes that support the components of the National Preparedness System exist and are currently in use, while others will need to be updated or developed. For example, FEMA has not yet developed national preparedness capability requirements based on established metrics for the core capabilities to provide a framework for national preparedness assessments. As I testified last year, until such a framework is in place, FEMA will not have a basis to operationalize and implement its conceptual approach for assessing federal, state, and local preparedness capabilities against capability requirements to identify capability gaps for prioritizing investments in national preparedness. Chairman Bilirakis, Ranking Member Richardson, and Members of the Committee, this completes my prepared statement. I would be pleased to respond to any questions that you may have at this time. Contacts and Staff Acknowledgments For further information about this statement, please contact William O. Jenkins Jr., Director, Homeland Security and Justice Issues, at (202) 512- 8777 or jenkinswo@gao.gov. Contact points for our Offices of Congressional Relations and Public Affairs may be found on the last page of this statement. In addition to the contact named above, the following individuals from GAO’s Homeland Security and Justice Team also made major contributions to this testimony: Chris Keisling, Assistant Director; Allyson Goldstein, Dan Klabunde, Tracey King, and Lara Miklozek. This is a work of the U.S. government and is not subject to copyright protection in the United States. The published product may be reproduced and distributed in its entirety without further permission from GAO. However, because this work may contain copyrighted images or other material, permission from the copyright holder may be necessary if you wish to reproduce this material separately.

From fiscal years 2002 through 2011, the federal government appropriated over $37 billion to the Department of Homeland Security’s (DHS) preparedness grant programs to enhance the capabilities of state and local governments to prevent, protect against, respond to, and recover from terrorist attacks. DHS allocated $20.3 billion of this funding to grant recipients through four of the largest preparedness grant programs—the State Homeland Security Program, the Urban Areas Security Initiative, the Port Security Grant Program, and the Transit Security Grant Program. The Post-Katrina Emergency Management Reform Act of 2006 requires the Federal Emergency Management Agency (FEMA) to develop a national preparedness system and assess preparedness capabilities—capabilities needed to respond effectively to disasters. FEMA could then use such a system to help it prioritize grant funding. This testimony addresses the extent to which DHS and FEMA have made progress in managing preparedness grants and measuring preparedness by assessing capabilities and addressing related challenges. GAO’s comments are based on products issued from April 2002 through February 2012 and selected updates conducted in March 2012. DHS and FEMA have taken actions with the goal of enhancing management of preparedness grants, but better project information and coordination could help FEMA identify and mitigate the risk of unnecessary duplication among grant applications. Specifically, DHS and FEMA have taken actions to streamline the application and award processes and have enhanced their use of risk management for allocating grants. For example, in November 2011, GAO reported that DHS modified its risk assessment model for the Port Security Grant Program by recognizing that different ports have different vulnerability levels. However, in February 2012, GAO reported that FEMA made award decisions for four of its grant programs—the State Homeland Security Grant Program, the Urban Area Security Initiative, the Port Security Grant Program, and the Transit Security Grant Program—with differing levels of information, which contributed to the risk of funding unnecessarily duplicative projects. GAO also reported that FEMA did not have a process to coordinate application reviews across the four grant programs. Rather, grant applications were reviewed separately by program and were not compared across each other to determine where possible unnecessary duplication may occur. Thus, GAO recommended that (1) FEMA collect project information with the level of detail needed to better position the agency to identify any potential unnecessary duplication within and across the four grant programs, weighing any additional costs of collecting this data and (2) explore opportunities to enhance FEMA’s internal coordination and administration of the programs to identify and mitigate the potential for any unnecessary duplication. DHS agreed and identified planned actions to improve visibility and coordination across programs and projects. FEMA has proposed consolidating the majority of its various preparedness grant programs into a single, comprehensive preparedness grant program called the National Preparedness Grant Program (NPGP) in fiscal year 2013; however, this may create new challenges. For example, allocations under the NPGP would rely heavily on a state’s risk assessment, but grantees have not yet received guidance on how to conduct the risk assessment process. FEMA has established a website to solicit input from stakeholders on how best to implement the program. DHS and FEMA have had difficulty implementing longstanding plans and overcoming challenges in assessing capabilities, such as determining how to validate and aggregate data from federal, state, local, and tribal governments. For example, DHS first developed plans in 2004 to measure preparedness by assessing capabilities, but these efforts have been repeatedly delayed. In March 2011, GAO reported that FEMA’s efforts to develop and implement a comprehensive, measurable, national preparedness assessment of capability and gaps were not yet complete and suggested that Congress consider limiting preparedness grant funding until FEMA completes a national preparedness assessment of capability gaps based on tiered, capability-specific performance objectives to enable prioritization of grant funding. In April 2011, Congress passed the fiscal year 2011 appropriations act for DHS that reduced funding for FEMA preparedness grants by $875 million from the amount requested in the President’s fiscal year 2011 budget. For fiscal year 2012, Congress appropriated $1.28 billion less than requested in the President’s budget.

