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[ "splenic pseudoaneurysms ( spa ) are a serious sequela of blunt traumatic injury to the spleen . they are very rare with fewer than 200 cases reported in english literature in 2006 and their incidence is virtually unknown . spa have a high risk of rupture and have been reported to be fatal if left untreated in adults . in the setting of trauma , spa can form 1 to 8 days after the blunt splenic injury and could be missed on the initial imaging . however , the management in pediatric patients is poorly defined . with regards to spa , although the american pediatric surgical association does not recommend routine follow - up imaging in pediatric trauma patients regardless of grade of injury , several reviews have described the potential benefits of follow - up ct imaging for detection of pseudoaneurysm . here , we share our experience at an urban level 1 trauma center of managing a hemodynamically stable pediatric patient who sustained a grade iii / iv splenic laceration following blunt trauma with follow - up imaging demonstrative of splenic pseudoaneurysm and proposed algorithm to manage such a case .", "a 15 year - old male with no significant past medical history presented to the emergency room complaining of severe abdominal pain of acute onset after sustaining injury to his left side while playing football . his vital signs were within normal limits on arrival to the emergency room with a gcs of 15 . the patient s physical exam was significant for mild tenderness to palpation of his abdomen without peritonitis . primary survey included a focused assessment with sonography for trauma ( fast ) , which was positive for free fluid in the abdomen . a ct abdomen / pelvis was obtained , demonstrating a grade iii / iv left splenic laceration with moderate to large hemoperitoneum . he was admitted to the surgical intensive care unit for further monitoring . over the next 24 h serial labs were drawn with a hematocrit as follows : 34.5% > 33.2% > 30.1% . serial abdominal exams and hemodynamic status remained stable throughout his admission . on hospital - day 3 , a follow - up cta abdomen / pelvis was obtained which demonstrated an approximately 6 mm ovoid hypervascular structure within the cleft of the splenic laceration suspicious for a small pseudoaneurysm ( fig . interventional radiology was consulted and on hospital - day 5 ; they performed a splenic arteriogram which demonstrated multiple pseudoaneurysms arising from the second order splenic artery branches . proximal coil embolization with 10 mm 20 cm boston scientific interlock coil was performed without complications . he was observed for another 24 h post - procedure without event and was discharged the next day after receiving the pneumococcal and meningococcal vaccines . the patient was seen for follow up in our trauma clinic 5 days post - discharge without complaints .", "splenic artery pseudoaneurysms ( spa ) remain a rare but potentially fatal pathology following blunt trauma which is managed non - operatively . studies have demonstrated the benefits of repeated follow - up imaging and early intervention upon diagnosis of spa . management of spa can be operative entailing splenectomy with or without pancreatectomy or non - operative , using endovascular techniques . in recent studies , utilization of angioembolization has been demonstrated to decrease non - operative management failure rate in adults with blunt splenic injuries . however in children , there are no strong evidence - based guidelines that exist to help guide management of spa . pediatric splenic trauma may be more suitable for non - operative management than that of adult because the differences in pathophysiology of the injury . the pediatric spleen has a fibrous capsule that is thicker than that of an adult , and lacerations in pediatric patients tend to occur in avascular planes . one major controversy in management of pediatric blunt trauma patient is the utilization of predischarge or postdischarge follow - up imaging , usually 37 days after initial presentation [ 68 ] . the recommendation from the american pediatric surgical association trauma committee against this practice is based on the fact that less than half of pediatric patient received follow - up imaging without reported complications . due to small cases presented in literature , . showed in a retrospective study that follow - up doppler ultrasound found spa in 7.7% of grade iii splenic injuries and 17% of grade iv . the study showed that follow - up imaging may be useful in identifying spa in grade iii and iv splenic injury . however , the study is limited by the size of the sample population . in 2008 , mayglothling et al . described their experience of managing 97 adolescent patients with blunt splenic injury . in the study , mayglothling presented their algorithm with aggressive angiography and embolization as a useful adjunct to non - operative management of adolescents with blunt splenic injuries . the trauma center performed splenic artery angiography screening for patients with grade iii , iv or v splenic injury , contrast blush or pseudoaneurysm on ct . the authors also favored follow - up imaging 4896 h post - injury in their treatment algorithm . when compared to the direct catheter angiography , our patient 's follow - up ct angiography of the abdomen demonstrated an underestimation of the size and severity of the pseudoaneurysms . also , dsa may be better at showing the smaller pseudoaneurysms that can not be visualized on the ct scan . direct catheter angiography of the splenic artery has been assumed to be the gold standard to diagnose splenic artery pseudoaneurysm . with the advancing technology of interventional radiology , advanced blood vessel fluoroscopy techniques such as digital subtraction angiography ( dsa ) can provide high spatial resolution that allows the imaging of small vessels . the use of doppler sonography has also been described in the diagnosis of splenic aneurysms . however , this mode of imaging may be limited by its operators and its low spatial resolution . the use of magnetic resonance imaging ( mri ) has been reported . multiple detector computer tomography ( mdct ) technology is the most utilized tool in critical care . ct can obtain high resolution images of the whole body in a short amount of time when compared to dsa and to mri . studies have shown that mdct is favorably comparable to catheter angiography in detecting stenosis and small blood vessels . cta has also been shown to be safe and effective alternative to dsa for making decisions in cerebral aneurysm management . , brenner et al . of columbia university have estimated that the lifetime risk of fatal malignancy from both abdominal and head ct examination is roughly 500 out of 600,000 . although the risk is present , benefit of ct scan may outweigh the small increase in cancer risk in acute situations . given that many of above sample populations received ct scans over a decade ago , the advancement of ct technology may have significantly decreased the radiation burden to children . even though there may be an association between incidence of spa and high grade splenic lacerations ( i.e. , grade iii and iv splenic laceration ) , there is no strong clinical evidence to show benefits of aggressive angiography of these patients . we concur with safavi et al . in the necessity of follow - up imaging before planned discharge because there is a potential risk of delayed splenic rupture in this population .", "the limited existing literature leaves us with a still unclear algorithm for the management of non - operative blunt splenic trauma in pediatric patients . questions still remain regarding the timing of repeat imaging for diagnosis of spa following non - operative blunt splenic trauma , which patients should be imaged , and how to manage spa upon diagnosis . more clinical study and basic science research is warranted to study the disease process of spa in pediatric patient . we believe that our proposed management algorithm timely detect formation of delayed spa formation and addresses the possible fatal disease course of pediatric spa .", "", "", "", "", "roger chen zhu review of literature , preparation of case report , and writing of the paper .", "", "" ]

[ "transitions in care are arguably one of the most important and challenging safety issues in healthcare today . transitions in care involve the transfer of duties and accountabilities from one person , or group of people , to others . transitions are a complex business that require a high degree of context - specific coordination and communication among different people with different backgrounds and skills . two years ago , as the time was approaching for my father to be discharged after emergency hip reconstruction surgery , and three months of non - weight bearing rehabilitation , my mother became increasingly concerned about his impending discharge . in the weeks leading up to his discharge , i had several meetings with his social worker to discuss my mother 's reluctance to take my father home and her concern about his ability to once again take care of his colostomy . the social worker arranged for a number of things to happen , prior to his discharge , which enabled a smooth transition to home . a home safety assessment was conducted and bath bars and toilet seat supports were installed a week in advance of his expected date of discharge . arrangements were made for him to attend a day hospital three times a week , including the provision of specialized transportation services . a personal care worker was arranged to bath dad three days a week and the nurses provided him with up - dated colostomy products to facilitate self - management . my mother had time to adjust to his return home and my dad thrived when he got there . just last week , my mother was discharged from the same hospital after spending three weeks in rehabilitation for a minor stroke . the day before her discharge i was still trying to meet with her social worker ( i.e. , a different social worker than the one who cared for my dad ) ; several attempts to reach her by telephone and pager were unsuccessful . on one of my daily visits , i met mom 's physiotherapist , who told me to adjust my dad 's old walker for mom when she got home . i never did talk with the social worker , or the occupational therapist , or the family physician . i saw nurses , who never seemed to know anything about an expected date of discharge . i was surprised when the social worker called me the day before mom 's discharge to ask me what time i was planning on picking her up the next day . we were given a discharge summary and mom was told to take it to her family doctor within a month . only when i inquired about homecare was i told that a home safety assessment had been requested , but they were not sure if one would be provided . the transition to home was very difficult for us , and a week later we are still waiting for a home safety assessment and for someone to assist her with activities of daily living . two years ago , as the time was approaching for my father to be discharged after emergency hip reconstruction surgery , and three months of non - weight bearing rehabilitation , my mother became increasingly concerned about his impending discharge . in the weeks leading up to his discharge , i had several meetings with his social worker to discuss my mother 's reluctance to take my father home and her concern about his ability to once again take care of his colostomy . the social worker arranged for a number of things to happen , prior to his discharge , which enabled a smooth transition to home . a home safety assessment was conducted and bath bars and toilet seat supports were installed a week in advance of his expected date of discharge . arrangements were made for him to attend a day hospital three times a week , including the provision of specialized transportation services . a personal care worker was arranged to bath dad three days a week and the nurses provided him with up - dated colostomy products to facilitate self - management . my mother had time to adjust to his return home and my dad thrived when he got there . just last week , my mother was discharged from the same hospital after spending three weeks in rehabilitation for a minor stroke . the day before her discharge i was still trying to meet with her social worker ( i.e. , a different social worker than the one who cared for my dad ) ; several attempts to reach her by telephone and pager were unsuccessful . on one of my daily visits , i met mom 's physiotherapist , who told me to adjust my dad 's old walker for mom when she got home . i never did talk with the social worker , or the occupational therapist , or the family physician . i saw nurses , who never seemed to know anything about an expected date of discharge . i was surprised when the social worker called me the day before mom 's discharge to ask me what time i was planning on picking her up the next day . we were given a discharge summary and mom was told to take it to her family doctor within a month . only when i inquired about homecare was i told that a home safety assessment had been requested , but they were not sure if one would be provided . the transition to home was very difficult for us , and a week later we are still waiting for a home safety assessment and for someone to assist her with activities of daily living . can we attribute them to differences in the way healthcare providers responded to each client 's needs ? do the existing regulatory standards of practice for nurses adequately address safety critical issues in relation to transitions in care ? in this paper , we report the findings of a study undertaken with one of canada 's provincial regulatory colleges to develop a greater understanding of the kinds of regulatory requirements needed to support safe care transitions . three assumptions guided this work : ( 1 ) the fundamental aim of any transition in care is the transfer of high - quality clinical information between health care providers and a clear understanding by each healthcare provider about who is responsible for which aspects of the client 's care ; ( 2 ) regulation is one element in the web of complex interprofessional relationships and safe handoff communication ; and ( 3 ) professional regulation sets the standards for skills , knowledge , and behavior for their members . we begin the paper with a review of the literature to gain a better understanding of the current knowledge related to safe transitions in care . we then consider the way in which a traditional and complexity lens helps us develop a fuller understanding of the way in which clients and families can have such dramatically different healthcare encounters , such as that illustrated in the story told above . next , we describe the complexity informed method used to generate a rich account of client transitions from nurses , physicians , and pharmacists currently engaged in direct client care in a variety of settings in the canadian province . following this , we examine regulatory requirements for safe client handovers , illuminating the tension between traditional and complexity informed approaches to professional standards of practice .", "handoffs , transfer of accountability , and handover are terms used to describe the transfer of information and the accountability for care . terms such as sign - over , sign - off , and sign - out are used to describe handoffs between on - call physicians . specific to nursing , terms such as shift change , change - over , and report are used to mark the end of a time period and to transfer the accountability for care to the next . regardless of the nomenclature , a client handoff involves the transfer of rights , duties , and obligations from one person , or group of people , to another . communication at the time of handover should result in a clear understanding about who is responsible for which aspects of the client 's care . the evidence tells us that major gaps in care occur during these critical exchanges [ 4 , 5 ] . frequent misunderstandings , errors , and lost information are related to higher rates of unexpected readmission , failure to adhere to care plans , delays or duplication of tests / treatments , delays in obtaining consents and authorizations , inappropriate use of medication , and potentially negative clinical outcomes [ 69 ] . in addition , several operational challenges , such as frequent interruptions , too much noise , and lack of space , have a negative impact on care transitions . increased client acuity results in stress related to insufficient staff time to complete the client handover [ 2 , 1013 ] . despite the reality that many clients are discharged from health care settings quicker and sicker , requiring complex treatment protocols from multiple providers across multiple settings , until recently , little attention has been paid to the role professional standards of practice and safe outcomes . overall , there is a dearth of empirical evidence that considers the link between regulatory standards of practice and safe transitions in care . yet , to have a positive impact on the quality of care , regulatory standards of practice have to be contextually relevant , evidence based , and keep pace with the rapidly changing health care environment .", "a complexity lens helps nurses and other health professionals to develop a fuller understanding of the ways in which people and processes of care are interconnected , emergent and how relationship - rich networks and shared sense - making enable them to manage uncertainty more effectively . newcomers to complexity science may imagine a picture of utter confusion when they hear someone refer to healthcare as chaotic or complex . for students of complexity science , however , chaos is not about utter confusion , but rather about the existence of patterns where we thought there were none . unlike traditional models of science , which are cast in the form of linear and proportional relationships between cause and effect , complexity models are nonlinear , and events are related within degrees of proportionality . in other words , in linear models , there is one cause for one effect . in nonlinear models , there is more than one cause for an effect and more than one effect with a cause . from a traditional , linear perspective , the roles and responsibilities for each healthcare provider involved in care transitions are viewed as centrally structured through each discipline 's professional standards of practice . in contrast , from a complexity perspective , healthcare providers respond to their own particular local context , producing diversity of agent behaviours and client and family experiences . our example illustrates how healthcare providers interact with others according to their own evolved principles of local interaction . complexity theory reminds us that healthcare providers enable and constrain each other in local interactions , and these patterns of interaction constitute control and order . and because complex systems interact with other complex systems , tension and paradox are a common theme seemingly opposing forces of competition and cooperation can work together in ways that can never be fully predicted or resolved . whereas traditional scientific thinking assumes all issues can be resolved , complexity theory is comfortable with and values inherent tension between different parts of the system . the integration of complexity and traditional approaches to understanding processes of care during transition points will help nurses develop more comprehensive understanding of their work environment and client and family needs .", "the appreciative inquiry ( ai ) approach was taken to gain insight into current practices associated with care transitions and to develop insight into whether existing regulatory standards of practice for care transitions provide a sufficient underpinning to clinical practice . what is working well around here and how do we build on what is working well ? ai is based on the assumption that in every group or organization , something works well . developed by david cooperrider and colleagues at case western university , ai involves the art of asking questions in ways that strengthen a system 's capacity to apprehend , anticipate , and exploit procedures and processes that are working exceptionally well . four generic ai processes were incorporated into design and execution of this study , including ( 1 ) using the positive as the focus of inquiry ; ( 2 ) inquiring into exceptionally positive moments ; ( 3 ) sharing the stories and identifying life - giving forces ; and ( 4 ) creating shared images of a preferred future . relationships are vital in ai and help make sense of emerging patterns across multiple settings and roles , and so we structured the project in a way that all members of the project committee ( i.e. , ten representatives from the three regulatory agencies that partnered to conduct the inquiry ) were directly involved in collecting and analyzing research data . at the outset of the project , we held a one - day workshop focused on ai philosophical approach and data collection methods . subsequently , the process for data collection required each member of the project team to contact between 8 and 12 individuals from various practice settings and to invite these individuals to be interviewed either in person or on the phone . the sample was opportunistic , and a standardized ai guide was used for the interviews . interviews lasted between thirty and sixty minutes and took place between the months of february and may 2009 . regularly scheduled teleconferences were conducted with the project team during data collection phase to address any questions and/or concerns . each member of the project team summarized their interviews and transcribed their notes using a standardized format ( approximately three pages per interview ) . we began our analysis by reading through the interview notes several times to comprehend their essential features . preliminary analysis of the data involved open coding to generate a range of key themes that emerged from the data . the initial codes were then organized into provisional categories to build a coding framework divided into major themes and subthemes . the author and a research assistant conducted all aspects of the coding , first independently and then we compared and discussed categories until consensus was reached . subsequently , we engaged the project team in a one - day workshop to review and validate preliminary findings and explore implications for each profession 's regulatory framework . sixty - six interviews were conducted with a wide range of health care providers ( e.g. , nurses , physicians , pharmacists , social workers , physio- and occupational therapy , and pastoral care ) from a variety of practice settings ( e.g. , acute care , long - term care , palliative care , mental health , and corrections ) . the number of participants from each jurisdiction is provided in table 1 . the study protocol received approval by toronto 's york university office of research ethics .", "overall , study participants emphasized the fact that clients and families are most vulnerable when they transition between health care providers and between organizations , and we heard that nurses and other healthcare providers need to know three things to ensure safe care transitions , including ( 1 ) know your client ; ( 2 ) know your team on both sides of the transfer ; and ( 3 ) know the resources your client needs and how to get them . transitions need to be timely , provide comprehensive client and family information , be customized to client needs , involve in - person exchange of information , and provide opportunities for ongoing dialogue as required . clients are placed at even more risk when there are many interruptions during the exchange of information , when there are insufficient human resources to conduct the transfer of information , when the transfer is unplanned and unprepared , when there is a lack of respect between providers , and when there is a lack of understanding about issues of client privacy and confidentiality . three key themes emerged from the interviews in response to our questions regarding successful client transfers , including ( 1 ) flexible structures ; ( 2 ) independence and teamwork ; and ( 3 ) client- and provider - focused care . we describe each theme here with examples from the best transfer experiences . this theme speaks of the process of client transitions , highlighting tension between the need for well - defined structures for successful transitions and the need to be flexible in order to meet unique client needs . at their best , client transitions were described as a structured process comprising several stages , including prehandoff planning , movement of the client , and posthandoff followup . they involve in - person reciprocal exchange of client information , including comprehensive data on the client 's history , medications , and diagnostic testing results . verbal report prior to physically receiving the client helps the team prepare for the client 's arrival , and written documentation helps to decrease reliance on memory , thus enhancing the accuracy of the client 's data . advanced notice of a transition is especially important when clients have complex clinical needs and are being discharged between acute and homecare settings.during a home visit , the community nurse determined that the client needed to be assessed in the emergency department ( ed ) . first , the community nurse provided a verbal report to the ed triage nurse telling her why she was sending the client to the ed . she also sent written documentation to the ed in the event that to a different nurse than the one to whom she provided the verbal report to ended up assessing the client . the written documentation also provided a detailed list of the correct dosages of the client 's medication . ( community ) during a home visit , the community nurse determined that the client needed to be assessed in the emergency department ( ed ) . first , the community nurse provided a verbal report to the ed triage nurse telling her why she was sending the client to the ed . she also sent written documentation to the ed in the event that to a different nurse than the one to whom she provided the verbal report to ended up assessing the client . the written documentation also provided a detailed list of the correct dosages of the client 's medication . ( community ) participants also told us that successful transitions involve a high degree of flexibility , or professional latitude . specifically , several participants spoke about how they stretch standard operating procedures in order to assist clients , a situation which happens frequently with handoffs between institutional and home settings . participants spoke of situations in which the hospital provided supplies and equipment for discharged clients until arrangements could be made with community providers . this flexibility contributes to successful handoffs while building a sense of good will between health care professionals working in hospital and community . this palliative care client was being discharged home and was prescribed iv mixture to be administered by an epidural which needed laminar flow compounding and several days to order ingredients . there was insufficient time to arrange for this medication from a community pharmacy , so the client was sent home with sufficient meds to cover the gap . this was not official hospital policy ; the communication and arrangements were made through the hospital pharmacist . ( pediatrics ) this palliative care client was being discharged home and was prescribed iv mixture to be administered by an epidural which needed laminar flow compounding and several days to order ingredients . there was insufficient time to arrange for this medication from a community pharmacy , so the client was sent home with sufficient meds to cover the gap . this was not official hospital policy ; the communication and arrangements were made through the hospital pharmacist . ( pediatrics ) this theme speaks of independent and collaborative roles health care practitioners play in relation to successful handoffs . specifically , participants discussed the importance of knowing their independent roles and responsibilities in any given situation , while respecting the skills and knowledge of teams of providers from different health care sectors and organizations . participants stressed the importance of mutual respect when negotiating across multiple organizational and team boundaries while remaining focused on complex client needs . my role [ as a nurse ] in transferring accountability for the client is to make sure that the doctor 's orders are correct , the charting is up to date , the time and date is set for transfer and to ensure that the receiving institution knows that the client is being transferred . they made it [ the handoff ] a great experience because the team was very fluid and every member contributed to the efficiency of the team ( mental health and corrections)this transfer would have been even better if there was a greater understanding of the role of health workers and parish nurses within the community , and more willingness to share health information with client consent . ( long - term care ) my role [ as a nurse ] in transferring accountability for the client is to make sure that the doctor 's orders are correct , the charting is up to date , the time and date is set for transfer and to ensure that the receiving institution knows that the client is being transferred . ( acute care ) everyone knew their role really well , especially the forensic team . they made it [ the handoff ] a great experience because the team was very fluid and every member contributed to the efficiency of the team ( mental health and corrections ) this transfer would have been even better if there was a greater understanding of the role of health workers and parish nurses within the community , and more willingness to share health information with client consent . ( long - term care ) participants also identified the importance of collaboration between multiple healthcare teams and organizations . collaboration is particularly important in situations of conflicting client goals , changing condition , and complex needs . in addition , several participants suggested that transfers need to be seen as a seamless continuum of care rather than independent points at which one kind of care is concluded and another starts . the following describes a situation in which multiple health and social services providers collaborated to improve the situation for one client who was living in substandard conditions : the individual had mental health issues and the family found it difficult to care for the individual , to the extent that they were neglectful . efforts to improve care his care involved partnership of various organizations , including coordinated police and emergency medical services who removed the client from the unsafe environment . when presented to emergency room , the link between the his community and acute care case manager ensured sharing of his history and current situation , which facilitated an expedited review of his case by the capacity board . while the change in his care took a month of planning , it was an extremely positive experience for all involved due to teamwork and in the end , the client was in a ( mental health and corrections ) the individual had mental health issues and the family found it difficult to care for the individual , to the extent that they were neglectful . efforts to improve care his care involved partnership of various organizations , including coordinated police and emergency medical services who removed the client from the unsafe environment . when presented to emergency room , the link between the his community and acute care case manager ensured sharing of his history and current situation , which facilitated an expedited review of his case by the capacity board . while the change in his care took a month of planning , it was an extremely positive experience for all involved due to teamwork and in the end , the client was in a better place . ( mental health and corrections ) the third theme speaks of the information needs of clients , families , and members of the healthcare team . clients are especially vulnerable during times of transfer and a successful handoff is measured by the degree to which the client is informed , and comfortable , the extent to which the family is informed and the degree to which staff are confident they have met client and family care needs . specifically , study participants spoke to the importance of making sure that the client and their family members understand the reasons for the transfer and what they can expect from healthcare providers in the new healthcare setting . as illustrated by this participant , family members play a critical role in ensuring that client information has been shared with and between health care providers , including between nurses at shift change.nurse going off shift brought the oncoming nurse to the bedside , introduced her to us and included us in a portion of her report as i d band and fluid levels checked . as a spouse , i had an opportunity to provide input regarding my husband 's limitations and needs . i felt much more confident / comfortable with care provided by on - coming nurse , and my husbands ' needs were better met on that shift ( acute care ) nurse going off shift brought the oncoming nurse to the bedside , introduced her to us and included us in a portion of her report as i d band and fluid levels checked . as a spouse , i had an opportunity to provide input regarding my husband 's limitations and needs . i felt much more confident / comfortable with care provided by on - coming nurse , and my husbands ' needs were better met on that shift ( acute care ) in addition , participants told us that transfers from institutional to home care are more successful when family members are involved in planning and when they have time to obtain needed supplies and equipment . this is especially true in the case of pediatric care when family members need to learn new ways of caring for their child , or when family members are entering long - term care . i want to share my experience of the transfer of my husband from medical floor to long - term care . found out preferences / needs / wants plan of care had been shared ahead of time . found out areas of chief concern , including the need to give rescue meds quickly when shortness of breath started . decision to transfer was made based on bed availability and palliative care teams identification that my husband 's needs were not being met on the medical floor . nurses gave me a tour and showed me what was available and opportunities for me to discuss my role as a contributor to my husband 's care . were allowed to go out on pre - planned pass and when we returned , even though it was a different team , they knew our story and our preferences . ( long - term care ) i want to share my experience of the transfer of my husband from medical floor to long - term care . found out preferences / needs / wants plan of care had been shared ahead of time . found out areas of chief concern , including the need to give rescue meds quickly when shortness of breath started . decision to transfer was made based on bed availability and palliative care teams identification that my husband 's needs were not being met on the medical floor . nurses gave me a tour and showed me what was available and opportunities for me to discuss my role as a contributor to my husband 's care . were allowed to go out on pre - planned pass and when we returned , even though it was a different team , they knew our story and our preferences . ( long - term care ) study participants also spoke of the importance of transferring complete and accurate information between all members of the sending and receiving healthcare team . as stated above , successful transfer involves the exchange of comprehensive client information , including the client 's medical history , a summary of the current situation , medications , test results , and those pending . participants told us that they were more confident about their ability to provide safe care when they receive accurate , complete , and appropriate information from other team members . in addition , they spoke of the importance of trusting the providers from whom they are receiving the information . the positive part of the experience was the verbal report received as well as the phone call prior to transferring the client to the critical care unit that seemed to help everyone be comfortable with the admission . the rn who took the report was able to ask questions once the client arrived because they already had that head start in the information about the client before they arrived which allowed time for the rn to use critical thinking and come up with appropriate probing questions . ( acute care ) the positive part of the experience was the verbal report received as well as the phone call prior to transferring the client to the critical care unit that seemed to help everyone be comfortable with the admission . the rn who took the report was able to ask questions once the client arrived because they already had that head start in the information about the client before they arrived which allowed time for the rn to use critical thinking and come up with appropriate probing questions .", "to goal of this project was twofold : to gain insight into the practice setting realities for nurses and other health care providers as they manage increasingly complex care transitions across multiple settings and with a wide variety of care providers and to determine if current regulations reflect practice setting realities . our findings demonstrate that healthcare providers responded to client and family needs in different ways , and how the outcomes of care can be dramatically different , depending on that responsiveness . here first , participants seem to be contradicting themselves as they reinforce the importance of well - defined structures for successful transitions and the need to be flexible in order to meet unique client needs . from a traditional perspective , we heard how rules , structures , and boundaries are important elements of establishing minimum expectations of performance . we believe that when treated from a traditional mechanistic framework , structures might inadvertently impede safe transitions in care by inhibiting our ability to adapt in the dynamic , ever - changing healthcare environment . from a complexity perspective , study findings alert us to the fact that successful care transitions , and safe client and family outcomes , depend on the ways in which individual healthcare providers learn from each other and respond effectively to novel situations as they arise . we heard of situations that , when facing exceptional situations , individual healthcare providers worked hard to overcome difficult dynamics and organizational policies that affect safe care transitions . in reality , the need for structures and flexibility coexists . standard operating procedures are critically important to establish general goals and boundaries , while at the same time , the ability to explore , act on experience , and interact and respond are key to the delivery of safe and effective care in the event of ambiguous objectives and divergent problems . second , while demonstrating the importance of discipline - specific knowledge and roles , our findings also demonstrate the ways in which successful transitions in care require a high degree of collaboration and teamwork with all stakeholders , and with those who have the most to gain ( or lose ) : clients and families . complexity science reminds us that successful care transitions involve multiple agents who are constantly interacting and changing in response to each other , and the strength of the partnership is a key factor in achieving desired outcomes . exemplar transitions in care illustrate how relationships between individual care providers and with clients , and family members , also known as clinical microsystems , are the essential building block of larger systems of care . clinical microsystems are the place where clients , families , and care teams meet , and as our findings demonstrate , are the place where interactions determine the outcome of care transitions . consistent with what stacey refers to as complex responsive processes of relating , our finding that , at times , nurses and other healthcare workers make extraordinary responses and engage in superb teamwork to ensure safe transitions , reinforces the social nature of healthcare processes and of how safe and effective outcomes of care emerge in the social act of call and response . we suggest that nurses and other healthcare professionals take different paths , depending on whether they use traditional and/or complexity informed approaches , to manage care transitions . do they use a for - granted approach when they interact with clients and with each other , seeing themselves as one person in a web of constrained and constraining relationships , not fully responsible for outcomes of care ? or do they fully engage with each client , making sense of their need and the opportunities to collaborate with other team members to become agents of possibility and transformation ? third , with respect to regulatory implication , our findings demonstrated that while professional standards of practice provide the underpinning for safe care transitions , the use of good clinical judgment in the context of each client situation is a key to successful outcomes for clients , families , and care providers . in addition , findings suggest that it is easier to transfer clients within systems you know and to people you have worked with before , and that increased flexibility is required in a less familiar context . cook and rasmussen provide a complexity informed model of system dynamics that helps us understand the adequacy of existing standards of nursing practice and of how nurses and other health professionals uphold professional accountability while coming up with innovative responses and strategies to ensure safe transitions in care . in the going solid model of system dynamics , cook and rasmussen differentiate between safe and unsafe operating space ; safe operating space is conceptualize as an envelope created by three boundaries : economic , workload , and performance . there is room within the safe operating space to analyze risk and respond to unique client needs with creative adaptation while still upholding practice standards . however , nurses and other health care providers often operate at the margins of acceptable performance , and as our findings demonstrate , flexibility with regulation and standards is often required in complex sociotechnical work to make the system more efficient and adaptive to changing circumstances . the dynamic modeling of care transitions raises the fundamental question of how tightly regulated nurses and other healthcare professionals should be so as not to cause a trap of overregulation , which inevitably leads to more safety violations because of the tendency of standards to restrict behaviours . given the complex and context - specific nature of transitions , and the importance of clinical judgment for client safety outcomes , we concluded that a regulatory standard of practice , designed specifically for care transitions is not necessary , nor would it likely be meaningful .", "the present study was limited to one canadian province and the results may , to some extent , reflect contextual factors that are not shared by other national and international jurisdictions . in addition , because this study is unique in its use of appreciative inquiry as a method of research , findings may be biased toward an optimistic view of care transitions . however , we adopted this approach because we were most interested in hearing about how nurses and other health providers contribute to safe care transitions and learning about ways to support exemplary care . members of the study team who were new to ai expressed concerns about the positive approach at the outset of the project ; however , by the end of the project , these same individuals endorsed its use because the approach allowed us to build on current strengths and avoid the trap of only focusing on deficiencies . moreover , the project provided an excellent and unique opportunity for nurses and other health professionals to gain insight into each others ' practice environments and explore ways to uphold professional accountability in exceptional situations where innovations are required to ensure safe transitions in care .", " examples of positive and successful transitions demonstrated universal features across settings and healthcare providers , including the involvement of the client and family in decision making and planning , comprehensive and concise client information , opportunity for questions and followup by client and family as well as health care providers , time for planning and availability of staff to execute the transfer , and interprofessional and interorganizational collaboration . our findings demonstrated that exemplar transitions in care are laden with paradoxes , or the coexistence of apparent opposites , including flexible structures ; independence and teamwork ; and client and provider focus . to ensure safe and effective care transitions , nurses and other healthcare professionals need to accept the paradoxes as a normal part of their practice environment and they need to engage in relationship - rich networks which foster creativity and responsiveness . all regulatory bodies establish expectations through policies , position statements , practice standards , guidelines and/or other documents , for how members do what they do in an effective , safe , and ethical manner . viewed through a complexity lens , we see how the social act of call and response can lead to improved client and family outcomes , and that flexibility with regulation and standards is often required in complex sociotechnical work to make the care more efficient and adaptive to changing circumstances . we conclude that the development and introduction of new regulations holds the risk of constraining professionals ' responsiveness at the point of care , thereby threatening client safety outcomes . rather than focusing on new standards of practice , priority needs to be given to creating conditions where nurses and other healthcare providers are free to creatively engage and respond in ways that will optimize safe care transitions ." ]

[ "positron emission tomography - computed tomography is useful for diagnosing primary and metastatic lesions of esophageal cancer . the sensitivity and specificity of a pet / ct are also relatively preferable to other diagnostic imaging tools . although the number of cases is few , false positives exist in which an inflammatory tumor mass and a nonspecific tumor mass are difficult to differentiate between , even when combined with other image findings . in cases of esophageal cancer , the types of treatment and their prognosis are greatly affected if there is a metastasis or recurrence . positron emission tomography - computed tomographic false positives can not simply be ignored even if they appear to be due to inflammation or some other factors . this case report discusses the reasons to conduct a pet / ct test and its limitations in diagnosing esophageal cancer .", "the subjects of this investigation had suspected metastasis from a pet / ct which occurred before surgery of the 129 resected cases of esophageal cancer at our department from june 2010 to march 2015 . however , after surgery , biopsy and/or follow - up observation , only the cases that were confirmed as metastasis negative were considered to be pet / ct false positives for this assessment .", "there were 3 cases of preoperative pet / ct false positives ( table 1 ) . two cases in which bone metastasis was suspected revealed almost normal range of squamous cell carcinoma ( scc)associated antigen , alkaline phosphatase , and serum calcium levels . abbreviations : ln , lymph node ; m , metastasis ; mt , middle thoracic lesion of esophagus ; n , node ; t , tumor ; ut , upper thoracic lesion of esophagus . in case 1 , a 59-year - old man , without particular medical history , had a diagnosis of esophageal carcinoma by endoscopy . multiple bone metastases were suspected by a pet / ct in the spine , sternum , ribs , and pelvic bone ( figure 1 ) . after conducting a ct - guided biopsy of the right iliac bone , the patient is alive with no recurrence 4 years and 10 months after surgery , although the false - positive lesions in pelvic bone are still detected by pet / ct . in the case 1 , multiple bone metastases were suspected by a positron emission tomography - computed tomography in the pelvic bone ( suvmax = 2.94 ) . in case 2 , a 71 year - old man , without particular medical history , abnormal accumulations of fluorodeoxyglucose ( fdg ) in the manubrium sterni , vertebra th5 , and the pedicle of vertebral arch were found by pet / ct . therefore , after the sufficient informed consent , the patient underwent a laryngopharyngeal esophagectomy , a transhiatal esophagectomy without thoracotomy , and reconstruction with the gastric tube through the posterior mediastinal route . pathological finding showed poorly differentiated scc of esophagus and moderately differentiated scc of hypopharynx with a cervical lymph node metastasis . the patient is alive with no recurrence 3 years and 11 months after the surgery . in case 3 , a 76-year - old man , without particular medical history , had a diagnosis of a superficial esophageal cancer by endoscopy . he was suspected of lymph node metastasis of right recurrent nerve lymph node ( figure 2 ) by preoperative pet / ct . . scattered silicotic nodules and inflammatory lymphadenopathy might be caused by pneumoconiosis ( figure 3 ) . case 3 was suspected of right recurrent nerve lymph node metastasis by preoperative positron emission tomography - computed tomography ( suvmax = 3.00 ) . pathological finding of case 3 revealed scattered silicotic nodules in resected lymph nodes with infiltration of histiocytosis to sinusoid .", "in esophageal cancer treatment , fdg - pet / ct is also used to diagnose metastases and to judge the effectiveness of treatment . especially , pretherapeutic there are cases when the surgery may not apply to a patient if remote metastasis or high - grade lymph node metastasis is found . many cases have already been reported claiming that fdg - pet is a useful modality for a pretherapeutic staging . esophageal cancer causes 30% to 40% of lymph node metastases among cancers that spread to the submucosa , and the prognosis of esophageal cancer is known to be defined by the presence of lymph node metastasis , how many metastases are present , and how much the metastasis spreads . therefore , evaluating lymph node metastasis is a very important factor in deciding the course of treatment . the diagnosability of lymph node metastasis using a fdg - pet for esophageal cancer is generally reported to have a sensitivity of 32% to 51.9% , a specificity of 94.2% to 100% , and an accuracy of 48% to 93% . in addition , fdg - pet / ct , which is a combination of a fdg - pet and a ct , improves the diagnosability even more . yuan et al have reported that they evaluated the diagnosability of lymph node metastasis using fdg - pet / ct for esophageal squamous cancer and found a sensitivity of 94% , a specificity of 92% , and an accuracy of 92% . however , the size of a lymph node that can be detected by pet / ct is said to be only 6 to 8 mm , thus it is difficult to detect micrometastasis . also , detecting metastasis to neighboring lymph nodes could be difficult in cases with a lot of fdg accumulation in the primary lesion , or accumulation in the intestinal tract such as in the stomach , or in the heart . the fdg accumulates in inflamed lymph nodes as well , so false positives often appear in hilar lymph nodes or in patients who have chronic lung disease . lymph node metastasis was suspected after a preoperative pet / ct for the case 3 , as described above , but it was actually an inflammatory lymphadenopathy caused by pneumoconiosis . dual - time - point fdg - pet has been reported to be useful in reducing this kind of false positive . it uses the time differences of peak fdg accumulation for inflammation versus for tumors . after injecting the fdg , pet has revealed occult distant metastases at nodal and non - nodal sites in 5% to 40% of patients . distant metastases of esophageal squamous cancer are mainly found in the lungs , liver , and bones . kato et al reported the diagnosability of fdg - pet for esophageal cancer as having a sensitivity of 92% , a specificity of 94% , and an accuracy of 93% . when comparing those numbers to bone scintigraphy , whose diagnosabilities are 77% , 84% , and 82% , respectively , the diagnosability of fdg - pet is superior , especially for osteolytic lesions . but 2 false positives out of 44 cases came from pet , showing that fdg - pet is not perfect . the suvmax of metastic tumor might be helpful to increase the accuracy of pet / ct . our previous study demonstrated that the suvmax of the primary tumor was positively correlated with tumor size and vessel invasion and was positively related to the suvmax of metastic tumor . in the report , the diagnostic accuracy of pet / ct ( 87.3% ) was higher than that of conventional ct scans . when diagnosing metastatic bone tumors , the positive predictive value of bone metastasis is known to be quite high if the findings for both pet and ct match . therefore , even when bone metastasis is suspected due to abnormal fdg accumulation after a fdg - pet , yet no bone destruction image is found in the same site by ct , then interventional radiology or a surgical biopsy should be conducted to obtain a pathological diagnosis .", "this case report was on 3 patients with suspected metastasis due to a false - positive pet / ct who were later confirmed to not have metastasis . the metastases were originally suspected after conducting a pet / ct before surgery to treat esophageal cancer or during the postoperative course . conducting a pet / ct is useful when diagnosing esophageal cancer metastasis , but there should be an awareness of the possibility of false positives . therapeutic decisions should be made based on appropriate and accurate diagnoses , and a pathological diagnosis should be actively introduced if necessary ." ]