gov_report

CROSS REFERENCE TO RELATED APPLICATION Reference is made to U.S. patent application Ser. No. 09/112,917 now U.S. Pat. No. 6,058,327, by Borgerding et al. for an IMPLANTABLE DEVICE WITH AUTOMATIC SENSING ADJUSTMENT, filed Jul. 9, 1998, which contains subject matter related to that of the present application. BACKGROUND OF THE INVENTION The present invention relates to medical stimulators and leads generally, and more particularly to implantable pacemakers, cardioverters and defibrillators. In the context of implantable pacemakers or other stimulators which stimulate and sense electrical activity in multiple chambers of the heart, it has been conventional to provide a blanking period for the amplifier associated with one chamber of the heart, during delivery of a pacing pulse to another chamber of the heart. An earlier example of this feature may be found in U.S. Pat. No. 4,312,355 issued to Funke. It is also conventional to provide a blanking period for the sense amplifier coupled to the chamber being paced, during delivery of the pacing pulse and to provide atrial refractory and/or blanking periods associated with sensed ventricular depolarizations, as in U.S. Pat. No. 5,027,815, issued to Funke and U.S. Pat. No. 5,123,412, issued to Betzold. Particularly in the context of devices which detect tachyarrhythmias, amplifiers have been developed which automatically adjust the effective sensing threshold, in order to facilitate sensing of the relatively lower amplitude depolarization wave forms that may be associated with tachyarrhythmias without sensing the repolarization wave forms associated with depolarizations occurring during normal sinus rhythm. The adjusting of the effective sensing threshold may be accomplished by adjusting the gain of the amplifier and comparing the amplified signal to a fixed threshold and/or by adjusting the threshold level of the detector associated with the amplifier, which adjustments should be understood to be equivalent alternatives in the context of the present invention. One such auto-adjusting amplifier is disclosed in U.S. Pat. No. 5,117,824 issued to Keimel et al, incorporated herein by reference in its entirety. An alternative implementation of an auto adjust amplifier is disclosed in U.S. Pat. No. 5,269,300 issued to Kelly et al., also incorporated herein by reference in its entirety. In these references, following a detected depolarization, the amplifier is automatically adjusted so that the effective sensing threshold is set to be equal to a predetermined portion of the amplitude of the sensed depolarization, and the effective sensing threshold decays thereafter to a lower or base sensing threshold. Following delivery of a pacing pulse, in the system disclosed in the Keimel et al patent, no adjustment is made to the sensing threshold, while in the Kelly et al. patent, following delivery of a pacing pulse the effective sensing threshold is set to a preset value and remains at this value for a defined period of time, after which the threshold decays to the lower or base value. In the context of a device which paces and senses in multiple chambers of the heart, employing blanking and refractory periods as described above, alone or in conjunction with auto adjusting amplifiers as described above, does provide a useful and workable device. However, this approach does not address the difficulties which arise when the signal associated with a depolarization in the ventricle is of sufficient amplitude to be sensed by the atrial sense amplifier, commonly referred to as far-field R-wave sensing. This problem is addressed to some extent by provision of atrial blanking or refractory periods following sensing in the ventricle, but at the cost of the ability to accurately respond to atrial depolarizations occurring within these periods. In addition, the far-field R-wave may sometimes be sensed in the atrium before the R-wave is sensed by the ventricular sense amplifier, prior to initiation of blanking or refractory periods associated with the R-wave. SUMMARY OF THE INVENTION The present invention addresses the problem of far field R-wave sensing by defining a time window associated with a ventricular event (sensed or paced R-wave) during which far field R-wave sensing is likely to occur (hereafter referred to as the “far-R window”) and by automatically increasing the atrial sensing threshold following a ventricular event until the expiration of the far-R window. The atrial sensing threshold is adjusted from a programmed base sensing threshold to a level which will allow for appropriate sensing of P-waves in the atrium while preventing inappropriate sensing of far field R-waves. The atrial sensing threshold may be adjusted as a function of the amplitude of one or more preceding P-waves as sensed by the atrial sense amplifier. Only sensed atrial events which exceed this increased sensing threshold are classified as P-waves. In one embodiment of the invention, the adjustment of the sensing threshold is accomplished in a hardware implementation, in which the atrial sensing threshold following a ventricular event preceded by a sensed P-wave is adjusted to a level selected as a function of the amplitude of the preceding sensed P-wave. In this embodiment, the atrial sensing threshold following a ventricular event preceded by a delivered atrial pacing pulse is adjusted to a level selected as a function of the programmed base sensing threshold. In a second, software based embodiment, the device defines a minimum amplitude which a sensed atrial event must exceed to be classified as a P-wave or more preferably a range of amplitudes in which a sensed atrial event must fall to be classified as a P-wave. The minimum amplitude or the range of amplitudes is set as a function of the amplitudes of previously sensed P-waves. In both embodiments, the device preferably employs only the amplitudes of sensed atrial events which are classified as P-waves for purposes of defining the sensing threshold or range of amplitudes indicative of a sensed P-wave. The present invention may be employed in the context of any implantable pacemaker which senses and/or paces in both the atrium and ventricle, including pacemakers capable of pacing in DDD, DDDR, VDD, VDDR, DDI, DDIR and VAT modes. The invention is particularly desirable in the context of a device such as a pacemaker/cardioverter/defibrillator which detects and/or treats atrial and/or ventricular arrhythmias. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 illustrates a first embodiment of an implantable defibrillator and lead according to the present invention. FIG. 2 is a functional schematic diagram of an implantable pacemaker/cardioverter/defibrillator in which the invention may usefully be practiced. FIG. 3 is a functional schematic diagram of an atrial sense amplifier according to a first embodiment of the present invention. FIG. 4 is a functional schematic diagram of the automatic sensing threshold adjustment circuitry of the amplifier of FIG. 3. FIG. 5 is a timing diagram illustrating the operation of the circuitry of FIGS. 3 and 4. FIGS. 6, 7 and 8 are functional flow charts illustrating the operation of a pacemaker according to a second embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT FIG. 1 illustrates a pacemaker/cardioverter/defibrillator and lead set according to the present invention. The ventricular lead includes an elongated insulative lead body 16, carrying three mutually insulated conductors. Located adjacent the distal end of the lead are a ring electrode 24, an extendable helix electrode 26, mounted retractably within an