[ "class ii malocclusion can be appraised as skeletal and dental class ii.1 the treatment of skeletal class ii malocclusion in non - growing individuals involves either surgical correction of the jaw abnormality , or orthodontic camouflage which usually requires the extraction of premolars or distal movement of the maxillary molars.23 for several years , extraoral appliances such as headgear were the most widely used distalizing appliances , but they are not esthetically desirable or socially acceptable , especially for adults . they are also removable and require patient compliance , which can compromise the results.4 several intraoral noncompliance devices for maxillary molar distalization have been recommended since the 1980s.5 these include but are not limited to the hilgers pendulum appliance , jones jig distalization apparatus , open nickel - titanium ( niti ) push coils , distal jet , repelling magnets , and molar slider.67891011 numerous side effects have been reported in association with these tooth - borne distalizing appliances , including anchorage loss of the maxillary premolars and flaring of the incisors , and a significant amount of relapse can occur . also , the distalized molars must be used for anchorage during retraction of the premolars and anterior teeth,5 resulting in a bite - opening effect that is not tolerable for most patients . temporary anchorage devices ( tads ) have been investigated , in an effort to overcome some of the side effects associated with tooth - borne distalizing appliances.1 dental implants , miniscrews , and miniplates have been used for skeletal anchorage.11112131415 tads have several advantages . they are relatively easy to place , inflict less trauma on the oral tissues , are stable under normal degrees of force , and can bear force immediately after placement . miniscrews , in particular , are relatively inexpensive and patient compliance is limited to maintaining good oral hygiene . given their small size , they can be inserted in a variety of sites on the alveolar and basal bones.316171819 however , they have been associated with damage to anatomical structures such as dental roots , nerves , and blood vessels , and there are the possibilities of screw breakage on placement and removal , and screw failure with peri - implant inflammation . the reported success rates of miniscrews range from 80% to 95%.20 the placement of miniscrews in the buccal interradicular bone is one of the most common approaches used to provide skeletal anchorage . the interradicular space is a potentially advantageous region for insertion , because there is less potential for complications related to soft tissue irritation , particularly if they are placed through the attached gingiva.20 although adjacent teeth may limit mesiodistal tooth movement , buccal interdental miniscrews are very useful for molar distalization , due to their ease of placement , and simple application during treatment . with a properly positioned tad , 3 mm of distal movement per side can be achieved.21 on the palatal side , a tad may be placed paramedian ( near the midline ) or in the interdental space . while paramedian appliances usually require more expensive and sophisticated attachments , the risk of root damage associated with them is minimal.161222 tads placed on the palatal interdental area can apply distalization forces directly to the molar . moreover , these tads can also control the mesiodistal axis of the molar via manipulation of the line of action.22 we have introduced a new appliance using palatal miniscrews \" mimi - implant - aided ( mia ) in the interdental area between the 1st molar and 2nd premolar root and a trans - palatal arch ( tpa ) to overcome some of the mechanical shortcomings of previous appliances , in contexts including anterior protrusion , extrusion of posterior teeth , and tipping of molar teeth ( mia - tpa ) . traction between tads in the palatal interdental area and a tpa can produce direct distalization force that travels through the center of resistance of molars ( figure 1 ) . the purpose of the present study was to measure the three - dimensional ( 3d ) movement of 1st molars after using this newly designed distalizing appliance .", "the sample group consisted of 26 patients ( 22 females , 4 males ) with an average age of 19.8 6.3 years ( range 12 - 36 years ) at the beginning of treatment ( table 1 ) . the main inclusion criterion was angle class ii malocclusion of no more than one cusp on each side . patients with missing teeth , substantial restorations , or any craniofacial malformation were excluded from the study . the study was approved by the university research and ethics committee , and registered as a clinical trial at iranian registry of clinical trials ( no . irct201302269085n3 ) . all patients ( and their parents in the case of minors ) were informed about the treatment procedures involved , and informed consent was provided . after preparing the initial diagnostic records including dental casts , a dentist placed two miniscrews , each with a range of 8 - 10 mm in length and 1.4 , 1.6 , or 2.0 mm in diameter in the palatal interdental area under local anesthesia . miniscrews were inserted between maxillary 2nd premolars and 1st molars with a self - tapping technique , approximately 5 - 6 mm from the gingival crest at an angle of 60 - 80 relative to the long axis of adjacent teeth . the heads of the screws were approximately 2 mm above the mucosal surface , to facilitate the attachment of elastic modules . a tpa was made on a cast with stainless steel wire of 0.8 mm in diameter , with two helices or hooks soldered into the lateral incisors area . miniscrews were loaded with an average force of 150 - 200 gram - force / side via elastic modules , and ligature wire was used to connect the tpa , almost parallel to the occlusal plane of the miniscrew . all of the other teeth were bonded from 2nd molar to 2nd molar with standard edgewise brackets and tubes ( dentaurum , ispringen , germany ) , and leveling and aligning were performed before the termination of distalization to reduce any possible anchorage loss after distalization . the final arch wire was 0.018-in stainless steel . a cone - beam computed tomography ( cbct ) scan ( newtom 3 g ; quantitative radiology , verona , italy ) was performed to evaluate the precise position of mini screws relative to adjacent teeth and the direction of the force relative to molar teeth . after maxillary 1st molars were driven to super class i relationship , distalization was terminated and a final impression was taken for final evaluation . digital model analysis of molars was used to evaluate changes in the 3d position of the maxillary molars . digital scans of models before and after distalization were prepared via a maestro 3d dental scanner ( age solutions s.r.l . , maestro ortho studio software version 2.5 ( age solutions s.r.l . ) was used to analyze the digital models . the palatal rugae possesses unique characteristics , and as it tends to exhibit reasonable stability during growth , it may serve as a suitable reference point for longitudinal cast analysis.23 as the patients in the current study were not in an active growth period and no potential incisor movement was anticipated during the distalization period , as in nalcaci et al.,1 the distance from the central point of the incisive papilla to the mesiopalatal cusp of the 1st maxillary molars was used to measure distalization . the distances between the mesiopalatal cusps of the left and right molars were measured to evaluate intermolar width . the angles between the incisive papilla and the distopalatal and mesiobuccal cusps of the 1st maxillary molars were measured to evaluate the rotation of maxillary molars1 ( figure 2 ) . interocclusal space at the mesiobuccal , distobuccal , and mesiopalatal cusps of the 1st maxillary molars was evaluated by occlusion inspection , and maestro 3d scanner and software was used to evaluate vertical movement . after putting the casts in the maximum intercuspal position , the spaces between the maxillary 1st molar cusps and the antagonist tooth at t1 and t2 were measured via a 0.5-mm interval scale with a color coded tool ( figure 3 ) . for cbct analysis , digital imaging and communications in medicine ( dicom ) files were imported to a dolphin 3d 11 ( dolphin imaging , chatsworth , ca , usa ) . the angle between this line and a line drawn parallel to the occlusal plane was measured on right and left 3d sagittal view . in 3d axial view , lines between the helix and the miniscrews on the right and left sides were drawn , and the angle between those lines ( axial angle ) was measured ( figure 4 ) . measurements were repeated one month after the initial measurements , and the reliability of the measurements was calculated via the intraclass correlation test . reliability was between 79.7 and 97.0 for linear measurements , and between 93.8 and 96.6 for angular measurements . the paired t - test was used to compare measurements acquired before and after distalization in a digital cast . a probability of p < 0.05 was deemed to indicate statistical significance . pearson 's correlation was used to evaluate the relationships between cbct parameters and digital cast parameters . statistical package for the social sciences ver . 13.0 ( spss inc . ,", "the palatal rugae possesses unique characteristics , and as it tends to exhibit reasonable stability during growth , it may serve as a suitable reference point for longitudinal cast analysis.23 as the patients in the current study were not in an active growth period and no potential incisor movement was anticipated during the distalization period , as in nalcaci et al.,1 the distance from the central point of the incisive papilla to the mesiopalatal cusp of the 1st maxillary molars was used to measure distalization . the distances between the mesiopalatal cusps of the left and right molars were measured to evaluate intermolar width . the angles between the incisive papilla and the distopalatal and mesiobuccal cusps of the 1st maxillary molars were measured to evaluate the rotation of maxillary molars1 ( figure 2 ) . interocclusal space at the mesiobuccal , distobuccal , and mesiopalatal cusps of the 1st maxillary molars was evaluated by occlusion inspection , and maestro 3d scanner and software was used to evaluate vertical movement . after putting the casts in the maximum intercuspal position , the spaces between the maxillary 1st molar cusps and the antagonist tooth at t1 and t2 were measured via a 0.5-mm interval scale with a color coded tool ( figure 3 ) . for cbct analysis , digital imaging and communications in medicine ( dicom ) files were imported to a dolphin 3d 11 ( dolphin imaging , chatsworth , ca , usa ) . the angle between this line and a line drawn parallel to the occlusal plane was measured on right and left 3d sagittal view . in 3d axial view , lines between the helix and the miniscrews on the right and left sides were drawn , and the angle between those lines ( axial angle ) was measured ( figure 4 ) .", "measurements were repeated one month after the initial measurements , and the reliability of the measurements was calculated via the intraclass correlation test . reliability was between 79.7 and 97.0 for linear measurements , and between 93.8 and 96.6 for angular measurements . the paired t - test was used to compare measurements acquired before and after distalization in a digital cast . a probability of p < 0.05 was deemed to indicate statistical significance . pearson 's correlation was used to evaluate the relationships between cbct parameters and digital cast parameters . statistical package for the social sciences ver . 13.0 ( spss inc . ,", "maxillary 1st molars were moved distally an average of 2.3 1.1 mm , and the difference between the right and left sides was not statistically significant ( p = 0.63 ) . the mean duration of distalization was 6.8 2.8 months ( range 2 - 14 months ) , thus the average rate of distalization achieved via the method utilized in the current study was 0.4 0.2 mm / month . maxillary 1st molars were slightly rotated mesially around the palatal root after distalization , though the amount of rotation was not statistically significant ( this rotation is shown with a minus sign in table 2 ) . the mean intermolar width increase was 2.9 1.8 mm ( range 0.6 - 6.8 mm ) . the examination of vertical movement of three cusps of the maxillary molars showed relative molar intrusion that was statistically significant , except in the left mesiopalatal cusp ( table 2 ) .", "orthodontists are frequently faced with mild to moderate class ii molar and canine relationships that they prefer not to treat via the extraction of premolars , and for which treatment via auxiliary methods is often unsatisfactory due to insufficient patient compliance . many studies on intraoral maxillary molar distalization without patient cooperation have been performed , in order to solve the patient compliance problem associated with extraoral distalization appliances.110111220222425262728 in the present study , maxillary 1st molars were distalized via mini - implant - aided tpa . in mia - tpa , direct traction from elastic modules spanning from a miniscrew to a tpa helix is used to achieve molar distalization . miniscrews were inserted into the palate in this study , wherein attached gingiva is not a major concern , in contrast to the buccal side . moreover , there is more interdental space in the palate relative to the buccal side , and larger miniscrews ( 2 mm in diameter ) can be inserted . in the present study , the mesiodistal axis of molar movement can be controlled by adjusting the vertical position of miniscrews and/or the direction of the line of action relative to furcation . thus , bodily tooth movement is promoted , rather than tipping movement . in the current study , a removable tpa was used which made possible both proper adjustment of the line of action , and the control of molar rotation . the appliance is of a reduced size relative to other similar devices , and was tolerated by all of our patients without any concerns . there were also no problems reported with regard to the maintenance of oral hygiene . in the treatment protocol used in the current study , the elastic properties of the modules were lost relatively quickly in the oral cavity however , so the force applied was intermittent in nature , and they needed to be changed at least every 3 weeks . while niti pull coils allow for the application of more continuous force for distalization , they require a distance of at least 20 mm between the miniscrew and helix , to produce an appropriate level of force . in this study , 3d models were used for the 3d analysis of maxillary molar movements . two - dimensional ( 2d ) analysis by superimposing pretreatment and posttreatment cephalometric tracings has commonly been used in orthodontic studies.26 there are some disadvantages of cephalometric radiographs and superimpositions . the superimposition of lateral cephalograms is not easily accomplished , because the relevant radiographic landmarks and structures are frequently difficult to accurately trace . also , the process of superimposing can be technique - sensitive.29 cephalometric superimposition can reveal positional changes in the maxillary and mandibular dentition in both vertical and sagittal dimensions , but not in the buccopalatal direction . in contrast to 2d analysis , 3d analysis of serial dental models can provide further information on tooth movements , particularly in the buccolingual or transverse direction . the projection of bilateral teeth on the midsagittal plane also causes greater tracing errors because of the difficulty in identifying the bilateral teeth . consequently , the separate investigation of the tooth movements on each side , which can be achieved relatively easily via 3d models , is seldom attempted via cephalometric analysis.26 in this study , the distal movement of molars was measured along the line connecting the central point of the incisive papilla and the mesiolingual cusp of the 1st molar . while this measurement does not reflect true distal movement , measuring the intermolar change can provide a clear determination of both the distal and buccal movement of maxillary molars according to best available anatomical reference points . applying a distal force to maxillary molars moved them in three dimensions . with the device used in the current study , maxillary 1st molars were distalized 2.3 1.1 mm . in other studies based on intraosseous anchorage appliances , distalization was between 1.3 and 6.4 mm.110202224262728 distalization was higher in studies that used tooth - borne appliances such as pendulums or dual force distalizers supported by mini - implants for molar distalization.222527 the main reasons for the reduced distal movement of maxillary molars in our study were related to characteristics of our sample . the majority of patients in our sample required less than one cusp of distal movement . nalcaci et al.1 distalized molars via miniscrews with open coil springs by an average 3.95 1.35 mm . gelgr et al.10 distalized molars by an average 3.9 1.6 mm via screws supported by tpas and niti open coil springs . yamada et al.20 used miniscrews and elastomeric chain or niti closed coils for distalization , and average molar movement was 2.8 1.6 mm . the mean duration of distalization in our study was 6.8 2.8 months , thus the rate of distalization was 0.4 0.2 mm / month . in other studies , the duration of distalization was between 4.5 and 10.2 months.11020222425262728 nalcaci et al.1 reported an average duration of 9.61 2.1 months , and a rate of 0.41 mm / month . yamada et al.20 reported a duration of 8.4 months and a rate of 0.33 mm / month . in this study , mean maxillary 1st molar rotation was -0.86 4.6 and -0.35 4.1 in the right and left sides respectively . nalcaci et al.1 reported 0.54 and 0.74 rotation in the right and left sides respectively . buccal movement of molars was evident in the current study , with a mean increase of 2.9 1.8 mm in intermolar width . many studies1112224 reported buccal movement of molars , while yamada et al.,20 and lai et al.,26 reported palatal movement of molars . tpas made up of 0.8 mm round stainless steel wire do not have enough strength to resist expansion due to distal traction force . intrusion was the hallmark of this appliance , and numerous authors reported intrusion of molars in their studies.1222425 in a study similar to the current study , maxillary molars were intruded by an average of 0.3 0.8 mm.1 on the other hand , yamada et al.20 and lai et al.26 reported extrusion of molars . in the majority of our cases , the line of action of force passed through the furcation and the center of resistance of molars in an apical direction , concomitant intrusion of 2nd molars and premolars could be masked by mandibular overclosure , if present at all . so , we did not measure absolute intrusion via this type of measurement , instead interocclusal discrepancy was measured . the absolute measurement of intrusion is more accurately achieved via cbct before and after distalization . however , a second cbct was not approved by the relevant research ethics committee . two miniscrews of 1.4 8.0 mm inserted in the same patient failed in the current study . they were found to be loose at the first visit after fitting . larger miniscrews ( 2.0 10.0 mm ) were then inserted in this patient , but these also became loose so the patient was removed from the study . among the remaining patients , 3 screws ( 5.7% ) became loose . in these cases , excessive contact between the tpa and the miniscrew was the probable cause of screw failure in these cases .", "the mia - tpa is an appliance that can achieve absolute anchorage , and successfully drive maxillary 1st molars distally ." ]

[ "acute liver failure ( alf ) is a rare but life - threatening illness which occurs mostly in young adults without any known underlying liver disease [ 1 , 2 ] . in the united states , the most common cause of alf is acetaminophen overdose ( 39% ) but in 13% of cases , the causes are indeterminate . epstein - barr virus ( ebv ) is a dna virus associated with infectious mononucleosis ( i m ) in children and young adults . it may cause moderate and transitory increase of liver enzymes ; however , in rare cases , severe liver injury and even fatal alf may occur . we report a case of successful orthotopic liver transplantation ( olt ) in a 67-year - old female who presented with ebv - associated alf .", "a 67-year - old woman with bilateral osteoarthritis of the knee , hypothyroidism and recurrent deep vein thrombosis presented with 3 weeks of fatigue and a 1-week history of jaundice . her medications include diclofenac , rivaroxaban , desiccated thyroid , alprazolam , selenium and glucosamine . four weeks prior to admission , the patient was found to have mildly elevated serum aminotransferases during a routine visit . one week prior to admission , she reported to become jaundiced and her aminotransferases were found to be elevated to about 1,000 one day prior to admission , her serum alanine aminotransferase ( alt ) was 3,542 u / l , aspartate aminotransferase ( ast ) was 3,610 u / l and her inr was 2.7 . on admission , her vital signs were normal , and a physical exam was positive only for jaundice . laboratory findings included a white blood cell count of 13,200/l ( neutrophils 84.9% , lymphocytes 12.4% , monocytes 3.5% and eosinophils 0.2% ) . u / l , ast was 1,236 u / l , alp was 215 u / l and inr was 2.9 . hepatic artery , hepatic veins , and portal vein were patent , with a normal direction of flow by doppler study . serum acetaminophen level was 7 g / ml . hav antibody , hbsag , hcv and hev antibody were all negative . blood tests for human immunodeficiency virus ( hiv , hiv1/2 antibody and p24 antigen ) , cytomegalovirus ( cmv ) , rubella and rubeola were negative . antinuclear antibody , anti - mitochondrial antibody and anti - smooth muscle antibody were negative . the patient was initially diagnosed with alf possibly , secondary to diclofenac use , because she had begun to take diclofenac potassium 50 mg twice daily since 5 months ago . she was admitted to the icu and received multiple transfusions of fresh - frozen plasma ( ffp ) to correct her coagulopathy . she was given midodrine and octreotide and underwent hemodialysis for hepatorenal syndrome . despite multiple transfusions of ffp , her coagulopathy worsened , and her inr was 3.7 the following day . her serum alt and ast decreased rapidly in the following 2 days and the patient became more lethargic and somnolent , and difficult to arouse . four days after admission , she underwent a successful liver transplantation from a cmv - negative , ebv ( igg ) positive cadaveric donor . the explanted liver was significantly smaller than normal indicating acute failure of the liver ( fig . after transplantation , she received thymoglobulin , methylprednisolone taper and was later switched to prednisone taper , and she also started tacrolimus 4 days after transplantation . ( a ) small explanted liver weighing 720 g. ( b , c ) histologically , the liver showing 75% of parenchyma extinction and massive necrosis with sinusoidal lymphocytosis and atypical lymphocytes ( h&e , b : 100 ; c : 400 ) . ( d ) ebv in situ hybridization ( left , 600 ) and cd20 immunostain ( right , 600 ) showing numerous ebv - positive atypical b lymphocytes . her histological report of the explanted liver showed that there was 75% massive hepatic parenchyma necrosis ( fig . there were also acidophilic bodies , microvesicular steatosis and mild macrovesicular steatosis present in the residual hepatic parenchyma . there was a mixed population of inflammatory cells with predominant lymphocytes with sinusoidal lymphocytosis ( fig . 1d , left ) and positive for cd20 immunostain ( 1:100 dilution , horse radish peroxidase technique ; dako , carpenteria , ca ) ( fig . hsv staining of the liver tissue was negative although the serum antibodies were slightly higher than normal . a review of the liver biopsy 5 days prior to admission showed about 25% hepatic parenchyma necrosis with bridging necrosis ( fig . a whole blood assay for ebv dna using pcr of edta anticoagulated peripheral blood showed 462,000 copies / ml . she was started on intravenous acyclovir immediately after the diagnosis of ebv infection , and later she was switched to oral acyclovir which was administered for 3 months . two weeks after transplantation , ebv viral capsid antigen ( vca)-igg test was positive and vca - igm was negative , ebv d early antigen ( ea - d ) and epstein - barr nuclear antigen ( ebna ) were negative . one month later , the transplanted liver underwent biopsy and showed negative cd20 and ebv rna ( data not shown ) . the patient is presently doing well 6 months after liver transplantation with no evidence of clinical ebv infection or lymphoproliferative disease . ( a , b ) liver showing 25% parenchyma necrosis with bridging necrosis , acidophilic bodies , mixed inflammatory infiltrate and sinusoidal lymphocytosis ( a : 100 ; b : 400 ) . ( c ) ebv in situ hybridization showing occasional ebv - positive b lymphocytes ( 400 , arrows ) . lt : liver transplant ; acv : acyclovir ; alp : alkaline phosphatase ; ast : aspartate aminotransferase ; alt : alanine aminotransferase .", "we describe a case of alf - associated with ebv infection in a previously healthy elderly woman that underwent successful emergent liver transplantation . fulminant liver failure , is a sudden and severe hepatic injury in patients without previous liver diseases . the clinical manifestation usually includes loss of hepatic function , coagulopathy , hepatic encephalopathy and in many cases , progressive multi - organ failure [ 1 , 2 ] . despite recent advances in intensive care management , it has been reported that 5-year survival rate is about 80% in patients who underwent emergency liver transplantation for alf . the etiologies of alf vary greatly by country and have evolved over time . in the unites states , drug - induced alf , mainly acetaminophen , accounts for about 58% of cases , and hepatitis a and b accounts for 14% of cases according to the us acute liver failure study group data . these cases often follow a relatively insidious presentation before patients quickly develop liver failure , and rates of survival are poor without transplantation . recent studies showed that the cause may be related to ebv infection . in our case , the cause of alf was initially thought to be diclofenac , because the patient had been taking diclofenac for about 5 months prior to admission , and other causes such as acetaminophen , infectious agents and metabolic diseases were excluded through serological tests ; however , the biopsy and blood ebv dna pcr results confirmed that ebv is the major cause of alf , and diclofenac may have played no or a minor role in this process . ebv - associated alf is very rare , accounting for only 0.21 % of adult alf cases . it usually occurs in adults younger than 40 years of age , which is consistent with the fact that young adult patients tend to have symptomatic infectious mononucleosis [ 10 , 11 ] . it has been reported that patients with neoplasm , cmv and hiv and patients on immunosuppressant drugs are at high risk for ebv - associated alf , although recent studies also showed that immunocompetent patients can also develop alf , indicating that other unknown risk factors may also be involved in the development of ebv - associated alf [ 12 - 14 ] . in adult aged 60 years or older , there were only two cases of ebv - associated alf in the literature ; however , both of them had impaired immune system : one had just started steroids and the other had recent heart surgery [ 15 - 17 ] . in our case , the patient does not have any of the above mentioned problems and did not take any immunosuppressant medications . this is the first case of ebv reactivation - associated alf in a healthy elderly patient . this indicates that in older immunocompetent patients presenting with alf , a primary ebv infection or ebv reactivation should be considered when other causes have been excluded . early diagnosis of ebv - associated hepatitis was difficult as reliable diagnostic criteria are not clearly defined . molecular approaches , such as ebv - dna pcr or eber - rish , are frequently used to diagnose ebv - related hepatitis [ 9 , 18 ] . serological ebv tests such as vca - igm , vca - igg , ea - d and ebna can also help to detect a recent infection or a prior infection , or a reactivated ebv infection , although they are not reliable , especially in the elderly , as they have significantly higher titers of the serum tests than young adults [ 9 , 18 ] . in our case , the liver biopsy 1 week before admission showed 25% hepatic necrosis , some atypical b lymphocytes with positive ebv rna . in explanted liver , there was over 75% necrosis , significantly increased atypical b cells and strongly positive expression of ebv rna , which is consistent with the progression of the liver failure . we also tested the blood ebv level by pcr 4 days after liver transplantation and it was extremely high , indicating a severe active ebv infection . the ebv serology tests 10 days after liver transplantation showed very high vca - igg but normal vca - igm , indicating that this is an ebv reactivation . moreover , the liver enzyme showed cholestatic enzyme changes after transplantation with continuously increased alp . we think that the increased alp was due to damage caused by ebv infection to the implanted liver . treatment with acyclovir should be strongly considered after liver transplantation for alf from ebv to prevent infection of the liver graft during the period of high level immunosuppression . first , we were not able to diagnose ebv hepatitis until after evaluation of the explanted liver . liver transplantation could be potentially avoided if an early diagnosis of ebv hepatitis could have been made . second , the ebv pcr test was done after transplantation , when patient had already been on steroids . the extremely high ebv pcr value may not reflect the severity of ebv infection , as steroids can suppress the immune system and ebv could replicate faster than expected . third , the serological test was done 10 days after we started acyclovir , which may not show the initial changes of ebv infection . in addition , the use of diclofenac in this patient since a few months ago might have caused some initial injury to the liver . diclofenac is reported to cause liver injuries by two main mechanisms , hypersensitivity and metabolic aberration [ 19 , 20 ] . acute liver injury caused by diclofenac is thought to be due to hypersensitivity and usually presents within days to a few weeks after starting diclofenac , which can result in fatal liver failure . metabolic aberration can cause persistent mild liver injury and once diclofenac is stopped , the liver can recover quickly . in our patient , diclofenac might have caused mild liver injury initially ; however , the reactivation of ebv plays a major role in developing alf . we are confident that ebv is the cause of alf in the patient based on liver enzyme changes , characteristic histopathological changes , and most importantly strong ebv rna expression in the explanted liver . in conclusion , our case demonstrates that ebv infection or reactivation may be under - recognized as a cause of alf . early recognition and diagnosis is extremely important , however difficult . in both healthy young and elderly adults with alf liver biopsy should be strongly considered in patients with unexplained alf with hepatitis pattern and ebv rna by in situ hybridization should be mandatory if sinusoidal lymphocytosis is present . emergent liver transplantation is the only definitive treatment option , with very favorable prognosis for such patients . treatment with acyclovir should be strongly considered after liver transplantation for alf from ebv to prevent infection of the liver graft during the period of high level immunosuppression ." ]

[ "cranial nerves were composed of two different histological portions , glial and non - glial portion . the transitional zone refers to the junction between glial portion and the non - glial peripheral portion . the most outer layer of brain cortex is coated by pia glial membrane called glial limiting membrane or glial limitans10 ) . skinner12 ) reported the extent of glial out growth into the various cranial nerves and glial out spread of facial nerve was extended as far as 2.6 mm in length from the origin of brain stem . in general , root exit zone or root entry zone ( rez ) of cranial nerves means the area nerves come from the brainstem . recently , some authors proposed to replace the term of rez by root exit point ( rexp ) , root detach point ( rdp ) , and root exit zone ( rexz ) in detail13 ) . the purposes of this study were to measure the length of the central myelin , especially from root detach point to transitional zone and determine histological structures of central myelin of facial nerve .", "twenty brain stems were obtained from formalin embedded cadavers . the 12 men and 8 women of cadavers ranged in age from 32 to 78 years ( mean , 58 years ) . during the brain stems were served , care should be taken to preserve the cisternal segment of facial nerve . we obtained 40 facial nerves but 17 facial nerves were put out of shape during preparation for histological study . twenty three facial nerves could be examined properly . to make a facial nerve - brainstem tissue block , we cut the midline of brain stem into halves and slice them along the facial nerve . first , one slice was stained with hematoxylin and eosin ( h&e ) for screening the shape of facial nerve and brain stem . and then , other slices were stained with periodic acid - schiff ( pas ) for peripheral myelin and glial fibrillary acid protein ( gfap ) for central myelin and glial membrane . after staining , photomicrographs of each section were taken and measure the distance of medial rez , from detach point of facial nerve at the brain stem to the most distal part of central myelin , and thickness of glial membrane of central myelin of rez . the thickness of glial membrane was obtained at two different points , the thickest point of proximal and distal area of central myelin . usually to correct for the shrinkage error during fixation , occulomotor nerve of same specimen was used as a standard . but we did not calculate the percentage of facial nerve shrinkage because of being used pre - fixed specimens with formalin .", "with the h&e staining , the shape of facial nerve was easily determined ( fig . special staining methods , such as pas and gfap , could be clearly distinguished the facial nerve into peripheral and central myelin . peripheral myelin was stained dark blue with pas staining and central myelin was stained dark brown with gfap ( fig . 2 ) . glial outgrowth from the brain stem , central myelin , ends in dome or cone formation . the outermost membrane surrounding the brain stem , glial limiting membrane , continues to the central myelin could be visible . the mean thickness of glial membrane of central myelin was 66.5 um ( 40 - 110 um ) at proximal and 7.4 um ( range of 5 - 10 um ) at distal ( table 1 ) . the length of medial rez was mean 2.6 mm ( range of 1.6 - 3.5 mm ) ( table 2 ) .", "there is no clear description of the relationship between the rez and the transitional zone . in the strict sense , rez means the area nerve comes from the brainstem and transitional zone refers to glia - schwann cell border . the term of rez was defined by jannetta as a junctional area between central and peripheral myelin6 ) . many authors used the terms of rez and transitional zone interchangeably1,3,11 ) . in a recent study , tomii et al.13 ) described the transitional zone as the region where the myelin sheath is composed of both central glial and schwann cell myelin . they have proposed the use of terms root exit point ( rexp ) , rdp ( medial detach point ) and transitional zone instead of rez . and they stressed the rdp appears to be a good landmark for the microvascular decompression surgery . in 1915 , henschen5 ) has been descried that cranial nerve was composed of two different segments , such as glial and non - glial portion . skinner12 ) also reported several characteristics of various cranial nerves as follows . from the brain there is an outgrowth of glia cells , astrocyte and oligodendroglia , along the cranial nerve trunks and the extent of glial outgrowth varies in different nerves . many investigators named the glial outgrowth segment of cranial nerve as cns segment of cranial nerve , central myelin , or central glial myelin2,10,13 ) . to measure the exact length of central myelin of facial nerve is difficult because of its irregular distal margin . skinner12 ) reported that the glial outspread in the facial nerve extended as far as 2.6 mm from the plane of superficial origin . adams1 ) described that the transitional zone is only 1 to 3 mm in length . jannetta6 ) also reported the distance to the peripheral boundary of the rez was 0.5 to 1 cm in the fifth , seventh , and ninth cranial nerves . in 2003 , he measured the length of the lateral and medial transitional zone were mean 1.9 mm and 0.58 mm , respectively . we measured the distance from medial detach point of brain stem to the most distal glial segment of facial nerve . glial portion of the cranial nerve , central myelin , is composed of fibrillary astrocyte and oligodendroglia cells12 ) . and it has a structure similar to that of white matter of the cns , consisting of parallel traveling nerve fibers , and lacks funicular structure . nerve fibers are embedded in supporting tissue , but endo- , peri , and epineurium are absent . de ridder et al.2 ) stated the layer of pia mater surrounds the central myelin . glial limiting membrane , pia glial membrane , or glial limitans , is a thin barrier of astrocyte foot process associated with the parenchymal basal lamina surrounding the brain10 ) . the main function of glial limiting membrane is to act as a physical barrier of the cns . we observed that the glial limiting membrane extending from the outermost layer of brain stem , strong stained with gfap , surrounds the central myelin . we called the layer closing the central myelin as glial sheath like epineural sheath of peripheral nerve . we also measured the thickness of glial sheath of facial nerve and it becomes thin at distal portion than proximal . the glial sheath of central myelin may act as a barrier of the nerve fiber from vascular compression . the cause of hemifacial spams is generally known as that facial nerve compressed by blood vessels located at the rez . the mechanism of hemifacial spasm is thought that thinning or defect of neural sheath of facial nerve because of vascular compression may result in developing cross talking of neural transmission4,9 ) . jannetta et al.7 ) reported that myelin defect exist in the rez in patient of hemifacial spasm .", "there is no clear description of the relationship between the rez and the transitional zone . in the strict sense , rez means the area nerve comes from the brainstem and transitional zone refers to glia - schwann cell border . the term of rez was defined by jannetta as a junctional area between central and peripheral myelin6 ) . many authors used the terms of rez and transitional zone interchangeably1,3,11 ) . in a recent study , tomii et al.13 ) described the transitional zone as the region where the myelin sheath is composed of both central glial and schwann cell myelin . they have proposed the use of terms root exit point ( rexp ) , rdp ( medial detach point ) and transitional zone instead of rez . and they stressed the rdp appears to be a good landmark for the microvascular decompression surgery .", "in 1915 , henschen5 ) has been descried that cranial nerve was composed of two different segments , such as glial and non - glial portion . skinner12 ) also reported several characteristics of various cranial nerves as follows . from the brain there is an outgrowth of glia cells , astrocyte and oligodendroglia , along the cranial nerve trunks and the extent of glial outgrowth varies in different nerves . many investigators named the glial outgrowth segment of cranial nerve as cns segment of cranial nerve , central myelin , or central glial myelin2,10,13 ) . to measure the exact length of central myelin of facial nerve is difficult because of its irregular distal margin . skinner12 ) reported that the glial outspread in the facial nerve extended as far as 2.6 mm from the plane of superficial origin . adams1 ) described that the transitional zone is only 1 to 3 mm in length . jannetta6 ) also reported the distance to the peripheral boundary of the rez was 0.5 to 1 cm in the fifth , seventh , and ninth cranial nerves . in 2003 , he measured the length of the lateral and medial transitional zone were mean 1.9 mm and 0.58 mm , respectively . we measured the distance from medial detach point of brain stem to the most distal glial segment of facial nerve .", "glial portion of the cranial nerve , central myelin , is composed of fibrillary astrocyte and oligodendroglia cells12 ) . and it has a structure similar to that of white matter of the cns , consisting of parallel traveling nerve fibers , and lacks funicular structure . nerve fibers are embedded in supporting tissue , but endo- , peri , and epineurium are absent . de ridder et al.2 ) stated the layer of pia mater surrounds the central myelin . glial limiting membrane , pia glial membrane , or glial limitans , is a thin barrier of astrocyte foot process associated with the parenchymal basal lamina surrounding the brain10 ) . the main function of glial limiting membrane is to act as a physical barrier of the cns . we observed that the glial limiting membrane extending from the outermost layer of brain stem , strong stained with gfap , surrounds the central myelin . we called the layer closing the central myelin as glial sheath like epineural sheath of peripheral nerve . we also measured the thickness of glial sheath of facial nerve and it becomes thin at distal portion than proximal . the glial sheath of central myelin may act as a barrier of the nerve fiber from vascular compression . the cause of hemifacial spams is generally known as that facial nerve compressed by blood vessels located at the rez . the mechanism of hemifacial spasm is thought that thinning or defect of neural sheath of facial nerve because of vascular compression may result in developing cross talking of neural transmission4,9 ) . jannetta et al.7 ) reported that myelin defect exist in the rez in patient of hemifacial spasm .", "our study shows the length of medial rez is mean 2.6 mm ( range of 1.6 - 3.5 mm ) and the rez is covered by glial sheath continued from glial limiting membrane of brain stem ." ]

[ "falls are both common and debilitating in patients with parkinson 's disease ( pd ) . they have devastating consequences for affected individuals , often leading to injuries , secondary immobility , and reduced quality of life . survival is reduced once falls have occurred , although a recent report failed to identify a relationship between falls and mortality risk in pd . falls are also important for the public health system , as the costs associated with falls and fall - related injuries are enormous . a prospective 20-year follow - up of 136 patients with newly diagnosed pd confirmed the high prevalence of falls ( 87% ) and resulting fractures ( 35% ) . for the management of environmental factors such as slippery floors , loose rugs , poor lighting , or inadequate footwear may also contribute . there is an increasing awareness that freezing of gait - a sudden and episodically occurring inability to generate effective forward stepping movements - is one of the leading causes of falls , presumably because patients are caught by surprise due to the often unexpected nature of freezing events . recent work has underscored the additional importance of cognitive impairment as a key factor contributing to both falls and freezing ; falls are an issue in demented patients in particular . possible explanations for this link include more prominent dopaminergic denervation of the caudate nucleus or more generalized cholinergic dysfunction . preventing falls is generally perceived as being difficult , but is not impossible . given the complex multifactorial nature of falls and the experience in elderly non - parkinsonian populations , a multidisciplinary approach appears preferable , but for patients with pd this strategy is not yet backed by scientific evidence . crucial elements in the therapeutic approach include optimizing pharmacotherapy - increasing pd medication for dopa - sensitive signs , and stopping sedative drugs - and tailored physiotherapy , based on evidence - based practice guidelines . here , we highlight a few important new developments in this field .", "the quest should not stop when one causative factor has been found because falls are typically multifactorial in origin . a recent prospective study in 101 patients with early - stage pd assessed the best prediction was reached by combining disease - specific measures ( such as pd severity and freezing of gait severity ) with balance measures ( such as the occurrence of symptomatic orthostatic hypotension , the tinetti total score , and the extent of postural anterior - posterior sway ) . however , even this combination attained a sensitivity and specificity for predicting falls of only around 80% . including cognitive measures that are more specific may further improve these predictive scores , but this remains to be examined . particular emphasis should be placed on testing for freezing of gait - using a series of provoking tests , including rapid 360 degree turns on the spot - and for frontal executive dysfunction . specific attention should be paid to fear of renewed falling as this is common in pd . fear of falling is not only a risk factor for renewed falls , it may also lead to secondary immobilization with all its related adverse consequences . most balance deficits are resistant to dopaminergic medication . however , gait problems - including freezing of gait - can improve with dopaminergic medication , although doses that are higher or more frequent than those typically needed to increase hand functioning may be required . recent work points to a possible role for cholinesterase inhibitors in the treatment of gait and balance deficits , both in pd and in alzheimer 's disease , given that many axial motor deficits may result from misbalance between central cholinergic and dopaminergic systems . it has become clear that stereotactic deep brain surgery should be reserved for patients whose gait and balance deficits are still levodopa - responsive pre - operatively . several studies are comparing bilateral stimulation of two different targets , namely the subthalamic nucleus ( stn ) and the internal globus pallidus . the initial results indicate that both approaches are effective in providing short - term relief of motor symptoms , but one study suggested internal globus pallidus stimulation may offer better long - term outcomes for gait and balance deficits . this difference was not evident in the largest study , although falls after a 2-year follow - up tended to be more common after stn stimulation , but this requires more study . in particular , stn stimulation has been associated with a worsening of gait and balance deficits , not only in the immediate postoperative phase , but also several years after follow - up . adjusting the stimulation parameters ( e.g. , markedly lowering the stimulation frequency ) may be helpful in such patients . the pedunculopontine nucleus has been studied as a promising new target , specifically for gait and balance deficits , but so far the results have not been very impressive . the evidence - based guidelines on physiotherapy for pd were recently updated , providing a menu of treatment modalities to improve mobility and reduce falls . examples of evidence - based physiotherapy strategies include cueing techniques , cognitive movement strategies , and the use of exercise . rhythmic auditory or visual cues can improve gait in pd , including freezing of gait . new inventive cueing approaches include walking glasses with different patterns of visual and auditory stimulation and mental singing while walking . another promising approach , especially for pd patients with freezing of gait , is the use of cycling , the skill for which can be remarkably preserved in some patients . evidence based mainly on the effects of cueing on laboratory measures of gait and balance remains ; the challenge is to ascertain an enduring clinical improvement in daily life , including a reduction of falls . fears that cues might worsen the tendency to fall - for example , by increasing overall mobility - have not been substantiated . a systematic review concluded that exercise in patients with pd results in improvement in postural stability and balance task performance . however , power and quality of exercise studies have hitherto been insufficient to make definitive recommendations . future randomized controlled trials will look into the ( cost ) effectiveness of exercise to reduce falls . there is a growing body of literature on the use of treadmill training for gait rehabilitation in patients with neurologic disorders in general and for patients with pd in particular . a recent cochrane review concluded that treadmill training may help to improve gait akinesia in pd , but the effect on falls remains unclear . another interesting new development is the use of robotics , which can assist pd patients in making medio - lateral anticipatory weight shifts in preparation for taking a step . the initial results are promising , and such new techniques now need to be implemented in situations that are more realistic to evaluate the clinical merits of these techniques in relation to everyday gait performance and falls . delivering such specific physiotherapy interventions to patients with pd a large cluster - randomized trial showed that a community - based professional network with trained expert physiotherapists improved the quality of physiotherapy care and reduced health care costs , but without health benefits for patients .", "the quest should not stop when one causative factor has been found because falls are typically multifactorial in origin . a recent prospective study in 101 patients with early - stage pd assessed the best prediction was reached by combining disease - specific measures ( such as pd severity and freezing of gait severity ) with balance measures ( such as the occurrence of symptomatic orthostatic hypotension , the tinetti total score , and the extent of postural anterior - posterior sway ) . however , even this combination attained a sensitivity and specificity for predicting falls of only around 80% . including cognitive measures that are more specific may further improve these predictive scores , but this remains to be examined . particular emphasis should be placed on testing for freezing of gait - using a series of provoking tests , including rapid 360 degree turns on the spot - and for frontal executive dysfunction . specific attention should be paid to fear of renewed falling as this is common in pd . fear of falling is not only a risk factor for renewed falls , it may also lead to secondary immobilization with all its related adverse consequences .", "most balance deficits are resistant to dopaminergic medication . however , gait problems - including freezing of gait - can improve with dopaminergic medication , although doses that are higher or more frequent than those typically needed to increase hand functioning may be required . recent work points to a possible role for cholinesterase inhibitors in the treatment of gait and balance deficits , both in pd and in alzheimer 's disease , given that many axial motor deficits may result from misbalance between central cholinergic and dopaminergic systems .", "it has become clear that stereotactic deep brain surgery should be reserved for patients whose gait and balance deficits are still levodopa - responsive pre - operatively . several studies are comparing bilateral stimulation of two different targets , namely the subthalamic nucleus ( stn ) and the internal globus pallidus . the initial results indicate that both approaches are effective in providing short - term relief of motor symptoms , but one study suggested internal globus pallidus stimulation may offer better long - term outcomes for gait and balance deficits . this difference was not evident in the largest study , although falls after a 2-year follow - up tended to be more common after stn stimulation , but this requires more study . in particular , stn stimulation has been associated with a worsening of gait and balance deficits , not only in the immediate postoperative phase , but also several years after follow - up . adjusting the stimulation parameters ( e.g. , markedly lowering the stimulation frequency ) may be helpful in such patients . the pedunculopontine nucleus has been studied as a promising new target , specifically for gait and balance deficits , but so far the results have not been very impressive .", "the evidence - based guidelines on physiotherapy for pd were recently updated , providing a menu of treatment modalities to improve mobility and reduce falls . examples of evidence - based physiotherapy strategies include cueing techniques , cognitive movement strategies , and the use of exercise . rhythmic auditory or visual cues can improve gait in pd , including freezing of gait . new inventive cueing approaches include walking glasses with different patterns of visual and auditory stimulation and mental singing while walking . another promising approach , especially for pd patients with freezing of gait , is the use of cycling , the skill for which can be remarkably preserved in some patients . evidence based mainly on the effects of cueing on laboratory measures of gait and balance remains ; the challenge is to ascertain an enduring clinical improvement in daily life , including a reduction of falls . fears that cues might worsen the tendency to fall - for example , by increasing overall mobility - have not been substantiated . a systematic review concluded that exercise in patients with pd results in improvement in postural stability and balance task performance . however , power and quality of exercise studies have hitherto been insufficient to make definitive recommendations . future randomized controlled trials will look into the ( cost ) effectiveness of exercise to reduce falls . there is a growing body of literature on the use of treadmill training for gait rehabilitation in patients with neurologic disorders in general and for patients with pd in particular . a recent cochrane review concluded that treadmill training may help to improve gait akinesia in pd , but the effect on falls remains unclear . another interesting new development is the use of robotics , which can assist pd patients in making medio - lateral anticipatory weight shifts in preparation for taking a step . the initial results are promising , and such new techniques now need to be implemented in situations that are more realistic to evaluate the clinical merits of these techniques in relation to everyday gait performance and falls . delivering such specific physiotherapy interventions to patients with pd a large cluster - randomized trial showed that a community - based professional network with trained expert physiotherapists improved the quality of physiotherapy care and reduced health care costs , but without health benefits for patients .", "asking about falls and their impact on daily functioning should be a standard part of the evaluation of patients with pd . while awaiting further evidence , neurologists should consider installing a multidisciplinary team approach to tackle the vexing problem of falls in patients with pd . management involves a systematic search for risk factors for falling , and a subsequent multifactorial approach aimed at eliminating or alleviating all patient - related and environmental risk factors for falling . apart from optimizing dopaminergic medication , cholinergic therapies are now beginning to enter the field of play as well . fear of falling must be addressed , and immobilization must be avoided as long as independent movements can still be made reasonably safely . the multidisciplinary team should ideally consist of trained and experienced professionals who treat large numbers of patients . using this integrated approach , the goal should be to at least reduce falls or perhaps prevent them altogether , restore mobility and independence , and thereby help to maintain the quality of life for patients with pd .", "" ]