insulative electrode head 28, and an elongated coil electrode 20. Each of the electrodes is coupled to one of the conductors within the lead body 16. Electrodes 24 and 26 are employed for cardiac pacing and for sensing ventricular depolarizations. At the proximal end of the lead is a bifurcated connector assembly 14 which carries three electrical connectors, each coupled to one of the conductors. The defibrillation electrode 20 may be fabricated from platinum, platinum alloy or other materials known to be usable in implantable defibrillation electrodes and may be about 5 cm in length. The atrial/SVC lead includes an elongated insulative lead body 15, carrying three mutually insulated conductors, corresponding generally to the structure of the ventricular lead. Located adjacent the J-shaped distal end of the lead are a ring electrode 21 and an extendable helix electrode 17, mounted retractably within an insulative electrode head 19. Each of the electrodes is coupled to one of the conductors within the lead body 15. Electrodes 17 and 21 are employed for atrial pacing and for sensing atrial depolarizations. An elongated coil electrode 23 is provided, proximal to electrode 21 and coupled to the third conductor within the lead body 15. Electrode 23 preferably is 5-10 cm in length or greater and is configured to extend from the SVC toward the tricuspid valve. In one preferred embodiment tested by the inventors, approximately 5 cm of the right atrium/SVC electrode was located in the right atrium, with the remaining 5 cm located in the SVC. At the proximal end of the lead is a bifurcated connector assembly 13 which carries three electrical connectors, each coupled to one of the coiled conductors. The coronary sinus lead includes an elongated insulative lead body 6, carrying one conductor, coupled to an elongated coiled defibrillation electrode 8. Electrode 8, illustrated in broken outline, is located within the coronary sinus and great vein of the heart. At the proximal end of the lead is a connector assembly 4 which carries an electrical connector, coupled to the conductor in lead body 6. The coronary sinus/great vein electrode 8 may be about 5 cm in length. An implantable pacemaker/cardioverter/defibrillator 10 is shown in combination with the leads, with the lead connector assemblies 4, 13 and 14 inserted into the connector block 12. Optionally, insulation of the outward facing portion of the housing 11 of the pacemaker/cardioverter/defibrillator 10 may be provided using a plastic coating, for example parylene or silicone rubber, as is currently employed in some unipolar cardiac pacemakers. However, the outward facing portion may instead be left uninsulated, or some other division between insulated and uninsulated portions may be employed. The uninsulated portion of the housing 11 optionally serves as a subcutaneous defibrillation electrode, used to defibrillate either the atria or ventricles. FIG. 2 is a functional schematic diagram of an implantable pacemaker/cardioverter/defibrillator in which the present invention may usefully be practiced. This diagram should be taken as exemplary of one type of device in which the invention may be embodied, and not as limiting, as it is believed that the invention may usefully be practiced in a wide variety of device implementations, including cardiac pacemakers which do not provide high voltage cardioversion and defibrillation therapies. The device as illustrated is provided with an electrode system including electrodes as illustrated in FIG. 1. The correspondence to the illustrated electrodes is as follows. Optional electrode 310 corresponds to the uninsulated portion of the housing 11 of the implantable pacemaker/cardioverter/defibrillator. Electrode 320 corresponds to electrode 20 and is a defibrillation electrode located in the right ventricle. Electrode 311 corresponds to electrode 23, and is located in the right atrium and/or SVC. Electrode 318 corresponds to electrode 8 and is a defibrillation electrode located in the coronary sinus and great vein. Electrodes 324 and 326 correspond to electrodes 24 and 26, and are used for sensing and pacing in the ventricle. Electrodes 317 and 321 correspond to electrodes 17 and 19 and are used for pacing and sensing in the atrium. Electrodes 310, 311, 318 and 320 are coupled to high voltage output circuit 234. High voltage output circuit 234 includes high voltage switches controlled by CV/defib control logic 230 via control bus 238. The switches within circuit 234 control which electrodes are employed and which are coupled to the positive and negative terminals of the capacitor bank including capacitors 246 and 248 during delivery of the defibrillation pulses. Electrodes 324 and 326 are located on or in the ventricle and are coupled to the R-wave amplifier 200, which preferably takes the form of an automatically adjusted amplifier according to the above-cited application by Borgerding et al., incorporated herein by reference in its entirety and corresponding to the operation of the sense amplifiers in Medtronic Model 7250 and Model 7271 implantable pacemaker/cardioverter/defibrillators. The R-wave amplifier 200 provides an adjustable sensing threshold as a function of the measured R-wave amplitude and provides an increased sensing threshold following pacing pulses delivered to the atrium. Operation of amplifier 200 is controlled by pacing circuitry 212 via control lines 201. A signal is generated on R-out line 202 whenever the signal sensed between electrodes 324 and 326 exceeds the present sensing threshold. Electrodes 317 and 321 are located on or in the atrium and are coupled to the P-wave amplifier 204, which in the hardware based implementation of the present invention preferably also takes the form of an automatically adjusted amplifier providing an adjustable sensing threshold following R-waves sensed by amplifier 200 as a function of a previously measured P-wave amplitude and also providing an increased sensing threshold following pacing pulses delivered to the ventricle. Operation of amplifier 204 is controlled by pacing circuitry 212 via control lines 205. A signal is generated on P-out line 206 whenever the signal sensed between electrodes 317 and 321 exceeds the present sensing threshold. The operation of amplifier 204 is discussed in more detail below in conjunction with FIGS. 3, 4 and 5. Switch matrix 208 is used to select which of the available electrodes are coupled to wide band (0.25-100 Hz) amplifier 210 for use in digital signal analysis. Selection of electrodes is controlled by the microprocessor 224 via data/address bus 218, which selections may be varied as desired. Signals from the electrodes selected for coupling to bandpass amplifier 210 are provided to multiplexer 220, and thereafter converted to multi-bit digital signals by A/D converter 222, for storage in random access memory 226 under control of direct memory access circuit 228. Microprocessor 224 may employ digital signal analysis techniques to characterize the digitized signals stored in random access memory 226 to recognize and classify the patient&#39;s heart rhythm employing any of the numerous signal processing methodologies known to the art. The remainder of the circuitry is dedicated to the provision of cardiac pacing, cardioversion and defibrillation therapies, and, for purposes of the present invention may correspond to circuitry known in the prior art. An exemplary apparatus is disclosed of accomplishing pacing, cardioversion and defibrillation functions follows. The pacer timing/control circuitry 212 includes programmable digital counters which control the basic time intervals associated with DDD, VVI, DVI, VDD, AAI, DDI and other modes of single and dual chamber pacing well known to the art. Circuitry 212 also controls escape intervals associated with anti-tachyarrhythmia pacing in both the atrium and the ventricle, employing any anti-tachyarrhythmia pacing therapies known to the art. Intervals defined by pacing circuitry 212 include atrial and ventricular pacing escape intervals, the refractory periods during which sensed P-waves and R-waves are ineffective to restart timing of the escape intervals and the pulse widths of the pacing pulses and all intervals associated with the automatic adjustments of effective sensing thresholds discussed in more detail below. The durations of these intervals are determined by microprocessor 224, in response to stored data in memory 226 and are communicated to the pacer timing/control circuitry 212 via address/data bus 218. Circuitry 212 also determines the amplitudes of the cardiac pacing pulses under control of microprocessor 224. During pacing, the escape interval counters within pacer timing/control circuitry 212 are reset upon sensing of R-waves and P-waves as indicated by signals on lines 202 and 206, and in accordance with the selected mode of pacing on time-out trigger generation of pacing pulses by pacer output circuits 214 and 216, which are coupled to electrodes 317, 321, 324 and 326. The escape interval counters are also reset on generation of pacing pulses, and thereby control the basic timing of cardiac pacing functions, including anti-tachyarrhythmia pacing. The durations of the intervals defined by the escape interval timers are determined by microprocessor 224, via data/address bus 218. The value of the count present in the escape interval counters when reset by sensed R-waves and P-waves may be used to measure the durations of R-R intervals, P-P intervals, P-R intervals and R-P intervals, which measurements are stored in memory 226 and used to detect the presence of tachyarrhythmias. Microprocessor 224 operates as an interrupt driven device, under control of a stored program in its read only memory and is responsive to interrupts from pacer timing/control circuitry 212 corresponding to the occurrence sensed P-waves and R-waves and corresponding to the generation of cardiac pacing pulses. These interrupts are provided via data/address bus 218. Any necessary mathematical calculations to be performed by microprocessor 224 and any updating of the values or intervals controlled by pacer timing/control circuitry 212 take place following such interrupts. For example, in response to a sensed or paced ventricular depolarization or R-wave, the intervals separating that R-wave from the immediately preceding R-wave, paced or sensed (R-R interval) and the interval separating the paced or sensed R-wave from the preceding atrial depolarization, paced or sensed (P-R interval) may be stored. Similarly, in response to the occurrence of a sensed or paced atrial depolarization (P-wave), the intervals separating the sensed P-wave from the immediately preceding paced of sensed atrial contraction (P-P interval) and the interval separating the sensed P-wave from the immediately preceding sensed or paced ventricular depolarization (R-P interval) may be stored. Preferably, a portion of the memory 226 (FIG. 4) is configured as a plurality of recirculating buffers, capable of holding a preceding series of measured intervals, which may be analyzed in response to the occurrence of a pace or sense interrupt to determine whether the patient&#39;s heart is presently exhibiting atrial or ventricular tachyarrhythmia. Detection of atrial or ventricular tachyarrhythmias, as employed in the present invention, may correspond to tachyarrhythmia detection algorithms known to the art. For example, presence of atrial or ventricular tachyarrhythmia may be confirmed by means of detection of a sustained series of short R-R or P-P intervals of an average rate indicative of tachyarrhythmia or an unbroken series of short R-R or P-P intervals. The suddenness of onset of the detected high rates, the stability of the high rates, or a number of other factors known to the art may also be measured at this time. Appropriate ventricular tachyarrhythmia detection methodologies measuring such factors are described in U.S. Pat. No. 4,726,380, issued to Vollmann, U.S. Pat. No. 4,880,005, issued to Pless et al. and U.S. Pat. No. 4,830,006, issued to Haluska et al., all incorporated herein by reference in their entireties. An additional set of tachycardia recognition methodologies is disclosed in the article “Onset and Stability for Ventricular Tachyarrhythmia Detection in an Implantable Pacer-Cardioverter-Defibrillor” by Olson et al., published in Computers in Cardiology, Oct. 7-10, 1986, IEEE Computer Society Press, pages 167-170, also incorporated herein in its entirety. Atrial fibrillation detection methodologies in particular are disclosed in Published PCT Application Ser. No. US 92/02829, Publication No. WO 92/18198, by Adams et al., and in the article “Automatic Tachycardia Recognition”, by Arzbaecher et al., published in PACE, May-June, 1984, pp. 541-547, both of which are incorporated by reference in their entireties. Particularly appropriate hierarchical rule-based tachyarrhythmia detection and classification methodologies for use in conjunction with a device as illustrated in FIG. 2 are set forth in U.S. Pat. No. 5,545,186, by Olson et al. and in U.S. Pat. No. 5,755,736, by Gillberg et al., both incorporated herein by reference in their entireties. However, one of the advantages of the present invention is that it is believed practicable in conjunction with most prior art tachycardia detection algorithms. Because the accurate detection of arrhythmias using measured intervals between R-waves and P-waves is dependent on accurate sensing of the occurrences of these depolarization signals, the automatic effective sensing threshold adjustment provided by the present invention is particularly valuable in the context of anti-tachyarrhythmia devices. However, the improved sensing accuracy is also valuable in the context of anti-bradycardia pacemakers as well, particularly in the context of mode-switching features intended to prevent such pacemakers from pacing the heart at inappropriately high rates. In the event that an atrial or ventricular tachyarrhythmia is detected, and an anti-tachyarrhythmia pacing regimen is desired, appropriate timing intervals for controlling generation of anti-tachyarrhythmia pacing therapies are loaded from microprocessor 224 into the pacer timing and control circuitry 212, to control the operation of the escape interval counters therein and to define refractory periods during which detection of R-waves and P-waves is ineffective to restart the escape interval counters. Alternatively, circuitry for controlling the timing and generation of anti-tachycardia pacing pulses as described in U.S. Pat. No. 4,577,633, issued to Berkovits et al. on Mar. 25, 1986, U.S. Pat. No. 4,880,005, issued to Pless et al. on Nov. 14, 1989, U.S. Pat. No. 4,726,380, issued to Vollmann et al. on Feb. 23, 1988 and U.S. Pat. No. 4,587,970, issued to Holley et al. on May 13, 1986, all of which are incorporated herein by reference in their entireties may also be used. In the event that generation of a cardioversion or defibrillation pulse is required, microprocessor 224 employs an escape interval counter to control timing of such cardioversion and defibrillation pulses, as well as associated refractory periods. In response to the detection of atrial or ventricular