[ "prostate cancer ( pca ) is one of the major \n causes of cancer death in men worldwide . the molecular basis of the disease involves an irregular behavior \n of the functions mediated by the androgen receptor ( ar ) . human ar \n belongs to the nuclear receptor ( nr ) superfamily of transcription \n factors , which regulate gene transcription upon ligand binding . the structure of nrs is extensively documented \n in the literature , and in general , nrs \n share the following common organization : a variable amino - terminal \n activation function domain ( af-1 ) , a highly conserved dna - binding \n domain ( dbd ) , a hinge region that contains the nuclear localization \n signal , a conserved c - terminal ligand - binding domain ( lbd ) comprising \n a 12 helical structure that encloses a central ligand binding pocket \n ( lbp ) , and a second activation function domain ( af-2 ) that is located \n at the carboxy - terminal end of the lbd and mediates ligand - dependent \n transactivation . ar is activated by the endogenous hormone testosterone \n ( tes ) and its more potent metabolite dihydrotestosterone ( dht ) , both \n of which bind in the lbp . the binding of these endogenous modulators \n induces a reorganization of helix 12 to the so - called agonist \n conformation , generating a structured hydrophobic surface ( af-2 ) suitable \n for the recruitment of tissue - specific nr coactivators . such nr coactivators \n can be thought of as master switches , directing and \n amplifying the subsequent transcriptional activity of the target nr . \n in a recent work , an additional secondary function site called binding \n function 3 ( bf-3 ) has been reported on the surface of the ar that \n could also play a relevant role in the allosteric modulation of the \n af-2 . nr drug development has traditionally \n focused on advancing full or partial agonists / antagonists interacting \n within the lbp of the lbd . pca has been \n treated by intervention at the early stages through utility of classical \n antiandrogens , which act by displacing the natural hormones from the \n pocket and inducing a conformational change of the helix 12 so that \n coactivators can not be recruited . tissue specificity , detrimental \n side effects , and a loss of the pharmacological effect ( acquired drug \n resistance ) over time are major and ongoing concerns with such lbp \n targeting treatment regimes . it has been demonstrated \n that it is possible to inhibit the transcriptional activity of the \n nrs by directly blocking the critical receptor : coactivator interaction . this alternative approach to traditional nr modulation may furnish \n greater pharmacological insight and afford opportunities to modulate \n not only under tissue specific circumstances but without adversely \n affecting natural ligand binding and so preserving the beneficial / nondisease \n linked functions of the receptors . specifically , the steroid receptor \n coactivator ( src ) family has been postulated as a feasible target \n for pharmacological intervention . the \n viability of targeting ar coactivator interaction using small \n molecules has been recently demonstrated . moreover , it \n has been postulated that circumventing the lbp will overcome the problem \n of drug resistance in pca . here we describe the discovery and characterization of a novel \n class of selective non - lbp true antiandrogens , characterized \n by full ar antagonism in inhibiting the recruitment of coactivators \n and lacking intrinsic partial agonistic properties . mechanistically , \n these compounds are totally differentiated from the recent description \n of true lbp antiandrogens like mdv3100 and rd162 , while their selectivity and druglike nature underpin the potential \n of a non - lbp intervention strategy in advanced prostate cancer resistant \n to classical therapy , first described for the true \n non - lbp targeting antiandrogens pyrvinium pamoate ( pp ) and harmol \n hydrochloride ( hh ) . the biological \n data obtained both on target with time - resolved fluorescence resonance \n energy transfer ( tr - fret)/fluorescence polarization ( fp ) assays and \n in cellular pca models demonstrate the non - lbp antagonist activity \n of the series and an alternative mechanism of inhibition , furnishing \n a new class of nonpeptidic , small molecule ar : coactivator selective \n disruptors as leads for the development of novel treatments for prostate \n cancer .", "a virtual ( computational ) screen \n of six vendor compound databases ( see experimental \n section ) was performed through a combination of 3d pharmacophore \n generation and docking . seven x - ray structures of coactivator peptide \n bound ar were used to define key ligand - derived pharmacophoric features \n of the most represented motifs occurring in known ar coactivators . initially , common key interaction motifs within \n the peptide of the form fxxlf , lxxll , or fxxlw were considered to \n generate a consensus af-2 pharmacophore . subsequently , a second site - derived \n pharmacophore model was advanced based on the specific characteristics \n of the androgen receptor af-2 region , which demonstrates known selectivity \n toward the fxxlf coactivator motif ( figure 1b ) . the cocrystallization of the ar lbd bound with \n dht in the presence of the fxxlf peptide ( pdb i d 1t7r ) provided the structural basis of the af-2 interaction for \n docking studies . virtual screening and identification of diarylhydrazide \n scaffolds . ( a ) a series of coactivator peptides cocrystallized in \n the af-2 groove was employed ; for illustrative purposes we present \n the fxxlf coactivator motif from pdb entry 1t7r . the af-2 groove is represented in dark \n gray . for clarity reasons , only lys720 and glu897 are shown and dht \n is not illustrated ; ( b ) a 3d pharmacophore \n model was derived containing the common features between ar coactivators \n and the two aromatic features of the fxxlf motif . pharmacophores were \n used to screen vendor compound databases and to guide the docking \n of putative hits into the af-2 site . ( c , d ) two first round \n actives 1 ( mdg173 ) and 2 ( mdg15 ) docked \n poses in the af-2 site , with the surface rendered and only key amino \n acids shown . partial mapping of initial hits to the pharmacophore \n suggested additional virtual screening to identify more potent family \n members . images were generated with molecular operating environment \n ( moe ) and pymol . from the virtual screen , a first series of compounds \n with predicted target affinity was selected from commercially available \n databases ( see experimental section ) and evaluated \n for biological activity using tr - fret and fp techniques . this initial \n screen ( figures 1c , d and 2 ) identified two small molecules , 1 and 2 , both diarylhydrazides , as possible non - lbp ar antagonists . \n non - lbp modulatory activity was evidenced by demonstration of an ic50 in the range of 50100 m in ar tr - fret coactivator \n displacement assay and their inability to displace bound fluorescently \n labeled ligand from the lbp through an fp assay . these first round \n hit molecules map only partially to the screening \n pharmacophore ( figure 1c , d ) . accordingly , an \n optimization round of screening was initiated to explore the utility \n of the scaffold for more effective disruption of ar : coactivator interaction . from these initial data , a simple molecular similarity search was \n performed ( tanimoto coefficient > 70% ) to furnish a new screening \n series of 37 compounds bearing the desired diarylhydrazide scaffold . \n this second round screen identified four small molecules ( figure 2 ) , 3 ( mdg 483 ) , 4 ( mdg \n 292 ) , 5 ( mdg 506 ) , and 6 ( mdg 508 ) , with \n improved activity ( ic50 < 50 m in an ar tr - fret \n assay ) . these ligands were taken forward for additional investigation \n and characterization . the series of diaryl - substituted \n hydrazides identified through the vs process ( figure 2 ) inhibited the recruitment of the fluorescent labeled d11-fxxlf \n coactivator peptide in the presence of an agonist ( dht ) concentration \n equal to ec80 using time - resolved fret assays . d11-fxxlf \n is a peptide developed from random phage display technology that resembles \n the src family of coactivator proteins in its flanking sequence but \n that also has an ar n - terminal interaction domain . thus , it is a biological mimic of the n - terminal and the \n src coactivator interactions with the lbd . a 12-point dose response \n curve was determined for those compounds that inhibited coactivator \n binding in the micromolar range , acting as full ar antagonists , 36 ( figure 3a \n and table 1 ) . the background \n signal , representing diffusion - enhanced fret in the absence of ar , \n was subtracted from the fret value of each compound and from the maximal \n signal , representing fxxlf - bound ar in presence of dht . diarylhydrazides inhibit the ar recruitment \n of a fluorescent - labeled d11-fxxlf peptide but do not displace a potent \n fluorescent ligand from the ar - lbp . ( a and b ) compounds were tested \n in a tr - fret assay across a concentration range from 100 m \n to 45 nm in the presence of a concentration of dht = ec80 in ar - lbd wt ( a ) and ar - lbd t877a ( b ) . error \n bars represent the standard error of the mean ( sem ) for n = 6 values . data was fitted using log antagonist concentration vs \n response ( variable slope ) with graphpad prism 5 ( see experimental section for details ) . ( c ) fluorescence polarization \n data is plotted as percent maximal activity represented by ar - lbd \n and fluorophore complex ( 0% inhibition ) . the minimum control value \n represents free fluorophore ( free f ) in solution ( 100% inhibition ) . \n the tr - fret assay can not differentiate between \n direct coactivator antagonists acting on the lbd surface and classical \n ar antagonists , which also functionally disrupt coactivator recruitment \n by displacing dht from the ligand binding pocket . to characterize \n the nature of the antagonist effect , compounds were tested for their \n ability to displace a potent fluorescent ligand ( fluorophore ) from \n the ar lbp through a fluorescence polarization ( fp ) assay at a single \n point concentration ( 50 m ) , using cyproterone acetate ( cpa ) \n at the same concentration as a reference , a known ar lbp - mediated \n antagonist . all compounds tested showed 0% inhibition of the ar - lbd \n and fluorophore complex , indicating a non - lbp - mediated mechanism of \n ar transactivation inhibition ( figure 3c ) . compound 3 gave an unusually high value of millipolarization \n units ( mp ) , 20% higher than the maximal control ( figure 3c ) . this could be indicative of solubility issues in the assay \n buffer and therefore could generate a false negative result . it is \n known that fp assay outcomes can be influenced by intrinsic fluorescence \n of the test compounds and/or light scattering phenomena due to poor \n solubility and precipitation . to minimize the possibility of such \n false negative or positive reporting , none \n of the compounds tested showed competing autofluorescence in the assay \n conditions or was shown to be a false negative . results are shown \n in the supporting information ( supplementary \n figure 2 ) . to further validate the utility of these ligands \n in pca , on - target binding experiments were also performed using the \n recombinant t877a ar mutant characteristic of advanced \n stage androgen - independent pca . in tr - fret , the compounds demonstrated \n similar activity to that observed in the wild type assays , indicating \n their potential in advanced phases of prostate cancer ( figure 3b ) . activity data are in agreement \n for 4 and 5 in both ar wt and art877a . the \n higher confidence mp values and experimental reproducibility obtained \n for 4 and 5 in coactivator studies were \n used as the basis to advance these compounds to cellular characterization \n and receptor subtype selectivity evaluations . we undertook to profile the selectivity of these compounds for \n ar over other members of the same phylogenetic branch of the steroidal \n nuclear receptor subfamily . compound binding affinities for progesterone \n receptor ( pr ) , glucocorticoid receptor ( gr ) , estrogen receptor \n ( er- ) , and estrogen receptor ( er- ) were determined \n using tr - fret ( table 2 ) . er- \n and estradiol er- complex and do not displace fluorescent - labeled \n coactivator src1 - 4 from dexamethasone gr complex at concentrations \n up to 100 m ( supporting information , supplementary figures 35 ) . compound 5 binds \n pr with comparable affinity to that observed for ar , while 4 demonstrates approximately 2-fold binding selectivity for the ar \n over pr . in functional evaluation we determined that the diarylhydrazide \n compounds are full ar antagonists , with a partial antagonistic profile \n demonstrated in pr , displacing src14 from progesterone the non - lbp nature of \n this interaction was confirmed by an fp assay ( supporting information , supplementary figure 6 ) . compounds were tested at a concentration range from 100 to \n 1 m in the presence of a concentration of progesterone = ec80 . error bars represent the standard error of the mean \n ( sem ) for n = 6 values . data was fitted using log \n antagonist concentration vs response ( variable slope ) with graphpad \n prism 5 ( see experimental section for details ) . \n ic50 values are shown in \n table 2 . to ascertain the translational ( clinical ) \n potential of these ligands , compounds were evaluated in cellular models \n of prostate cancer ( lncap , an androgen - dependent \n cell line and pc-3 , an androgen - independent \n cell line ) and in normal prostatic epithelia cell line pwr-1e . cell viability was assessed after 24 h of incubation \n with the test compounds at three different concentrations ( figure 5 ) . the classical antiandrogen cpa was used as a \n reference , showing a minor effect at 50 m in the androgen independent \n cell line pc-3 . at 50 m 4 reduces cell viability \n to a 5060% , whereas 5 acts consistently across \n the three cell lines , retaining cell viability at around 80% . compounds were tested at 5 10 , 1 10 , and 5 10 m. error bars represent the sem of two independent experiments done \n in triplicate ( n = 6 ) . the diarylhydrazides were evaluated for their effects \n on the ar signaling pathway and on hormone - dependent cellular growth \n of lncap cells . compound 5 was well - tolerated after 5 \n days of treatment at 10 and 20 m concentrations and enabled \n observation of a specific reduction in dht - treated cell count ( figure 6a ) . prostate specific antigen ( psa ) \n is a serine protease normally secreted by the prostate epithelia . \n psa is widely used as a \n marker for pca , as its serum levels are \n increased in this condition . compound 5 was shown to \n reduce dht - induced psa secretion in a dose response fashion \n as quantified by an elisa experiment in lncap cells ( figure 6b ) . it is well - documented that classical antiandrogens \n ( i.e. , those binding within the lbp / competing with endogenous ligands ) \n have partial agonistic properties , which make them less useful in \n the management of advanced prostate cancer . arising from this inherent agonism , in an androgen - deprived lncap \n cell line , antiandrogens such as cpa can actually activate the ar pathway and stimulate cell growth . in direct contrast to the behavior of traditional antagonists , 5 shows no detectable agonist or partial agonist activity \n at tested concentrations , consistent with an alternative mechanism \n to that of the classical antiandrogens . finally , treatment with 5 at 10 m was found to antagonize cpa partial agonist \n activity ( measured as secreted psa levels in the cellular media in \n an elisa experiment ) , suggesting its potential benefit in combination \n therapy for advanced stages of prostate cancer ( figure 6c ) . to further challenge this hypothesis , the compounds also demonstrated similar effects in this alternate \n system , supporting the hypothesis of their functioning as true antiandrogens \n ( supporting information , supplementary \n figure 7 ) . ( a and b ) 5 reduces androgen - stimulated \n cell growth and dht - dependent ar signaling measured as psa levels \n secreted in the cellular media in a dose - dependent fashion . ( c ) 5 at 10 m reduces cpa - induced ar signaling ( in absence \n of androgens ) measured as psa levels secreted in the cellular media \n in a dose - dependent fashion . data are presented as the mean of two \n independent experiments , and bars show sem for n = \n 6 values ( a ) . secreted psa ( ng / ml ) was measured considering the optical \n density at 450 nm minus the optical density at 540 nm and interpolating \n the values from the standard curve . data are presented as mean of \n two independent experiments , and bars show sem for n = 4 values ( b and c ) .", "classical antiandrogen therapy is known \n to have limited beneficial effects in hormone - insensitive pca . alternative \n ar inhibitors are therefore needed in the treatment of pca . in this \n study , we demonstrate the successful implementation of a virtual screening \n approach in the identification of small molecule ar modulators , where \n the structural motif of ar coactivators was included in a 3d pharmacophore . \n we report the discovery , identification , and characterization of a \n novel series of diarylhydrazide non - lbp - binding antiandrogen compounds , \n with demonstrated ability to displace ar coactivators and with established \n potency in ar - dependent prostate cancer cell lines . activity was measured \n with a tr - fret assay and a non - lbp - mediated mechanism of inhibition \n was confirmed by fp assay . these compounds are shown to function without \n any demonstrated intrinsic or partial agonist activity in ar and therefore \n can be classified as true non - lbp antiandrogens . the nature of nr coactivators and the high homology of nr coactivator \n binding sites are such that , to more fully profile the potential utility \n of these ligands , their selectivity was evaluated across members of \n the subclass of steroid receptors , including er- and er- , \n gr , ar , and pr . the selectivity of the diarylhydrazide scaffold \n for the ar was demonstrated through tr - fret evaluation in the estrogen \n and glucocorticoid receptors , where agonist bound receptor recruitment \n of coactivator was unimpaired at screening concentrations up to 100 \n m . we additionally investigated the potential cytotoxicity \n of the diarylhydrazides in three different cell lines , selecting 5 for its favorable cytotoxic profile ( cell viability was \n retained at around 80% in different prostatic cellular models ) . unmodified diarylhydrazide screening hits were also shown to have \n 2-fold selectivity for ar over pr , with partial antagonist activity \n demonstrated for the scaffolds in a pr functional assay , remarkable \n given the high ( > 60% ) homology of these nr family members . futhermore , given the established utility of mifepristone ( a pr \n modulator which also has antiandrogenic activity ) in the treatment \n of castration resistant prostate cancer , the narrower \n selectivity window observed for these ar ligands in pr over the other \n nr s assessed is not a significant concern in the context of \n the therapeutic area under consideration . classical antiandrogens \n can be also distinguished for their different behaviors at a cellular \n level . save for two recent examples , all lbp antiandrogens \n described to date have also intrinsic partial agonist activity , demonstrated by induction of psa in the absence \n of hormone stimulation in lncap cells . in this study , the novel non - lbp \n diarylhydrazide antiandrogen 5 did not induce psa expression \n in absence of hormone stimulation when compared to cpa . in androgen - deprived \n lncap cells , 5 reduces psa expression in combination \n with cpa , antagonizing its partial agonist activity in a dose responsive \n fashion . this result supports the hypothesis of a nonclassical mechanism \n of ar inhibition for these diarylhydrazide ligands and it also demonstrates \n the potential application of these and other non - lbp antiandrogen \n small molecules targeting alternative ar sites in combination with \n existing prostate cancer therapy .", "through application of virtual screening \n methodologies , we present and characterize novel diarylhydrazide scaffolds \n as true antiandrogens displacing ar coactivator interaction \n and having a full antagonistic profile on ar ( both wt and t877a ) , \n partial antagonistic profile for pr , and selectivity for the other \n members of the nr-3 family ( gr , er- , and er- ) . the initial small molecule non - lbp true ar modulators provided by \n this study will be used to further characterize the ar coactivator \n interface , to understand the basis of selectivity , and to further \n guide rational drug design in the search of other novel scaffolds \n directed at this interface .", "black , low volume , 384-well assay plates ( corning , ny , cat . no . 3676 ) \n were used to perform the assay ( total volume 20 l ) , and tr - fret \n signal was measured with pherastar equipment ( bmg labtech ) using a \n lanthascreen optic module ( excitation , 335 nm ; emission , 520 nm channel \n a and 495 nm channel b ) . tr - fret values were calculated at 10 \n flashes per well , using a delay time of 100 s and integration \n time 200 s as recommended by the invitrogen assay guidelines . \n a serial dilution of compounds was first prepared in \n 100 dmso ( sigma - aldrich ) starting from the maximum desired concentration \n to achieve a 12 point range concentration using 96-well polypropylene \n plates ( nalgene nunc , rochester , ny ) . each 100 solution was \n diluted to 2 concentration with tr - fret coregulator buffer a \n ( invitrogen proprietary buffer ) , yielding a final concentration of \n 1% dmso in each well . ten microliters of 2 solution was then \n added to the 384-well plate , following addition of 5 l of 4 \n ar - lbd and 5 l of d11-fxxlf / tb anti - gst antibody in agonist \n mode and 5 l of d11-fxxlf / tb anti - gst antibody / dht ( included \n at a concentration equal to ec80 as determined by running \n the assay in agonist mode first).d11-fxxlf and tb antibody were premixed in \n light protecting vials prior to use . a final concentration of 5 mm \n dtt was used in the assay buffer in order to prevent protein degradation . \n all plates ( agonist and antagonist mode ) were incubated between 2 \n and 4 h at room temperature protected from light prior to tr - fret \n measurement . ic50 values were determined by testing each \n ligand at concentrations ranging from 100 m to 45 nm using \n 2- and 3-fold dilutions to generate a 12 point dose data was fitted using the sigmoidal dose response ( variable \n slope ) available from graphpad prism 5.the z factor for these assays \n was > 0.5 , as calculated by the equation provided by zhang et al . in line with the assay protocol , a \n known agonist , dihydrotestosterone ( dht , cat no . a8380 , sigma ) , and \n a known antagonist , cyproterone acetate ( cat no . a control with no ar - lbd present was \n included to account for diffusion - enhanced fret or ligand - independent \n coactivator recruitment . a negative control with 2 dmso was \n present to account for any solvent vehicle effects . the \n assay was adapted to exclude possible nonspecific aggregation mechanism \n of inhibition by adding very low concentration of detergent triton \n x-100 ( 0.01% ) to the assay buffer following the shoichet review guidelines ( supporting information , supplementary figure 1 ) . p3018 ) was \n used to investigate the binding of the test compound to the lbp site , \n occupied by a high - affinity fluorophore ligand ( fluormone ) . the 100 test compound solutions in dmso were diluted in ar \n green buffer ( invitrogen ) to achieve 2 concentrations and placed \n in a 384-well plate ( corning , cat no . ar - lbd and fluormone ( 2 ) mix were prepared separately \n and then added to each compound dilution to achieve a final concentration \n lbd - fluormone of 50 and 2 nm , respectively . plates were incubated \n protected from light for at least 4 h. controls included a maximum \n mp positive control , which consists of the ar - lbd and fluormone mix \n ( 2 ) , and a minimum mp control , containing only fluormone ( 2 ) . \n a vehicle control was added to account for dmso effect , and a blank \n control containing buffer only . fluorescence polarization was measured \n with pherastar equipment ( bmg labtech ) using an optic module with \n excitation at 485 nm and emission at 530 nm . lncap cells ( androgen - dependent ) , pc-3 \n ( androgen - independent ) , and pwr-1e ( normal prostatic epithelia ) were \n cultured in rpmi-1640 glutamax ( invitrogen ) , f12k ( invitrogen ) , and \n k - sfm media ( invitrogen ) . the first two were supplemented with 10% \n fetal bovine serum ( fbs ) , penicillin ( 100 units / ml ) , and streptomycin \n ( 100 g / ml ) . k - sfm was supplemented with 5 ng / ml epidermal growth \n factor ( egf ) and 0.05 mg / ml bovine pituitary extract ( bpe ) . cells \n were propagated at 1:3 or 1:6 dilutions at 37 c in 5% co2 . for cell \n viability ( end point ) assays lncap , pc-3 , and pwr-1e cells were seeded \n at 2.5 10/ml density in 200 l volume of \n a 96-well plate in triplicate and incubated for 24 h prior testing . \n test compounds were included at different concentrations to achieve \n a final concentration of 0.5% dmso in each well . cell viability was assessed \n after 24 h of treatment using 10% alamarblue reagent ( invitrogen ) \n for each well . cell viability was monitored by the reduction of resazurin , \n a blue , cell - permeable , nontoxic compound , to resorufin , a red and \n highly fluorescent product . viable cells continuously convert resazurin \n to resorufin , increasing the overall color and fluorescence of the \n media surrounding cells . fluorescence intensity can be quantitatively \n determined with a fluorescence microplate reader at excitation / emission \n 544 nm/590 nm ( spectramax gemini ) . for hormone - dependent cell \n proliferation assays in androgen - deprived lncap cells , cells were \n seeded at 2 10 cells / ml in a 24-well plate in triplicate . \n cells were plated in phenol red free rpmi glutamax ( invitrogen ) supplemented \n with 10% charcoal - stripped fbs to deplete endogenous steroids 48 h \n prior to the assay , as described in previous reports . the optimal condition for the treatment was found to be \n 5 days and the concentration of dht included to stimulate the cells \n was 0.1 nm . cells were treated with different concentrations of test \n compounds with or without 0.1 nm dht to achieve a final concentration \n of 0.1% dmso in each well . a control for the vehicle was included \n to ensure that no effect on viability could be detected . media and \n treatments were replaced every second day , after washing the cells \n twice with 1 pbs . supernatants were collected after 5 days for \n evaluation of secreted psa levels , and cell proliferation was assessed \n for the same plate using alamarblue in order to exclude nonspecific \n effects due to toxicity issues . secreted levels \n of prostate specific antigen were evaluated with a commercially available \n kit ( quantikine human kallikrein 3/psa immunoassay , r&d systems ) . \n , 50 l of standards and cell culture samples were added \n to precoated wells containing assay diluent rd1w ( r&d systems ) \n and incubated for 2 h at room temperature . unbound material was washed \n several times and 200 l of horseradish peroxidase ( hrp ) labeled \n psa conjugate antibody was added to each well and further incubated \n for 2 h at room temperature . wells were washed and treated with colored \n substrate ( tetramethylbenzidine ) for an additional 30 min , after which \n 50 l of stop solution ( 2 n sulfuric acid ) was added per well \n and optical density ( 450 nm with correction at 540 nm ) was read with \n a plate reader within 30 min ( versamax ) . a virtual screen was designed to \n select compounds mapping onto the peptide binding surface ( af2 ) of \n the ar receptor , based on an ensemble of documented x - ray crystal \n structures ( pdb i d 1t73 , 1t74 , 1t76 , 1t79 , 1t7f , 1t7 m , 1t7r , and 1t7 t ) . molecular operating environment ( moe ) software was employed to preprocess the proteins and \n to remove the coactivator peptides from the complexes . an initial \n pharmacophore was generated using the moe pharmacophore elucidator \n and considering the most significant features , which involved hydrophobic , \n donor , and acceptor features . a second pharmacophore was developed \n including two additional hydrophobic / aromatic features to represent \n the phe side chains present in the fxxlf coactivator motif ( 1t7r ) , so as to increase \n the selectivity for ar over other families of nuclear receptor . these \n pharmacophore models were then applied for in silico screens of small - molecule \n commercial libraries to identify compounds that resemble the active \n principle of the starting peptides . a number of vendor databases \n were selected for screening of ligands , including amsterdam ( 5389 compounds ) , peakdale ( 8188 ) , asinex platinum collection \n ( 75 258 ) , specs ( 175 800 ) , \n maybridge ( 56 870 ) , and zinc ( 4.6 million ) compounds . a bayesian analysis was performed on the \n peptide structures to estimate parameters of an underlying distribution \n based on the observed distribution . the above databases were then \n filtered for those compounds with properties similar to the peptides , \n thus focusing the search on the ar ligand chemical space . all molecules were standardized for stereochemistry \n and charges and ionized at a ph of 7.4 and all calculable tautomers \n were enumerated . at this stage the conformational flexibility of the \n screening compounds was explored using the omega software ( openeye scientific package ) . a maximum of 50 \n conformations were generated for each molecule in the data set . the virtual molecules were overlaid \n on and compared to the generated pharmacophore of the active ligands , \n and those molecules that compared favorably were advanced for additional \n virtual screening and scoring . the fast rigid exhaustive docking ( fred ) software as implemented in openeye scientific s \n package was used to exhaustively examine all possible poses within \n the protein site , filtering for shape complementarity and scoring . \n the smaller databases ( amsterdam and \n peakdale ) were screened on all 13 crystal \n structures and only ligands scoring well on more than one crystal \n structure were considered . the larger databases specs , asinex , maybridge , and zinc were screened on the 1t7r crystal structure . a structural similarity search was \n conducted on 1 and 2 using a tanimoto coefficient \n of > 70% on the specs compound database . thirty - seven compounds were purchased and four small molecules were \n selected for optimization and characterization studies based on their \n improved on - target activity determined by tr - fret . all screening compounds \n described in this work were purchased as commercial samples from specs \n nv . compound purity in all instances \n was greater than 95% as determined by lcms and nmr ." ]

[ "during the last years tyrosine kinase inhibitors ( tkis ) have changed the natural history of metastatic non - small - cell lung cancer ( nsclc ) harboring epidermal growth factor receptor ( egfr ) mutations . eight important studies were conducted to evaluate the efficacy and tolerability of tkis on advanced nsclc in comparison with standard platinum - based chemotherapy . not surprisingly , the use of tkis was correlated with a higher response rate , a longer progression - free survival and a better quality of life in patients with advanced nsclc activating egfr mutation . the iressa pan - asia study ( ipass ) , which enrolled 1,217 patients , was the largest trial in which patients were randomized to receive gefitinib or standard chemotherapy , and in the group of tkis therapy the primary endpoints were reached obtaining a statistically significantly higher response rate , a longer progression - free survival and better symptom control . similar results were reported by first - signal and by west japan thoracic oncology group ( wjtog 3405 ) studies . the north - east japan study group ( nej002 ) trial was stopped early because gefitinib showed a significantly higher progression - free survival in comparison with standard chemotherapy in patients with advanced lung adenocarcinoma activating egfr mutation . impressive results were also reported with the use of other tkis such as erlotinib or afatinib versus chemotherapy in patients carrying the same egfr mutations . better responses were observed in patients with mutations in exons 1821 of the tyrosine kinase domain of egfr . however , egfr gene mutations were also identified in small - cell lung cancer ( sclc ) [ 3 , 4 ] and in large - cell neuroendocrine carcinoma ( lcnec ) of the lung . lcnec is a high - grade carcinoma ( > 10 mitoses/2 mm ) belonging to the neuroendocrine tumors of the lung . it represents about 3% of all pulmonary malignancies and is characterized by neuroendocrine cytologic features ( formation of rosettes , trabeculae and perilobular palisading pattern ) and markers ( neuron - specific enolase , cd56 , synaptophysin , chromogranin and leu7 ) . in fact , the cytologic and biologic features of lcnec are different from those of large - cell carcinoma . the molecular alterations that are commonly found in lcnec are p53 , bcl-2 overexpression and rb mutation . to our knowledge , few cases of lcnec with egfr gene mutation have been described up to now , and only one case was treated with gefitinib , with a good response [ 7 , 8 ] .", "a 47-year - old caucasian woman with no family history of neoplastic diseases and no comorbidities was examined by a general practitioner after the appearance of back pain unresponsive to usual non - steroidal anti - inflammatory drugs . standard chest x - ray showed a left lung perihilar lesion , probably suggesting pneumonia . as a consequence , thus , after 2 weeks , chest x - ray was repeated and showed persistence and stability of the left lung lesion . about 1 month later , the patient came for the first time to our attention for appearance of vomiting , dyspnea , fatigue and abdominal pain ( visual analog scale 7 ) . abdominal physical examination revealed a painful hepatomegaly . she underwent a total body computed tomography ( ct ) scan that showed multiple focal liver lesions , solid left lung tissue and multiple secondary brain lesions ( two left frontal cerebral lesions , one right parietal lesion and two cerebellar lesions ) ( fig . 1 ) . as a result , a liver biopsy was performed . since all investigated tumor markers ( carcinoembryonic antigen , carbohydrate antigen 19 - 9 , carbohydrate antigen 125 , neuron - specific enolase , glycoprotein hormones alpha polypeptide ) were increased , it was not possible to identify the primary site of localization of the tumor and to reach a definitive diagnosis . given the rapidly progressive impairment of her clinical conditions and performance status , we administered an empirically not targeted chemotherapy with gemcitabine 1,000 mg / m die 1 and oxaliplatin 100 mg / m die 2 q 2 weeks although we did not yet have definitive histopathological results . although the sample was poor , the diagnosis was evocative of lung adenocarcinoma ( ttf-1 positive , cytokeratin 7 positive ) . however , since a further deterioration of her clinical condition was observed , a biopsy was repeated in order to have an additional sample for molecular analysis . therefore , we started tki therapy and gefitinib was administered at 250 mg p.o . once a day . the patient reports a good quality of life and no relevant side effects ( skin toxicity grade 1 ) have been registered . a restaging total body ct scan showed a significant improvement of disease , with a partial response > 50% . particularly , the brain ct scan showed a significantly reduced volume of the lesions ( valuable only 2/5 lesions ) and complete resolution of edema ( fig .", "lcnec is a rare tumor which is usually treated with cisplatin - based chemotherapy as sclc as it shares many characteristics with it . usually , the administration of tkis is considered only for advanced lung adenocarcinoma because the most important studies demonstrating higher activity of tki , in comparison with standard platinum - based chemotherapy , were conducted in patients affected by nsclc with egfr mutations . some features are associated with a higher possibility that egfr mutations are present in nsclc . the research of egfr mutations is not performed routinely on lcnec since this mutation is not commonly present in this subtype . the prognosis of this tumor with standard chemotherapy is poor , thus we need to evaluate alternative diagnostic strategies such as the investigation of egfr mutational status in order to explore new effective treatments . we report the case of a patient affected by lcnec carrying all of the predictive characteristics associated with egfr mutation ( caucasian , female , never - smoker ) . when the patient came to our attention she was very symptomatic , so in an attempt to reach a better and faster response , we decided to test egfr mutational status . strikingly , the mutation was present , and we were allowed to administered gefitinib , obtaining an impressive and objective response . therefore , we suggest the research of egfr mutations also in patients with lcnec to offer them one more therapeutic option .", "" ]

[ "tetralogy of fallot ( tof ) accounts for approximately 5% to 7% of all cases of congenital heart disease . complications related to surgical repair of tof are clinically relevant and include pulmonary regurgitation , residual obstruction of the right ventricular ( rv ) outflow tract , rv systolic dysfunction , and arrhythmias . among these , residual rv outflow tract obstruction with accompanying pulmonary regurgitation is the most frequently reported sequela after initial surgical correction of tof . therefore , a systolic murmur that is audible over the base of the heart is frequently interpreted as a residual rv outflow tract lesion . however , this is not always true . we herein report a rare case of subaortic stenosis in association with a previous tof surgical repair that was not considered to be a differential diagnosis . discrete subaortic stenosis is generally thought of as a disease of children or young adults ( < 25 years of age ) and is thus rarely reported in subjects older than 25 years , particularly in association with tof .", "a 29-year - old woman presented for a routine check - up of surgically corrected congenital heart disease . at 7 years of age , she had undergone surgical repair of tof with excision of the infundibular stenosis and closure of a 10-mm ventricular septal defect . she had been asymptomatic until her first decade of life , and only a 5-year clinical follow - up had been performed after the operation . approximately 6 months prior to presentation , she began to feel exertional dyspnea that was progressively aggravated . on physical examination , she was alert and well - oriented . her blood pressure was 100/60 mmhg , pulse 60 beats / min , respiration rate 18/min , and body temperature 36.6. the left ventricular ( lv ) impulse was almost normal in location . on auscultation , a grade 3 of 6 mid- to late - systolic murmur was audible over the base of the heart and heard clearly on both sides of the sternum . routine blood examinations , including a complete blood count , chemistry panel , coagulation panel , and c - reactive protein level , were all within normal ranges . transthoracic and transesophageal echocardiograms both demonstrated severe subaortic stenosis due to a subvalvular membrane , on which a mobile mass - like structure that was suspicious of a vegetation was noted ( fig . there was a mild degree of tricuspid regurgitation , but no clinically significant obstruction of the rv outflow tract . the maximum velocity measured across the tricuspid valve was 2.8 m / sec , and thus the maximal pressure gradient was estimated to be 31.4 mmhg using the simplified bernoulli equation . due to the up to 20 mmhg pressure gradient across the pulmonary valve her rv was not dilated , and its systolic function was good . due to the suspicion of infective endocarditis despite the absence of clinical evidence , blood cultures were performed three times ; however , no pathogen was cultured . the patient underwent resection of the subaortic membrane and myomectomy of the lv outflow tract along with aortic valve commissurotomy . the membrane , with the exception of the noncoronary cusp area , was located beneath the aortic annulus ( i.e. , inverted u shape ) . the mobile mass - like structure described on transesophageal echocardiography was proven to be loose tissue attached to the subaortic membrane .", "although subaortic stenosis is the second most - common form of lv outflow tract obstruction , it is predominantly found in children or adolescents [ 4 - 8 ] . subaortic stenosis is strongly associated with other congenital heart defects ; however , subaortic stenosis in association with tof , especially in women , is extremely rare . according to a medline database review of the last 30 years furthermore , all were diagnosed in childhood or adolescence ( < 20 years of age ) with two exceptions . the present case is peculiar in that subaortic stenosis manifested more than 20 years after the initial tof surgical correction . although we could not definitively say that the subaortic stenosis in the present case was acquired , we consider it an acquired or progressive form given the long interval ( > 20 years ) before echocardiographic confirmation . thomas and foster suggested that acquired subaortic stenosis can develop gradually due to hemodynamic disturbances derived from coexisting lesions or after surgical correction . moreover , the mass - like mobile structure observed on transesophageal echocardiography could be regarded as a degenerative byproduct of flow convergence induced by the subaortic membrane , further supporting the concept of an acquired or progressive form of the disease . discrete subaortic stenosis is well - known to be linked to important complications , such as bacterial endocarditis and significant aortic regurgitation . flow turbulence distal to the obstructing subaortic membrane plays a crucial role in progressive damage to the aortic valve with resultant thickening of its leaflets and regurgitation . the secondary jet lesion also predisposes the patient to bacterial endocarditis . in this context , in particular , patients with surgically corrected tof tend to be misinterpreted as having only residual rv outflow obstruction without consideration of the lv outflow tract lesion . transthoracic and transesophageal echocardiography can assist differentiation of the rv from the lv outflow tract obstruction . therefore , periodic performance of echocardiography should be kept in mind during follow - up of patients with surgically corrected tof ." ]

[ "pregnancy - associated plasma protein - a ( papp - a ) is a metzincin metalloproteinase primarily produced by the placental syncytiotrophoblast during pregnancy . papp - a is also synthesized by fibroblasts , osteoblasts , vascular smooth muscle cells ( vsmcs ) , and endothelial cells ( ecs ) . in vitro , papp - a functions to cleave insulin - like growth factor - binding protein 4 ( igfbp-4 ) , an inhibitory igfbp , consequently increasing igf bioavailability for receptor activation [ 14 ] . in vivo , several studies have shown a similar role for papp - a in modulating site- and event - specific igf signaling during injury repair processes . recent studies have indicated that papp - a is a novel biomarker for plaque instability and inflammation useful in early diagnosis , risk stratification , and prognostic prediction in patients with acute coronary syndrome ( acs ) [ 5 , 6 ] . papp - a was found abundantly expressed in ruptured and eroded human atherosclerotic plaques , colocalized with activated smooth muscle cells and macrophages [ 7 , 8 ] . since plaque - derived papp - a is being considered as a new biomarker that may potentially play a role in the development of atherosclerotic lesions [ 9 , 10 ] . a better understanding of its cellular source and regulation is important to the future development and implementation of therapeutics utilizing this biomarker . previous studies have indicated that proinflammatory cytokines , interleukin- ( il- ) 1 , and tumor necrosis factor- ( tnf- ) were potent stimulators of papp - a expression in cultured human fibroblasts , osteoblasts , coronary artery smooth muscle cells , and endothelial cells ( ecs ) [ 9 , 11 ] . despite these significant findings , little is known about the effect of c - reactive protein ( crp ) and tnf- on papp - a expression in human peripheral blood monocytes ( pbmcs ) . the current study investigates the ability of crp and tnf- to induce papp - a expression in the pbmcs of healthy volunteers . furthermore , inhibitor experiments have been designed to explore the underlying intracellular signaling pathways involved in papp - a expression . the focus of these studies is nuclear factor- ( nf- ) b pathways , a major pathway associated with cytokine stimulation in various cell types .", "peripheral blood was collected from the forearm vein of healthy volunteers enrolled in the current study . for each experiment , 30 ml samples were freshly collected from 6 healthy subjects for use in pbmc preparations . each of these 6 subjects provided 4 blood donations over the course of the study . the study was approved by the ethics committee of the beijing friendship hospital and conforms to the principles outlined in the declaration of helsinki . human pbmcs were isolated from 30 ml fresh blood samples obtained from healthy volunteers by ficoll - paque ( amersham bioscience , uppsala , sweden ) centrifugation . resultant cells were washed 3 times with pbs and subsequently resuspended in rpmi 1640 ( gibcobrl , grand island , ny , usa ) supplemented with 10% fetal calf serum ( gibcobrl , grand island , ny , usa ) , penicillin ( 100 u / ml ) , and streptomycin ( 100 g / ml ) . cells were then cultured for 24 hours in plastic dishes at 37c in a humidified atmosphere of 5% co2 . upon observation of subconfluent growth , the medium was replaced with fresh medium . all nonadherent lymphocytes were discarded during the medium change , reserving only healthy adherent monocytes . after 24 hours , trypan blue exclusion indicated that 95% of cultured pbmcs were living . the pharmacological agents recombinant human tnf- ( perprotech , rocky hill , ct , usa ) , crp , actinomycin d , and bay11 - 7082 ( sigma chemicals , deisenhofen , germany ) were dissolved into solution according to the manufacturer 's instructions . resultant solutions were added to cultured pmbcs at defined time intervals ( 2 , 8 , 16 , 24 hours ) and concentrations ( crp : 5 , 10 , or 20 mg / l ; tnf- : 25 , 50 , or 100 ng / ml ; bay11 - 7082 : 20 m ) in the absence or presence of actinomycin d ( 1 g / ml ) , as further described in the following sections . individual controls were determined for each experiment , as described in figures 14 . briefly , total rna was isolated using trizol ( invitrogen , calsbad , ca , usa ) according to the manufacturer 's instructions . reverse transcription - generating cdna was performed using the superscript iii first - strand synthesis system ( invitrogen , calsbad , ca , usa ) . papp - a cdna was amplified using forward ( 5-ata tct cac gtg acc gag ga-3 ) and reverse ( 5-aga tga tgg tgc tgg aag tc-3 ) primers , which produce a 529 bp product . amplification was performed at 94c for 2 min for preheating , followed by 30 cycles of 94c for 45 s , 65c for 45 s , 72c for 60 s , and a final extension of 72c for 10 min . -actin was amplified using forward ( 5-gca tgg agt cct gtg gca t-3 ) and reverse ( 5-cta gaa gca ttt gcg gtg g-3 ) primers , which produce a 320 bp product . amplification was performed at 94c for 2 min for preheating , followed by 28 cycles of 94c for 30 s , 60c for 30 s , 72c for 30 s , and a final extension of 72c for 20 min . the resulting bands were photographed under ultraviolet light and analyzed using a gel imaging system ( gel doc2000 , bio - rad , hercules , ca , usa ) . the relative intensity of bands of interest was expressed as the ratio to -actin mrna bands . for cell lysates , cells were washed twice with ice - cold pbs and lysed in ripa buffer . total protein was quantified using the bca assay ( pierce , rockford , il , usa ) . equal amounts of protein ( 40 g ) were separated by sds - page in 14% tris - glycine gels ( tefco , tokyo , japan ) . after electrophoresis , the proteins were blotted onto a nitrocellulose membrane and blocked with 5% skim milk powder diluted in tris - buffered saline ( tbs ) with 0.05% tween 20 . rabbit polyclonal antibodies against human papp - a ( 1 : 1000 , abcam systems , cambridge , usa ) were used as the primary antibody . membranes were incubated with diluted antibody preparations overnight at 4c . after washing the next day , membranes were incubated with horseradish peroxidase- ( hrp- ) conjugated affinity - purified goat anti - rabbit igg antibody ( 1 : 3000 , santa cruz . papp - a levels were determined using the ultrasensitive elisa kit ( diagnostic systems laboratories , webster , tx ) . the assay was calibrated using recombinant papp - a calibrated against the world health organization 's international reference preparation 78/610 for pregnancy - associated proteins , by definition containing a papp - a concentration of 100 minimum sensitivity was 0.24 miu / l with intra- and interassay coefficients of variation of 4.7% and 4.2% , respectively . all statistical analyses were carried out using the spss statistical package , version 13.0 ( spss inc . , p - values less than 0.05 were considered statistically significant ( p < 0.05 ) .", "the time course of papp - a mrna expression in pbmcs under basal and cytokine - stimulated conditions is presented in figure 1(a ) . little papp - a expression was observed in pbmc cultures under basal conditions after 24 hours . treatment with crp ( 20 mg / l ) or tnf- ( 100 ng / ml ) significantly increased paap - a mrna expression at all time points ( 2 , 8 , 16 , 24 hours ) . papp - a mrna levels increased 2 hours after stimulation with crp ( 20 mg / l ) and remained elevated by approximately 3.7-fold up to 24 hours . pappp - a mrna expression , however , rapidly increased and peaked at approximately 6.8-fold 2 hours after tnf- ( 100 ng / ml ) stimulation . a subsequent decrease was then observed , though levels remained elevated at approximately 4.5-fold up to 24 hours . maximal papp - a protein expression in pbmcs and concentrations in culture supernatants were achieved with crp stimulation by 24 hours and tnf- stimulation by 8 hours ( figures 1(b ) and 1(c ) ) , reflecting the changes in papp - a mrna expression . as shown in figure 2 , dose - response experiments confirmed crp or tnf- treatment elicited dose - dependent increases in papp - a mrna expression , protein expression in pbmcs , and secretion in the supernatant after 24 hours . crp showed half - maximal effectiveness at approximately 5 mg / l , with maximal effectiveness at approximately 20 mg / l ( figure 2 ) . tnf- showed half - maximal effectiveness at approximately 25 ng / ml , with maximal effectiveness at approximately 100 ng / ml . the dependence of papp - a expression on mrna synthesis was explored in the following three experiments . figure 3 showed that the effects of these proinflammatory cytokines appeared to be at the level of transcription , as the dna - directed rna polymerase inhibitor , actinomycin d , completely prevented crp or tnf- induction of papp - a mrna expression , protein expression , and concentrations in culture supernatants . these results showed that crp or tnf- was responsible for new protein synthesis of the papp - a protein . furthermore , papp - a protein was actively secreted into the supernatant . as indicated in our previous experiments , treatment of human pbmcs with crp ( 20 mg / l ) or tnf- ( 100 ng / ml ) significantly increased papp - a mrna expression , protein expression , and concentrations in culture supernatants . to confirm the role of nfb activation , bay11 - 7082 , which inhibits inducible phosphorylation of ib , was shown to effectively inhibited crp and tnf--stimulated papp - a expression ( figures 4(a ) , 4(b ) , and 4(c ) ) . it is well known that the nfb pathway is the critical mediator of prooxidant stimuli , such as inflammatory cytokines . the basic mechanism by which nfb is activated is through phosphorylation of intrinsic inhibitors , with the ib , subsequently freeing nfb to translocate into the nucleus where it regulates gene expression .", "novel markers of coronary artery disease progression have been confirmed in recent years , with circulating levels of papp - a standing out as one of the most prominent indicators of this profile . the current study indicates that papp - a expression in human pbmcs may be regulated by crp and tnf- through the nf-b pathway , a mechanism that may play a critical role in increases in serum papp - a levels during acute coronary syndrome ( acs ) . these findings are consistent with previous reports indicating that papp - a is a marker of atheromatous plaque instability as well as extent and prognosis of cardiovascular disease [ 1315 ] . serum papp - a levels increase in patients with acs , indicating that papp - a may also be a marker of adverse events [ 1618 ] . moreover , in chronic stable angina ( csa ) patients , papp - a is an independent predictor for the extent of vessel stenosis , where it has been shown to correlate with the presence of vulnerable coronary artery stenosis [ 19 , 20 ] . thus , papp - a levels have demonstrated a firm association with angiographic plaque complexity in csa patients . the stimulation of papp - a expression by tnf- has been observed previously in human fibroblasts , osteoblasts , vsmcs , and ecs [ 4 , 11 , 22 ] . using specific monoclonal antibodies , bayes - genis et al . reported that papp - a was abundantly expressed in both eroded and ruptured plaques , but was only minimally expressed in stable plaques . moreover , in plaques with large lipid cores and cap rupture , staining for papp - a occurred mostly in the inflammatory shoulder region , in areas surrounding the lipid core , and in areas with localized cd68-positive cells . thus , papp - a levels have been associated with inflammation in regions of atherosclerotic plaques , potentially contributing to progression and poor outcomes in patients . most evidence of monocyte involvement of papp - a expression has been completed through analysis of atherosclerotic plaques , where monocytes are the predominant leukocyte contributing to the development , progression , and instability of atherosclerotic lesions , they often contain high levels of papp - a . because samples of plaques may contain a mixture of leukocytes and circulating compounds , these tests have left the source of papp - a observed in these monocytes a point of debate among researchers . double immunofluorescence confocal microscopy ( icm ) has been used to characterize cell types expressing papp - a , suggesting that monocytes are the primary source of papp - a in plaques and the target cells for cytokines . it has also been speculated that papp - a may be produced by activated monocytes or macrophage cells in unstable plaques and released into the extracellular matrix and circulation . conversely , conover et al . suggested that in vivo macrophages actually failed to produce papp - a , but instead internalized the compound through their membranes , thus accounting for the accumulation of circulating papp - a produced by other sources in macrophage cells associated with plaques . the current study , however , indicates that the specific mrna expression associated with papp - a production increases in macrophages in vitro , resulting in expression and supernatant secretion of papp - a . these findings indicate that papp - a production occurs in macrophages rather than being internalized through the membrane , as suggested by conover et al . further in vivo studies will be required to assess the affect of circulating papp - a levels on macrophage papp - a expression and secretion , which may account for the discrepancies between these two studies . cumulatively , the findings of the current study indicate that macrophages in cultured human pbmcs can synthesize and secrete papp - a . furthermore , crp and tnf- were indicated to be potent stimulators of papp - a gene expression and protein secretion in human pbmcs , suggesting a link between the increase in local inflammatory cytokine production and papp - a during acs . while further studies of the in vivo effects in acs patients will be required to confirm these results , these findings suggest that serum papp - a , hscrp , and tnf- levels may be significantly higher in acs patients than in patients with stable angina pectoris and that increasing papp - a mrna levels in patients with acs may also have a positive association with serum hscrp and tnf- mrna expression . exploration of these effects is a topic being explored in our current research based on the initial positive findings of the current research . furthermore , the time course of stimulation by crp and tnf- differed , with papp - a expression increasing 2 hours after stimulation with crp , peaking at approximately 3.7-fold by 24 hours , whereas a rapid increase to approximately 6.8-fold was seen in only 2 hours with tnf- stimulation . the current study provides significant evidence for papp - a production in pmbcs and stimulation by the cytokines crp and tnf- ; however , further in vivo studies will be required to verify these findings and assess the effects of circulating papp - a on pmbc papp - a production . actinomycin d was observed to complete block the induction of papp - a mrna expression by crp and tnf- , indicating potential regulation at the level of transcription . the rapid increase in papp - a mrna levels after treatment with these cytokines further confirms the hypothesis of transcriptional regulation . as expected , increases in papp - a protein expression and secretion into the supernatants were paralleled by increases in gene expression . based on this observation , it is possible that the biological consequence of crp- and tnf--induced papp - a expression in human pbmcs was enhanced igf - i bioactivity mediated by papp - a proteolysis of igfbp-4 , thus contributing to the progression of both coronary atherosclerosis and restenosis . it is well known that the free fraction of circulating and locally synthesized igf - i stimulates vsmc proliferation , migration , and extracellular matrix synthesis . in macrophages , igf - i also promotes excess ldl cholesterol uptake , production of proinflammatory cytokines , and chemotaxis [ 26 , 27 ] . crp and tnf- were also identified as potential regulators of papp - a expression , operating through a mechanism involving the activation of the nfb system in pbmcs of healthy volunteers . nfb is a ubiquitous transcription factor that is activated by inflammatory cytokines , infection , oxidative stress , and shear stress . ritchie reported that nfb was activated in peripheral monocytes using an electromobility shift assay in patients suffering from unstable angina . the nfb family of transcription factors plays a critical role in coordinating and regulating the expression of a wide variety of inflammatory genes that have been linked to the pathologies of acs . some studies have even demonstrated that crp induces nfb activity in various cell types , including peripheral monocytes , saphenous vein endothelial cells , and human aortic endothelial cells [ 23 , 33 , 34 ] . moreover , resch et al . reported that tnf- induced ib degradation and papp - a - regulated expression in human fibroblasts by tnf- was mediated by nfb activation . the current study also demonstrated that bay11 - 7082 was a potent inhibitor of both crp- and tnf--stimulated papp - a expression in human pbmcs . the view of nfb as a transcription factor for papp - a gene expression , however , will require further study to identify the promoter region and validate these findings . the current study provides a variety of evidence to support the expression on papp - a by leukocytes in pmbcs , including increased papp - a - associated mrna expression , papp - a expression , and papp - a secretion into the supernatant of fresh in vitro samples collected from healthy subjects . furthermore , the cytokines crp and tnf- were shown to stimulate papp - a expression in these cells . based on these findings and the previously observed association between pmbcs and acs , it is likely that activated monocytes or macrophages in pmbcs surrounding developing artherosclerotic plaques may secrete proinflammatory cytokines , thus stimulating the expression and secretion of papp - a . further studies will , however , be required to assess the effects of elevated papp - a concentrations on autocrine and paracrine mechanisms for the exacerbation of atherosclerosis procession and plaque rupture through igfbp-4 cleavage and enhanced local igf - i bioavailability . the current results , however , provide the fundamental mechanistic groundwork for further understanding of the entire mechanism associated with cytokine regulation of papp - a expression and igf bioavailability . this understanding may lead to the future development of novel therapeutic targets for the treatment of acs ." ]

[ "there is an increasing interest in the study of the consultation process and patients ' satisfaction with it . the core activity in primary care is the consultation irrespective of whether patients consult for cure , services , counseling , prevention , or care . a widely accepted model views the consultation as a dialogue involving elements of negotiation to create a common reality to which agenda setting is paramount . in the medical consultation the doctor and patient meet on common grounds with tolerance for each other 's rights . this consultation by necessity requires a doctor who is expected to possess the requisite knowledge which will be useful in solving the problems the patient presents with the assumption that the doctor will act in the best interest of the patient . guided by rules of professional conduct , objectivity , and being emotionally detached the doctor is guaranteed the right to examine the patient physically and to enquire into intimate areas of the patient 's physical and emotional life . during the consultation , the reason for attendance is defined and an appropriate action is chosen . this process aims at achieving a shared understanding , involving the patient in management and using time and resources appropriately . physicians have been noted to have fixed ideas about what is best for a patient , and this inflexibility leaves little room for negotiation . quite often animosity is expressed when the patient attempts to negotiate . however , as demonstrated by a study in the netherlands , interindividual and intra - individual variability does occur among physicians , who were noted to adjust their styles according to the situation . this study sought to identify the factors contributing to patients ' satisfaction , shed more light on the burden of patients ' dissatisfaction with the consultation in our environment , and help devise strategies for practicing physicians to strive for an improvement in the overall patient care in our cultural context . the findings would provide some of the information needed to further fill the knowledge gap about our patient needs in our environment and highlight the need to teach the consultation process at both undergraduate and postgraduate medical training . this study comes from a background where patient awareness of their opportunities in the patient doctor encounter is still in its early days , and thus this study brings to the literature a unique perspective of the patients ' views from this environment .", "this study was conducted in the general outpatient clinic ( gopc ) of the university of calabar teaching hospital , ( ucth ) calabar . the university of calabar teaching hospital is a tertiary hospital located within calabar metropolis , which lies along latitude 4 , 58 north of the equator and longitude 820 east of the greenwich meridian . margaret 's annex , maternity annex , the permanent site , and the comprehensive health centre ( chc ) okoyong . the general outpatient clinic ( gopc ) is situated at the permanent site and has fourteen outpatient consulting rooms in which about 1014 doctors ( family physicians or resident doctors in family medicine ) consult from 8 am4 pm on a daily basis . three consulting rooms are for consultants , three for doctors dedicated to the hiv clinic , one for consultancy patients , one for patients who are staff , and six consulting rooms are for outpatient consultation by other doctors . the department also had 16 nurses , 6 records ' personnel , 5 orderlies , 3 counselors , and 5 administrative staff who usually assisted the doctors during the consultation . all adults between the ages of 18 and 65 years who consented to participate in the study were recruited . informed consent was obtained from the patients before they were given a questionnaire to complete . the average attendance of patients at the ucth gopc in the three months preceding the study was 74 patients per day . therefore , the number of patients estimated to attend the clinic was 74 22 = 1628 . from the calculated sample size of 430 subjects , a sampling interval of 4 was used to systematically select subjects who were recruited to participate in the study . the patients ' attendance register for each day was used as the sampling frame from which patients were selected . the first subject was chosen randomly from this sampling frame , and subsequently every fourth patient was selected and invited to participate . if a selected subject did not meet the inclusion criteria or refused to participate in the study , the next patient was approached until the recommended sample size was recruited . a self - administered , pretested questionnaire adapted from the general practice assessment questionnaire the general practice assessment questionnaire was developed in the united kingdom and used to study certain components of the consultation . the questionnaire consisted of 21 questions divided into five sections that investigated the proportion of patients that are satisfied with their patient - doctor encounter , which patient - factors are associated with patients ' satisfaction or lack of satisfaction with the consultation . some of the questions had options from which the patient selected the response while others made room for a narrative response . prior to the commencement of the consultation , all the patients waiting to be consulted were addressed on the possibility of being approached to join an ongoing study . selected patients on leaving the consulting rooms were approached by the trained assistants and requested to complete the questionnaire . data generated in the study was analyzed using the epi info software for analyzing medical data from the centre for disease control , atlanta , georgia , usa . privacy of the patients was maintained during the study , and all information provided by the patients was treated with utmost confidentiality . patients ' consents were sought and formally obtained after a detailed explanation of the intention of the author concerning the research findings . ethical approval for this study was sought and received from the ethical committee of the university of calabar teaching hospital .", "the age distribution of the respondents varied with the highest proportion being young adults aged 2640 years ( 44% ) , adolescents aged 1825 years ( 34% ) , middle - aged persons aged 4160 years ( 18% ) , and elderly patients aged 60 + 9 ( 4% ) , table 1 . two hundred and fifty - five ( 59.3% ) were satisfied with their patient - doctor encounter . the average age of the respondents was 29 years while the average age of all the patients who presented to the hospital during the study period was 31 years . the sex ratio was almost equal with males accounting for 201 ( 47% ) and females 229 ( 53% ) of the respondents ( table 1 ) . there was a wide variation among the occupational characteristics with patients who had any form of paid employment accounting for 27% , students 32% , retired persons 5% , unemployed 9% , housewives 7% , and others 22% ( table 1 ) . among the others were artisans , self - employed businessmen , and farmers . sex and occupational distribution of the respondents were shown to be similar to those of all the patients who presented in the clinic during the study period . majority of the patients 230 ( 53% ) felt the time they spent with the physician was adequate or very adequate ( table 2 ) . only 26 ( 6% ) respondents assessed the time they spent with the physician as inadequate ( table 2 ) . two hundred and twenty - five ( 52% ) respondents felt they understood the illness much more than when they came to visit the doctor ( table 2 ) . seventy - eight percent of the patients who participated in the study and perceived that the encounter had made it possible to cope with the illness were satisfied with their encounter ( table 2 ) . a good majority of the patients perceived that their ability to cope with the illness after the visit influenced the patients ' satisfaction with the encounter ( p < 0.001 ) . three hundred and fifty ( 81% ) of patients who found an improvement in their ability to maintain their health were satisfied ( table 2 ) with their encounter ( p < 0.001 ) . \n table 3 shows that the frequency of visits did not statistically influence the patients ' satisfaction with the consultation ( p > 0.25 ) . the patient 's assessment of time spent in the consultation was shown to have a statistically significant influence on the patients satisfaction with the consultation ( p < 0.001 ) . this table also shows that the patients preference for a particular physician did not statistically influence the patients satisfaction with the consultation ( p > 0.05 ) .", "none of the sociodemographic variables studied were found to have any statistically significant relationship with a patient satisfaction in a consultation . this study could not demonstrate any statistical significance between a patient age and their satisfaction with the consultation . this agrees with some studies which demonstrated similar findings [ 8 , 9 ] but differs from other studies which demonstrated that patients ' satisfaction rates usually improve with advancing age [ 10 , 11 ] . the elderly patients included in this study were only 18 , and perhaps with a larger elderly population the study could have demonstrated an age - related effect on satisfaction in the consultation . many elderly patients did not agree to complete their questionnaires , and this was probably due to the influence of the accompanying persons who often insisted they had to return to work as soon as possible . the living arrangements in our society possibly make us share similar illness perceptions of what is good or bad accompanied by a shared cultural understanding of wellness or illness . this study could not demonstrate any statistically significant influence of a patient 's sex on his / her satisfaction with a medical encounter ( table 1 ) . the patients ' frequency of visits to the gopc was not found to statistically influence the patient 's satisfaction with the consultation ( table 3 ) . it was believed that the higher the number of visits , the higher the level of dissatisfaction because this was thought to be related to the higher likelihood of social factors not being addressed in these frequent users of the hospital services . . patients often request to see particular doctors , but this was not shown to influence their satisfaction in this study ( table 3 ) and is supported by a study among israeli patients . this finding differs from other studies which have shown that continuity and being seen by a particular doctor improve concordance and satisfaction [ 10 , 13 , 14 ] . the difference in this study may be accounted for by the fact that the patients were in a teaching hospital and many were usually referred to other clinics when the need arose . this may explain the fact that 95% of patients in this study did not insist on seeing a particular doctor as many patients often see the clinic as a transit route to other specialist clinics . only about 32% of patients had been to the clinic on at least three previous visits with the majority ( 44% ) having attended just 1 - 2 times or with no previous visits ( 25% ) in the last 12 months . it is possible they had not cumulatively spent enough time with the doctors to form an opinion . there is also the practice of doctors changing rooms , duties , and postings in between patients ' visits . this makes patients wary of requesting for a particular doctor who may not be on duty . usually in the study area clinic , patients are not given the choice of selecting a doctor and may be rebuked by the nurses who do the sorting if they request for a particular doctor . also patients probably did not request for a particular doctor because they did not know the doctors , were not aware if a particular doctor was on duty , or how long they needed to wait to see a preferred doctor . the use of time in the consultation has been shown to be crucial to consultation satisfaction ratings . this study demonstrates a statistical significance between patients ' perception of time spent in the consultation and satisfaction ( table 2 ) , but not all studies agree . fifty - three percent of the patients rated the time spent in their consultation as adequate or very adequate , with 47% describing the time spent as either fair or inadequate ( table 2 ) . patients ' assessment of the adequacy of time is crucial in gauging their satisfaction as it has been linked to satisfaction with psychosocial issues in the consultation . patients ' assessment of time spent in the consultation may be influenced by certain individual traits such as age . in this study , patients often confused the time spent in the consultation with the time spent in the waiting room , and throughout the data collection patients were encouraged to make this distinction as they completed their questionnaires . the patient perception of time is crucial in the consultation , and this influenced whether the patient was satisfied with the consultation or not . duration of a patient illness has been shown to have an influence on the consultation by a study of chronic illnesses while a patient satisfaction with a consultation can also affect the duration of his illness . however , this study could not demonstrate any statistically significant influence of chronic illness on patients ' satisfaction ( table 3 ) . patients with chronic illnesses are expected to know more about their illness than those with acute illness and are thought to require more attention . chronic illnesses are usually not what doctors expect to manage when they graduate , and their management may be a form of psychological burden to the physician . this study did not demonstrate any effect on satisfaction rates by the presence or absence of a chronic illness ( table 3 ) . this finding may be explained by the fact that many patients in our environment are not well informed about their illnesses , so their knowledge of the illnesses does not necessarily increase as the durations of their illnesses increase . this study could not demonstrate any significant influence of occupation on a patient 's satisfaction with the consultation ( table 1 ) . students made up 32% of the respondents , employed persons 27% , and housewives 7% . despite this spread the patient 's occupational status is closely linked to the person paying for their medical expenses . this was also found not to have any statistically significant influence on patients ' satisfaction with their consultation ( table 1 ) . fifty - two percent of the patients were paying for their medical expenses themselves while families were paying for 30% . ten percent of the patients including two males were being paid for by their spouses . however , 3% of the patients did not know who would pay and an equal number was being sponsored by their employers . the number being sponsored by their employers was unexpectedly low ( 2.6% ) considering the nigerian government 's efforts at promoting a national health insurance scheme ( table 1 ) . the effect of managed care in this study can not be discussed owing to the low number of patients who were using health care insurance , but studies in the united states have demonstrated that managed care affects neither the perception of time used in the consultation nor patients ' satisfaction with it . there was also a possibility that the number of students might be lower than the observed figures because some young people in calabar town often claimed to be students when they were not . this finding suggests that people from different occupational backgrounds in our practice environment may not bring their psychological expectations to influence their satisfaction with the consultation . the more a consultation contributes to a patient 's understanding of his illness , the higher the likelihood for the patient to be satisfied with the consultation [ 18 , 19 ] but very often patients get less information than they expect . fifty - nine percent of the patients had a satisfying consultation , while 52% had some improvement in the understanding of their illness . however , of the 41% of patients with unsatisfying consultations , 28% of them still had an improvement in their illness understanding ( table 2 ) . this suggests that , despite the lack of illness understanding there is still some satisfaction with the consultation . however this study clearly demonstrates that a patient understanding of his / her illness has a statistically significant effect on the patient 's satisfaction with the consultation ( table 2 ) . it seems clear that the more informing a consultation is , the more likely a patient is to be satisfied with the consultation . this finding supports the call by one report for the patient to be more involved in decision making . many patients were observed in this study to have shown great interest when they found the doctor to be willing to provide some explanation about their illness . a patient 's ability to cope with his illness can be helped or marred by a consultation , and this is more evident in chronic illnesses . patients ' abilities to cope with their illness based on the information received have been demonstrated by this study to statistically affect patients ' satisfaction with the consultation ( table 2 ) . it is increasingly clear that better informed patients have better outcomes , choose less risky procedures , and avoid equivocal treatments . a patient 's ability to maintain health after a consultation would be addressing one of the core issues in family medicine by promoting prevention of illnesses . this study demonstrates a statistically significant effect of a patient 's ability to cope with his illness on his / her satisfaction with the consultation ( table 2 ) . this is vital in our environment considering that the bulk of illnesses we manage is due to preventable diseases . in conclusion , factors influencing the patient - doctor consultation are numerous , and the exact influence of any of these factors is not easily isolated , but together these factors influence the interaction either positively or negatively . however , the study has shown that , despite the various factors that are considered to encourage client satisfaction at primary care consultation , a few of such factors contributed to end of consultation satisfaction in our environment . this calls for a refocusing if improvement in the overall patient care in our cultural context is to be achieved with the aim of meeting patient needs . teaching of consultation process must take these factors into consideration . to further address effort towards improving patient satisfaction rates in the study centre , it is recommended that the physician - related factors that influence the doctor - patient encounter should be further studied . in particular the effect of sociodemographic variables such as same - sex consultation , cultural / language diversity , and experience should also be further explored ." ]

[ "acute pancreatitis is an inflammatory intra - abdominal process , which in approximately 1520% of patients presents in a severe form , with a gradual establishment of multiple organ dysfunction or local complications , including necrosis , pseudocyst , and abscess . severe acute pancreatitis is a condition associated with high mortality , which is characterized by a complex and incompletely understood pathophysiological mechanism [ 2 , 3 ] . the deficit in our understanding of the mechanism driving the inflammatory process in acute pancreatitis is a reason why our therapeutic strategy has failed to reduce mortality , despite ongoing research . when acute pancreatitis leads to the establishment of acute kidney injury , there is a 5- to 10-fold rise in mortality , which can reach 70% [ 46 ] . the prevention of acute kidney injury can be a useful strategy in the prevention of the morbidity and mortality associated with acute pancreatitis . eugenol ( 1-allyl-4-hydroxy-3-methoxybenzene ) is a naturally occurring substance , found in the essential oil of commonly consumed spices such as clove oil as well as cinnamon , basil , and nutmeg oils . it has many pharmacological properties which are mainly analgesic , anti - inflammatory , antioxidant , and vasodilatory action , while it has been shown to ameliorate kidney injury in a model of gentamycin - induced nephrotoxicity . the aim of this study is to assess the possible reduction in the extent of acute kidney injury after administration of eugenol in an experimental model of acute pancreatitis .", "106 male wistar rats , aged 3 - 4 months and weighing 220350 gr , were used in this study . they were housed in cages under standard laboratory conditions ( 12 hr light - dark cycles , 2225c room temperature , and 5558% humidity ) , with free access to food and water . the animals were procured from the hellenic pasteur institute ( athens , greece ) . the experiment took place at the elpen experimental research center ( pikermi , greece ) , while the histological analysis was carried out at the lab of histology , embryology , medical school , democritus university of thrace . the experimental surgical procedures and the general handling of the animals conformed to the international guidelines of directive 86/609/eec on the protection of animals used for experimental and other scientific purposes . the animals were randomly assigned in 3 groups : sham ( n = 20 ) , control ( n = 46 ) , and eugenol ( n = 40 ) . the animals were anaesthetized initially by being placed in a glass box containing isoflurane and then through administration of 0.25 ml of butorphanol ( dolorex ; intervet / schering / plough animal health , boxmeer , holland ) by subcutaneous injection . the animals were intubated with a 16 g venous catheter , which was then connected to a ventilator set at 70 breaths / min and a tidal volume of 3 ml . after confirmation of the success of intubation , anaesthesia was maintained by a mixture of 93% o2 , 5% co2 , and 2% isoflurane . briefly , after induction of anaesthesia and preparation of the surgical site , the abdomen was entered via a 3 cm midline incision under sterile conditions . the biliopancreatic duct was identified and ligated near the duodenal wall with a 4 - 0 silk sutures ( in the control and eugenol groups , but not in the sham group ) . 1 ml of normal saline and 1 ml of 5% d5w were instilled in the abdominal cavity . the abdomen was closed with vicryl 2 - 0 sutures . in the eugenol group , eugenol was administered by a nasogastric catheter in a dose of 15 mg / kg , while the sham and control groups received corn oil solution without eugenol . postoperatively , analgesia was maintained through subcutaneous administration of 2 ml / kg butorphanol ( dolorex ; intervet / schering / plough animal health , boxmeer , holland ) . euthanasia was performed at a predetermined time for each animal with the use of ketamine ( narcetan ; vetoquinol , buckingham , uk ) 0.30.6 ml and xylazine ( rompun ; bayer , uxbridge , uk ) 0.10.3 ml , followed by a midline laparotomy and exsanguination of the abdominal aorta . time points for analysis were 6 , 12 , 24 , 48 , and 72 hours postoperatively . serum samples for measurement of urea and creatinine as well as specimens from both kidneys for histopathological examination were acquired . louis , mo , usa ) was purchased and prepared in an oily solution in the chemical laboratory of elpen pharmaceutical co. inc . this was achieved with the admixture of pure eugenol in a corn oil solution in a concentration of 1.5 mg eugenol / ml . samples were placed in 10% buffered formalin solution , and 4 m paraffin - embedded sections were stained with hematoxylin / eosin . all specimens were evaluated by a pathologist blinded to the sequence of the biopsy specimens . slides were evaluated with regard to 5 histopathological parameters and with the use of a semiquantitative scoring system as depicted on table 1 . the scores of each individual parameter for each slide were added and a histopathological score was obtained for each specimen . immunohistochemical staining was applied to detect the possible expression of inflammatory cytokines like il-6 , tnf- , and myeloperoxidase . the following antibodies were used : myeloperoxidase ( rabbit polyclonal ) , dako ( a 0398 ) , diluted 1 : 400 tnf- ( rabbit polyclonal ) , abnova ( pab8016 ) , diluted 1 : 1000 , il-6 ( rabbit polyclonal ) , and abcam ( ab6672 ) , diluted 1 : 500 . the buffers , blocking solutions , secondary antibodies , avidin - biotin complex reagents , and chromogen were supplied in a detection kit ( envision hrp , mouse / rabbit detection system ( k 5007 ) , dako ) . to inhibit endogenous peroxidase , the specimens were incubated with 3% h2o2 ( 200 ml h2o and 6 ml h2o2 ) for 15 min in a dark room . before the primary antibody was applied , the sections were immersed in 10 mm citrate buffer ( ph 6.0 ) , rinsed in tris - buffered saline , and subsequently heated in a microwave oven ( 650800 w ) for three cycles of 5 min . the slides were washed with tris - buffered saline before application of the primary antibody in order to reduce nonspecific binding of antisera . sections were then briefly counterstained with mayer 's hematoxylin , mounted , and examined under a nikon eclipse 50i microscope ( nikon instruments inc , ny , usa ) . the average labeling index was assessed according to the proportion of positive cells , after scanning the entire section of the specimen . the results were graded as negative ( 0 ) for < 10% of stained cells , low ( 1 ) for > 10% and < 30% of cells stained , moderate ( 2 ) for > 30% and < 70% cells stained , and high expression ( 3 ) for > 70% cells stained ( table 1 ) . the statistical analysis of the results was completed with the use of the 20th version of spss ( statistical package for the social sciences , spss inc . , we performed an analysis in which the data were treated as qualitative using fisher 's exact test ( this test was preferable to x \n because of the small number of animals in each subcategory / time point ) . evaluation of the different variables was performed to determine whether they were normally distributed ( kolmogorov - smirnov are shapiro - wilk ) . the three different groups were then analyzed using the kruskal - wallis one - way analysis of variance test . finally , the mann - whitney u test was further used to compare the groups in pairs . these tests were applied to the overall sample and for each individual subgroup corresponding to individual time points ( 6 , 12 , 24 , 48 , and 72 hours postoperatively ) .", "the difference between the eugenol and control groups is apparent at 48 and 72 hours after induction of pancreatitis ( figures 1 and 2 ) . the histological score for these two groups is higher compared to the sham group at 48 and 72 hours and for the whole sample . eugenol administration lowers hyperemia and dilation of renal parenchyma capillaries and the difference was statistically significant for the 48 and 72 hour time points and for the whole sample . the eugenol group exhibited lower values than the control group and both exhibited higher values than the sham group . the same was true for hyperemia and dilation of renal corpuscles capillaries for the 48- and 72-hour time points , but not for the whole sample . there were no inflammatory infiltrations in any of the animals in our experimental model and measurement of this factor did not produce any results . edema was reduced through the administration of eugenol and , again , the difference to the control group was significant for the 48- and 72-hour time points and the whole sample . the control group had higher values than the sham group at 48 hours and also higher values than both the sham and eugenol groups at 72 hours . when values of the whole sample were considered , the control group had higher values than the eugenol group , which in turn had higher values than the sham group . analysis of the whole sample showed only higher values for the eugenol and control groups when compared to the sham group . there was no clear difference regarding il-6 expression between the different groups ( figures 3 and 4 ) . on the contrary there was a statistically significant difference between the eugenol and control groups 72 hours after induction of pancreatitis , while both groups exhibit higher tnf- expression than the sham group . there was no statistically significant difference between the eugenol and control groups for mpo expression , although there was a trend toward higher expression for the control group after 72 hours . eugenol administration resulted in lower serum levels of urea and creatinine especially at the 48- and 72-hour time points , compared to the control group . urea and creatinine levels were higher for both the eugenol and control groups , when they were compared to the sham group ( figure 4 ) .", "the results of this study suggest that eugenol attenuates the intensity of the histopathological changes and the expression of tnf- and mpo in the renal parenchyma , while lowering the values of serum urea and creatinine when administered in a rat acute pancreatitis experimental model . to evaluate the extent of kidney injury , we decided to evaluate serum urea and creatinine levels and the histopathological changes in the kidney , as well as the expression of tnf- , il-6 , and mpo in the renal parenchyma . the role of cytokines , such as tnf- and il-6 , in the pathophysiology of acute pancreatitis has been studied extensively and they have been found to contribute to the activation of the systematic inflammatory response process and multiorgan failure , which is a hallmark of severe acute pancreatitis and is , ultimately , correlated with the observed high mortality rates [ 10 , 11 ] . the role of cytokines in acute kidney injury has been found to be equally important . the cytokine - mediated inflammatory response has a central role in the pathophysiology of acute renal failure irrespective of its cause . mpo has been used as a marker of neutrophil migration in acute pancreatitis studies and has been correlated to the severity of kidney injury [ 1214 ] . the histopathological evaluation showed that the histologic score was lower for the eugenol group in comparison to the control group at 48 and 72 hours from the initiation of the inflammatory process ( means : 3.75/6.5 and 4.12/7.62 , resp . ) and this difference was statistically significant . this difference between the two groups was also present for individual histological changes such as hyperemia and dilation of renal parenchyma and renal corpuscles capillaries and edema . the difference observed in the degree of acute tubular necrosis and inflammatory infiltration was not statistically significant . regarding the expression of inflammatory mediators , tnf- levels were higher for the control group in comparison to the eugenol group with the difference reaching statistical significance at the 72-hour time point , while there was a trend for higher mpo expression in the control group at 72 hours , which was , however , not statistically significant . in contrast , il-6 levels did not show the same correlation and there were no statistically significant differences between the eugenol and control groups . we chose the bile - pancreatic duct ligation model as it is a well - characterized model of acute pancreatitis , which mimics acute pancreatitis caused by biliary obstruction , which is a frequent clinical scenario and results in multiorgan failure similar to that observed in humans [ 15 , 16 ] . we have previously used this experimental model and we were able to show that it generates acute pancreatitis with histopathological changes in the pancreatic tissue including hemorrhage and necrosis . out of a total of 106 animals , 6 died and the fact that they were all in the control group could be seen as further evidence supporting the protective role of eugenol . it is possible that these animals would have exhibited signs of severe kidney injury , if they had survived until the predetermined time of euthanasia . however , since the distal bile - pancreatic duct ligation model is not usually fatal , we can not directly attribute the death of these animals to the severity of acute pancreatitis . eugenol has been shown to possess a multitude of pharmacological effects , some of which make it a likely candidate for use in the setting of acute pancreatitis and can explain the results observed in our study . the analgesic action of eugenol has been well documented and doses in the range of 40100 mg / kg have been shown to be effective in rat experimental models [ 1820 ] . in addition , eugenol acts as an anti - inflammatory substance inhibiting cyclooxygenase and reducing the release of proinflammatory mediators such as il-1 , tnf- , and pge2 [ 2224 ] . the antioxidative potential of eugenol has been studied in a number of , mainly in vitro , studies where it has been shown to bind to free oxygen radicals and attenuate the action of oxidative substances [ 2528 ] , while a recent study of gentamycin - induced nephrotoxicity offers insight into how eugenol can prevent kidney injury by reducing oxidative damage . these combined properties of eugenol can be used to explain the observed reduction in tnf- expression , as well as the reduction of kidney inflammation . eugenol administration causes a dose - dependent , reversible vasodilation through its effect on the endothelial cells [ 29 , 30 ] , which is comparable to nifedipine . the potential of eugenol to inhibit the vasoconstriction that is associated with kidney injury points to another potential mechanism for its effect in the model of acute pancreatitis . a number of authors have proposed strategies to reduce kidney injury caused by acute pancreatitis . zhang et al . have tried dexamethasone administration in an experimental model of retrograde injection of sodium taurocholate in the pancreatic duct . the dexamethasone group exhibited milder congestion of the glomerular capillary , swelling of the renal tubular epithelial cells , and less inflammatory cell infiltration than that of the control group , which was shown by the lower histological score at the 6- and 12-hour time points . the same authors found a significant difference in the serum levels of tnf- in favor of the dexamethasone group , while expression of nf-b in the renal tissue was more pronounced in the dexamethasone group . the same model has been used to study octreotide and baicalin ( 5,6,7-trihydroxyflavone-7-o - d - glucuronic acid ) . the administration of these substances had a protective effect on the kidney and both the histological score and renal parenchyma nf-b expression were lower in comparison to the control group . il-6 were reduced compared to the control group in another study with the same experimental protocol . there have been a number of studies of plant derived substances , used in traditional chinese medicine . ligustrazine proved to be protective for the kidney as was demonstrated by the lower creatinine levels and the milder histopathological changes in comparison to the control group . in another study , the administration of 3 traditional chinese medicine substances ( ligustrazine , kakonein , and panax notoginsenosides ) resulted in reduced mortality and milder histopathological changes in the rat kidney . finally , the model of induction of acute pancreatitis through sodium taurocholate administration was used for the study of poly(adp - ribose ) polymerase inhibition , through 3-aminobenzamide ( 3-ab ) administration . the administration of 3-ab resulted in reduced mortality and a reduction in the increase of creatinine , tnf- , il-1b , and il-6 , milder histopathological changes , and reduced mpo expression in the kidney . the half life of eugenol in the rat has been determined to be 18,3 hours ; therefore , at 72 hours , most of the initial dose would have been cleared from the circulation . it is possible that a repeat administration of eugenol could further increase the therapeutic result . moreover , the time frame of our protocol reached 72 hours , which was not adequate for the complete evaluation of the effect of eugenol . indeed , a difference in the extent of kidney injury between the eugenol and control groups is first observed 48 hours after the onset of acute pancreatitis and it is greater at 72 hours . in conclusion , the administration of eugenol in a rat model of acute pancreatitis was protective for the kidneys in our experimental model . further research is necessary to determine the possible role of eugenol in the management of acute pancreatitis ." ]