fibrillation or tachyarrhythmia requiring a cardioversion pulse, microprocessor 224 activates cardioversion/defibrillation control circuitry 230, which initiates charging of the high voltage capacitors 246 and 248 via charging circuit 236, under control of high voltage charging control lines 240 and 242. The voltage on the high voltage capacitors is monitored via VCAP line 244, which is passed through multiplexer 220 and in response to reaching a predetermined value set by microprocessor 224, results in generation of a logic signal on Cap Full (CF) line 254, terminating charging. Thereafter, timing of the delivery of the defibrillation or cardioversion pulse is controlled by pacer timing/control circuitry 212. Following delivery of the fibrillation or tachycardia therapy the microprocessor then returns the device to cardiac pacing and awaits the next successive interrupt due to pacing or the occurrence of a sensed atrial or ventricular depolarization. One embodiment of an appropriate system for delivery and synchronization of ventricular cardioversion and defibrillation pulses and for controlling the timing functions related to them is disclosed in more detail in commonly assigned U.S. Pat. No. 5,188,105 by Keimel, issued Feb. 23, 1993, incorporated herein by reference in its entirety. Embodiments of appropriate systems for delivery and synchronization of atrial cardioversion and defibrillation pulses and for controlling the timing functions related to them are disclosed in more detail in U.S. Pat. No. 5,269,298 by Adams et al., issued Dec. 14, 1993 and in U.S. Pat. No. 4,316,472 by Mirowski et al., issued Feb. 23, 1982, both incorporated herein by reference in their entireties. However, any known cardioversion or defibrillation pulse control circuitry is believed usable in conjunction with the present invention. For example, circuitry controlling the timing and generation of cardioversion and defibrillation pulses as disclosed in U.S. Pat. No. 4,384,585, issued to Zipes on May 24, 1983, in U.S. Pat. No. 4,949,719 issued to Pless et al., cited above, and in U.S. Pat. No. 4,375,817, issued to Engle et al., all incorporated herein by reference in their entireties may also be employed. In the illustrated device, delivery of the cardioversion or defibrillation pulses is accomplished by output circuit 234, under control of control circuitry 230 via control bus 238. Output circuit 234 determines whether a monophasic or biphasic pulse is delivered, the polarity of the electrodes and which electrodes are involved in delivery of the pulse. Output circuit 234 also includes high voltage switches which control whether electrodes are coupled together during delivery of the pulse. Alternatively, electrodes intended to be coupled together during the pulse may simply be permanently coupled to one another, either exterior to or interior of the device housing, and polarity may similarly be pre-set, as in current implantable defibrillators. An example of output circuitry for delivery of biphasic pulse regimens to multiple electrode systems may be found in the above cited patent issued to Mehra and in U.S. Pat. No. 4,727,877, incorporated by reference in its entirety. An example of circuitry which may be used to control delivery of monophasic pulses is set forth in commonly assigned U.S. Pat. No. 5,163,427, by Keimel, issued Nov. 17, 1992, also incorporated herein by reference in its entirety. However, output control circuitry as disclosed in U.S. Pat. No. 4,953,551, issued to Mehra et al. on Sept. 4, 1990 or U.S. Pat. No. 4,800,883, issued to Winstrom on Jan. 31, 1989 both incorporated herein by reference in their entireties, may also be used in conjunction with a device embodying the present invention for delivery of biphasic pulses. In the event that, as in FIG. 1, both atrial and ventricular defibrillation are available, ventricular defibrillation may be accomplished using higher pulse energy levels than required for atrial defibrillation and may employ the same or a different electrode set. For example, electrodes 310, 311, 318 and 320 or only electrodes 311, 318 and 320 may be employed for atrial defibrillation. Electrodes 311, 320 and 310 might be employed for ventricular defibrillation, with electrode 311 (right atrium/SVC) coupled to electrode 310 (device housing). Alternatively, electrodes 310, 318 and 320 may be employed, with electrode 318 (coronary sinus/great vein) coupled to electrode 310. As a further alternative, electrodes 311, 310, 318 and 323 might all be employed for ventricular defibrillation, with electrodes 310, 311 and 323 coupled in common. As yet another alternative, only electrodes 310 and 320 might be employed for ventricular defibrillation added or substituted for either of electrodes 311 or 318 for treating ventricular fibrillation. One particularly desirable embodiment of the invention employs only the right atrial/SVC electrode 311, the coronary sinus/great vein electrode 318 and the right ventricular electrode 320. During atrial defibrillation, electrodes 320 and 318 are coupled in common with one another, and the atrial defibrillation pulse is delivered between these electrodes and electrode 311. During ventricular defibrillation, electrodes 311 and 318 are coupled in common with one another, and the ventricular defibrillation pulse is delivered between these electrodes and electrode 320. This particular set of electrodes thus provides optimized defibrillation pulse regimens for both atrial and ventricular defibrillation, by simply switching the connection of the coronary sinus/great vein electrode. In modern implantable cardioverter/defibrillators, the particular therapies are programmed into the device ahead of time by the physician, and a menu of therapies is typically provided. For example, on initial detection of an atrial or ventricular tachycardia, an anti-tachycardia pacing therapy may be selected and delivered to the chamber in which the tachycardia is diagnosed or to both chambers. On redetection of tachycardia, a more aggressive anti-tachycardia pacing therapy may be scheduled. If repeated attempts at anti-tachycardia pacing therapies fail, a higher level cardioversion pulse may be selected thereafter. Therapies for tachycardia termination may also vary with the rate of the detected tachycardia, with the therapies increasing in aggressiveness as the rate of the detected tachycardia increases. For example, fewer attempts at anti-tachycardia pacing may be undertaken prior to delivery of cardioversion pulses if the rate of the detected tachycardia is above a preset threshold. The references cited above in conjunction with descriptions of prior art tachycardia detection and treatment therapies are applicable here as well. In the event that atrial or ventricular fibrillation is identified, the typical therapy will be delivery of a high amplitude defibrillation pulse, typically in excess of 10 joules in the case of ventricular fibrillation and about 1 joule or less in the case of atrial defibrillation. Lower energy levels will be employed for cardioversion. As in the case of currently available implantable pacemakers/cardioverter/defibrillators, and as discussed in the above-cited references, it is envisioned that the amplitude of the defibrillation pulse may be incremented in response to failure of an initial pulse or pulses to terminate fibrillation. Prior art patents illustrating such pre-set therapy menus of anti-tachyarrhythmia therapies include the above-cited U.S. Pat. No. 4,830,006, issued to Haluska, et al., U.S. Pat. No. 4,726,380, issued to Vollmann et al. and U.S. Pat. No. 4,587,970, issued