[ "periodontal health can be described as a dynamic state where the activity of proinflammatory / antimicrobial cytokines to control infection is optimally balanced by anti - inflammatory mechanisms to prevent unwarranted inflammation . in subjects susceptible to periodontal disease ( pd ) , an imbalance of the inflammatory response results in excessive production of proinflammatory cytokines and the subsequent loss of periodontal attachment . on the other hand , furthermore , the release of tissue regenerating factors may contribute to periodontal regeneration by regulating the function of periodontal ligament cells , endothelial cells , and cementoblasts . in this setting , neurotrophin brain - derived neurotrophic factor ( bdnf ) has been reported to enhance periodontal tissue regeneration [ 4 , 5 ] . bdnf is a member of the neurotrophin family which is expressed by vascular endothelium and osteoblastic , immune , and neuronal cells . bdnf is reported to be involved in the joint inflammatory process and its production is increased in response to proinflammatory cytokines . although a role for bdnf in periodontal regeneration has been proposed , no information is available concerning bdnf and periodontal disease . the aim of this study was to measure the levels of bdnf in periodontal tissues from patients with chronic periodontitis . the presence of polymorphisms rs6265 and rs4923463 of the bdnf gene and its correlation with inflammatory and clinical parameters were also assessed .", "twenty - eight patients with cp , treated at the periodontal clinic , school of dentistry , at universidade federal de minas gerais ( ufmg , brazil ) , were enrolled in this study . patients in this study met the following inclusion criteria : previous history of cp , diagnosed according to previously described criteria : ( 1 ) exhibiting more than one tooth with probing depth higher than 5 mm , ( 2 ) exhibiting more than two sites with clinical attachment loss deeper than 6 mm , and ( 3 ) exhibiting lesions distributed in more than two teeth in each quadrant . patients who met the following criteria were excluded : ( 1 ) having a history of smoking , ( 2 ) use of antibiotic , ( 3 ) usage of anti - inflammatory and/or immunosuppressive medications during the 6 preceding months , and ( 4 ) a history of any systemic diseases ( i.e. , immunologic and autoimmune disorders , diabetes mellitus ) . the control group ( hc ) comprised 29 age and gender matched periodontally healthy patients enrolled for third molar removal surgery . periodontal examination was performed in both groups of patients , cp and hc , at the initial visit to determine probing depth ( pd ) , clinical attachment loss ( cal ) , and bleeding on probing ( bop ) . measurements were performed full - mouth at 6 sites per tooth ( mesiobuccal , midbuccal , distobuccal , mesiolingual , midlingual , and distolingual ) . all the measurements were performed by the same examiner . at the time of the examination a peripheral blood sample periodontal tissue samples from periodontal pockets or healthy oral mucosa extracted during surgery of impacted third molars were fixed in 10% buffered formalin , embedded in paraffin wax , and cut longitudinally ( 3 m ) . the sections were deparaffinized , rehydrated , and stained with h&e for evaluation of the inflammatory infiltrate . inflammatory cells were counted in four fields in two independent sections , using a light microscope ( axioskop 40 zeiss ; carl zeiss , gottingen , germany ) at 400x magnification . the concentrations of the il-17a , bdnf , il-10 , and tnf- and the chemokine cxcl10 were measured in periodontal tissues by enzyme - linked immunosorbent assay ( elisa ) using commercially available kits ( r&d systems , minneapolis , mn , usa ) . the lower limit of detection for each cytokine was 15 pg / ml , 3.9 pg / ml , 5.5 pg / ml , 20 pg / ml , and 4.5 pg / ml , respectively , for il-17a , il-10 , tnf- , bdnf , and cxcl10 . the data were determined using a standard curve prepared for each assay and expressed as picograms of cytokine / chemokine per 100 mg of tissue . periodontal tissue samples were also used for determination of myeloperoxidase ( mpo ) activity , a neutrophil enzyme marker , as described earlier . the mpo activity in homogenized periodontal tissues was evaluated by enzymatic reaction and absorbance was measured at 450 nm . the mpo content was expressed as relative units calculated from standard curves based on mpo activities from 5% casein peritoneal - induced neutrophils assayed in parallel . total genomic dna was extracted from blood samples using qiaamp dna blood mini kit ( qiagen , valencia , ca , usa ) according to manufacturer 's instructions . quality , integrity , and quantity of dna were analyzed by nanodrop spectrophotometer ( thermo scientific , wilmington , de , usa ) . all amplifications were carried out in an abi 7900h thermal cycler ( applied biosystems , foster city , ca , usa ) using taqman genotyping master mix and following manufacturer 's recommended amplification conditions . chi - square test analysis was used to test for deviation of genotype frequencies from hardy - weinberg equilibrium . the levels of cytokines in periodontal tissues and the frequency of gene polymorphisms were compared by the student 's t - test and chi - square tests .", "the sample included in the current study was composed by age and gender matched groups . the clinical features pd , cal , and bop were significantly higher in the cp than in the hc group ( p < 0.0001 ) ( table 1 ) . the levels of il-17a , cxcl10 , il-10 , tnf- , and bdnf in periodontal tissues were greater in cp patients than in controls ( figure 1 ) . moreover , the mpo activity and the inflammatory infiltrate in the periodontal tissues , characterized by polymorphonuclear and mononuclear leukocytes , were significantly higher in the cp than in the hc group ( figure 2 ) . the bdnf and il-10 levels in periodontal tissues were negatively correlated ( r = 0.691 , p = 0.002 ) , whereas no correlation between bdnf and il-17a , tnf- , cxcl10 , or clinical parameters was observed ( pd , cal , and bop ) . following the clinical investigation , the frequencies of polymorphisms ( bdnf ) were assessed in blood samples of hc and cp subjects ( table 2 ) . the frequency of these genotypes agreed with the hardy - weinberg equilibrium ( p > 0.05 ) . the distribution of the bdnf polymorphisms was similar between the groups ( table 2 ) . we also investigated whether some of these polymorphisms were associated with worse clinical periodontal parameters . as shown in table 3 , no differences in clinical parameters were found when comparing the genotypes . the levels of bdnf and the inflammatory mediators cxcl10 and tnf- were increased in gg genotype of bdnf rs6265 polymorphism ( figures 3(a ) , 3(b ) , and 3(c ) ) , but mpo levels did not alter significantly ( figure 3(d ) ) . in bdnf rs4923463 polymorphism the levels of bdnf and mpo did not differ , but the levels of cxcl10 and tnf- were higher in patients with aa genotype ( figures 3(e)3(h ) ) .", "a wide range of nonneural cells in peripheral tissues or in the immune system expresses neurotrophins and their receptors . thus , the mitogenic and immune regulatory functions of neurotrophins have been discussed [ 6 , 1012 ] . the neurotrophin bdnf is reported to be involved in inflammatory reactions , and its production is increased in response to proinflammatory cytokines . the present study is the first to demonstrate that bdnf levels were increased in periodontal tissues from chronic periodontitis compared to healthy subjects . in agreement with our findings , bdnf was found in high levels in the plasma of patients with osteoarthritis and in patients with rheumatoid arthritis . while some authors reported that bdnf levels were significantly correlated with self - reported pain , others did not find association between bdnf and clinical parameters of arthritis . several studies analyzed bdnf rs6265 polymorphisms in psychiatric disorders [ 1618 ] . to date , few studies examined the rs4923463 polymorphism [ 16 , 1820 ] . while one study found correlation between snp rs4923463 and attention - deficit / hyperactivity disorder , schizophrenia , and risk of suicide in bipolar disorder , another study did not find any correlation between schizophrenia and rs4923463 polymorphism . previously , two studies have evaluated the effects of bdnf polymorphisms in bone [ 21 , 22 ] , but there are no available studies in periodontal disease . in the present study we did not find differences in bdnf genotype distribution between patients with cp and controls . nevertheless , we found that subjects with gg ( rs6265 ) genotype expressed higher levels of bdnf in periodontal tissues , in agreement with a previous report showing that bdnf - m66 variant alters intracellular trafficking and impairs bdnf secretion . on the other hand , rs6265 polymorphism interestingly , the snp rs6265 was reported as a phossnp , which means this snp regulates protein phosphorylation . the snp rs6265 affects substrate - kinase interaction between bdnf protein and chek2 kinase and regulates bdnf phosphorylation at site t62 . subjects aa genotype carriers exhibited lower bone mineral density compared to g carriers . specifically , bdnf - v66 ( major allele g at rs6265 ) transfection significantly increases expression of osteoblast specific markers ( opn , bmp2 , and alp ) and promotes osteoblast differentiation and maturation in cell culture . an association of rs6265 with bone metabolism was also suggested in the largest meta - analysis involving 32,961 individuals of european and east asian ancestry . they found that homozygous minor allele a carriers ( aa ) have significantly decreased bmd compared to major allele g carriers ( ga and gg ) . it has been reported that bdnf is able to induce an increase in il-10 expression . however , a negative correlation between the production of bdnf and il-10 was observed in samples from patients with periodontitis . il-10 can inhibit the release of proinflammatory cytokines from monocytes / macrophages and can therefore inhibit the lipopolysaccharide- and ifn--induced secretion of inflammatory cytokines ( e.g. , tnf- , il-1 , il-6 , cxcl8 , and others ) . in periodontal disease , il-10 is thought to be associated with lower disease severity . previous studies demonstrated that bdnf induces periodontal tissue regeneration by activation of cementoblasts differentiation , vascular endothelial cell migration , and also has a positive effect on bone remodeling [ 5 , 28 ] . this data together suggested that bdnf has a role in bone remodeling and any change in this neurotrophin levels could have an impact in bone repair . finally , we observe that cp subjects with gg ( rs6265 ) and aa ( rs4923463 ) genotypes demonstrated increased levels of tnf- and cxcl10 . cxcl10 has several roles , such as chemoattraction of macrophages , t cells , nk cells , and dendritic cells . in addition , previous studies showed that exposure to bdnf substantially and synergistically enhanced tnf- levels in vitro , and tnf- preconditioning increased proliferation , mobilization , and osteogenic differentiation in vitro . we can hypothesize that the concomitant increase of bdnf , tnf- , and cxcl10 in patients with the gg genotype may be an attempt of the host to induce periodontal healing . so , maybe if these patients were examined after periodontal treatment , they could display higher and better levels of tissue regeneration compared to patient who do not exhibit the gg genotype . in conclusion , bdnf seems to be related to periodontal pathogenesis and also involved in tissue repair . the results obtained here provide a benchmark for future studies with a large cohort of patients to help strengthen and understand the influence of neurotrophins in periodontal disease ." ]

[ "we analyzed information about 634 immigrants from latin america seen at the tropical medicine unit of the ramn y cajal hospital in madrid , spain , during april 1989june 2008 . we used 5 strict criteria for diagnosing vlm : 1 ) positive serologic test for toxocara sp . roundworm infection , performed by using a commercial elisa toxocara immunoglobulin ( ig ) g ridascreen ( r - biopharm gmbh , darmstadt , germany ) , following the manufacturer s recommendations ; 2 ) absolute peripheral blood eosinophil count > 500 cells / mm ; 3 ) exclusion of other parasites causing eosinophilia , such as intestinal nematodes , particularly strongyloides stercoralis ( excluded by larval culture and serology by elisa igg ) , schistosoma sp . , fasciola hepatica , trichinella spiralis , taenia solium , echinococcus granulosus , and cutaneous and blood microfilariae ; 4 ) symptoms associated with vlm ( respiratory signs , such as asthma , dyspnea , and eosinophilic pneumonia ; dermatologic symptoms , including pruritus and recurrent urticaria ; and abdominal symptoms , including abdominal pain and hepatomegaly ) ; and 5 ) response to treatment with albendazole ( 1015 mg / kg / d in 2 doses orally for 5 days ) assessed 6 months after treatment , decreased titers to toxocara sp . the most frequent countries of origin for patients were ecuador 221/634 ( 34.9% ) , bolivia 176/634 ( 27.8% ) , peru 71/634 ( 11.2% ) , and colombia 56/634 ( 8.8% ) . median age was 32 years ( range 440 years ) ; 421 ( 66.4% ) patients were male . the median number of months from arrival in spain to first consultation at the tropical medicine unit was 19 months . concomitant serologic results positive for toxocara sp . roundworm infection and eosinophilia were found in 28 ( 4.4% ) patients ; 606 patients were excluded . of these 28 patients , 11 were excluded because of other concomitant parasitic infections that also can cause eosinophilia : 8 patients had positive elisa results for s. stercoralis nematodes ( not detected in fecal samples or larval culture ) ; 1 had ascaris lumbricoides eggs in feces ; 1 had a positive indirect hemagglutination result but negative elisa result for e. granulosus tapeworm ; and 1 had a positive elisa serologic result for t. spiralis nematodes . only 4 of the 5 remaining cases fulfilled the strict inclusion criteria ( table ) ; 1 patient was asymptomatic . after 6 months of treatment with albendazole , titers for toxocara sp . roundworm infection and eosinophil count decreased , and symptoms improved or resolved for the 4 patients . all patients were treated with albendazole ( 1015 mg / kg / d in 2 doses orally for 5 days ) . clinical toxocariasis is rarely diagnosed in western countries as previously described despite evidence of environmental exposure ( 1 ) . results of seroprevalence surveys performed in healthy adults in france were positive for 2%5% of persons in urban areas , compared with 14%37% in rural areas ( 2 ) . in latin america , rates vary from 1.8% to 51.6% ( 3,4 ) . however , literature references to vlm imported by immigrants are scarce ( 5 ) , and the disease may be underdiagnosed in the immigrant population , partly because of nonspecific symptoms and the limitations of serologic diagnosis . in our study , toxocariasis is a common cause of eosinophilia in peripheral blood , although its absence does not exclude infection by toxocara sp . roundworm infection without eosinophilia ( 6 ) ; similarly , 27% of patients with high antibody titers had eosinophil counts within the reference range ( 7 ) . by including only patients with eosinophilia , our study applied more stringent criteria . thus , 28 ( 90% ) of 31 patients who had positive serologic results showed an elevated eosinophil count , in accordance with previously described high toxocara sp . roundworm seroprevalence ( < 68% ) in patients with eosinophilia of unknown cause ( 8) . eleven of the 28 patients with positive serologic results for toxocara sp . roundworm and eosinophilia also had positive serologic results for other parasites that cause eosinophilia . one patient who was infected with a. lumbricoides roundworm had asthma , hepatomegaly , and pruritus . the latter is not usually associated with this parasite , which suggests possible co - infection . serologic tests for toxocara sp . roundworm infection should be interpreted with caution because commercial elisa kits that use excretory and secretory antigens derived from second - stage larvae of toxocara sp . roundworms exhibit a sensitivity of 91% and a specificity of 86% ; cross - reactivity has also been described with other nematode infections . the positive serologic results for t. spiralis nematodes and e. granulosus tapeworms may have been caused by cross - reactivity ( 9 ) . these patients had asthenia and asthma , respectively , and symptoms resolved after treatment with albendazole . eight patients with strongyloides antibodies were also excluded ; however , this finding does not exclude co - infection by both parasites . finally , a limitation of the study was that we could not definitively exclude cryptic strongyloidiasis for 12 patients because of the difficulty in finding s. stercoralis threadworms in feces and because detection of strongyloides antibodies was not possible . this study illustrates the difficulties in diagnosing vlm in immigrants from tropical and subtropical areas of latin america because only a very small proportion of patients in the series ( n = 4 ) had vlm . the most common symptoms were respiratory ( 3/4 ) ; 2 patients had asthma - like syndrome and 1 had chest pain followed by abdominal pain ( 2/4 ) . typical manifestations of vlm are abdominal symptoms ( pain , hepatomegaly ) and respiratory symptoms ( severe asthma , eosinophilic infiltrates ) . in addition to this , evidence points to toxocara sp . roundworm infection as a risk factor for asthma in some populations ( 11,12 ) . albendazole is the treatment of choice for vlm ; for practical purposes , it could be recommended for presumptive treatment in immigrants from latin america with eosinophilia in whom strongyloidiasis is suspected ( 13 ) . however , the superiority of ivermectin over albendazole has been documented in the treatment of chronic strongyloidiasis ( 14 ) .", "vlm may be difficult to diagnose , especially in immigrants from regions in latin america where polyparasitism is endemic . positive serologic test results , marked eosinophilia , absence of other helminthic infections , compatible clinical signs , and disappearance of symptoms after specific treatment can help establish a vlm diagnosis , especially in areas of low parasitism . vlm should be included in the differential diagnosis of eosinophilia in immigrants ( children and adults ) from tropical areas if respiratory or abdominal symptoms are evident . albendazole is an effective and relatively safe drug that could be used to treat suspected vlm and other concomitant nematode infections , including cryptic s. stercoralis threadworm infections . empirically described treatment may lead to resolution of clinical symptoms , even though ivermectin is a better treatment for chronic strongyloidiasis ." ]

[ "although lbp has generally been believed to be uncommon before the age of 20 , the prevalence of lbp among school children and adolescents has been reported to be high in different parts of the world , mostly in western countries , where it varies from 1040% . the nhanes ii ( national health and nutrition examination survey series ) reported the onset of lbp before the age of 20 in 11% of the general population . lbp prevalence rates were found to be 2831% in 2 studies carried out on school children in kuwait and tunisia . these studies show that the prevalence of lbp in children is high , equaling that of adults by the end of the growth period . lbp has a significant economic impact on individuals , society , and quality of life . many studies have analyzed the risk factors associated with lbp in children and adolescents to describe risk factors profiles [ 3,58 ] . to our knowledge there have been no reports on nlbp and related factors among school children in turkey . the aim of this study was to investigate possible factors associated with pain intensity among turkish school children with nlbp , aged 1018 years , and to determine the relationship between related factors and pain intensity using the regression tree method .", "this study was conducted on school children aged 1018 years in the city of denizli , located in the western part of turkey . out of 88 primary and high schools in the city , fifth and eleventh grade classes were selected from each of 10 schools ( 8 governmental and 2 private ) , using a simple random sampling method . the city department of education gave written permission , and all parents of students gave informed consent . the exclusion criteria were having any kind of musculoskeletal , rheumatic , orthopedic , somatic or psychiatric disorders . all of the exclusion criteria were considered to define nlbp more clearly because we studied nonspecific low back pain . in the sampled schools , 624 children were interviewed in total and 292 ( 46.8% ) were reported as having nlbp . after excluding the students who displayed the aforementioned disorders , 222 students ( 116 girls and 106 boys ) the visual analog scale ( vas ) was used for measuring the intensity of pain . the vas is designed to present to the respondent a rating scale with minimum constraints . this scale , shown below , was reported as the number of cm . from left of line , with range 010 : the interviewers used in the study were selected from the final year students in the physical therapy school of pamukkale university . the dependent variable was pain intensity measured in cm , and the independent variables were bmi , sex , regular exercise habit , studying posture , transportation to / from school , duration of studying , bag handling and type of bed the participants were asked to select 1 of the following studying postures : ( 1 ) sitting on chair and studying on a table , ( 2 ) sitting on ground without chair and table , and ( 3 ) lying in prone or supine position on a bed . two methods of enquiry about nlbp were used , namely a direct question and a pre - shaded manikin question : have you ever had low back pain ? ( look at the drawing ) have you experienced pain in the shaded area which lasted for 1 week , a month , or longer ? students who answered both of the questions as the regression tree method ( rtm ) was used to determine risk factors that may affect pain intensity . rtm is a tree - based model , and is more useful than traditional statistical methods when a data set is large and when there are many variables . moreover , the rtm takes into consideration interactions among variables , and is not affected by high correlations between risk factors . there is no assumption about distribution shapes of risk factors , but outcome variable should be numeric . in rtm the association between risk factors ( x ) and outcome variable ( y ) ( pain intensity , in this study ) are examined by a schematic representation . homogeneous groups are constituted according to the adequate cut - off values of the risk factors . at the beginning , all individuals are collected in 1 group called a root node . homogeneous groups that come into being based on recursive binary splitting are termed terminal node . the homogeneous group means that this group is sufficiently homogeneous and can not be split any more . splitting continues until the tree reaches maximum size , and then passing the selection of adequate tree structure stage , called pruning . the maximum tree is not used for every data set because of its overfit structure . after pruning , the values that take place in the terminal nodes give the mean and variance of that group .", "two methods of enquiry about nlbp were used , namely a direct question and a pre - shaded manikin question : have you ever had low back pain ? ( look at the drawing ) have you experienced pain in the shaded area which lasted for 1 week , a month , or longer ? students who answered both of the questions as ", "the regression tree method ( rtm ) was used to determine risk factors that may affect pain intensity . rtm is a tree - based model , and is more useful than traditional statistical methods when a data set is large and when there are many variables . moreover , the rtm takes into consideration interactions among variables , and is not affected by high correlations between risk factors . there is no assumption about distribution shapes of risk factors , but outcome variable should be numeric . in rtm the association between risk factors ( x ) and outcome variable ( y ) ( pain intensity , in this study ) are examined by a schematic representation . homogeneous groups are constituted according to the adequate cut - off values of the risk factors . at the beginning , all individuals are collected in 1 group called a root node . homogeneous groups that come into being based on recursive binary splitting are termed terminal node . the homogeneous group means that this group is sufficiently homogeneous and can not be split any more . splitting continues until the tree reaches maximum size , and then passing the selection of adequate tree structure stage , called pruning . the maximum tree is not used for every data set because of its overfit structure . after pruning , the values that take place in the terminal nodes give the mean and variance of that group .", "the descriptive statistics are shown ( table 1 ) as mean sd and as frequencies and percentages . the overall mean and standard deviation of pain intensity was 2.580.86 ( minimum=1 , maximum=5 ) . among the risk factors used in this study , duration of studying , type of bed , transportation to / from school , and bmi score were found to have a significant effect on pain intensity , while sex , studying posture , regular exercise habit , and bag handling were not significant ( figure 1 ) . as will be seen from figure 1 , 6 homogeneous groups are defined by the rtm according to pain intensity , with an increasing order . these are as follows : group ( id=124 ) : studies less than 4 hours , uses school bus for transportation and has 17.13 < bmi < 20.9 ( mean pain score=1.74 ) . this group is the lowest risk group among all 6 groups , giving the lowest mean . group ( id=4 ) : studies more than 4 hours and sleeps on orthopedic bed ( mean pain score=2.48 ) . group ( id=44 ) : studies less than 4 hours and uses public transportation or walks to / from school ( mean pain score=2.60 ) . group ( id=125 ) : studies less than 4 hours , uses school buses for transportation and has bmi > 20.9 ( mean pain score=2.70 ) group ( id=120 ) : studies less than 4 hours , uses school buses for transportation and has bmi < 17.13 ( mean pain score=2.86 ) . group ( id=5 ) : studies more than 4 hours and sleeps on wool or cotton beds ( mean pain score=3.11 ) . this group is the highest risk group among the 6 groups , giving the highest mean .", "it has become clear that a high prevalence of lbp occurs not only in adults , but also in children / adolescents . more recently , cross - sectional and longitudinal studies have focused on nlbp in children . the prevalence has been reported to vary from 10% to 40% in the literature , but the authors found it to be 46.7% in their previous cross - sectional study of 624 school children / adolescents 1018 years old . among the 8 risk factors included in the study , these factors are as follows : transportation to / from school : prista et al found that school children walking > 30 min per day to and from school are associated with an increased risk factor of lbp . they also showed that long distance walking to / from the school might lead to muscle fatigue resulting in back pain . in our study , the transportation to / from school had an important effect on lbp . bmi : the bmi score was also a significant risk factor affecting pain intensity in our study . as is well known , increased bmi score increases pain intensity in subjects with low back pain . type of bed : jacobson et al . in 2002 reported that an experimental bedding system ( ameri - spring ) reduced back pain and improved the quality of sleep . in our study we found also a significant relationship between the type of bed and low back pain intensity . this shows that quality of sleep is a very important factor affecting pain intensity for subjects who suffer from low back pain . duration of studying : we also found a significant relationship between duration of studying and nlbp intensity . korovessis et al . in 2004 reported that dorsal pain increased with increasing backpack weight among children . lee and chiou found that poor sitting habit were statistically associated with lbp . in our study , studying posture was not found to be an important factor . we also found that the sex of the children was not an important factor in nlbp intensity .", "results from the literature , as well as our study , show that taking parents and teachers concerns seriously is of vital importance . therefore , health care providers should evaluate school children carefully and make accurate observations in terms of risk factors , including duration of studying , type of bed , transportation to / from school , and obesity , to predict any severe musculoskeletal problems , especially nlbp . finally , physical factors and musculoskeletal risk factors are especially important in terms of nlbp in school children . further studies are needed to investigate psychosocial risk factors and their relationships with nlbp in school children ." ]

[ "this study was developed and approved by the steering committee of the translating research into action for diabetes ( triad ) study and conducted in one of triad 's six translational research centers , kpnc . kpnc is a group practice , prepaid health plan that provides comprehensive medical services through 17 hospitals and 23 outpatient clinics to > 3 million members located in a 14-county region in northern california ( 30% of the general population in the geographic areas covered ) . the kpnc membership closely approximates the population living in the same geographic area demographically except with respect to income : kpnc members underrepresent the very poor and the very wealthy ( 9,10 ) . upon comparison with regional birth certificates over a 14-year period , there were no meaningful differences between women who delivered at a kpnc hospital and women who delivered in the underlying region regarding age at delivery or race , except that women who delivered at a kpnc hospital were slightly less likely to be hispanic ( 25.8 vs. 32.0% ) . we used the kpnc gdm registry ( 11 ) to identify women with gdm who delivered between 1 january 1995 and 31 december 2006 . during this 12-year period , the proportion of women who had been screened for gdm with a 50-g , 1-h oral challenge test during the second trimester increased by 3% over time ( age- and race / ethnicity - adjusted proportions 92.5 [ 95% ci 92.392.7 ] in 19951997 vs. 95.7 [ 95.695.9 ] in 20042006 ) . if results were abnormal ( 1-h plasma glucose levels 7.8 mmol / l [ 140 mg / dl ] ) , this test was followed by a standard diagnostic 100-g , 3-h ogtt . we identified 14,448 pregnancies that had a diagnosis of gdm from a health provider and with plasma glucose results during the index pregnancy that met the national diabetes data group ( nddg ) criteria on the 3-h 100-g ogtt for gdm , i.e. , 2 glucose values at or exceeding the following thresholds : fasting , 105 mg / dl ; 1 h , 190 mg / dl ; 2 h , 165 mg / dl ; and 3 h , 145 mg / dl ( 12,13 ) without recognized preexisting diabetes ( 14 ) . we included only women who met the nddg criteria of gdm because , in this clinical setting , the nddg criteria were used for diagnosis of gdm until january 2007 . approximately 550 women had no diagnosis of gdm but met the nddg criteria ( equivalent to 4% of the size of our gdm cohort with a diagnosis ) ; women without a diagnosis of gdm were excluded from the analysis . for the first and second aims , the primary outcome was performance of postpartum screening for diabetes by either an fpg test alone or a 75-g , 2-h ogtt . because several weeks may elapse before glucose metabolism returns to normal in most women with gdm ( 15 ) , the american diabetes association ( 16 ) and the american college of obstetricians and gynecologists ( 17 ) both recommend that postpartum glucose screening be performed at 6 weeks postpartum or later . we considered the postpartum screening performed only if it was done during the first year after delivery starting from 6 weeks and if the woman was not pregnant again . for the third aim , the outcome was the proportion of women identified with either ifg ( defined as an fpg 100 mg / dl but < 126 mg / dl ) or diabetes diagnosed by fpg 126 mg / dl ( 18 ) . among women who had postpartum screening , therefore , when we report on trends in postpartum conversion to diabetes , we report only on the fasting values detected by either an fpg or an ogtt . for the fourth aim , we examined the proportion of women who were identified as having pre - diabetes on the postpartum screen ( that included ifg as defined above and impaired glucose tolerance [ igt ] defined as a 2-h plasma glucose value 140 mg / dl ) or diabetes ( defined as an fpg > 126 mg / dl or a 2-h plasma glucose value 200 mg / dl ) ( 18 ) . age , gestational age at delivery , race / ethnicity , maternal body weight during the beginning of the second trimester , and gestational age at gdm diagnosis were ascertained from the computerized medical records at birth . use of insulin or glyburide during pregnancy was obtained from the pharmacy database . because we had data on body weight but not on height , a woman was considered obese if her weight was 90th percentile of the weight distribution of women of her race / ethnicity in this study population . macrosomia was defined as birth weight > 4,000 g. data on annual household income were based on census block data . education and parity were obtained by linkage with the state of california birth certificate database . because the lag time before state birth certificates became available is 3 years , we have these variables for women who delivered between 1995 and 2004 . the yearly age- and race / ethnicity - adjusted proportion of women with gdm who had postpartum glucose screening and 95% cis were calculated by the direct method , in which the age and race / ethnicity distribution of the entire study population was used as the standard . among women who had postpartum screening , the direct method was used to calculate the yearly age- and race / ethnicity - adjusted rates of ifg and diabetes diagnosed by fpg . predictors of postpartum screening were examined in a multivariable logistic regression model adjusted for age , race / ethnicity , education , income , obesity , parity , gestational age at gdm diagnosis , glyburide and insulin use during pregnancy , a macrosomic infant at the index pregnancy , visits to an internal medicine or obstetrics / gynecology provider during the postpartum period , year of delivery , and medical facilities . sas ( version 9.1 ; sas institute , cary , nc ) was used for all analyses . this study was approved by the human subjects committee of the kaiser foundation research institute .", "the yearly age- and race / ethnicity - adjusted proportion of women with gdm who had postpartum glucose screening and 95% cis were calculated by the direct method , in which the age and race / ethnicity distribution of the entire study population was used as the standard . among women who had postpartum screening , the direct method was used to calculate the yearly age- and race / ethnicity - adjusted rates of ifg and diabetes diagnosed by fpg . predictors of postpartum screening were examined in a multivariable logistic regression model adjusted for age , race / ethnicity , education , income , obesity , parity , gestational age at gdm diagnosis , glyburide and insulin use during pregnancy , a macrosomic infant at the index pregnancy , visits to an internal medicine or obstetrics / gynecology provider during the postpartum period , year of delivery , and medical facilities . sas ( version 9.1 ; sas institute , cary , nc ) was used for all analyses . this study was approved by the human subjects committee of the kaiser foundation research institute .", "we identified 14,448 pregnancies complicated by gdm occurring between 1995 and 2006 among kpnc members who were aged 1544 years and delivered live infants or had still births . these pregnancies occurred among 13,547 women , because 901 had more than one pregnancy during the 12-year study period . the percentage of women with gdm pregnancies who received a postpartum glucose screening test increased from 1995 ( 20.3% ) to 2006 ( 55.9% ) . between 1995 and 2006 , the age of women with gdm increased slightly ( 28.2 5.7 to 28.8 6.0 years ) and the proportion of women with gdm who were hispanic increased markedly . the race / ethnicity distributions in 1995 versus 2006 were as follows : 60.7 and 43.4% white , 14.5 and 15.4% asian , 14.1 and 24.9% hispanic , 6.7 and 6.7% african american , 2.0 and 4.4% other , and 2.0 and 5.2% unknown . changes in the demographics represent changes in the entire population with gdm , regardless of performance of diagnostic screening . after adjustment for age and race / ethnicity , the proportion of women with gdm who received a postpartum glucose screening increased steadily over time from 20.7% ( 95% ci 17.823.5 ) in women who delivered in 1995 to 53.8 ( 51.356.3 ) in women who delivered in 2006 ( fig . unadjusted characteristics of women by postpartum screening status are illustrated in table 1 . in a multiple - adjusted logistic regression model ( table 2 ) , older age , asian or hispanic race / ethnicity , higher education , at least two prior births , earlier gestational age at gdm diagnosis , use of insulin or glyburide during pregnancy , and visits to an internal medicine or obstetrics / gynecology provider during the postpartum period were independent and significant predictors of postpartum screening . obesity and higher parity were significantly associated with less frequent screening . on the basis of fpg ( either performed alone or as part of the ogtt ) , 3.5% ( n = 191 ) had diabetes and 22.0% ( n = 1,228 ) had ifg . the proportion of women with ifg postpartum remained similar over time , but the proportion of women with diabetes decreased from 19951997 to 19982000 and then leveled off ( fig . 564 were screened at postpartum , and 131 had ifg and 32 had diabetes by fpg ( age- and race / ethnicity - adjusted rates 24.2 [ 95% ci 20.27.8 ] and 6.1 [ 4.28.1 ] , respectively ) . among women who delivered in 20042006 , 2,381 women were screened , and 583 had ifg and 80 had diabetes by fpg ( age- and race / ethnicity - adjusted rates 24.3 [ 22.626.0 ] and 3.3 [ 2.64.0 ] , respectively ) . the proportion of women receiving an ogtt increased from 1995 ( 5.0% ) to 2005 ( 16.6% ) and markedly increased in 2006 ( 71.5% ) . in 2006 , kpnc instituted a nurse managed care program that included greater attention to postpartum screening guidelines . among the 600 women who underwent a 75-g ogtt in 2006 , 16 had diabetes at postpartum . diabetes was diagnosed in 4 ( 25% ) by fpg alone . of the remaining 12 women , of the 188 women who were found to have pre - diabetes ( i.e. , either ifg or igt according to the fasting or 2-h glucose values measured during the 75-g ogtt ) , only 114 ( 60% ) were diagnosed with ifg ; 74 ( 40% ) would have been classified as having normal glucose tolerance on the basis of fpg alone . therefore , 78 ( 38% ) of the 204 women with either diabetes or pre - diabetes were identified only by the 2-h glucose measurements .", "we identified 14,448 pregnancies complicated by gdm occurring between 1995 and 2006 among kpnc members who were aged 1544 years and delivered live infants or had still births . these pregnancies occurred among 13,547 women , because 901 had more than one pregnancy during the 12-year study period . the percentage of women with gdm pregnancies who received a postpartum glucose screening test increased from 1995 ( 20.3% ) to 2006 ( 55.9% ) . between 1995 and 2006 , the age of women with gdm increased slightly ( 28.2 5.7 to 28.8 6.0 years ) and the proportion of women with gdm who were hispanic increased markedly . the race / ethnicity distributions in 1995 versus 2006 were as follows : 60.7 and 43.4% white , 14.5 and 15.4% asian , 14.1 and 24.9% hispanic , 6.7 and 6.7% african american , 2.0 and 4.4% other , and 2.0 and 5.2% unknown . changes in the demographics represent changes in the entire population with gdm , regardless of performance of diagnostic screening . after adjustment for age and race / ethnicity , the proportion of women with gdm who received a postpartum glucose screening increased steadily over time from 20.7% ( 95% ci 17.823.5 ) in women who delivered in 1995 to 53.8 ( 51.356.3 ) in women who delivered in 2006 ( fig .", "unadjusted characteristics of women by postpartum screening status are illustrated in table 1 . in a multiple - adjusted logistic regression model ( table 2 ) , older age , asian or hispanic race / ethnicity , higher education , at least two prior births , earlier gestational age at gdm diagnosis , use of insulin or glyburide during pregnancy , and visits to an internal medicine or obstetrics / gynecology provider during the postpartum period were independent and significant predictors of postpartum screening .", "on the basis of fpg ( either performed alone or as part of the ogtt ) , 3.5% ( n = 191 ) had diabetes and 22.0% ( n = 1,228 ) had ifg . the proportion of women with ifg postpartum remained similar over time , but the proportion of women with diabetes decreased from 19951997 to 19982000 and then leveled off ( fig . 564 were screened at postpartum , and 131 had ifg and 32 had diabetes by fpg ( age- and race / ethnicity - adjusted rates 24.2 [ 95% ci 20.27.8 ] and 6.1 [ 4.28.1 ] , respectively ) . among women who delivered in 20042006 , 2,381 women were screened , and 583 had ifg and 80 had diabetes by fpg ( age- and race / ethnicity - adjusted rates 24.3 [ 22.626.0 ] and 3.3 [ 2.64.0 ] , respectively ) .", "the proportion of women receiving an ogtt increased from 1995 ( 5.0% ) to 2005 ( 16.6% ) and markedly increased in 2006 ( 71.5% ) . in 2006 , kpnc instituted a nurse managed care program that included greater attention to postpartum screening guidelines . among the 600 women who underwent a 75-g ogtt in 2006 , 16 had diabetes at postpartum . diabetes was diagnosed in 4 ( 25% ) by fpg alone . of the remaining 12 women , 8 ( 50% ) had ifg and 4 ( 25% ) had normal fpg . of the 188 women who were found to have pre - diabetes ( i.e. , either ifg or igt according to the fasting or 2-h glucose values measured during the 75-g ogtt ) , only 114 ( 60% ) were diagnosed with ifg ; 74 ( 40% ) would have been classified as having normal glucose tolerance on the basis of fpg alone . therefore , 78 ( 38% ) of the 204 women with either diabetes or pre - diabetes were identified only by the 2-h glucose measurements .", "in a managed care plan with a large number of women with gdm pregnancies , we found that between 1995 and 2006 , screening for postpartum diabetes increased from 20.7 to 53.8% . the increase in screening performance is not likely to be due to advancing maternal age or changes in the racial / ethnic composition of women with gdm , as this trend in screening performance was similar after adjustment for age and race / ethnicity , and almost the entire population of pregnant women was screened for gdm between 1995 and 2006 . although the proportion of women with ifg on their postpartum screen did not significantly change over time , the proportion of women with diabetes ( diagnosed by fpg levels ) at postpartum decreased by 50% . this observed decrease in diabetes among women with postpartum screening is not likely to be a consequence of the small increase ( 3% ) in gdm screening over time . the decrease is more likely because of better identification of diabetes before pregnancy , as suggested by the reported increase in postpartum screening among women with gdm and because of an increase in glucose screening in postpartum women without gdm ( 1.9% in 1995 vs. 8.2% in 2006 ) . as in other reports ( 58 ) , the majority of women in our cohort did not undergo postpartum diabetes screening in the early years of the study . it is possible that health care providers might have recommended more postpartum screening among these racial / ethnic groups , given their higher prevalence of diabetes ( 19 ) . it is also possible that asian women were more likely to have had a recent physical examination , giving the health care provider the opportunity to recommend screening , as suggested by racial / ethnic differences in access to care among gdm women ( 20 ) . similar to other reports , we found that greater contact with medical care , either through a postpartum visit or other contacts , was associated with greater postpartum screening and may have provided additional opportunities to perform screening ( 6,7 ) . women who were more likely to be screened also were older and had higher educational attainment . although reasons are speculative , these women may have had greater awareness of their diabetes risk and the recommendation for screening . in women who were screened postpartum , gdm was diagnosed earlier in their index pregnancy , and they were more likely to have been treated with medications , which may have increased their and their provider 's awareness of their diabetes risk . glucose levels on the diagnostic 3-h ogtt during pregnancy were similar in women who were and were not screened , suggesting that these were not used to guide testing . as shown by others ( 21 ) , there was a suggestion that some of the women with a history of gdm who might have had a higher risk of developing diabetes during the postpartum period , such as those who were obese or with higher parity , were less likely to perform postpartum screening . the american diabetes association ( 16 ) , the american college of obstetricians and gynecologists ( 17 ) , and the fifth international congress workshop for gestational diabetes ( 15 ) endorse the postpartum ogtt and fpg to different extents . if only fpg were used in postpartum screening , 74 ( 40% ) cases of igt and 16 ( 75% ) cases of diabetes would have been missed . kitzmiller et al . ( 19 ) reported that among 527 women with gdm , at postpartum 16.5% had isolated igt , only 16% of women in whom diabetes was diagnosed met the criteria for both elevated fpg and 2-h values , and 21 of 25 women met the criteria for diabetes according to their 2-h values alone . hunt and conway ( 21 ) also reported that one - third of their postpartum gdm cohort undergoing the ogtt and who had diabetes or pre - diabetes had isolated 2-h elevations . their results are very similar to those found in this study : 78 of 204 women compared with 41 of 117 ( or 38% vs. 35% ) . therefore , the greater convenience of the fpg needs to be weighed carefully against its decreased sensitivity , particularly among women with a history of gdm . we were not able to distinguish whether the lack of screening occurred because of a lack of provider order or other reasons . however , provider orders for screening might occur only after negotiation with the patient , and a lack of provider order may , at least in part , reflect women 's objections to the test . we defined obesity by using race / ethnicity - specific percentiles , rather than height - to - weight ratios , thus introducing the possibility for misclassification and artificially decreasing the association between obesity and screening to the null . information on other confounders , such as family history of diabetes , was not available from electronic records . because the population of women with gdm is of reproductive age , postpartum screening and subsequent diagnoses of diabetes affect not only the mothers but also future pregnancies . the risk of complications , particularly stillbirths and congenital abnormalities , may be reduced with optimal glycemic control before the subsequent pregnancy ( 1 ) . prepregnancy glycemic control might also reduce the risk of the infant to the in utero exposure to hyperglycemia that might lead to childhood obesity and diabetes ( 22 ) . a diagnosis of pre - diabetes would identify women at high risk of future maternal diabetes , but this risk could be reduced through the application of interventions such as thiazolidinediones , metformin , or intensive lifestyle modification ( 3,4 ) . we conclude that , among women with a gdm history , postpartum diabetes screening has increased , but screening is still suboptimal . performance of an fpg alone , as opposed to the ogtt , will miss a subpopulation of women at risk ." ]

[ "protein phosphorylation and dephosphorylation are central events in cell recognition of external and internal signals , leading to specific responses . while protein kinases transfer a phosphate group from atp to a protein ( i.e. , phosphorylate ) , protein phosphatases catalyze the removal of phosphate groups from specific residues of proteins ( i.e. , dephosphorylate ) [ 1 , 2 ] . the balance between the antagonistic activities of protein kinases and phosphatases are responsible for many cellular functions , including metabolic pathways , cell - cell communication , proliferation , and gene transcription . the complete genome sequencing of various microorganisms made it possible to assemble the kinome and phosphatome of a few trypanosomatids [ 4 , 5 ] . these strategies have brought new perspectives of researches in the areas of biochemistry , physiology , and genetics , providing knowledge about the microorganisms ' life cycles , as well as predicting diagnostic biomarkers , novel drug targets and vaccine candidates against parasitic infections . parasites engage a plethora of surface and secreted molecules in order to attach and enter mammalian cells . some of these molecules are involved in triggering specific signaling pathways both in the parasite and the host cell , which are critical for parasite entry and survival . plasma membranes of cells contain enzymes that are oriented with their active sites facing the external medium rather than the cytoplasm , which are important for host - parasite interactions [ 7 , 8 ] . in the case of an ectoenzyme other criteria can be included as : ( 1 ) the enzyme has to act on extracellular substrate , ( 2 ) cellular integrity is maintained during enzyme activity , ( 3 ) the products are released extracellularly , ( 4 ) the enzyme is not released to the extracellular environment ; and ( 5 ) the enzyme activity can be modified by nonpenetrating reagents [ 7 , 8 ] . supporting this idea , the presence of surface - located phosphatases , called ecto or extracytoplasmic phosphatases have been characterized in several microorganisms . however , the physiological roles of these enzymes in these cells are not well established yet . in eukaryotes , thus , catalytic signature motifs and substrate preferences classified these proteins into four major groups : phosphoprotein phosphatases ( ppps ) , metallo - dependent protein phosphatases ( ppms ) , aspartate - based phosphatases with a dxdxt / v motif ( the members of these three groups are ser / thr specific phosphatases ) and the distinct group of protein tyrosine phosphatases ( ptps ) . protein tyrosine phosphatases belong to three evolutionarily unrelated classes : protein tyrosine phosphatases ( ptps ) , cdc25 and low molecular weight phosphatases ( lmw - ptps ) , which have a common motif ( cx5r ) in their catalytic sites . the classical ptps are classified , depending on the presence or absence of transmembrane domains , into receptor or nonreceptor type phosphatase groups . the use of inhibitors , divalent cations , metal chelators and different ph range has also been an important tool for classification of these enzymes . likewise , phosphatases may be acid or alkaline according to their ph range for activity . the optimum ph for acid ectophosphatases lies on the acid range ( ph values between 4.5 and 5.5 ) , while the optimum ph for alkaline ectophosphatases lies on the alkaline range ( ph values between 8.0 and 9.0 ) [ 9 , 10 ] . the inhibitors classically used include : phosphotyrosine phosphatase inhibitors ammonium molybdate and sodium orthovanadate ; acid phosphatase inhibitor sodium fluoride ( naf ) ; secreted phosphatase inhibitor sodium tartrate ; alkaline phosphatase inhibitor levamisole and phosphoserine / threonine phosphatases inhibitors okadaic acid and microcystin - lr [ 1113 ] . these enzymes may provide microorganisms with a source of inorganic phosphate by hydrolyzing phosphomonoester metabolites [ 1315 ] protect them upon entering the macrophage by suppressing the respiratory burst , as well as play a role in cell differentiation , infection of host cells [ 1820 ] and protecting the cells from acidic conditions by buffering the periplasmic space with phosphate released from polyphosphates . some protein phosphatases have been described as being active towards low molecular weight nonproteic phosphoesters , such as alkyl and aryl phosphates , including the phosphotyrosine analog , p - nitrophenylphosphate . from a general standpoint , the surface accessibility of ectophosphatases , along with protein phosphorylated on serine / threonine / tyrosine residues at the cell surface make this set of enzymes a key tool for the survival of pathogens in hostile environments and escaping the host immune responses [ 19 , 2224 ] . in this review , we describe the role of ectophosphatase activities in host - parasite interactions , particularly ectophosphatases in parasitic protozoa and fungi .", "little is still known about the physiological role of protein phosphatase activity in trypanosomatids , even though the first demonstration of this activity in trypanosoma brucei and t. cruzi took place in 1972 . the kinetoplastid parasites have complex life cycles and some of their life forms are difficult to grow in culture , which may represent a problem for studying ectophosphatases . pathogenic trypanosomatids have at least two different host environments in their life cycles , an insect vector and a mammal . also , each trypanosomatid genus has different abilities to survive and reproduce in such hosts . t. cruzi invades and replicates in many cell - types , including macrophages , fibroblasts and myocytes . t. brucei is an exclusively extracellular parasite that resides in the bloodstream of the mammalian host . as the life cycles of these parasites take place through widely different environments , frequent and substantial adaptive changes are required in many cell processes , resulting in changes in gene expression , protein levels and protein modifications [ 26 , 27 ] . along with those , cell surface components play a key role in the survival of protozoan parasites in hostile environments and in confrontation with host immune responses . since , these flagellates have an unusual composition of phosphatases with the ptp family being greatly reduced while the stp family is expanded by comparison with human phosphatases . the low similarity to their vertebrate counterparts indicates that these enzymes may be potentially suitable targets for development of potent inhibitors with minimal effects on the physiology of mammalian hosts . under these conditions , ectophosphatases play an important role in the interaction of cells with their surroundings , especially because their catalytic sites face the extracellular milieu . ecto - phosphatases has been reported in some protozoa parasites , such as t. rhodesiense , t. congolense , t. brucei [ 30 , 31 ] , t. cruzi , t. rangeli [ 13 , 33 ] , some leishmania species [ 11 , 34 ] , herptomonas muscarum muscarum , phytomonas spp . [ 36 , 37 ] , entamoeba histolytica , giardia lamblia and trichomonas vaginalis . in general , these ectoenzymes are usually reported to have optimum activities in the acidic ph range , and they are therefore also known as membrane - bound acid phosphatases [ 28 , 29 ] . in trypanosomatids , the low optimum ph and the surface location of these enzymes suggest its role in an acidic microenvironment and/or a close relationship with lysosomal digestion , possibly reflecting an adaptation of the parasite to the intracellular or phagosomal environment [ 41 , 42 ] . cloning and purification of an acidic phosphatase in t. brucei suggest that these enzymes may represent a new ectophosphatase class lacking homology to other known phosphatases . it seems that these proteins are related to the regulation of t. brucei development , since these acidic phosphatases are expressed in bloodstream forms , but not in the insect procyclic form . likewise , an ectophosphatase activity on the surface of intact procyclic and bloodstream forms of t. brucei was demonstrated by fernandes et al . [ 43 , 44 ] . similarly , an ectophosphatase was also cloned and purified in l. mexicana , where it was located in the endosomal / lysosomal compartment between the flagellar pocket and the nucleus in wild - type promastigotes , and the overexpression of this protein leads to its abundant exposure on the cell surface [ 45 , 46 ] . the same was seen with membrane - bound acid phosphatase from the bloodstream form of t. brucei , where the enzyme is supposed to participate in the maintenance of endocytosis / exocytosis and in differentiation to the insect stage . the wide distribution of acid phosphatases on the cell may reflect some physiological adaptation for parasite survival within the host . in this scenario , ectophosphatase activities were identified at the cell surface of all t. cruzi development stages : epimastigote , trypomastigote and amastigote forms [ 18 , 32 ] . it seems that in amastigote forms these enzymes are magnesium - dependent and can hydrolyse phosphoaminoacids and phosphoproteins under physiological conditions [ 18 , 32 ] . this behavior could facilitate the interaction between parasite and host cells , once t. cruzi phosphatases leads to dephosphorylation of proteins important in the signal transduction pathway or cycle regulation of this protozoan parasite . supporting this idea , y strain presents mg - dependent ectophosphatase activity , while colombiana strain expresses mg - independent activity . among other characteristics , members of these two groups parasites from the colombiana strain appeared to be more infective to myoblasts than those from the y strain , while the latter is more infective towards macrophages than the parasites of the colombiana strain . intriguingly , platelet - activating factor ( paf ) , a phospholipid mediator involved in differentiation cellular in t. cruzi , induces the secretion of an ectophosphatase in these parasites , associating this event with the infectivity of the parasite . addition of sodium orthovanadate ( a protein tyrosine phosphatase inhibitor ) in the interaction medium from l. amazonensis and macrophages significantly increased parasite binding and internalization , suggesting that leishmania induces tyrosine phosphorylation [ 24 , 51 ] . under these conditions , protein tyrosine kinase - linked pathways regulate the leishmania promastigote invasion , which ectophosphatase activity upregulate l. amazonensis binding ligands for macrophage receptors and intracellular survival within these cells [ 24 , 51 , 52 ] . it seems that during macrophage infection by leishmania the parasite attenuates map kinase signaling , as well as c - fos and inos expression in macrophages , stimulating the phosphotyrosine phosphatase activity in these cells [ 5355 ] . possibly by other intracellular pathogens as a strategy of the parasites to interact and survive within their hosts . in l. donovani tyrosine phosphatase activity was also detected , suggesting that tyrosine phosphorylation occurs , though not via receptor tyrosine kinase or tyrosine kinase - like activities but very likely due to the activity of atypical and/or dual specific kinases . futhermore , a membrane - bound ptp has been describe in l. major metacyclic promastigote forms , which is translocated to the cytoplasm in promastigotes . in spite of the increased level of the molecule in metacyclic promastigotes compared to the procyclic forms , the specific activity of the enzyme was lower in metacyclic than in procyclic promastigotes . interestingly , a protein tyrosine phosphatase , has been identified in l. major ( lmptp1 ) that allows amastigotes forms to survive in mice . although its biological function is unclear , this may be an important factor in virulence , enabling the invading pathogen to survive in a host . ecto - phosphatase isolated from l. donovani promastigotes inhibits the production of superoxide anions in intact human neutrophils . this activity could contribute to the survival of the parasite within the host , we can hypothesize that parasites with greater ectophosphatase activity would be more resistant to oxidative bursts from the host 's immune system . the role of ectophosphatases in invasive amoebiasis is still unknown , even though two acid phosphatases have been characterized in these parasites : a membrane - bound acid phosphatase ( map ) [ 58 , 59 ] and a phosphatase that is secreted to the culture medium ( sap ) , as well as to the cell interface in amoebic liver abscess [ 60 , 61 ] . these enzymes may be associated with cellular adhesion processes , since the invasive e. histolytica showed much higher ectophosphatase activity when compared to the noninvasive counterpart and the free - living e. moshkovskii .", "the fungal cell wall is a compact albeit dynamic structure that plays important roles in several biological processes determining cell shape , morphogenesis , reproduction , cell - cell and cell - matrix interactions , osmotic and physical protection . several different cell wall components have been characterized such as specific enzymatic activities , heat - shock proteins , glycosphingolipids ( gsl ) , melanin , histone and integrin - like proteins . even though the roles of ectophosphatases in fungi are still largely unknown , the cellular distribution of ectophosphatases , together with their ability to interfere with physiologic processes through the removal of phosphate groups of regulatory proteins , suggest a task for these molecules during the infection of host cells . the presence of surface - located acid phosphatases , called ecto or extracytoplasmic phosphatases has been demonstrated in nonpathogenic yeast saccharomyces cerevisiae and in pathogenic species such as candida albicans , candida parapsilosis [ 19 , 65 ] , sporothrix schenckii , aspergillus fumigatus , fonsecaea pedrosoi [ 22 , 68 ] , cryptococcus neoformans and pseudallescheria boydii . futhermore , most of the phosphatases synthesized under pi - limiting conditions are either located on the extracellular medium or are associated with the plasma membrane or cell wall [ 15 , 22 ] . corroborating with this hypothesis , kneipp et al . demonstrated that conidial forms of f. pedrosoi has an ectophosphatase activity modulated by exogenous phosphate . it seems that in f. pedrosoi , conidial cells that were cultivated in a pi - depleted medium had an ectophosphatase activity significantly higher than that of fungal cells grown in the complete medium . these cells expressing high phosphatase activity were significantly more capable of adhering to epithelial cells and fibroblasts than fungi expressing basal levels of enzyme activity . it was then proposed that the removal of phosphate groups from surface proteins in host cells could result in conformational transitions and in an attenuated electrostatic repulsion between fungal and epithelial cells . probably , the removal of inorganic phosphate could therefore expose at the host surface additional sites for interaction with infectious agents . it seems that ectophosphatases may contain adhesive domains that could directly promote the attachment of fungal cells to their hosts , therefore functioning similarly to the well - characterized microbial adhesins . probably , they could regulate the functional activation of surface adhesins , which would be the key structures mediating fungal attachment . intriguingly , known activators of signaling pathways and cell differentiation , paf and propanolol , promoted an enhancement of f. pedrosoi ectophosphatase activity suggesting that f. pedrosoi ectophosphatase may be considered a surface marker for morphological transition and infection . in the fungus c. neoformans a thick capsule composed of neutral and charged polysaccharides , can be modulated by different environmental conditions , including the sites of fungal infection inside the host . it seems that the molecules coating the outer layer of the cell wall could be relevant during the interaction of poorly encapsulated cells with host tissues . ectoenzymes possibly have their accessibility to external receptors masked by the capsule polysaccharides of c. neoformans , diminishing the potential of these structures to be surface molecules influencing the interaction between fungal and host cells . . however , the levels of enzyme activity , varied considerably among the isolates and no correlation between enzyme activity and capsular size or serotype was observed . evidences show that isolates with capsular polysaccharides of the same serotype varied greatly in ectophosphatase activity . in addition , the strain , which is poorly encapsulated , removed phosphate groups much more efficiently than strain , which expresses a large capsule , indicating that the presence of the capsule impairs enzyme activity in this process . on the other hand , some encapsulated strains presented levels of ectophosphatase activity higher than that observed in the acapsular mutant . moreover , some strains that had very similar levels of enzyme activity , but differ greatly in capsule size were also found . taken together , these data indicate that differences observed in enzyme activity should be derived from natural variation of ectophosphatase expression in different c. noeformans strains . corroborating with the previous findings , kiffer - moreira et al . investigated three different isolates of c. parapsilosis , including a laboratory - adapted strain ( cct 3834 ) and two recently isolated strains ( rfo and h297 ) . they observed that the rfo strain exhibits the highest levels of enzyme activity and adhesion to cho cells , followed by the h297 and the cct 3834 isolates . pretreatment of yeasts with the irreversible inhibitor sodium orthovanadate caused a significant reduction in the ability of these fungi to attach to epithelial cells . although sodium orthovanadate can affect different biological processes and inhibit atpases involved in cation transport [ 72 , 73 ] , its major biological activity in living cells occur on the cell surface , as the oxidation reduction reactions that take place in the cytoplasm diminish its inhibitory effect . similarly , c. albicans isolate from oral cavities of hiv - infected children ( hiv ) present an ectophosphatase activity significantly higher than the hiv - negative children ( hiv ) . the c. albicans yeasts from hiv patients showed higher indices of adhesion to epithelial cells , which suggests that the activity of fungal acidic surface phosphatases may contribute to the early mechanisms required for disease establishment . it is reasonable the hypothesis that ectophosphatases represent a virulence marker , since these enzymes represent part of the outer layer and are linked to cell differentiation and host cell - pathogen interactions .", "the balance of phosphorylation - dephosphorylation of serine , threonine and tyrosine residues modulates signaling pathways critical for determining the outcome of multiple cellular functions . further studies are warranted to resolve the roles of ectophosphatases in host - pathogen interactions , as well as the possible correlations between the expression of these enzymes and the clinical manifestation of the diseases ." ]

[ "during the recent decade , a number of researchers have described the growing psychosocial risk factors for coronary heart disease ( chd ) , which include depression , anxiety , stress , and alexithymia that decrease the health - related quality of life and protective factors such as social support . studies carried out internationally highlight that besides negative psychological factors like depression , anxiety , and stress , positive factors such as social support play a protective role in the onset and progress of chd . in 1973 , sifnoes proposed the term alexithymia to imply a cognitive affective disturbance , characteristic of persons who can not describe their feelings or elaborate their fantasies . it has been characterized more generally as an emptiness of feeling , a poverty of imagination or a life of fantasy , difficulty in communicating with others , and a lack of positive emotions with a high prevalence of negative emotions . alexithymia is associated with states of negative affect . which increase the possibility of somatic complaints . the assumption is that these characteristics would lead to a deficiency in cognitive processing and regulation and adjustment of emotions , and are associated with starting and/or continuing some of the psychiatric and medical disorders . some researchers believe that dysfunction in the limbic system , brain abnormal lateralization , and/or difficulty in the efficiency of hemispheric communication are the causes of its emergence . the prevalence of alexithymia in working age populations has been shown to be about 9 - 17% for men and 5 - 10% for women . at the population level , alexithymia is associated with older age , lower socioeconomic status , fewer years of education , single marital status , and poorer perceived health . alexithymia has been shown to be associated with several medical conditions and mental health problems . an association between alexithymia and dissatisfaction with life has been found in some studies of coronary heart disease patients . one study investigated factors associated with alexithymia in patients ( n = 153 ) with coronary heart disease verified by coronary angiography . in chd patients , alexithymia was unrelated to cardiovascular diseases , but was related to depression and decreased life satisfaction . in the kuopio ischemic heart disease risk - factor study , 2682 middle - aged men were studied using the 26-item toronto alexithymia scale ( tas ) . alexithymia was associated with greater self - reporting of symptoms during exercise - tolerance testing and it was found that the extent of carotid atherosclerosis was inversely related to the degree of alexithymia , suggesting a biological connection to atherosclerosis . in this study , men in the highest alexithymia quintile were at a two - fold greater risk of death from any cause , while no association was found between behavioral or physiological risk factors and mortality . lumley et al . , observed that the degree of alexithymia ( tas-20 ) was associated with the presence or absence of chest pain during exercise testing , but not with chd ( n = 180 ) . they concluded that alexithymia was associated with an increased risk of illness - related behavior , but not necessarily with the presence or severity of chd . one study to testing the validity of existing conceptualizations of the alexithymia concept in the general adult population described that older adults engaged in less introspective thoughts , traditionally thought to denote increased alexithymia . difficulty in identifying and describing emotions did not differentiate older and younger adults , but they were both associated with heightened depression , anxiety , and poor perceived quality of life . alexithymia in somatoform and depressive disorders ( 2003 ) was investigated in one study ( two groups of 30 subjects each , bearing diagnoses of somatoform disorder and depressive disorder , respectively , and one group of 30 normal controls ) and the results showed that the mean of alexithymia scores in the somatoform ( 60.4 ) and depressive disorder groups ( 62.5 ) were higher than in normal subjects ( 54.2 ) . even as the total alexithymia scores did not differentiate somatoform from depressive disorders , the depressed subjects had greater difficulty in expressing their feelings . kauhanen et al . , noted more physical symptom - reporting with high alexithymia , and myers observed alexithymia - elevated anxiety scores . linden et al . , noted that high alexithymia showed smaller heart rate responses to the stress task and more anger in behavior . 2009 ) investigated the relationship between alexithymia and the health - related quality of life ( hrqol ) in a nationally representative population sample of 5,418 subjects , of age 30 to 97 years , and concluded that alexithymia could be a predisposing factor to a poorer hrqol . a study investigated the relationship between alexithymia and the health - related quality of life ( hrqol ) and concluded that the alexithymic group had significantly ( p < 0.001 ) lower mean scores on every dimension of hrqol , even after controlling for confounding demographic variables , somatic diagnoses , and depressive and anxiety disorders .", "this research was a cross - sectional study that examined the causal relationship between alexithymia and dass ( depression , anxiety , stress ) , quality of life , and social support . the sample consisted of 398 chd patients ( 166 females and 232 males and age : 40 - 70 years ) , who were randomly selected from the isfahan chamran heart center and isfahan cardiovascular research center ( icrc ) ( 2013 ) and the other heart clinical center . the patients filled in five questionnaires assessing depression , anxiety , stress ( dass-21 ) , alexithymia ( tas-20 ) , health - related to quality of life ( whoqol-26 ) , multiple scale of perceived social support ( mspss-12 ) , and another questionnaire , which obtained demographic information . before attending the study , each patient had been examined by a cardiologist , who had confirmed the diagnosis as coronary heart disease . alexithymia - tas-20 scale : the tas is a 20-item instrument that is one of the most commonly used measures for alexithymia . the tas-20 has three subscales : 1.1 . - difficulty describing feelings subscale ( ddf ) is used to measure the difficulty in describing emotions . , which has five items : 2 , 4 , 7 , 12 , 17 . 1.2 . - difficulty identifying feelings subscale ( dif ) is used to measure the difficulty in identifying emotions . , which has seven items : 1 , 3 , 6 , 11 , 9 , 13 , 14 - externally - oriented thinking subscale ( eot ) is used to measure the tendency of individuals to focus their attention externally , which has eight items : 5 , 8 , 10 , 15 , 16 , 18 , 19 , 20 . the items are rated using a five - point likert scale whereby 1 = strongly disagree and 5 = strongly agree . the total alexithymia score is the sum of responses to all the 20 items , while the score for each subscale factor is the sum of the responses to that subscale . the tas-20 uses cutoff scoring : equal to or less than 51 = non - alexithymia , equal to or greater than 61 = alexithymia . it demonstrates good internal consistency in the normal iranian sample ( cronbach 's alpha = 0.81 ) and test retest reliability ( 0.77 , p < 0.01 ) . the internal consistency reported in a clinical sample from iran , using cronbach 's alpha for the total tas-20 scale and subscales of dif , ddf , and eot , were . 66 , respectively , and the total validity using test retesting for tas-20 and its subscales dif , ddf , and eot were . 2-dass scale-21 : the dass scale is a set of three self - report scales designed to measure the negative emotional states of depression , anxiety , and stress . each of the three dass scales contains seven items , divided into subscales of two to five items with a similar content . the depression scale assesses dysphoria , hopelessness , devaluation of life , self - deprecation , lack of interest / involvement , anhedonia , and inertia . the anxiety scale assesses autonomic arousal , skeletal muscle effects , situational anxiety , and subjective experience of anxious affect . 2.3 . it assesses difficulty in relaxing , nervous arousal , and being easily upset / agitated , irritable / over - reactive and impatient . subjects are asked to use the four - point severity / frequency scales to rate the extent to which they have experienced each state over the past week . scores for depression , anxiety , and stress are calculated by summing the scores for the relevant items . the assumption on which the dass development has been based ( and which has been confirmed by the research data ) is that the differences between depression , anxiety , and stress experienced by normal subjects and the clinically disturbed , are essentially differences of degrees . the validity and reliability of this scale was reviewed by samani and joker ( 2007 ) , who reported that reliabilities for depression , anxiety , and stress scale were . 3 . multiple scale perceived social support : the 12-question multidimensional scale of perceived social support ( mspss ) was used to measure the degree of social support each participant felt he or she had . high levels of perceived social support are generally associated with low levels of depression and anxiety . family ; 2 . friends ; 3 . significant others ) based on the seven - point likert measures , which measures the respondents perceptions of the availability of various types of social support such as family , friends , and significant others the remaining 24 items comprise four dimensions of health including , physical , psychological , social , and environmental . all scores are transformed to reflect 4 - 20 for each domain , with the higher scores corresponding to a better qol . the individual 's perception of quality of life is measured by summing the total scores for each particular domain . all domain scores are scaled in a positive direction ( higher score indicates a higher qol ) . this questionnaire was standardized on a sample of 2,956 healthy and 2,936 unhealthy rural and urban inhabitants ( age 30 years and above ) from isfahan , najaf - abad , and arak , who participated in the isfahan healthy heart program ( ihhp ) from two dissimilar iranian provinces , during 2006 . demographic questionnaire : this questionnaire consists of questions about age , sex , marital status , education , and so on .", "table 1 shows the patients sociodemographic characteristics based on age , sex , marital status , and education . patients sociodemographic characteristics ( n = 398 ) table 2 shows patients descriptive characteristics such as mean and standard deviation . patients descriptive characteristics cronbach 's alpha was calculated as 0.74 for the toronto alexithymia scale ( tas ) , 0.89 for the depression , anxiety , and stress scale ( dass ) , 0.87 for the multidimensional scale of perceived social support ( mspss ) , and 0.90 for the world health organization quality of life questionnaire ( whoqol ) . based on the conceptual model [ figure 1 ] of the study , the structural model of variables ( i.e. , tas , dass , mspss , and whoqol scores ) was presented using the outputs of amos . although in traditional statistical methods , the researcher uses a single measure to verify the null hypothesis , the complexity of the structural equation modeling eliminates this single measure . therefore , three main groups of indices ( absolute fit , comparative fit , and parsimonious fit indices ) are used to fit a model . the conceptual model of alexithymia according to table 3 [ relative chi - square ( cmin / df ) = 2.580 and df = 58 for default model ] , the model fitted well . even as most experts consider relative chi - square values of 2 - 3 to show a good fitting model , schumacher and lomax expanded the range to 1 - 5 . chi - square ( cmin ) values and number of parameters table 4 shows the absolute fit indices . goodness of fit index ( gfi = 0.94 ) and adjusted goodness of fit index ( agfi = 0.91 ) suggested good fitting of the default model ( values between 0 and 1 for these two indices indicate good fitting ) . table 5 shows the comparative fit index ( cfi ) , parsimony comparative fit index ( pcfi ) , and root mean square error of approximation ( rmsea ) . as is seen , the calculated values ( cfi = 0.96 and pcfi = 0.71 ) suggest a good fit for the default model ( cfi > 0.90 and pcfi > 0.50 are favorable ) . rmsea can be calculated for confidence intervals , to determine the significance of the difference between the obtained value for the model and zero . as rmsea < 0.08 is acceptable , our default model fitted well ( rmsea = 0.06 ) . in addition , the holter index of 204 was again indicative of a well - fitting model . comparative and parsimonious fit indices according to all the mentioned indicators , the default model presented a good fit for the relationship between alexithymia and depression , anxiety , stress , quality of life , and social support .", "based on the conceptual model [ figure 1 ] of the study , the structural model of variables ( i.e. , tas , dass , mspss , and whoqol scores ) was presented using the outputs of amos . although in traditional statistical methods , the researcher uses a single measure to verify the null hypothesis , the complexity of the structural equation modeling eliminates this single measure . therefore , three main groups of indices ( absolute fit , comparative fit , and parsimonious fit indices ) are used to fit a model . the conceptual model of alexithymia according to table 3 [ relative chi - square ( cmin / df ) = 2.580 and df = 58 for default model ] , the model fitted well . even as most experts consider relative chi - square values of 2 - 3 to show a good fitting model , schumacher and lomax expanded the range to 1 - 5 . chi - square ( cmin ) values and number of parameters table 4 shows the absolute fit indices . goodness of fit index ( gfi = 0.94 ) and adjusted goodness of fit index ( agfi = 0.91 ) suggested good fitting of the default model ( values between 0 and 1 for these two indices indicate good fitting ) . table 5 shows the comparative fit index ( cfi ) , parsimony comparative fit index ( pcfi ) , and root mean square error of approximation ( rmsea ) . as is seen , the calculated values ( cfi = 0.96 and pcfi = 0.71 ) suggest a good fit for the default model ( cfi > 0.90 and pcfi > 0.50 are favorable ) . rmsea can be calculated for confidence intervals , to determine the significance of the difference between the obtained value for the model and zero . as rmsea < 0.08 is acceptable , our default model fitted well ( rmsea = 0.06 ) . in addition , the holter index of 204 was again indicative of a well - fitting model . comparative and parsimonious fit indices according to all the mentioned indicators , the default model presented a good fit for the relationship between alexithymia and depression , anxiety , stress , quality of life , and social support .", "alexithymia , a condition of inability to use words to understand the emotional states of oneself or others , prevents individuals from adjusting to their emotions . the present study has examined the predictive value of alexithymia in changes in depression , anxiety , stress , social support , and quality of life among patients with coronary heart disease . after fitting all the data , alexithymia shows significant positive relationships with depression , anxiety , and stress , as previous research has described . in other words , increasing levels of alexithymia and its two dimensions would worsen depression and anxiety in patients . sarrijarri has shown that exacerbation of depressive symptoms will cause difficulty in identifying , describing , and sharing their feelings with others . similarly , research has shown that alexithymia is more common in people with anxiety disorders and that anxiety is related to the two mentioned aspects of alexithymia . alexithymia is a cognitive - emotional disorder that impairs the adjustment of emotions , concentration , processing , and evaluation of cognitive - emotional information . therefore , individuals with alexithymia fail to manage their cognitive - emotional system and properly deal with stressful situations , particularly diseases . this strengthens the feelings of inadequacy and insufficiency in this group and intensifies negative emotions such as anxiety and depression . moreover , since emotional arousal is associated with physiological arousal , negative emotions ( e.g. anxiety and depression ) will bring about physical symptoms including pain , fatigue , and dizziness . finally , disruption of psychological defenses in subjects with alexithymia leads them to deny their emotions and makes them incapable of describing them . we also found difficulty in identifying feelings and difficulty in describing feelings to be related to depression and anxiety . matn and jenakar suggested difficulty in identifying feelings as the only aspect of alexithymia that predicts depressive symptoms . furthermore , they reported difficulty in both identifying and describing feelings as predictors of anxiety symptoms . previous studies have indicated consistent results in terms of the relationship between alexithymia and stress and inadequacy of coping styles with stress . this finding can be justified by considering the poor emotional intelligence of people with alexithymia , which combines with difficulties in identifying and describing feelings and reduces the ability of dealing with stressful situations . as the inability to cope with difficult situations imposes a lot of stress on the individual , a positive relationship exists between alexithymia and stress . another finding of this study was the negative relationship between alexithymia and social support and its aspects , that is , increasing alexithymia decreases social support . in fact , patients with alexithymia can not receive adequate social support from their family , friends , and/or significant others . seemingly , impaired emotional intelligence in these individuals results in their poor interpersonal skills and inability to receive social support . the results indicated that there is a negative relationship between alexithymia and quality of life . the strongest relationships were identified between difficulty in identifying feelings and psychological aspect of the quality of life ( r = - 0.48 ; p < 0.01 ) , physical aspect of quality of life ( r = - 0.43 ; p < 0.01 ) , social aspect of quality of life ( r = - 0.34 ; p < 0.01 ) , and environmental aspect of quality of life ( r = -0.24 ; p < 0.01 ) . available evidence about the association between the difficulty in identifying feelings and somatoform disorder can explain the negative effects of alexithymia on the quality of life . it also seems that difficulty in identifying feelings results in a false interpretation of physical feelings and decreases the health - related quality of life . in other words , alexithymia is not only an inner state , but is also associated with health outcomes . research has shown that people with alexithymia do not use normal coping styles , and may hence , have a higher tendency toward unhealthy behaviors such as suppression of emotions and drinking . patients might experience problems in different aspects of life , that is , negative emotions may cause psychological problems , wrong interpretation of physical health may lead to physical problems , social relations may be affected by difficulty in establishing good social relations , and environmental quality of life can be decreased due to inappropriate interactions with the environment . according to the results of the present study it is important to pay attention to alexithymia as an important variable associated with ( depression , anxiety , stress ) , social support , and quality of life of patients with coronary heart disease . it can be concluded that alexithymia is connected with the pathological indicators of cardiac disease ( depression , anxiety , and stress ) and its protective parameters ( social support and quality of life ) . most patients with heart disease are elderly and alexithymia is also more common among older people . therefore , conducting a longitudinal study to determine the precise role of alexithymia in the psychiatric pathology of cardiac diseases is essential . this research showed the relation between alexithymia and ( depression , anxiety , stress ) , social support , and quality of life , as a psychological structural model in chd . most of the relationships were associated with alexithymia , depression , anxiety , and stress ( dass : 49% ) and the lowest relationship was between alexithymia and social support ( mspss : 09% ) . of particular interest in the findings if alexithymia is considered to be a personality trait or state - dependent trait , alexithymic individuals are at risk for medical or psychiatric disorders and negative emotions . increasing our knowledge regarding the role of a negative affect may have a major implication on how future interventions are targeted , for example , if evidence supports the lack of emotional awareness ( e.g. depression , anxiety , and stress ) as an underlying factor for a subset of patients seeking interdisciplinary treatment , then intervention can be modified to focus on processes related to increasing emotional awareness and identification , with a hope to improve the quality of life in patients with coronary heart diseases . the limitations of the study were : inability to control the behaviors that were associated with alexithymia and could thus affect the relationship between alexithymia and depression , anxiety , stress , social support , and quality of life . as the study population was limited , generalization of the results to other patients had to be made with caution .", "the present study showed the structural relations between the psychological factors associated with alexithymia in coronary heart disease . the results highlight the importance of alexithymia as a risk factor associated with neuroticism ( anxiety , depression , and stress ) and social support which reduces the quality of life among coronary heart disease patients . in this regard , psychological interventions are recommended to reduce the consequences of this disorder , especially among coronary heart disease patients . conducting longitudinal studies in this area may lead to more interesting and favorable results , which is recommended to the researchers interested in this field ." ]

[ "tumor - induced osteomalacia ( tio ) is a rare paraneoplastic disease in which causative tumor ( generally of mesenchymal origin ) gives rise to an oncogenic osteomalacial syndrome , whose underlying pathogenetic mechanism is related to the tumor s production of a phosphaturic factor that reduces the renal reabsorption of phosphates and causes renal phosphate leak . the tumors are generally small , slowly growing and non - invasive . most are localized at long bone level , but they are frequently impossible to locate and the correct diagnosis may take 5 - 7 years . the clinical picture is characterized by non - specific symptoms , such as hyposthenia , worsening myalgia and bone pain ( mainly affecting load - bearing areas ) , and gait alterations ( anserine gait ) . until a correct diagnosis has been made patients usually undergo symptomatic treatments without success . those patients history is characterized by numerous pathological fractures , periprothesical fractures and prothesis mobilizations . the elective surgical treatment of tio is tumor resection , which normally leads to a complete regression of the osteomalacial syndrome . the consequent rehabilitation treatment must consider the complexity of the clinical - functional picture of the patient , especially if a substitution megaprothesis surgery is needed .", "the clinical picture is characterized by non - specific symptoms , such as hyposthenia , worsening myalgia and bone pain ( mainly affecting load - bearing areas ) , and gait alterations ( anserine gait ) . until a correct diagnosis has been made patients usually undergo symptomatic treatments without success . those patients history is characterized by numerous pathological fractures , periprothesical fractures and prothesis mobilizations . the elective surgical treatment of tio is tumor resection , which normally leads to a complete regression of the osteomalacial syndrome . the consequent rehabilitation treatment must consider the complexity of the clinical - functional picture of the patient , especially if a substitution megaprothesis surgery is needed .", "the clinical history of pm ( born in 1945 ) began in 2000 , when she consulted her general practitioner because of widespread costal and vertebral pain of no known traumatic cause , mainly localized in the dorso - lumbar region . the x - ray findings were negative , and she was prescribed non - steroidal anti - inflammatory drugs treatment without receiving any benefit . the picture remained unchanged until 2003 , when bone scintigraphy revealed areas of the pathological accumulation of the radiolabeled tracer in the proximal third of the right femoral diaphysis , some costal arches and an area of bone rarefaction in the right humeral diaphysis . further laboratory tests revealed hypocalcemia , and so the patient started pharmacological treatment with bisphosphonates , calcium and vitamin d3 for suspected advanced osteoporosis . because of the persistence of the painful symptoms , a bone biopsy of the humeral lesion was performed ; that led to the diagnosis of a spindle cell mesenchymal tumor , morphologically arising from a glomus tumor . given the suspicion of tio , the patient underwent octreotide scintigraphy , which revealed increased tracer uptake at the level of the medio - proximal third of the right humerus . in january 2005 , she underwent a first surgical intervention that involved curettage of the lesion , an autograft from the ipsilateral tibia , and a bone biopsy , thus allowing the final diagnosis of a phosphaturic mesenchymal tumor . as a result of an accidental fall in july 2005 , the patient suffered a subcapital fracture of the right femur and a bifocal fracture of the previously treated right humeral diaphysis . laboratory tests once again revealed hyperphosphaturic hypophosphatemia , and so she therefore underwent dual surgery involving the implantation of a bi - articular endoprosthesis cemented to the femur , an osteosynthesis of the humerus with a screwed plate and then sent to the rehabilitation department . in 2007 , worsening humeral pain associated with disease recurrence ( confirmed by means of instrumental investigations ) led to the patient being readmitted to hospital to undergo to a segmental resection of the diseased humerus and its reconstruction by means of the insertion of a cemented 8-holed plate which , soon afterwards , were replaced with a new ipsilateral tibial graft in order to provide medial reinforcement after a distal screws displacement ( figure 1a ) . the patient s clinical condition remained stable until april 2009 when , during an outpatient follow - up examination , she complained pain in her right hip radiating to the knee and accentuated by weight bearing . x - rays revealed loosening of the femoral stem with bone reabsorption around the cemented area . the prosthetic stem was revised and a new femoral stem was implanted ( figure 1b ) . in march 2010 the pain in the right hip and femur reappeared and subsequent x - rays showed partial progressive subsidence of the prosthetic stem to a depth of about 2.5 cm ( figure 1c ) . furthermore , examinations of the humerus revealed the partial mobilization of the proximal screws ( figure 1d ) . in september 2010 the patient underwent to a new revision surgery , and due to a poor cortical bone resistance a 22 cm resection from the apex of the great trochanter were executed and a large - resection modular prosthesis was implanted . the stem was cemented and trevira mesh was used proximally to attach the extra - rotary thigh musculature in order to reconstruct the articular capsule ( figure 1e - f ) . the postoperative course was normal and the patient started rehabilitation training as per ad hoc protocol ( table 1 ) . the functional outcomes and efficacy of the rehabilitation program were evaluated using the following scores ( table 2 ) : range of motion , visual analogue scale , barthel s index , toronto extremity salvage score , and musculoskeletal tumor society scale . since the 1 week of permanence in the g. pini oncological orthopedic surgery the patient started distal mobilization exercises of the treated limb to prevent venous and lymphatic pooling , selective isometric exercises for maintaining the tone and trophism of the main anti - gravitational muscles ( gluteal muscles from day 3 - 4 ) , and self - mobilization and strengthening exercises of the body districts not involved in the surgery . from the 2 week , the patient has substituted a lower limb splint with a newport hip orthosis ( figure 2 ) locked in position 0. the patient and her relatives were trained in correct wearing of the support by technical staff and nurses . from day 12 , the by now clinically stable patient started the training for the recovery of orthostasis and short - distance transfers ( from bed to wheelchair ) with no weight bearing . the back of the wheelchair used was fully reclined in order to avoid the patient to flex the hip . it was necessary to make these movements for keeping the hip in position 0 , the on - bed transfer has been made on the operated limb side , with maximum assistance of the therapist in order to avoid any rotation of the lower limb . together with the re - education of postural transfers , the patient was trained in how to reach and maintain a standing position with the support of a 4-legged walking frame ( to guarantee the greatest stability ) with the treated limb just touching the floor . from the 3 week were added exercises using a 3-stage technique . the forward movement of the limb was compensated by a sagittal oscillation of the trunk to avoid hip flexion - extension . in order to allow the patient to adequately oscillate the operated limb without flexing the hip , she was initially asked to provide contralateral support in an equine manner ( to avoid a swinging forward movement ) balanced by the counter - oscillation of the trunk on the coronal plane . the therapeutic exercises proposed to the patient in a prone lying position had the aim of consolidating the objectives set from the 1 week and mobilizing the knee while keeping the hip extended . in addition to guaranteeing the early recovery of walking abilities , the knee mobilization exercises had the aim of improving the tone and trophism of the femoral quadriceps and the stiffness of the ilotibial band , which , as this was the site of surgical access , was at risk of adherences that could compromise the knee movement . on the 21 day after surgery , the patient was transferred to gaetano pini rehabilitation centre where the orthosis was gradually released ( starting on post - surgery day 30 ) until it reached 90 of flexion by means of passive and assisted active sagittal mobilization under the pain threshold . she also performed progressive exercises of assisted active mobilization with the hip in abduction , and strengthening exercises for her right arm overloaded by the heavy use of walking aids . the walking re - education exercises continued with the gradual replacement of the walking frame by elbow crutches : her gait showed slight abduction of the hip ( which gradually regressed ) during both the advancement and fleetingly light weight - bearing phases . once capable of walking autonomously for albeit short distances , the patient was given functional exercises in preparation for her return home , including stairs training , re - education in the activities of daily living , and the postural movements involved in entering and leaving a car . a few days before being discharged , she began the weaning from the orthosis and started step training exercises in water ( with the water up to her chest - 25% of her weight borne by the limb ) . this hydrokinetic therapy gave the treated limb an important proprioceptive stimulus in terms of weight bearing without aids , but in absolute safety conditions . throughout her stay in the rehabilitation center , when she was discharged 60 days after surgery , she was able of walking autonomously for long distances with the aid of two elbow crutches . she was instructed to continue the exercises at home in order to maintain and consolidate her acquired abilities . at the orthopedic follow - up examination 2 weeks after discharge the 6-months physiatric follow - up examination showed a clear improvement in the functional outcome measures ( table 2 ) .", "the tumor - induced osteomalacia is a paraneoplastic syndrome , which causes osteomalacy , causing small dimension lesions , characterized by a slow growth without any specific anatomic localization . those lesions can lead to pathologic fractures , which , if treated without removing the neoformation or with a partial excision , may recur subsequently , along with loosening or displacement of prothesis components . the lesions can be studied with x - rays , computed tomography , magnetic resonance imaging , concreotide tri - phasic whole - body scintigraphy and f-18 fluorodeoxyglucose positron - emission tomography . having confirmed the nature of the lesion , treatment of a phosphaturic - secreting mesenchymal tumor is its surgical removal with broad margins by means of unconventional techniques such as a large resection and prosthetic implantation . in absence of a correct diagnosis , the rehabilitation treatment of patients with tio was initially based on reducing / containing the painful symptoms and asthenia , and is naturally not beneficial . for this reason , the worsening pain and progressive loss of functional capacities can reduce the compliance with treatment and affect patient mood . the loss of bone mass due to this type of tumor is not associated with the muscle tissue loss , but the overall balance is usually characterized by asthenia of the main anti - gravitational , stabilizing and mobilizing muscles as a result of long pre- and post - surgery periods of immobility . the adherence of scars due to repeated revision surgeries can also give rise to articular and functional limitations ( figure 3 ) unless they are appropriately treated . the time necessary to resume everyday living activities is longer than after conventional surgery , and requires specific techniques strictly related to the highly invasive surgery . the requirements are : i ) special care during the first four weeks of rehabilitation and , in any case , a longer period of hospitalization that that required after a normal hip replacement procedure ( the protocol lasts of 60 days ) ; ii ) the use of specific aids ( hip orthosis and reclinable wheelchair ) and training the patient and caregivers by nurses and technical staff in how to use them ; iii ) learning specific movement techniques to get an early recovery of a standing position and making positional changes aimed at protecting the implant stability while developing the patients residual functional capacities also by means of compensatory strategies ; iv ) particular care in treating the surgical wounds , which are more extensive than those associated with traditional prosthetic implants .", "the functional pictures which result are often complex for the long diagnostic - therapeutic pathway and for the highly invasive surgery needed . the scientific research has not validated any standard rehabilitation protocol or any specific guidelines yet . the protocol developed at the gaetano pini orthopedic institute with the collaboration of the rehabilitation and orthopedic team has demonstrated to be effective in the case above described ." ]