to Holley et al. FIG. 3 is a functional block diagram of amplifier 204 illustrated in FIG. 2. This diagram illustrates the basic functional components of the amplifier and their interconnection to the pacer timing/control circuitry 212. Signals from the ventricular electrodes 317 and 321 first pass through blanking switches 350, which operate to disconnect the amplifier from the electrodes during delivery of an atrial pacing pulse, during the duration of a ventricular input blanking signal on line AINB, which extends through the delivered atrial pacing pulse and during the fast recharge period thereafter. Depolarization signals passing through blanking switches 350 are amplified by preamp 352 and then pass through a first high pass filter 354. The high pass filtered signal is passed through an adjustable gain amplifier 356 which amplifies the signal by one of eight available multiplication factors under the control of digital signals on lines ASEN:0, ASEN:1 and ASEN:2. The degree of amplification determines the programmed base sensing threshold, as discussed below. The amplified signal is passed on through a first low pass filter 358, a second low pass filter 360, a second high pass filter 362 and an absolute value circuit 364 which produces at its output the absolute value of the previously filtered and amplified signal. In response to a blanking signal on line AHP2BLK, passage of signals through high pass filter 362 is prohibited for defined periods of time following delivery of atrial and ventricular pacing pulses, providing an additional blanking function. The duration of blanking in conjunction with a delivered atrial pacing pulse is preferably the same as the blanking interval defined by the blanking signal on line AINB following delivery of an atrial pacing pulse. In conjunction with the delivery of an atrial pacing pulse, the blanking period may be greater, for example, forty or more milliseconds. The output of the absolute value circuit 364 is provided to the detector circuit 366 which compares it to a defined sensing threshold to determine whether an P-wave is to be detected or not. If the signal exceeds the threshold, detector circuit 366 provides an output on P-OUT line 206, which is provided to pacer timing and control circuit 212 (FIG. 2 ). The sensing threshold defined by detector circuit 366 is variable, and is adjusted in response to sensed and paced ventricular events, delivered ventricular pacing pulses and delivered atrial pacing pulses. The detector 366, in conjunction with the programmed gain factor of amplifier 356 defines the programmed base sensing threshold which is normally in effect, and a variable sensing threshold effective after sensed ventricular events and delivered atrial and ventricular pacing pulses. In order to be detected as an P-wave, the signal from absolute value circuit 364 must exceed the greater of the base sensing threshold and the variable threshold, as discussed in more detail below. The duration and selection of the increased effective sensing threshold following delivered atrial pacing pulses is controlled by pacer timing/control circuitry via the ABDET and ASW lines. Adjustment of the effective sensing threshold following a delivered atrial pacing pulse and following a ventricular event preceded by a delivered atrial pacing pulse is accomplished by means of a reference voltage applied to the input of the detector 366 the line VREF and by the signal levels on the ASEN:0, ASEN:1 and ASEN:2 lines. Adjustment of the sensing threshold following a sensed P-wave and following a ventricular event preceded by a sensed P-wave is a function of the amplitude of the preceding sensed P-wave as reflected by the output of absolute value circuit 364 on the ABSVAL line. FIG. 4 illustrates detector 366 in more detail, and illustrates the manner in which the detector works in conjunction with amplifier 356 to define the various effective sensing thresholds employed by the device. The amplified filtered P-wave signal on the ABSVAL line from absolute value circuit 364 (FIG. 3) is applied to a comparator 414 which, in conjunction with the remainder of the illustrated circuitry defines an adjustable sensing threshold. If the signal from the absolute value circuit exceeds the currently effective sensing threshold, a signal is generated on P-OUT line 206, which in turn is provided to the timing and control circuitry 212. The programmed base sensing threshold “S” is defined by the DC offset of the comparator circuit 414 in conjunction with the programmed amplification of the signal by amplifier 356. The effective sensing threshold is increased from the base threshold following sensed P-waves, ventricular events and delivered atrial pulses as follows. As noted above, the signal on the ABSVAL line is applied to comparator 414 and, if it exceeds the then established sensing threshold, causes generation of a signal on P-OUT line 206 indicative of the sensing of a P-wave. In addition, the signal on line ABSVAL is also employed to set the sensing threshold after a sensed P-wave. The signal on line ABSVAL is applied to amplifier 400, configured as a non-inverting voltage follower, via switch 402 which is normally closed. The output of amplifier 400 is applied to capacitor C 1, which, via resistors R 1 and R 2 defines the sensing threshold for comparator 414. The values of the components are chosen such that the voltage stored in capacitor C 1 discharges over a time constant T 1, which may be, for example, 100 milliseconds. Resistors R 1 and R 2 are sized so that the sensing threshold defined by capacitor C 1 in conjunction with resistors R 1 and R 2 is a desired proportion, e.g. approximately 75%, of the peak amplitude of the sensed P-wave. In addition, the signal on the ABSVAL line is also applied to a resettable peak follower 410 which in response to a signal on P- OUT line 206 stores the peak amplitude of the signal,. Thus, in response to sensing of a signal which is identified as a P-wave, its amplitude is stored in peak follower 410, which retains the stored amplitude until a subsequent sensed P-wave. In response to a signal on P-OUT line 206, pacing timing and control circuitry 212 (FIG. 2) sets the line ABDET high for a period of time, for example 100 milliseconds, in turn opening switch 402 and closing switch 406. If not already low, pacing timing/control circuit 212 also sets line ASW low, in turn closing switch 412. The voltage stored in peak follower 410 is applied through switch 412 and switch 406 to amplifier 400 and thence to capacitor C 1. Thus, following a sensed P-wave, the effective sensing threshold is set equal to a desired proportion of the peak amplitude of the P-wave for a period of time of approximately 100 milliseconds. At the expiration of this time interval, the line ABDET goes low, opening switch 406 and allowing capacitor C 1 to discharge, whereby the effective sensing threshold thereafter decays back to the programmed base sensing threshold S. Should a ventricular event be sensed following a preceding sensed atrial event, pacing timing and controller circuit 212 again sets the ABDET line high, for example for a period of 120 milliseconds, defining an increased sensing threshold thereafter proportional to the amplitude previously sensed P-wave. In response to a delivered atrial pacing pulse, pacing/timing controller 212 sets line ASW high until a subsequent P-wave. This in turn opens switch 412 and closes switch 408. The pacing/timing controller also sets line ABDET high for a period, for example, of 100 milliseconds, closing switch 406 and opening switch 402. Instead of