[ "human neurocysticercosis ( ncc ) is caused by larval stage of zoonotic tapeworm taenia solium ( pork tapeworm ) which remains a major public health problem in developing and some developed countries . porcine cysticercosis is the cause of human taeniasis and neurocysticercosis is a consequence of taeniasis . based on the available information , a very conservative and rough economic estimate indicates that the annual losses due to porcine cysticercosis in 10 west and central african countries amount to about 25 million euros . . also stated that , in china , the amount of pork discarded in the whole country due to cysticercosis annually has been estimated as 200,000,000 kg with a value of more than us $ 120,000,000 . stated that notably data on myanmar are lacking , although there are several reports of porcine cysticercosis based on meat inspection in the abattoirs in neighboring countries , 9.3% in india , 32.5% in nepal , 5.4% in china , 0.022.63% in indonesia , and 0.04 to 0.9% in vietnam . although most of myanmar culinary habits are based on thorough cooking , new food style such as barbecue and dishes based on raw or undercooked pork or pork product becomes popular among customers . moreover , small - scale pig husbandry has become one of the major sources of income in myanmar farmers . so it may be high risk of getting food - borne zoonotic diseases according to the new food style and traditional husbandry method . due to lacking of information on porcine cysticercosis in myanmar up to now , it is important to investigate the prevalence and associated risk factors . nay pyi taw area , the capital of myanmar , has big population of pigs ( about 200,000 pigs ) to support the demand of pork consumption in this area . most of the pig farmers are smallholders and most of pig husbandry systems are free ranging or semi - intensive with lack of proper sanitation . one of the main obstacles to control the t. solium infections is the lack of adequate epidemiological data on cysticercosis / taeniasis . therefore , the objectives of this community - based study were to investigate the prevalence of porcine cysticercosis and associated risk factors in pigs within study area . moreover , findings of this study will assist to develop the control strategies of porcine cysticercosis for the public health aspect .", "the cross - sectional studies were conducted from january to march and june to july 2014 , to investigate the prevalence of taenia solium cysticercosis in slaughtered and farmed pigs within pyinmana , lewe , and tatkon townships , nay pyi taw area . it is located between latitude 1945n and longitude 966e and with climate data ; the altitude is 115 m above sea level , annual rainfall is 115 mm , and annual temperature is 21.232.5c . an expected prevalence of 30% with a confidence level of 95% was used in this unit . in this study , 300 slaughtered pigs and 364 farmed pigs from the study area were examined although calculated samples were 298 and 323 , respectively ( table 1 ) . blood collected from the jugular vein of farmed pig was conducted for the seroprevalence and a structured questionnaire with both closed and open - ended questions was administered to owners to obtain management practices in pig husbandry . piglets younger than two months , pregnant sows , and nursing sows with litters less than two months old were excluded from this study to overcome the stress which causes adverse effect in animals . meat inspection was carried out as described by boa et al . in the three slaughterhouses of these townships . there were 300 randomly selected pigs recruited and 9 different muscles ( tongue , masseter , brain , shoulder , diaphragmatic , heart , skeletal , fore limb , and hind limb muscle ) from each pig in meat inspection . briefly , long and parallel incision into the masseter muscles on both sides of face in an upward direction was made . a deep longitudinal incision covering about 3/4 the thickness of the tongue and covering the whole length of the tongue was made to examine the cysts . after opening the pericardium , the heart was also visually examined for the presence of cysts . the heart was cut open and a deep ( 3/4 the thickness of septum ) incision into the septum was made to expose any metacestodes . all the other muscles were viewed , palpated , incised by surgical blade , and visually examined . the pig was kept under restraint at standing position and blood samples were obtained from the external jugular vein by using sterile disposable syringes and put into vacutainers with clot activators . those vacutainers were kept in cold boxes with ice and transported to department of pharmacology and parasitology , university of veterinary science , nay pyi taw , and allowed overnight at 4c to clot . to obtain serum , the clear sera were transferred to 1.5 ml microvial tubes and stored in labeled wails and kept at 20c until analysis . detection of igg antibody of t. solium cysticerci was carried out by using antibody - elisa kit ( novatec immundiagnostica gmbh co. , belgium ) according to manufacturer 's instruction . briefly , all thawed samples were diluted as 1 + 100 with igg sample diluent ( phosphate buffer ) before assaying . the 100 l controls and diluted samples were dispensed into their respective wells and the foil was covered . after incubation for 1 hour at 37c and the foil being removed , the contents of the wells were aspirated and washed three times with washing solution . and the 100 l protein a conjugate ( horseradish peroxidase ) was dispensed into all wells except a1 and covered with foil and incubated for 30 min at room temperature . after washing three times , 100 l tmb ( 3,3,5,5-tetramethylbenzidine ) substrate solution was dispensed into all wells and incubated for exactly 15 min at room temperature in the dark . the reaction was stopped by adding 100 l stop solution ( 0.2 m h2so4 ) . the absorbance was determined at 450/620 nm using an elisa reader ( stat fax ) . in each elisa kit testing , there are two cut - off controls ( c1 and d1 ) . the mean absorbance of these cut - off controls was used as cut - off value . samples are considered positive if the absorbance value is higher than 10% over the cut - off and samples are considered negative if the absorbance value is lower than 10% below the cut - off . the sensitivity and specificity of these kits to diagnose swine cysticercosis are 93.8% and > 95% , respectively . a questionnaire was developed and used to collect information on hypothesized risk factors and other related pieces of information from sampled pig owners . households in each township were selected by using the snowballing technique from those farmers willing to participate in the study . it is a technique for developing a research sample where existing study subjects recruit future subjects from their acquaintances . the questionnaire interviewed data were analyzed for the relationship between the prevalence of t. solium cysticercosis and hypothesized risk variables such as age , gender of pigs , husbandry system , feed type , environment of pig farm ( accessibility of human feces ) , personal hygiene of owners , pork consumption , cooking and eating habit of pork , use of anthelmintics in pigs and owners , and knowledge on taeniasis . they were examined for testing its significance by pearson chi - square test at = 0.05 .", "seroprevalence of porcine cysticercosis in farmed pigs was 15.93% ( 58/364 ) in the study area . prevalence of households with pigs infected with t. solium cysticerci by ab - elisa examination was 23.15% ( 47/203 households ) . the households with porcine cysticercosis in pyinmana , lewe , and tatkon were 0/12 ( 0% ) , 13/124 ( 10.48% ) , and 34/67 ( 50.75% ) , respectively . all the infected pigs presented parasites located in the tongue . only in one pig , the prevalence in slaughterhouses of pyinmana , lewe , and tatkon townships was 22% ( 44/200 ) , 23.33% ( 7/30 ) , and 28.57% ( 20/70 ) , respectively . univariate analysis of hypothesized risk factors of gender ( or = 3.0 ; 95% ci = 1.75.4 ) , increased age ( or = 2.3 ; 95% ci = 1.24.2 ) , husbandry system ( or = 5.1 ; 95% ci = 2.411.2 ) , feed type ( or = 16.9 ; 95% ci = 2.3124.3 ) , no hand washing habit before feeding ( or = 31.5 ; 95% ci = 4.3230.9 ) , not using anthelmintic in pigs ( or = 11.9 ; 95% ci = 5.028.5 ) and owner ( or = 2.5 ; 95% ci = 1.44.4 ) , and pork consumption of owner ( or = 37.4 ; 95% ci = 9.0156.1 ) was significantly associated with cysticercus cellulosae infection ( p < 0.05 ) . the distribution and odds ratio of significant risk factors concerning porcine cysticercosis are shown in table 2 .", "in southeast asia , pigs are an important source of food and economic important for smallholder farmers . older pigs may be penned or tethered although common raising practice of pigs is freely roaming in the village . in myanmar , most of the pig farmers are smallholders and practice as free - range or backyard farming . in myanmar , most of the pig farmers usually keep the weaned pigs until six to eight months of age and then send to slaughterhouse . in the village , every household keeps at least one pig not only for table waste feeding to pigs but also for extra income . most farms are having the habit of feeding waste materials such as swill and kitchen leftover , broken rice , rice bran , groundnut meal , sesame meal and local forage , and poor sanitation . the present study is the first report of t. solium cysticercosis in pigs in myanmar . pigs in the study area positive for cysticercosis have been exposed to t. solium eggs . among the 17 hypothesized risk factors , the gender of pigs ( being female ) was significantly associated with porcine cysticercosis in this study . it can be explained that female pigs were for kept long time for breeding purpose than male and so they have more risk to get exposed to t. solium eggs . however , jayashi et al . reported that gender was not a significant risk factor for porcine cysticercosis . the present study demonstrated that the older the pigs , the greater the chance to get infection . these results are in agreement with those reported by pouedet et al . , jayashi et al . , sarti et al . , garca et al . , and pondja et al . older pigs might also have greater chance to get exposed to t. solium eggs than younger ones . they might have much time to develop cyst and trigger the production of circulating antibodies . besides , it could be possible that younger pigs are protected during their first months of life against parasite infection , due to the presence of maternal cysticercus antibodies and they become susceptible later after the slow clearance of those antibodies . the result showed that pigs from households practiced semi - intensive system ( the pigs are allowed to roam freely in the environment and only panned or tethered at feeding time and night ) were more likely to have porcine cysticercosis than intensive ( the pigs are kept in the backyard or corral and not allowed to roam ) pigs . therefore , semi - intensive management system represented as an important risk factor for porcine cysticercosis in the study area as the pigs in this practice could access the infected human faeces . accessibility of infected human faeces is the main source for porcine cysticercosis [ 17 , 19 , 20 ] . among the feed types used in pig farms , this might be contaminated with t. solium eggs from infected food preparers of swill collected houses . so the collected swill should be cooked thoroughly before feeding to prevent infection including cysticercosis . use of anthelmintic in pigs and owners was significantly associated in this study . by interviewing the farmers and township veterinary officers , although ivermectin can not kill any larvae of cestode , albendazole can kill these larvae . although all the farmers wash their hands after feeding the pigs , only 21.2% famers ( 43/203 ) wash their hands before feeding . all cysticercosis positive samples were from those who do not practice hand - washing habit . pork consumption of owners is also one of the risk factors in survey of porcine cysticercosis . nine hypothesized risk factors not included in analysis were breed of pigs , place of purchase , presence of latrine , hand - washing after feeding the pigs , source of water for pigs , cleanliness of water , knowledge on taeniasis and cysticercosis , and occurrence of cyst in pork . in this study , all pigs are indigenously bred . all farmers have latrines using water , but the children do not use latrine and are used for defecation out of latrine . some farmers washed the hands before feeding the pigs and all farmers washed their hands after feeding . all farmers did not have the knowledge on taeniasis and cysticercosis and they have never seen the cysts in the pork in the study area . the presence of zoonotic agent , cysticercus cellulosae , may depend on intrinsic factors : age , gender , and extrinsic factors : pig husbandry system , hand - washing habit of owner , use of kitchen waste as pig feed , not using anthelmintic in pigs and owners , and pork consumption of owner in the study area . presence of this infection is of public health importance because it may lead to the occurrence of neurocysticercosis in human . although the occurrence of human neurocysticercosis has not been reported yet in myanmar , all public should take awareness of potential risk factors due to the prevalence with high percentage observed in this study . myanmar has no national monitoring program for t. solium cysticercus spp . in these animals yet . therefore , it is advisable to monitor whether there is high or low prevalence of t. solium cysticercosis in the whole country . it could also be suggested that confinement housing system should be developed in pig industry of myanmar to efficiently prevent porcine cysticercosis . for practicing sanitary and culinary habit , thorough cooking education programs should also be implemented for both swine breeders and consumers so as to prevent taeniasis in human and porcine cysticercosis and also other zoonotic helminth diseases in myanmar . this prevalence with relatively high percentage of porcine cysticercosis ( 15.93% ) in ab - elisa and 23.67% in slaughtered pigs indicates the presence of human taeniasis and it also leads to the associated risk of human cysticercosis and neurocysticercosis ." ]

[ "organized by nei dan school , european school of taiji quan and by the tao and science studies centre , under the aegis of the provincia di bologna and the partnership of asi ( alleanza sportiva italiana ) and luni editrice . goodwill of the conference is to act as a starting point to develop a net of experts , doctors and scientists , who will investigate the dynamic interactions between spiritual insight and scientific analysis to come to the creation of a new paradigm of modern science . science , philosophy , medicine and body arts of the ancient east are reunited together to create a new ecological awareness of body and mind . modern science , which paved the way for an outlook of reality considering the universe as a whole , in which all parts and phenomena are connected among them , can be integrated to the ancient eastern wisdom for the control of the mind and to the body arts ( taiji quan , qi gong , yoga ) to develop a new ecological awareness , an awareness based on nature and on the dynamic relation among all living creatures . the conference was divided in two sections : a gathering of experiences , of paths where science meets metaphysics to have a new language born , made of images and movement , and a panel to understand how taiji quan , the arts of movement and meditation , can prolifically meet cognitive sciences and neurosciences . speakers during the tao and science conference were : andrea pezzi ( presenter and tv author ) ; professor edwin l. cooper professor , laboratory of comparative neuroimmunology , department of neurobiology , david geffen school of medicine , university of california los angeles , editor - in - chief , the journal : evidence based complementary and alternative medicine , oxford university press ; professor carlo ventura ( professor of molecular biology at the faculty of medicine , university of bologna ; director of the laboratory of molecular biology and stem cell bioengineering , national institute of biostructures and biosystems by the institute of cardiology of santorsola malpighi hospital in bologna ) ; professor angelo marzollo ( professor of systems theory , faculty of sciences , university of udine ; vice general secretary of the international centre for mechanical sciences ; unesco ex - person in charge for mathematics and now consultant ) ; professor giovanni sambin ( professor of logic mathematics , university of padova ) ; dottor matteo luteriani ( publisher , journalist and master of martial arts ) , dottor massimo mori ( doctor , poet and master of taiji quan ) and eng . flavio daniele ( writer and master of taiji quan ) the following guests took part in the conference through their representatives : professor james k. gimzewski ( department of chemistry and biochemistry , university of los angeles ucla ; director of the pico lab laboratory at ucla ) ; professor aldo stella ( teacher of medical psychology , university of urbino ; teacher of psychology of cognitive processes , university for foreigners of perugia ) ; professor carmelo di stefano ( teacher of teaching didactics of the adapted movement and sports activity , faculty of motor sciences , university of bologna ) .", "carlo ventura , md , phd : western science has long been entangled with increasing reductionism and the development of field restricted approaches to understand cell biology and the molecular basis of disease . it is now becoming increasingly evident that reductionism is a remarkable bias in pursuing some of the major goals of modern biology and medicine . complex problems , including cell growth and differentiation under normal or malignant conditions ( cancer ) , and the adaptive mechanisms of humans to multiple changes in cell signaling networks now pose the need for holistic approaches both at the molecular biology and medical levels . such a requirement is even more urgent in spite of the emerging interest in stem cell biology , since taking a glimpse at the mechanisms underlying cell commitment and fate specification may hold promises for a revolutionary field , the so - called regenerative medicine. while moving from reductionism to holistic approaches , the cell is studied as an integrated system , behaving as a neural network with complex and sophisticated logics . awareness of these features has progressively led to wide - ranging strategies in the investigation of gene and protein expression . techniques such as the dna microarray and the serial analysis of gene expression ( sage ) are now able to follow the expression of thousands of genes and signaling molecules at a time , attempting to uncover the overall plans that underlie molecular patterning and cellular decisions . omics era ( genomics , proteomics ) and will hopefully form the scientific underpinning for moving from basic science to a clinical practice in which physicians will learn how to deal with illness rather than disease ( or even worst , diseased organs ) . a major sign of these cultural changes is provided by the ongoing development of nanobiotechnologies . in both the philosophical and visual sense , seeing is believing does not apply to nanotechnology , for there is nothing even remotely visible to create proof of existence . on the atomic and molecular scale , data is recorded by sensing and probing in a very abstract manner , which requires complex and approximate interpretations . more than in any other science , visualization and creation of a narrative becomes necessary to describe what is sensed , not seen . we have growing needs for separating the informational content of life from its material substrate. information is thought to be the essence of life , as in the dna code ( james k. gimzewski , university of california at los angeles , department of chemistry and biochemistry , director of the pico lab at ucla ) . edwin cooper highlighted how different alternative medicines if integrated can be useful to reclaim that holistic view of the diseased person , that an excessive specialization has made modern western medicine lose and he illustrated the work done in this direction by the biomedical journal ecam . disease has always been of enormous concern in human society . from prayers and spells to the birth of medicine as a rational science , man has developed all sorts of medical treatments to combat different illnesses and chronic ailments : according to the chinese proverb : life is worth more than a thousand gold pieces. the first objective in a serious approach to complementary and alternative medicine ( cam ) should be to obtain a broad understanding , with a minimum of detail , of how cam fits into the pattern of biology of the way in which the nervous , endocrine and immune systems coevolved , their function and coordination with other body systems , and their development from the embryo onwards including aging . at the same time , such an outline should provide an adequate background for easy application of cam ideas to the detail of practical cam work in public health , clinical and medical practice , and yet not stray far away from its essence , the very biology that under girds it . cam is organismic , considers the whole individual and is inclusive , not reductionist nor exclusive . concerning senescence and age - associated diseases , that accompany longer living populations , substantial attention is now focused on searching for : ( i ) mechanisms of aging and ( ii ) approaches to ameliorate or lessen the effects through the use of therapies , some of which utilize natural products from aquatic and terrestrial plants and animals . clearly there are numerous treatments to explore and to understand long past the anecdotal information that has been passed on through centuries . there is evidence for treatment of diseases of an ever - aging society especially in developed countries . it is of great interest that these remedies now being refined by ecam approaches derives almost entirely from primitive societies where there are minimal facilities essential for analyses by an evidence - based approach . massimo mori said : [ ] if the scientific research is free , the same is not valid for its technological applications , which have to serve an ecology of culture marked by wisdom , an ecology of the mind , as wrote gregory bateson , in harmony with nature ; nature , where the uncertainty principle of werner heisenberg has scientific and philosophical value ; harmony , comprising the clash and entropy of ilya prigogine as factors of transformation . a holistic view recomposing the divided self of ronald d. laing , giving deepness to the one - dimensional man of herbert marcuse. prof . angelo marzollo highlighted the importance of combining the art of to say with the art of to do , i.e. , that the learned man , the intellectual is direct evidence of the ideas carried on by him , that the body does not contradict the mind , that the body - mind unit remains not only a theoretical statement . giovanni sambin said : although the theme is science , i can speak only about my specialization . i am fascinated by oriental wisdom , but i know almost nothing about it because of lack of time . i am here only because i am trying to learn tai ji quan , with master roberto benetti. mathematics is important in western science . galilei was the first to propose that the laws of nature are written in the language of mathematics . this has been the base for inventions characterizing industrial revolution in xixth century . by using mathematics so mathematics is at the base of present - day western technological supremacy . in modern times , debate concerning foundations of mathematics has been most lively at the beginning of xxth century . one of its outcomes was the invention of programmable computers , which speaks of its importance . i am not a believer , but i find constructivism much more convincing than the now dominating theory , which is called classical and is considered by most scientists as an absolute truth . to do modern mathematics one needs an abstract notion of concept , or set . in the classical approach , one has classical logic ( by which all propositions are either true or false , any third possibility like abstinence is excluded ) and axiomatic set theory ( all sets are there already , a static universe containing now , and ever , all possible concept - sets ) . the mental scheme looks as something like : classical mathematics is at the base of western science , which is the reason for technological superiority , which makes western people the owners of the world . hence it must be that western mathematics , and all what follows from it , is an absolute truth , which can be imposed on others by force . needless to say , this view can be extremely dangerous . here and in all my life , my aim is to show that a different foundation of mathematics is possible , which has the same applications , but which avoids any form of fundamentalism . a way of doing mathematics is possible , in which the will of power is replaced by harmony with nature , control is replaced by knowledge , brute external force is replaced by internal energy , the search for the i want to emphasize that originally this was not motivated by some ethical or political principles , rather by the search for a better foundation of mathematics . master flavio daniele emphasized as most advanced research of the last decades of the past century in the field of cognitive sciences and neurosciences led to a new theory , revolutionizing the traditional cartesian concept of mind . this theory , known as the santiago theory of cognition , claims that mind can no longer be regarded as a thing but as a process . process , which is cognition to which belong perceptions , emotions and actions , the language , the conceptual thought and all the attributes of conscience , which is peculiar to man . this view , perfectly in line with the eastern traditional thought , entails that mind with its cognitive processes goes beyond the rational aspect as it includes the whole process of life . a further implication of this theory , which will show its vast potential , when it will be absorbed at the general cultural level , is that mind and matter are no longer regarded as separated dimensions , but as complementary aspects of the sole phenomenon of life : the process ( the mind ) and the structure ( the brain ) . mind and matter , process and structure are indivisibly connected at all levels of life : from the simplest cell to the most complex organism . this connection is so deep , as most recent studies in the field of cognitive sciences has demonstrated , that we can state , conceptual thought , on the whole , is physically incarnated in the body and in the brain. this goes beyond the simple consideration that to think we need a brain and leads to state that human reason does not transcend body , but it is structurally shaped by our physicality and body experience. a further discovery of cognitive sciences , consequent to it that : ( 1 ) mind is deeply incarnated in the body , and the other that , ( 2 ) thought is mainly unconscious , is that ( 3 ) abstract concepts are to a large extent metaphorical. for the time being , present researchers have not explained in detail the neurophysiological dynamics underlying the formation of abstract concepts ; however , the scientists lakoff and johnson state that the neural and cognitive mechanisms enabling us to sense and move are the same to create also our conceptual structures and our ways of reasoning. this statement that conceptual structures and ways of reasoning come from the same neural and cognitive structures of perception and movement , is extremely important for the practitioners of the arts of movement ( taiji quan , yoga , sacred dances , ritual gestures or mudra ) , as it demonstrates and confirms the creative power of movement , which no longer acts as simple instrument at the service of mind , to play the fundamental role of shaper of the cognitive capabilities ( conceptual thought , speech , conscience ) of the human mind . by paraphrasing maturana and varela cognition , the process of knowledge , and it is identical to the process of life self . nevertheless , beyond the dialectics coming from the differences , it is more and more important to reintroduce a principle , acting as basis for the man universally , a principle which is internal and unconventional , an equal able to give the equal to all psycho - biological behaviors of the human being . in italy this kind of research has always taken place , and in recent years has made its way into the experimental evidence of a psychology able to touch this dimension ." ]

[ "cardiovascular disease is the most common cause of mortality in dialysis patients that is responsible for about 60% of their mortality and is also 30 times more common than in general population . different cardiovascular disorders , such as left ventricular hypertrophy ( lvh ) , coronary artery diseases , congestive heart failure ( chf ) , and arterial hypertension are commonly seen in these patients . in addition , calcification of cardiac valves are common and may cause valvular and annular thickening that in turn could lead to valvular stenosis or regurgitation . some predisposing factors of cardiac disorders in dialysis patients are secondary hyperparathyroidism , long term hypertension , and anemia . different electrocardiographic abnormality may be seen in dialysis patients , such as st , t change , ventricular and supra - ventricular arrhythmia and qt interval prolongation . in the electrocardiogram , qt interval is the time between the start of the q wave and the end of the t wave . when heart rate increased then qt interval was decreased , so qt interval could be corrected with heart rate . there are a number of different correction formulas , but the standard clinical correction is to use bazett 's formula ( qt/r - r , where r - r is the rr interval in seconds ) . the normal corrected qt ( qtc ) interval for males and females are 430 ms and 450 ms , respectively . the borderline qtc for a male is 431 - 450 and borderline qtc for a female is 451 - 470 and the abnormal qtc range for a male is > 450 and for a female is > 470 . the qtc interval could be prolonged due to electrolyte abnormalities ( hypomagnesaemia and hypokalemia ) , drug consumption ( antihistaminic , antiarrhythmic , and antibiotics ) , brain trauma and genetic abnormalities ( long qt syndrome ) . patients with chronic renal failure and dialysis patients had a greater qtc interval and qt dispersion ( qtd ) ( qtd = qtmax qtmin ) compared with the normal population . a single session of hd could be increased qtc in patients undergoing hd . moreover , qt interval may be a predictor of ventricular arrhythmia and cardiovascular mortality in chronic kidney disease and dialysis patients . although there are some studies about qtc and qtc dispersion in hemodialysis ( hd ) patients with different results ; however , there are only a few studies concerning relationship of qtc internal and qtd with echocardiography findings , so the aim of this study was the evaluation of these relationship in dialysis patients .", "in a cross - sectional study , 60 hd patients with age > 18 years and the dialysis duration > 3 months were enrolled . exclusion criteria were : antiarrhythmic drugs consumption , history of cardiac diseases such as arrhythmia , heart block or chf . before dialysis session , electrocardiography ( ecg ) and echocardiography also , qtd was measured by mentioned formula ( qtd = qtmax qtmin ) in 12 leads ecg . the patients were on hd , by fresenius digital machine ( 4008b , germany ) and gambro digital machine ( ak95 and ak96 , sweden ) , 2 - 3 times / week , as a regular method ( 4 - 4.5 h with blood flow of 250 - 350 ml / min , dialysate flow of 500 ml / min and ultra - filtration based on the patient 's condition ) . the used buffer was bicarbonate powder , and the type of filters were intermediate and high efficient polysulfone membrane ( r5 , r6 ) made by soha factory under license of fresenius company . serum creatinine , hemoglobin , ca , parathyroid hormone ( pth ) , na , k , hco3 , and ph were checked by ra1000 machine ( made in italy ) . all laboratory tests were done before the dialysis session and were checked in a single laboratory . echocardiography and ecg were done before the dialysis session and by single cardiologist and technician respectively . all data and information were confidential , and for ecg and echocardiography taking an informed consent was taken from each patient . in echocardiography , left ventricular ejection fraction ( lvef ) , lvh , pulmonary artery pressure ( pap ) and valvular disorders were evaluated . at the end of the study , data were analyzed using spss software ( version 19 , ibm corporation ) . pearson correlation coefficient , two - independent samples t - test and anova were used for statistical analysis . this study was approved by ethical committee of shahrekord university of medical sciences with the grant number of 904 .", "mean body mass indexes ( bmis ) ( post dialysis ) and duration of dialysis were 21.77 3.6 mean qtc interval of the patients was 0.441 0.056 s ; however , qtc interval in men and women were 0.43 0.04 s and 0.45 0.07 s , respectively ( p > 0.05 ) . qtd in all of the patients was 64.17 25.93 ms , however in men and women were 62.70 28.05 ms and 66.52 22.48 ms respectively ( p = 0.87 ) . mean urea reduction ratio in men and women were 68.14% 9.34% and 68.22% 3.88% ; however , kt / v was 1.45 0.17 and 1.49 0.16 among men and women , respectively . characteristics of patients and their association with qtc and qtd sd = standard deviation , bmi = body mass index , pth = parathyroid hormone , qtc = corrected qt , qtd = qt dispersion there was no statistically significant relationship between qtc interval or qtd with duration of dialysis , bmi , age , and gender ( p > 0.05 ) . in ecg , lvh was seen in 23 ( 38.3% ) patients and st change in 14 patients ( 23.3% ) . in echocardiography , mitral regurgitation ( mr ) , tricuspid regurgitation ( tr ) , and aortic insufficiency ( ai ) were found in 54 , 47 , and 11 patients respectively [ table 2 ] . in addition , no significant relation was found between qtc interval and qtd with mr , tr , ai , lvh , septal thickness ( st ) and pap [ table 3 ] . qtc interval and qtd has also no correlation with serum pth or ca , k or hco3 . severity of valvular disorders in the patients mr = mitral regurgitation , tr = tricuspid regurgitation , ai = aortic insufficiency echocardiographic findings and their association with qtc and qtd qtc = corrected qt , qtd = qt dispersion , pap = pulmonary artery pressure , lvef = left ventricular ejection fraction , lvh = left ventricular hypertrophy , mr = mitral regurgitation , tr = tricuspid regurgitation , ai = aortic insufficiency , pe = pericardial effusion", "our findings revealed that , in hd patients , qtc interval and qtd had not any correlation with valvular disorders ( mr , ai , and tr ) , lvh or other echocardiographic findings such as pap and st . furthermore , there was no relationship between qtc interval and qtd with serum ca , k , hco3 , pth . in hd patients , cardiac abnormalities such as vascular calcification are common and are associated with the development of lvh , that may cause increased cardiac arrhythmias and mortality . elevation of calcium - phosphorus product can also cause valvular calcification and stenosis in these patients . mitral and aortic calcification and stenosis are common in these patients ; however , tricuspid and pulmonic valve calcification is rare . qtc interval prolongation in chronic kidney disease and hd patients was shown in some studies such as covic et al . and ljutic et al . studies which postdialysis qtc interval was 434 29 ms and 445.7 36.9 ms respectively , while in our patients qtc interval was 444.6 54.5 ms . selby and mcintyre in a review article reported that hd can increase qtc interval and qtd and is also capable of inducing arrhythmias and increasing mortality especially in patients with ischemic heart disease . in nakamura study on 48 dialysis patients , throughout the follow - up period , there was a higher incidence of cardiovascular death in patients with prolongation of qtc dispersion after hd . showed that the serum potassium was significantly higher in hd patients when compared to continuous ambulatory peritoneal dialysis ( capd ) patients and rate of qt interval dispersions was significantly higher in hd and capd patients when compared with healthy controls . they concluded that there is a tendency to cardiac arrhythmias in hd patients during the postdialysis period . maule et al . showed that after the hd session , qtc increased in 56% and decreased in 43% of the patients after dialysis session , cell - associated mg levels and qtd increased significantly in averbukh et al . study , so he concluded that excess daily mg intake and increased concentrations of cell - associated mg could be responsible for qtc prolongation in these patients . change in serum electrolytes during hd may also be responsible at least partially , to increase qtc or qtd . for example , increase of qtc interval after a dialysis session , and its correlation with plasma calcium level , postdialysis blood pressure , lvef and st was shown in covic et al . showed that qtd increased during hd due to serum k depletion and then return to baseline 2 h after the end of dialysis . qt prolongation during dialysis may also predispose to arrhythmia especially in the cardiac disease patients , nppi et al . demonstrated that hd increases qtd if a low - calcium dialysate is used . therefore , use of a low - calcium dialysate may predispose hd patients to ventricular arrhythmias . 16 hd patients showed that qtc interval has a reverse correlation with k and ca concentrations of dialysate . however , in hd children , ozdemir et al . found that qtc interval has no correlation with the patients sex , age and presence of hypertension or lvh but patients with left ventricular systolic dysfunction had significantly greater qtc dispersion . similar to our study , the changes in serum ca or k of the patients during dialysis were not associated with the qtc interval in this study . moreover , in hekmat et al.s study on 49 hd patients , qtc interval had not correlation with serum electrolytes and blood gas findings . in voiculescu study , there was no statistically significant correlation between qt interval and serum concentrations of mg , po4 in hd patients too . in addition , qtc interval was not dependent from lvef , arrhythmias or sudden death . our results had some similarities with some of above - mentioned studies as well as some differences with the others . the observed controversy between our study and some of these studies might be due to the discrepancy in the number of cases or racial differences of the patients , and more importantly , the fact that we did not compare predialysis and postdialysis qtc interval . we could not find any study on the correlation of qtc interval with lvh or valvular heart disease in hd patients ; however , there are some studies related to the correlation of qtc and lvh with other diseases or conditions . for example , mayet et al . reported that qtd has correlated with left ventricular mass index in hypertensive individuals . found that in athletes , lvh induced by physical training activity is not associated with an increase in qtd , whereas pathological increase in lvh secondary to hypertension could increase qtd . the study of dimopoulos et al . on the evaluation of qtd and left ventricular mass index in elderly hypertensive and normotensive patients reported that hypertensive patients had greater left ventricular mass index and higher qtd . the study had some limitations such as small sample size and lack of qtc measurement after hd session and comparison of it before and after procedure .", "by our knowledge , this is one of the first studies on the correlation of qtc or qtd with valvular abnormality and lvh in hd patients . there were a few studies about the relationship of qtc and lvh in nonrenal patients , so we could not compare our results with other studies . based on our results , in hd patients , qtc interval or qtd was not correlated with echocardiographic findings or laboratory exam results . therefore , it can be concluded that qtc interval prolongation probably has not any correlation with cardiac mortality of the hd patients ." ]