applying the voltage stored in peak follower 410 to define an elevated sensing threshold after a delivered atrial pacing pulse, multiplier circuit 404 operates, under control of lines ASEN:0, ASEN:1 and ASEN:2 to define an output signal which is a predetermined multiple of the voltage on line VREF. This voltage is passed via switch 408 and switch 406 to amplifier 400, where it is applied to capacitor C 1 to define a sensing threshold proportional to the output of the multiplier circuit 404. The value of the defined threshold preferably increases as a function of the programmed lower or base sensing threshold S. In a most preferred embodiment, the value of the defined increased threshold decreases a proportionally greater amount in conjunction with lower programmed base sensing thresholds, and increases by a relatively lesser amount, with respect to higher programmed base sensing thresholds. For example, at a programmed base sensing threshold of 0.1 millivolts, the defined increased threshold may be, for example, 0.9 millivolts. At a programmed sensing threshold of 0.6 millivolts, the defined increased sensing threshold may be 1.8 millivolts. At a programmed sensing threshold of 2.1 millivolts, the defined sensing threshold may also be 2.1 millivolts. This increased threshold persists for as long as the signal on line ABDET is high, and decays thereafter at a time constant T 1 in a fashion analogous to the decay of the sensing threshold following a sensed atrial event. In the event of a paced or sensed ventricular event following a preceding atrial pacing pulse, the sensitivity threshold is again temporarily increased in response to the timing/control circuitry 212 setting line ABDET high, for example for a period of 120 milliseconds or more. The increased sensing threshold again persists until line ABDET goes low, after which the sensing threshold decays according to time constant T 1 defined by capacitor C 1 and resistors R 1 and R 2. FIG. 5 is a timing diagram illustrating the operation of the detector 366 (FIG. 3) to define the various variable sensing thresholds described above. The upper portion of FIG. 5 is a diagram illustrating the variable sensing threshold A s (t). The variable effective sensing threshold A s (t) is shown decaying to the base or lower sensing threshold S at 500. At 501, a signal is generated on line P-OUT, 506, indicative of a sensed P-wave. This signal, as discussed above, causes pacemaker timing/control circuitry 212 to set line ABDET high at 504 and set line ASW low at 506. At 502, the effective atrial sensing threshold A s (t) is set equal to a predetermined percentage of the amplitude of the sensed P-wave which caused generation of the signal on line POUT at 501. This increased sensing threshold persists as discussed above until line ABDET goes low, 100 milliseconds later. Thereafter, the sensing threshold decays exponentially back to the lower or base sensing threshold S. A ventricular event 508 is illustrated occurring prior to the effective sensing threshold A s (t) returning to the base sensing threshold S. In response to the sensed or paced ventricular event at 508, pacing timing control circuitry sets line ABDET high at 512, for example for a period of 120 milliseconds. The sensing threshold A s (t) is increased at 510 to the same value as following the preceding sensed P-wave at 501, and persists at this level until pacing timing and control circuitry 212 sets line ABDET low again, after which the sensing threshold decays exponentially back to the base sensing threshold S. At 514, an atrial pacing pulse is delivered which in turn causes pacing timing and control circuitry 212 to set line ABDET line high at 518 and line ASW high at 520. Line ASW will stay high until a subsequent sensed P-wave. As discussed above, as long as line ABDET is high, the effective sensing threshold is set to a preset multiple of the base or lower sensing threshold at 516, and stays at this increased threshold level until line ABDET goes low, for example 100 milliseconds thereafter. At 522, a ventricular event, sensed or paced, occurs again, increasing the effective sensing threshold A s (t) to the present multiple of the base sensing threshold S, and causing the pacing timing circuitry 212 to set line ABDET high at 524. The increased sensing threshold persists until line ABDET goes low, for example 120 milliseconds thereafter, after which the effect of sensing threshold decays back to the base sensing threshold S. The operation of the amplifier according to the first embodiment of the present invention produces several benefits. By providing for an increased atrial sensing threshold following a delivered atrial pacing pulse as opposed to simply blanking the atrial amplifier, inappropriate sensing of the pacing pulse itself and of any post pacing polarization of the atrial electrodes is prevented, while sensing of P-waves closely spaced to the delivered atrial pacing pulse is facilitated. By providing for an increased atrial sensing threshold following a ventricular event, as opposed to simply blanking the atrial amplifier, inappropriate sensing of far-field R-waves is avoided, while sensing of P-waves is facilitated. By providing an increased sensitivity threshold level following a ventricular event which varies as a function of the preceding sensed atrial event, the accuracy of the amplifier in rejecting far-field R-waves is enhanced. By providing for an exponential decay of the increased threshold values, inappropriate atrial oversensing which might otherwise be associated with an abrupt change in sensing threshold is avoided. FIG. 6 is a functional flow chart illustrating the operation of a second embodiment of a device practicing the present invention, implemented in a device as in FIG. 2. In this device, however, rather than implementing the far field R-wave rejection mechanism in hardware, the mechanism is instead implemented in the form of software stored in the read only memory associated with microprocessor 224 (FIG. 2 ). In this implementation of the invention, the atrial sense amplifier 204 (FIG. 2) may be a conventional sense amplifier, having a programmable sensing threshold correspond to the programmable base sensing threshold discussed above and provided with a blanking period following delivery of an atrial or ventricular pacing pulse. The atrial sense amplifier 204 may also adjust sensing thresholds following sensed and paced atrial events in the same manner as the amplifier in the first embodiment of the invention, discussed above. Adjustment of the effective P-wave sensing threshold in conjunction with occurrences of ventricular events is controlled by microprocessor 224 (FIG. 2) The effective sensing threshold is adjusted according to the mechanism illustrated in the flow chart of FIGS. 6-8. In conjunction with these flow charts, it should be understood that the device is operating as a conventional DDD type pacemaker, pacing and sensing in both the atrium and ventricles. Details of operation of the device not illustrated in the flow chart should be understood to conform to conventional DDD pacemakers, and are not discussed in detail herein. In the implementation illustrated in FIGS. 6-8, the device defines a far-R window beginning X milliseconds prior to a ventricular event and extending for a period of Y milliseconds thereafter. For example, the time interval may span 50 milliseconds prior to the ventricular event and extend for 100-150 milliseconds thereafter. During the portion of the far-R window following the ventricular event, the device defines an effective increased P-wave sensing threshold in an effort to reject far field R-wave signals as described in conjunction with the hardware implementation above. The P-wave sensing threshold is defined as a function of the amplitude of preceding P-waves, sensed outside of far-R windows, and may either be a simple minimum amplitude above which an atrial sensed event is considered to be a P-wave or may be a range of acceptable amplitudes, between which the amplitude of an atrial event sensed during the defined window must fall in order for the event to be considered to be a P-wave. The determination as to whether an event sensed by the atrial amplifier is a far field R-wave or a P-wave may be employed in conjunction with the arrhythmia detection algorithms. If the event is sensed within the Far-R time window, following a ventricular event, the sensed event will not be employed to initiate a new AV interval. For convenience of description, the flow chart may be considered first at 600, concurrent with the initiation of the VA escape interval. During this time period, the device awaits either the expiration of the VA interval at 602, sensing of a ventricular event at 606 or sensing of an atrial event at 610. In the event the VA interval expires without sensing in the atrium or ventricle, an atrial pacing pulse is delivered at 604, and the AV interval is initiated at 614. In the event a ventricular depolarization is sensed by the ventricular sense amp at 606, the device determines at 622 whether it is appropriate to update the effective P-wave sensing threshold at 608, and the VA interval is reinitialized at 600. In the event that an atrial event is sensed at 610, the device checks at 612 to determine whether the signal might be indicative of a far field R-wave. In particular, the device determines whether the sensed atrial event occurred within the defined far-R window following the immediately preceding ventricular event and if so, whether it exceeds the minimum amplitude or falls within the defined amplitude range indicative of a P-wave. If the atrial event is sensed within the far-R window and is less than the minimum amplitude or outside the defined range, it is identified as a likely to be a far field R-wave and does not initiate an AV interval. The device continues to await expiration of the VA interval at 602, ventricular sensing at 606 and atrial sensing at 610. If the atrial event is sensed after the far-R window associated with the preceding ventricular event or is sensed within the window but is greater than the minimum amplitude or within the defined range, it is identified as a likely to be a P-wave and does initiate an AV interval at 614. The amplitude and time of the atrial event is stored the value may be used to update the effective P-wave sensing threshold employed during the far-R windows, as discussed below in conjunction with FIG. 7. During the AV interval, the device awaits expiration of the AV interval at 616 or ventricular sensing at 620. In response to expiration of the AV interval at 616, the device delivers a ventricular pacing pulse at 618 and initiates the VA interval at 600. In response to ventricular sensing at 620, the device checks to determine whether an update to the effective P-wave sensing threshold during the far field R-wave window is appropriate at 622 and initiates the VA interval at 600. FIG. 7 illustrates in more detail the mechanism by which the device determines at block 622 (FIG. 6 ), whether updating the effective P-wave sensing threshold for the far-R window is appropriate. Following the sensing of an R-wave or delivery of a ventricular pacing pulse, its time of occurrence is stored at 624, and the device checks at 626 to determine whether the preceding sensed atrial event was outside the far-R windows associated with the ventricular preceding and following the sensed atrial event. If so, the value of the preceding stored P-wave is employed to update the effective atrial sensing threshold applicable during the far field R-wave window. For example, the minimum amplitude indicative of a P-wave during the far field R-wave window may be set at a percentage, for example 75%, of the average amplitude of the preceding four to eight sensed atrial events sensed outside of the far field R-wave windows or a range extending from 75% of the average amplitude to 150% of the average amplitude may be defined. Like the hardware based embodiment described above, the device attempts to avoid employing the amplitudes of far field R-waves in setting effective atrial sensing thresholds. FIG. 8 illustrates the operation of the device in determining whether a sensed P-wave is likely to be a far field R-wave, illustrating the operation of block 612 in FIG. 6 in more detail. At 630, the time and amplitude of the sensed event on the atrial lead is stored. At 632 the device checks to determine whether the atrial event fell within a far-R window associated with the preceding ventricular event. If so, the device further checks at 634 to determine whether the amplitude of the sensed atrial event exceeds the defined effective P-wave threshold for the far field R-wave window, or falls within the defined acceptable range of amplitudes. If so, the device treats the sensed event as a P-wave, initiating the AV interval at 614. While not discussed in detail herein, it should also be understood that the ability of a device operating according to the flow chart of FIGS. 6 through 8 to distinguish between P-waves and far field R-waves is also of particular value in the context of arrhythmia detection functions. While the invention is described above in the context of a device which employs an atrial sense amplifier which provides for an automatically adjusted sensing threshold following sensed atrial events and following atrial pacing pulses, the present invention may also be usefully employed in the context of devices which do not adjust the effective sensing thresholds following either or both of such events. Further, while the disclosed embodiment of the invention takes the form of a microprocessor controlled device, the invention is of course equally useful in the context of a device in which the various time intervals employed to control the sensing thresholds are determined by hardware, for example by a digital circuit employing dedicated logic, or by analog timers. The specific mechanism by which the time intervals associated with the operation of the adjustable threshold function are defined is not critical to successful use and enjoyment of the present invention. As such, the above disclosure should be taken as exemplary, rather than limiting, with regard to the claims which follow.

A system and method is provided to view an anatomical structure such as a blood vessel in high contrast with its surrounding tissue. The system and method may be used to produce an image of an anatomical structure using reflected electromagnetic radiation singularly scattered from target tissue. The system and method may also provide same-side illumination and detection of reflected electromagnetic radiation in a convenient integral imaging device. The system and method may also provide helmet mounted imaging technology in a single integral helmet which allows the wearer to view an anatomical structure located within a patient such that the image is continuously oriented according to the orientation of the helmet wearer&#39;s head. The system and method may also be used in the performance of venipuncture. The system and method may provide for improved contrast between any anatomical structure and its surrounding tissue for use in any imaging system.

big_patent