[ "asthma is a chronic inflammatory condition of the airways , characterized by limited airflow and punctuated by acute symptoms related to hyperresponsiveness to a variety of triggers , such as allergens or fumes ( 1 ) . chronic inflammation of the airways can lead to persistent alterations in airway structure , including sub - basement membrane fibrosis , mucus hypersecretion , injury to epithelial cells , smooth muscle hypertrophy , and angiogenesis ( 2 ) . asthma symptoms include wheezing , dyspnea , coughing , and chest tightness associated with broad but variable airflow obstruction in the lungs ( 3 ) . during acute asthma events , symptom severity may range from mild to life - threatening , the latter being due to severe bronchospasm , airway edema , impaired gas exchange , and ultimately , respiratory failure . worldwide , an estimated 300 million people suffer from the disease and prevalence is increasing ( 47 ) . of the 25 million americans with asthma , 12 million experience acute symptoms ( 8) and asthma was linked to 3447 deaths ( about 9 per day ) in 2007 ( 5 ) . asthma symptoms account for about 500,000 hospitalizations and nearly 2 million emergency room visits per year in the united states ( 6 ) . asthma - associated morbidity remains high despite improvements in diagnosis and the availability of comprehensive national and international clinical practice guidelines for managing the disease ( 2,3,9 ) . avoiding triggers is an important first step in asthma management , but may require lifestyle changes that patients find difficult or unacceptable ( e.g. , giving away a family pet ) , and adherence to lifestyle modifications is poor ( 1,5 ) . if lifestyle changes do not successfully prevent and control asthma symptoms , pharmacologic therapy can reduce the frequency and severity of asthma exacerbations , and reverse airflow obstruction during acute attacks ( 2 ) . most asthma medications are delivered as orally inhaled products in order to achieve local effects in the lung and to minimize systemic adverse effects . inhaled asthma medications are categorized into two general classes : long - term control medications ( also known as preventive or maintenance medications ) , which are taken regularly to achieve and maintain control of persistent asthma , and rapid - acting drugs ( also known as rescue medications ) taken as needed to provide prompt reversal of acute airflow limitation and relieve bronchospasm . onset of action of inhaled drugs for rescue from acute bronchospasm is approximately 510 minutes . rescue medications are typically short - acting 2-adrenergic agonists ( sabas ) , such as albuterol , but may be a long - acting beta agonist with rapid onset of action , such as formoterol ( 10 ) . bronchodilators provide relief of bronchoconstriction by relaxing bronchial smooth muscle and functionally enlarging the luminal diameter of the airways . sabas are delivered via wet nebulization or metered dose inhaler ( mdi ) ( 1 ) . mdis may provide better clinical outcomes and fewer adverse effects compared with nebulizers ( 11 ) ; however , nebulizers are useful for young children , older adults , and for patients who are unable to use an mdi . among other factors , patient nonadherence to therapy is an important contributor to poor asthma control . while often volitional , nonadherence can also be inadvertent when prescribed medications are taken improperly . for example , patients may think they are taking their asthma medication when they actually are activating a nearly empty or an altogether empty mdi to deliver orally inhaled asthma medication ( 12 ) . mdis deliver a limited number of effective medication doses , as listed in the prescribing information for each product . after the manufacturer - recommended number of doses is expelled , the mdi will continue to actuate many more times ( 13 ) . accurately assessing the doses in an mdi is critically important for bronchodilating medications used for rescue from acute asthma symptoms . and significantly overestimate ( or less commonly , underestimate ) the remaining amount of active asthma rescue medication in mdis without dose counters , that the techniques used to guesstimate whether a recue mdi is effectively empty are unreliable , and how having a rescue mdi with an integrated dose counter mechanism can improve the health and quality of life of patients with asthma .", "we searched embase and medline english literature up to july , 2012 with search terms dose counter and asthma and dose counter and metered dose inhaler . when pertinent articles were identified , we searched for relevant references used in those papers . for environmental reasons , inhalers with chlorofluorocarbon ( cfc ) propellant were no longer sold in the us from 2009 , and the propellant in current mdis is hydrofluoroalkane ( hfa ) ; however , much of the research reviewed here was conducted on mdis with cfc propellant .", "activating a nearly empty or an altogether empty mdi to deliver orally inhaled asthma medication appears to be a common occurrence when patients use an mdi without a dose counter ( 12,14,15 ) . mdis contain more drug formulation than the labeled number of drug doses to ensure dosing consistency in each actuation , up to the labeled number ( 16 ) . short of recording every actuation , there is no accurate and practical way to gauge the remaining number of effective doses in an mdi without the addition of a dose - counting mechanism . the invention of the metered - dose valve led to development of mdis in the 1950s ( 13 ) . the dosing and performance and , consequently , drug efficacy may be directly dependent on the design of the mdi . mdis have 3 main components : the canister containing the drug formulation , the metering valve , which determines the quantity of formulation dispensed upon actuation , and an actuator ( mouthpiece ) which directs the aerosol into the patient s lungs . in addition to the bronchodilating drug , the formulation contains a liquefied gas propellant ( hfa ) and may contain nonactive excipients . with current valve designs , it is not possible for an mdi to cease delivering drug doses completely at an exact point . mdis continue to deliver a spray , which may not be within the labeled specifications for the active drug , for up to twice the nominal number of recommended doses . in one study , mdis with cfc propellant had , on average , 86% more actuations than the labeled dose number , and mdis with hfa propellant had 52% more ( 17 ) . importantly , the amount of drug in those additional actuations is variable ; propellant and excipients form up to 99% of an asthma drug formulation ( 16 ) . with continued use beyond the recommended number of doses , drug delivery per actuation becomes inconsistent and unpredictable , with the amount of active drug eventually becoming negligible , a phenomenon known as tail - off ( 13,18 ) . thus , after the recommended number of doses , the mdi may appear to be delivering a therapeutic spray when , in fact , it is not . tail - off may be rapid ( e.g. , within 5 actuations ) , or erratic , requiring 1020 actuations before the canister is finally empty of its drug contents , depending on several factors , including the valve design ( 13 ) . mdis with valves that have formulation fill holes at the base of the retaining chamber have more rapid and less erratic tail - off ( figure 1a ) than mdis with valves with fill holes at higher levels ( figure 1b ) . if the height of the liquid formulation falls below the fill hole , at the time of actuation there may be incomplete filling of the chamber ( 13 ) . tail - off is particularly problematic when the medication delivered by the mdi is formulated as a suspension rather than a solution . all short - acting asthma rescue medications currently available in an mdi are formulated as suspensions . if the drug substance adheres to the walls of the container or valve components , dose delivery and particle size distribution could be inconsistent ( 19 ) . for this reason , shaking a rescue inhaler before actuation is an important part of correct mdi use . \n panel a shows tail - off from 3 separate mdis with fill holes located at the base of the retaining cup that allow formulation to enter the valve ( valve - down orientation ) . panel b shows more erratic tail - off characteristics of 3 separate mdis with valve design in which the fill holes are located at higher levels relative to the base of the retaining cup ( adapted from schultz ( 13 ) ) . \n shown here is a proair metered - dose inhaler ( manufactured by teva pharmaceutical industries , ltd . panel a shows tail - off from 3 separate mdis with fill holes located at the base of the retaining cup that allow formulation to enter the valve ( valve - down orientation ) . panel b shows more erratic tail - off characteristics of 3 separate mdis with valve design in which the fill holes are located at higher levels relative to the base of the retaining cup ( adapted from schultz ( 13 ) ) . shown here is a proair metered - dose inhaler ( manufactured by teva pharmaceutical industries , ltd . studies conducted to assess the accuracy of asthma patients ability to gauge doses remaining in their mdis have consistently shown high error rates ( 14,15,20,21 ) . as noted , the only way to reliably determine the number of remaining doses in an mdi with no dose counting feature is by carefully and consistently tracking each dose , then subtracting it from the labeled number of doses . however , many people are not even aware of the recommended number of drug doses in their mdis . in a study conducted to investigate how asthma sufferers determined when to replace their mdis , 54% did not know the maximum number of actuations recommended by the manufacturer for their particular inhaler ( 15 ) . even when recommended dosing is known , patient records or diary entries of doses taken are rarely kept . in a study by ogren et al . , only 8% of patients kept any record of their mdi actuations ( 15 ) . most patients did not receive instructions from their health care providers to keep a count of doses taken ; in a survey conducted by sander et al . , only 36% of respondents reported having been instructed to keep track of the number of mdi - delivered doses of their asthma medications ( 14 ) . other methods by which patients estimate remaining medication in their mdis are not reliable ( 1315 ) . estimations of whether an mdi requires replacement based on the weight of the inhaler , or on the force , sounds , and taste of the actuation , are often made based on the volume of the remaining propellants and excipients and not on the remaining doses of active medication meeting therapeutic specifications ( 15,17 ) . in a study by rubin and durotoye of pediatric asthma patients , 72% of the children ( or their parents ) reported using an mdi until they could no longer similarly , in the study by sander and colleagues , 1 in 5 surveyed patients reported that they assessed their albuterol inhaler to be empty when it stopped spraying , unaware that propellant continues to spray long after the albuterol has run out ( 14 ) . many patients report shaking their mdis to assess the amount of drug remaining in them ( 15,20,22 ) . in one study , patients who adopted this method overestimated remaining medication by about 40 doses ( 21 ) . accordingly , 84% of mdis evaluated in this study had been used well past the recommended number of actuations . it has been suggested that floating an inhaler in water , known as a float test , can provide a measure of the inhaler s useful contents ( 20 ) . however , no universal flotation status accurately reflects when a device has reached the maximum recommended number of actuations ( 15 ) . moreover , this test is not only unreliable since some inhalers will float when they are full , it may damage the mdi by obstructing the metering valve ( 1517 ) . in practice , patients who are not tracking rescue medication doses often either discard an mdi that may still contain acceptable metered doses of drug , or continue to use a product that may no longer contain the labeled within - specification dosage . the former is wasteful and expensive and the latter is potentially dangerous ( 16 ) . holt and colleagues asked patients receiving asthma treatment to return their mdis , when deemed empty , to their physicians ( 21 ) . of 109 returned mdis , 11% of empty mdis still contained more than 20% ( > 40 doses ) of the recommended metered doses ( table 1 ) ( 21 ) . sander et al . reported that more than half ( 53% ) of the asthma patients they surveyed who used an inhaled bronchodilator ( n = 342 ) refilled their bronchodilator prescriptions more frequently than recommended in national guidelines ( 2,14 ) . a bronchodilator prescription typically requires refill only a few times a year , yet almost 20% of patients reported refilling their inhaler at least once a month . the authors speculated that the excessive number of bronchodilator refills might be due , in part , to throwing away partially used inhalers to forestall the prospect of finding an inhaler empty during an acute asthma event ( 14 ) . \n table 1.discrepancies in patient perceptions of the number of doses of rescue medication remaining in an mdi with no dose counter . seventeen patients with asthma who regularly use an mdi estimated the number of salbutamol doses remaining in mdis that had been partially emptied to different degrees ( adapted from holt et al . ( 21)).actual number of remaining salbutamol doses in mdis which had been partially emptied40 doses30 doses20 doses10 doses0 dosesrange of patient estimations of the number of doses in the mdi ( min max)0180019001400180080mean difference between the actual and the estimated number of doses in the mdi76.881.063.054.823.6proportionate overestimations of the remaining doses in the mdi+36.8%+51.0%+43.0%+44.8%+23.6% \n discrepancies in patient perceptions of the number of doses of rescue medication remaining in an mdi with no dose counter . seventeen patients with asthma who regularly use an mdi estimated the number of salbutamol doses remaining in mdis that had been partially emptied to different degrees ( adapted from holt et al . ( 21 ) ) . while discarding an mdi with effective doses remaining is both costly and wasteful , relying on rescue medication from a depleted mdi during acute bronchospasm can be life - threatening . in the study by holt and colleagues , collected mdis labeled to contain 200 salbutamol doses two separate studies found that more than 40% of asthma patients replaced their mdi when it was completely empty and over 20% said they could never tell when their mdi was running out of medication ( 22,23 ) . in the sander et al . study , 87 asthmatic patients25% of those surveyed discovered that their albuterol inhaler was empty when needed for relief from acute asthma symptoms , and of them , 8% had to call for emergency assistance ( 14 ) . most of these 87 patients ( 82% ) considered their mdi empty only when nothing came out of it , making it likely that they were inhaling only propellant for many doses , thereby increasing their risk of prolonged bronchoconstriction and airflow limitation requiring urgent care . when asked , many patients report that not knowing how much medication is left in their mdis makes them anxious about receiving a subtherapeutic dose or no medication at all ( 22,24 ) . dose counters are , therefore , likely to alleviate patient anxiety about running out of asthma medication in emergency situations . thus , it is not surprising that patient satisfaction studies consistently show wide acceptance and approval of dose counters on mdis ( 2224 ) . patients rate dose counters among the top 5 best features of their mdi ( 14,23 ) . in addition to relieving anxiety , reliable knowledge of when to replace an mdi can improve asthma management and , in turn , improve patients quality of life ( 25 ) . because they are preferred by patients ( 22 ) moreover , physician inspection of dose counters at office visits may provide a means of evaluating patient adherence to treatment and present an opportunity to discuss appropriate use of the mdi and proper inhalation technique .", "the invention of the metered - dose valve led to development of mdis in the 1950s ( 13 ) . the dosing and performance and , consequently , drug efficacy may be directly dependent on the design of the mdi . mdis have 3 main components : the canister containing the drug formulation , the metering valve , which determines the quantity of formulation dispensed upon actuation , and an actuator ( mouthpiece ) which directs the aerosol into the patient s lungs . in addition to the bronchodilating drug , the formulation contains a liquefied gas propellant ( hfa ) and may contain nonactive excipients . with current valve designs , it is not possible for an mdi to cease delivering drug doses completely at an exact point . mdis continue to deliver a spray , which may not be within the labeled specifications for the active drug , for up to twice the nominal number of recommended doses . in one study , mdis with cfc propellant had , on average , 86% more actuations than the labeled dose number , and mdis with hfa propellant had 52% more ( 17 ) . importantly , the amount of drug in those additional actuations is variable ; propellant and excipients form up to 99% of an asthma drug formulation ( 16 ) . with continued use beyond the recommended number of doses , drug delivery per actuation becomes inconsistent and unpredictable , with the amount of active drug eventually becoming negligible , a phenomenon known as tail - off thus , after the recommended number of doses , the mdi may appear to be delivering a therapeutic spray when , in fact , it is not . tail - off may be rapid ( e.g. , within 5 actuations ) , or erratic , requiring 1020 actuations before the canister is finally empty of its drug contents , depending on several factors , including the valve design ( 13 ) . mdis with valves that have formulation fill holes at the base of the retaining chamber have more rapid and less erratic tail - off ( figure 1a ) than mdis with valves with fill holes at higher levels ( figure 1b ) . if the height of the liquid formulation falls below the fill hole , at the time of actuation there may be incomplete filling of the chamber ( 13 ) . tail - off is particularly problematic when the medication delivered by the mdi is formulated as a suspension rather than a solution . all short - acting asthma rescue medications currently available in an mdi are formulated as suspensions . if the drug substance adheres to the walls of the container or valve components , dose delivery and particle size distribution could be inconsistent ( 19 ) . for this reason , shaking a rescue inhaler before actuation is an important part of correct mdi use . \n panel a shows tail - off from 3 separate mdis with fill holes located at the base of the retaining cup that allow formulation to enter the valve ( valve - down orientation ) . panel b shows more erratic tail - off characteristics of 3 separate mdis with valve design in which the fill holes are located at higher levels relative to the base of the retaining cup ( adapted from schultz ( 13 ) ) . \n shown here is a proair metered - dose inhaler ( manufactured by teva pharmaceutical industries , ltd . panel a shows tail - off from 3 separate mdis with fill holes located at the base of the retaining cup that allow formulation to enter the valve ( valve - down orientation ) . panel b shows more erratic tail - off characteristics of 3 separate mdis with valve design in which the fill holes are located at higher levels relative to the base of the retaining cup ( adapted from schultz ( 13 ) ) . shown here is a proair metered - dose inhaler ( manufactured by teva pharmaceutical industries , ltd . , horsham , pa ) .", "studies conducted to assess the accuracy of asthma patients ability to gauge doses remaining in their mdis have consistently shown high error rates ( 14,15,20,21 ) . as noted , the only way to reliably determine the number of remaining doses in an mdi with no dose counting feature is by carefully and consistently tracking each dose , then subtracting it from the labeled number of doses . however , many people are not even aware of the recommended number of drug doses in their mdis . in a study conducted to investigate how asthma sufferers determined when to replace their mdis , 54% did not know the maximum number of actuations recommended by the manufacturer for their particular inhaler ( 15 ) . even when recommended dosing is known , patient records or diary entries of doses taken are rarely kept . in a study by ogren et al . , only 8% of patients kept any record of their mdi actuations ( 15 ) . most patients did not receive instructions from their health care providers to keep a count of doses taken ; in a survey conducted by sander et al . , only 36% of respondents reported having been instructed to keep track of the number of mdi - delivered doses of their asthma medications ( 14 ) . other methods by which patients estimate remaining medication in their mdis are not reliable ( 1315 ) . estimations of whether an mdi requires replacement based on the weight of the inhaler , or on the force , sounds , and taste of the actuation , are often made based on the volume of the remaining propellants and excipients and not on the remaining doses of active medication meeting therapeutic specifications ( 15,17 ) . in a study by rubin and durotoye of pediatric asthma patients , 72% of the children ( or their parents ) reported using an mdi until they could no longer similarly , in the study by sander and colleagues , 1 in 5 surveyed patients reported that they assessed their albuterol inhaler to be empty when it stopped spraying , unaware that propellant continues to spray long after the albuterol has run out ( 14 ) . many patients report shaking their mdis to assess the amount of drug remaining in them ( 15,20,22 ) . in one study , patients who adopted this method overestimated remaining medication by about 40 doses ( 21 ) . accordingly , 84% of mdis evaluated in this study had been used well past the recommended number of actuations . it has been suggested that floating an inhaler in water , known as a float test , can provide a measure of the inhaler s useful contents ( 20 ) . however , no universal flotation status accurately reflects when a device has reached the maximum recommended number of actuations ( 15 ) . moreover , this test is not only unreliable since some inhalers will float when they are full , it may damage the mdi by obstructing the metering valve ( 1517 ) .", "in practice , patients who are not tracking rescue medication doses often either discard an mdi that may still contain acceptable metered doses of drug , or continue to use a product that may no longer contain the labeled within - specification dosage . the former is wasteful and expensive and the latter is potentially dangerous ( 16 ) . holt and colleagues asked patients receiving asthma treatment to return their mdis , when deemed empty , to their physicians ( 21 ) . of 109 returned mdis , 11% of empty mdis still contained more than 20% ( > 40 doses ) of the recommended metered doses ( table 1 ) ( 21 ) . sander et al . reported that more than half ( 53% ) of the asthma patients they surveyed who used an inhaled bronchodilator ( n = 342 ) refilled their bronchodilator prescriptions more frequently than recommended in national guidelines ( 2,14 ) . a bronchodilator prescription typically requires refill only a few times a year , yet almost 20% of patients reported refilling their inhaler at least once a month . the authors speculated that the excessive number of bronchodilator refills might be due , in part , to throwing away partially used inhalers to forestall the prospect of finding an inhaler empty during an acute asthma event ( 14 ) . \n table 1.discrepancies in patient perceptions of the number of doses of rescue medication remaining in an mdi with no dose counter . seventeen patients with asthma who regularly use an mdi estimated the number of salbutamol doses remaining in mdis that had been partially emptied to different degrees ( adapted from holt et al . ( 21)).actual number of remaining salbutamol doses in mdis which had been partially emptied40 doses30 doses20 doses10 doses0 dosesrange of patient estimations of the number of doses in the mdi ( min max)0180019001400180080mean difference between the actual and the estimated number of doses in the mdi76.881.063.054.823.6proportionate overestimations of the remaining doses in the mdi+36.8%+51.0%+43.0%+44.8%+23.6% \n discrepancies in patient perceptions of the number of doses of rescue medication remaining in an mdi with no dose counter . seventeen patients with asthma who regularly use an mdi estimated the number of salbutamol doses remaining in mdis that had been partially emptied to different degrees ( adapted from holt et al . ( 21 ) ) . while discarding an mdi with effective doses remaining is both costly and wasteful , relying on rescue medication from a depleted mdi during acute bronchospasm can be life - threatening . in the study by holt and colleagues , collected mdis labeled to contain 200 salbutamol doses two separate studies found that more than 40% of asthma patients replaced their mdi when it was completely empty and over 20% said they could never tell when their mdi was running out of medication ( 22,23 ) . in the sander et al . study , 87 asthmatic patients25% of those surveyed discovered that their albuterol inhaler was empty when needed for relief from acute asthma symptoms , and of them , 8% had to call for emergency assistance ( 14 ) . most of these 87 patients ( 82% ) considered their mdi empty only when nothing came out of it , making it likely that they were inhaling only propellant for many doses , thereby increasing their risk of prolonged bronchoconstriction and airflow limitation requiring urgent care .", "when asked , many patients report that not knowing how much medication is left in their mdis makes them anxious about receiving a subtherapeutic dose or no medication at all ( 22,24 ) . dose counters are , therefore , likely to alleviate patient anxiety about running out of asthma medication in emergency situations . thus , it is not surprising that patient satisfaction studies consistently show wide acceptance and approval of dose counters on mdis ( 2224 ) . patients rate dose counters among the top 5 best features of their mdi ( 14,23 ) . in addition to relieving anxiety , reliable knowledge of when to replace an mdi can improve asthma management and , in turn , improve patients quality of life ( 25 ) . because they are preferred by patients ( 22 ) moreover , physician inspection of dose counters at office visits may provide a means of evaluating patient adherence to treatment and present an opportunity to discuss appropriate use of the mdi and proper inhalation technique .", "in 2003 , the us food and drug administration issued a guidance to industry emphasizing the importance of ( but not requiring ) integrated dose counters or dose indicators on mdis for orally inhaled medications targeted to the lungs ( 16 ) . dose indicators that rely solely on a color coded display or indicator symbol to signal when the mdi is nearing the end of its recommended doses are less precise than dose counters that use a numeric display ( figure 2 ) . typically , discarding an mdi is recommended when the dose counter reads 0 or by the expiration date , whichever comes first . the expiration date for a rescue inhaler is generally 1 year after the prescription is filled ( 26 ) or after the foil pouch is opened ( 27 ) . at present , the only available albuterol rescue inhalers with integrated dose counting mechanisms are proair hfa ( teva pharmaceutical industries , ltd . ) ( 26 ) and ventolin hfa ( glaxosmithkline ) ( 27 ) . if there is a down - side to integrated dose counting mechanisms on mdis , it may be that they are more expensive to produce than mdis without them . after 2008 , generic albuterol inhalers stopped being made because they contained cfc propellant ( it is unknown when generic albuterol inhalers may be available again ) . the estimated annual expenditure related to health care and lost productivity due to asthma was more than $ 20 billion according to the national heart , lung , and blood institute ( 28 ) . nevertheless , dose counters may reduce health care costs by decreasing asthma - related morbidity and attendant medical costs ( 14,17 ) . for example , inhalation of rescue medication rather than propellant to control acute bronchospasm and quell acute asthma symptoms may avoid the cost of an emergency room visit ( 29 ) .", "although there have been relatively few studies conducted to assess the use of mdis , they consistently show patients frequently overestimate the amount of active asthma rescue medication in mdis without dose counters . dose counters provide the only accurate and practical method of ascertaining the remaining number of effective doses in an mdi . by ensuring that patients do not use a rescue mdi beyond the recommended number of actuations and that they are receiving the appropriate metered dose of asthma medication , dose counters can improve asthma management and potentially decrease asthma - related morbidity and mortality , and improve patients quality of life . for these reasons , integrated dose counting mechanisms should be required on mdis that deliver asthma rescue medication ." ]

[ "from november 2008 through may 2010 , m. tuberculosis isolates were cultured during routine care of adults > 18 years of age with primary tb and no history of treatment . the patients were from 2 regional referral centers , the irkutsk regional tb - prevention dispensary and the research practice center for phthisiatry ( yakutia ) ; the study was approved by the institutional review boards at the university of virginia and irkutsk state medical university . initial pretreatment isolates were grown on lowenstein - jensen agar slants and identified to species in accordance with world health organization recommendations . drug susceptibility was tested by absolute concentration method on agar slants ; drugs tested were rifampin ( critical concentration 40 g / ml ) , isoniazid ( 1 g / ml and 10 g / ml ) , ethambutol ( 2 g / ml ) , streptomycin ( 10 g / ml ) , ethionamide ( 30 g / ml ) , and kanamycin ( 30 g / ml ) . dna extraction was performed on all isolates , followed by 12-loci mycobacterial interspersed repetitive unit variable number tandem repeat ( miru - vntr ) analysis ( 7 ) and further lineage definition by region of difference deletions , or for ural strains as described ( 5 ) . phylogenetic tree construction was based on the miruvntrplus database ( 8) , and vntr international type numbers were confirmed on the sitvit database ( 9 ) . dna from mdr isolates was amplified and sequenced for the known drug - resistance determining regions katg , inha , rpob , embb , gyra , rrs , and eis by using methods described by the centers for disease control and prevention ( 10 ) . sequences were compared with published sequences for m. tuberculosis h37rv by using genedoc version 2.7.0 . among 235 patients with primary tb ( 130 from yakutia , 105 from irkutsk ) , isoniazid monoresistance was found in isolates from 16 ( 12% ) from yakutia and 19 ( 18% ) from irkutsk ( p = 0.27 ) . multidrug resistance was found for 61 patients ( 36 [ 28% ] from yakutia and 25 [ 24% ] from irkutsk ) ( p = 0.55 ) . mean age ( sd ) for these 61 patients was 33 ( 12 ) years , 40 ( 66% ) were male , and these characteristics did not differ significantly between patients from irkutsk and from yakutia . however , no hiv - infected patients were identified from yakutia compared with 11 ( 44% with mdr tb ) from irkutsk ( p<0.001 ) . twelve mdr tb patients from irktutsk died ( outcome unknown for the other 13 patients ) , including all with hiv , compared with 4 ( 11% ) from yakutia who died ( p = 0.002 ) . follow - up varied and was limited mostly to inpatients . among all 235 patients with primary tb , strains of the beijing family were significantly more common among those from irkutsk ( 70 [ 67% ] ) than from yakutia ( 40 [ 31% ] ) ( p<0.001 ) . however , strains found in yakutia ( s 256 [ 11% ] , t 8 [ 7% ] , and ural 171 [ 5% ] ) were not found in irkutsk ( table 1 ) . the cluster of s 256 ( miru profile 233325153325 ) was the most common among primary mdr tb isolates from yakutia and was fully 86% mdr ( table 1 ; technical appendix ) . * miru - vntr , mycobacterial interspersed repetitive unit variable number tandem repeat ( original 12-loci profile ) . included genotypes found in > 5 isolates only ; mit , miru vntr international type ; mdr , multidrug - resistant tuberculosis ( conventional resistance to isoniazid and rifampin ) ; na , not applicable . significance determined by analysis with yates correction or fisher exact test when appropriate . among isolates from patients with primary mdr tb , 51 ( 84% ) were available for dna sequencing : 27 from yakutia and 24 from irkutsk ( table 2 ) . among isoniazid - resistant isolates , the mutation in codon 315 of katg was present in 91% . among rifampin - resistant isolates , mutations in the resistance - determining region of rpob ( codons 511533 ) were present in only 79% . the pnca mutation was common across genotypes from both sites , occurring in 62% of isolates amplified . notably , both isolates with mutation in eis from yakutia occurred in mdr strains with the s 256 genotype and without rrs mutation . * all inha mutations were associated with a ser315thr mutation in katg except for 1 isolate in which katg did not amplify . excluding 25 silent pnca mutations ( ser32ser most common , n = 14 ) . for all 3 mutations of eis associated with kanamycin resistance , rrs was wild type .", "in eastern siberia , > 25% of primary tb was mdr , equivalent to the highest proportion reported from the russian federation ( 2 ) . however , regionally specific genotypic patterns and resistance mutations were identified . as expected , in irkutsk primary mdr tb was driven by strains of beijing lineage ( 5,6 ) . yet in the more geographically isolated population of yakutia , a strain previously unidentified in the russian federation , s 256 , had a miru profile recently found among canadian aboriginal populations ( 11 ) . in yakutia , s 256 was highly drug resistant and was the most common genotype among patients with primary mdr tb . although rpob mutations were found in only 79% of rifampin - resistant isolates , these findings are consistent with those in a recent report from novosibirsk oblast , which similarly included non - beijing and s - family strains and found a sensitivity of only 63% for the rpob mutation ( 12 ) . lack of phenotypic correlation can result from alternate mechanisms of resistance or imperfect conventional susceptibilities in lowenstein - jensen medium or from use of old drug stock . such discrepancy necessitates urgent clarification because substitution of conventional susceptibility testing with molecular probe based methods such as genexpert mtb / rif ( cepheid , ca , usa ) has been strongly advocated but would lead to dramatically different results and treatment regimens ( 13 ) . of note , isolates of the s 256 strain accounted for a proportion of the cases in which mutation in the promoter region of eis was associated with kanamycin resistance , but rrs was wild type . commercial assays have focused on the rrs locus , which has greater sensitivity for amikacin , as the sole target for the class of injectable agents ( 14 ) , yet in eastern siberia , the injectable agent available is kanamycin . furthermore , we found a range of reported and unreported mutations across the entire pnca gene ; most were point mutations resulting in amino acid substitution , but some strains had mutations that resulted in deletion or frameshift . phenotypic methods and assays of functional pyrazinamidase activity should be performed in this region because results might have major implications for novel mdr tb drugs that work best with pyrazinamide ( 15 ) . we were unable to obtain detailed clinical information about all patients with primary tb , thus preventing adequate comparison of nongenotypic risk factors for mdr tb or establishment of definitive epidemiologic links among clustered isolates . furthermore , lack of conventional fluoroquinolone or pyrazinamide susceptibility testing limited comparison with gyra and pnca mutations , respectively . despite these limitations , this work characterizes severe isoniazid monoresistant and mdr tb in eastern siberia among patients with no history of tb treatment . the regionally distinct phylogenetic patterns and certain drug - resistance mutations necessitate careful application of novel diagnostics and empiric therapeutic strategies .", "phylogenetic trees of mycobacterium tuberculosis from patients with primary tuberculosis , yakutia and irkutsk , russian federation ." ]

[ "studies over the past four decades have shown that 3.9%-16% of patients with pancreatic cancer ( paca ) have a family history of the disease , which is referred to as familial pancreatic cancer ( fpc ) . fpc describes families with at least two first - degree relatives with confirmed exocrine paca that do not fulfil the criteria of other inherited tumor syndromes with increased risks of paca , such as peutz - jeghers syndrome , hereditary pancreatitis , and hereditary breast and ovarian cancer . the first studies to explore what is now referred to as fpc were case reports of families in which there were multiple cases of paca . in 1973 , macdermott and kramer described a family , in which paca developed in four out of the six siblings . a small fraction of this aggregation can be accounted for inherited cancer syndromes , including familial atypical multiple - mole melanoma ( famm ) , peutz - jeghers syndrome , hereditary breast - ovarian cancer ( hboc ) , hereditary pancreatitis ( hp ) , and hereditary nonpolyposis colorectal cancer ( hnpcc ) . these syndromes arise as a result of germline mutations in the cdkn2a ( famm ) , stk11 ( peutz - jeghers syndrome ) , brca1 , brca2 , atm ( hboc ) , prss1 ( hp ) , mlh1 , and msh2 ( hnpcc ) genes . in addition , heredity plays an important role in certain patients with apparently sporadic paca . many patients with pacas caused by a germline mutation in a cancer - causing gene do not have a pedigree suggestive of a familial cancer syndrome . although much has been learned over the past few decades regarding the aggregation of paca in some families , the genetic basis for most of this familial aggregation is still poorly understood . ten founder mutations in four cancer predisposing genes have been identified in poland [ 4 - 6 ] . these include three founder mutations in brca1 ( 5382insc , 4153dela , and c61 g ) , four in chek2 ( 1100delc , ivs2 + 1g > a , del5395 , and i157 t ) , one variant allele in nbs1 ( 657del5 ) , and two in palb2 ( 509_510delga and 172_175delttgt ) [ 4 - 6 ] . the results of our previous case / control studies showed that these mutations in brca1 , chek2 , nbs1 , and palb2 are associated with the cancer risk at different sites of origin [ 4 - 12 ] . a recent study evaluated the prevalence of these founder mutations among sporadic paca patients and showed that the risk of sporadic paca is associated with the slavic founder mutation of nbs1 657del5 ( odds ratio [ or ] , 3.80 ; p=0.002 ) . a hereditary predisposition to different cancers , which is characteristic of the aforementioned genes , suggests that there could be a potential link between polish founder mutations and a predisposition to fpc . therefore , the aim of this study was to establish the prevalence of ten polish founder mutations in brca1 , chek2 , nbs1 , and palb2 among individuals from families with familial paca syndrome and to assess their possible association with the fpc risk in poland . a total of 400 individuals from fpc syndrome families were genotyped and the frequencies of the founder mutations were compared with a set of healthy control subjects representative of the polish population .", "the recruitment of individuals for this study was conducted between 2002 and 2014 . the majority ( 80% ) were patients of our center , living in north - western poland , as well as in other cities of the country : biaystok , krakw , ld , olsztyn , opole , rzeszw , widnica , zielona gra , kielce . the study involved 400 probands from families with a diagnosis of fpc syndrome , who were either healthy individuals ( n=295 ) or fpc patients with cancer ( n=105 ) . table 1 lists the characteristics of the individuals included in the study . each of the 400 study participants came from a family , in which there were two or more cases of paca among the first - degree relatives . the mean age of the participants enrolled in this study was 49.44 years ( range , 19 to 91 years ) . a family history of cancer was taken either by the construction of a family tree or the completion of a standardized questionnaire . this study was conducted in accordance with the declaration of helsinki and all participants signed an informed consent document prior to donating the blood samples . furthermore , this study was approved by the ethics committee of the pomeranian medical university in szczecin , poland . the first consisted of 2,000 newborn children from 10 hospitals throughout poland ( szczecin , biaystok , gorzw , katowice , wrocaw , pozna , opole , ld , and rzeszw ) collected between 2003 and 2004 . samples of cord blood from unselected infants were forwarded to the study center in szczecin . the second group was taken from adult patient lists of three family doctors practicing in the szczecin region . a total of 1,000 controls were selected at random from the patient lists of these family doctors . the third group consisted of 1,000 adults from szczecin who submitted blood for paternity testing . a sample of dna was forwarded to the reference laboratory without any identifying information . to ensure comparability of the control groups , the allele frequencies of the 10 alleles was computed separately for the adult and neonatal control groups and compared to determine if there were any differences in allele frequencies . ten founder mutations in the brca1 , chek2 , nbs1 , and palb2 genes were genotyped , as described previously . briefly , brca1 mutations , 4153dela and 5382insc were detected using the allele - specific oligonucleotide polymerase chain reaction ( pcr ) , and c61 g was detected using the restriction fragment length polymorphism pcr . the ivs2 + 1g > a and i157 t variants in chek2 were detected using restriction fragment length polymorphism pcr analysis , and the 1100delc mutation was analyzed using allelespecific oligonucleotide pcr . the two recurrent truncating mutations of palb2 ( 509_510delga and 172_175delttgt ) were detected using a taqman assay ( life technologies , carlsbad , ca ) using a lightcycler 480 real - time pcr system ( roche life science , mannheim , germany ) . sanger direct sequencing was undertaken to confirm the presence of mutations , using a bigdye terminator v3.1 cycle sequencing kit ( life technologies ) , according to the manufacturer s protocol . all ten mutations were analyzed successfully in 398 of the 400 fpc cases ( 99.5% ) . the two that failed to be analyzed due to the poor quality of dna were discarded from statistical analysis . the ors were generated from two - by - two tables and the statistical significance was assessed using a chi - square or fisher exact test with a yates correction .", "the recruitment of individuals for this study was conducted between 2002 and 2014 . the majority ( 80% ) were patients of our center , living in north - western poland , as well as in other cities of the country : biaystok , krakw , ld , olsztyn , opole , rzeszw , widnica , zielona gra , kielce . the study involved 400 probands from families with a diagnosis of fpc syndrome , who were either healthy individuals ( n=295 ) or fpc patients with cancer ( n=105 ) . table 1 lists the characteristics of the individuals included in the study . each of the 400 study participants came from a family , in which there were two or more cases of paca among the first - degree relatives . the mean age of the participants enrolled in this study was 49.44 years ( range , 19 to 91 years ) . a family history of cancer was taken either by the construction of a family tree or the completion of a standardized questionnaire . this study was conducted in accordance with the declaration of helsinki and all participants signed an informed consent document prior to donating the blood samples . furthermore , this study was approved by the ethics committee of the pomeranian medical university in szczecin , poland .", "the first consisted of 2,000 newborn children from 10 hospitals throughout poland ( szczecin , biaystok , gorzw , katowice , wrocaw , pozna , opole , ld , and rzeszw ) collected between 2003 and 2004 . samples of cord blood from unselected infants were forwarded to the study center in szczecin . the second group was taken from adult patient lists of three family doctors practicing in the szczecin region . a total of 1,000 controls were selected at random from the patient lists of these family doctors . the third group consisted of 1,000 adults from szczecin who submitted blood for paternity testing . a sample of dna was forwarded to the reference laboratory without any identifying information . to ensure comparability of the control groups , the allele frequencies of the 10 alleles was computed separately for the adult and neonatal control groups and compared to determine if there were any differences in allele frequencies .", "ten founder mutations in the brca1 , chek2 , nbs1 , and palb2 genes were genotyped , as described previously . briefly , brca1 mutations , 4153dela and 5382insc were detected using the allele - specific oligonucleotide polymerase chain reaction ( pcr ) , and c61 g was detected using the restriction fragment length polymorphism pcr . the ivs2 + 1g > a and i157 t variants in chek2 were detected using restriction fragment length polymorphism pcr analysis , and the 1100delc mutation was analyzed using allelespecific oligonucleotide pcr . the two recurrent truncating mutations of palb2 ( 509_510delga and 172_175delttgt ) were detected using a taqman assay ( life technologies , carlsbad , ca ) using a lightcycler 480 real - time pcr system ( roche life science , mannheim , germany ) . sanger direct sequencing was undertaken to confirm the presence of mutations , using a bigdye terminator v3.1 cycle sequencing kit ( life technologies ) , according to the manufacturer s protocol . all ten mutations were analyzed successfully in 398 of the 400 fpc cases ( 99.5% ) . the two that failed to be analyzed due to the poor quality of dna were discarded from statistical analysis .", "the ors were generated from two - by - two tables and the statistical significance was assessed using a chi - square or fisher exact test with a yates correction .", "a founder mutation in one of the four genes studied was observed in 44 out of 398 fpc cases ( 11.06% ) and in 283 out of 4,000 controls ( 7.08% ) ( p=0.011 ; or , 1.56 ; 95% confidence interval , 1.12 to 2.18 ) . table 2 provides details of the prevalence of founder mutations studied among individuals from fpc families and in the population control group . in the group of all fpc individuals , the largest and statistically significant difference compared to the population control group was observed for palb2 172_175delttgt ( 0.5% ; or , 10.05 ; p=0.048 ) . when the genotyping results were classified according to the health status of the probands , statistically significant differences were observed for the brca1 , palb2 and chek2 genes only in the group of fpc cases with cancer . two ( 5382insc , c61 g ) of the three polish founder brca1 mutations were detected in three of the 105 fpc cases with cancer ( 2.86% ) ( or , 6.72 ; p=0.006 ) compared to 17 of the 4,000 controls ( 0.42% ) . palb2 mutations were observed in two of the 105 fpc cases with cancer ( 1.90% ) ( or , 9.52 ; p=0.014 ) compared to eight of the 4,000 controls ( 0.20% ) . chek2 mutations ( all four mutations combined ) were detected in 14 of the 105 fpc cases with cancer ( 13.33% ) ( or , 2.26 ; p=0.008 ) compared to 236 of the 4,000 controls ( 5.9% ) . the chek2 \n i157 t missense mutation contributed the largest and was present in 11 of the 105 fpc cases with cancer ( or , 2.17 ; p=0.026 ) compared to 193 of the 4,000 controls . a single nbs1 mutation ( 657del5 ) was detected in three of the 389 unselected fpc cases ( 0.75% ) : two healthy fpc probands ( 0.68% ) , and one of the 105 fpc cases with cancer ( 0.95% ) , compared to 22 of the 4,000 controls ( 0.55% ) , but none of the associations were statistically significant .", "this study observed a significant correlation between the prevalence of the polish founder mutation 172_175delttgt of palb2 gene and the fpc . in addition , an increased incidence of changes was observed in genes brca1 ( three founder mutation combined : p=0.006 ; or , 6.72 ) and chek2 ( four chek2 mutations combined : p=0.008 ; or , 2.26 ) , particularly the chek2 \n i157 t mutation ( p=0.03 ; or , 2.17 ) among the cancer - affected individuals from the fpc syndrome families . the fact that brca1 and palb2 mutations might be causative of fpc in a small subset of families is relatively new [ 14 - 16 ] . furthermore , the results are consistent with the observations that palb2 and brca1 mutations are reported in fpc , especially in the families with a history of breast cancer . breast cancer was also observed among the first degree relatives of one of the palb2 172_175delttgt and one of the brca1 5382insc mutation carriers from fpc . the most valuable observation of the present study is the increased risk of fpc among carriers of chek2 mutations , particularly the chek2 \n i157 t missense variant . chek2 is a human homologue of the yeast cds1 and rad53 genes , which encodes the cell cycle checkpoint kinase . therefore , chek2 plays an important role in the network of cell cycle regulation and dna damage repair , which are crucial processes in preventing cancer development . four founder alleles of chek2 are present in poland ; three of these result in a truncated chek2 protein ( 1100delc , ivs2 + 1g > a and del5395 ) and the fourth ( i157 t ) is a missense substitution of an isoleucine by a threonine in exon 3 . the feature of the founder mutations in the chek2 gene is the multiorgan cancer risk among carriers . positive associations with protein - truncating alleles in the chek2 gene are observed for cancers of the prostate and thyroid . the large germline deletion in chek2 the missense variant , i157 t , is associated with an increased risk of colon , kidney , prostate , thyroid , and stomach cancers . in light of existing research , it can be assumed that chek2 gene mutations are more specific to a certain percentage of cancers of the gastrointestinal tract . in a study conducted at our center including 463 individuals , it has been shown that the i157 t variant of chek2 increases the risk of colorectal cancer among mismatch repair negative hnpcc / hnpcc - related families from poland ( or , 2.1 ; p=0.0004 ) . polish founder mutations in the chek2 gene were also observed in 57 out of 658 unselected patients with gastric cancer ( 8.7% ) compared to 480 out of 8,302 controls ( 5.8% ) ( or , 1.6 ; p=0.004 ) . overall , it can be concluded that fpc is another malignant tumor from the spectrum of digestive system cancers associated with mutations of the chek2 gene . the results of a previous study conducted among 383 paca cases did not show this association for sporadic cancers of the pancreas . the current relationship between the chek2 mutation and the occurrence of fpc can be explained by the multiorgan nature of the familial cancer predisposition of chek2 mutations . note that among the fpc cancer cases with detected chek2 mutations , there were cases not only with paca , but also with cancer of the breast , cervix , thyroid , and melanoma . thus far , the possible relationship between chek2 mutations and fpc has not been evaluated extensively . in one existing german study based on the genotyping of 35 patients from families with fpc syndrome , the chek2 1100delc mutation was identified in one out of 35 fpc cases ( 3% ) . the authors of that study suggested a possible contribution of the chek2 mutation in a small subset of fpc . in addition , it is believed that mutations of the chek2 gene , similar to the mutations in the other aforementioned genes , cause only a small percentage of fpc cases . therefore , it can be assumed , that the search for genes that predispose to fpc , will bring to light new genes , but none of them will be responsible for a large percentage of families with fpc . note that mutation carriers with cancer in the present study were affected by cancers at different sites ( supplementary table 1 ) . among those , chek2 mutation carriers had the largest variety of different neoplasms , i.e. , multiple breast cancers , cervical cancers , and single cases of thyroid cancer , paca , and malignant melanoma . three brca1 carriers with cancer were observed , two of which had breast cancer and one ovarian cancer . these three brca1 mutation carriers had no other family member with breast and/or ovarian cancer . the nature of the chek2 gene as a multiorgan cancer predisposition gene has been reported [ 4,5,8 - 11 ] , which explains the occurrence of cancer at different sites among the chek2 mutation carriers in the present study . despite the single cases of different cancers in these families with paca , they only fulfil the criteria for fpc and did not meet the criteria for any other defined inherited tumor syndrome except for three carriers of a brca1 mutation ( hboc , brca1 type ) . our previous study conducted among 383 paca patients reported that the polish founder mutation in nbs1 ( 657del5 ) is associated with an increased risk of paca ( or , 3.80 ; p=0.002 ) . while the results of the current study , including almost 400 cases from fpc families presented here , did not show a relationship between the nbs1 and fpc syndrome . it might mean that a nbs1 mutation is not associated with the risk of fpc . on the other hand , it is equally possible that a relationship between the nbs1 and fpc risk was not observed because of the relatively small number of cases with cancer ( n=105 ) present in this study , in addition to the low penetrance of nbs1 mutation . indeed , more cases of cancer from fpc families should be analyzed in the future to resolve this suspicion . the major limitation of this study was the small number of cases with cancer from fpc families subjected to genotyping . these results should be verified with a larger series of fpc cases from both the polish and other populations .", "this case control study indicated that mutations in palb2 ( 172_175delttgt ) appear to increase the risk of fpc in heterozygous carriers from the polish population . in addition , the fpc risk appears to be increased in carriers of brca1 ( two of the three occurring : 5382insc , c61 g ) and chek2 founder mutations ( particularly chek2-i157 t missense variant ) . therefore , in the polish population , founder mutations in the genes , brca1 , palb2 , and chek2 , cause a small percentage of familial paca . studies in slavic and other populations should be performed to confirm this association and evaluate the utility of testing these genes for fpc in cancer genetic counselling .", "" ]

[ "the earliest mammals were shrew - like creatures who could search for food and hide in places that were too cool and dark for many of their lizard contemporaries ( 1 ) . in addition to being warm blooded , mammals also were adept at performing adaptive thermogenesis , giving them the ability to further increase their metabolic rates when conditions would otherwise result in hypothermia . from the perspective of humans in 2015 , it is easy to forget that thermoregulation , in all of its dimensions , was a critical part of mammalian history thermogenesis comes from chemical reactions in which the liberated free energy is not captured in other molecules ( e.g. , atp or creatine phosphate ) or used for work . the best known example of a heat - generating pathway is the futile cycle of proton pumping in brown fat and beige fat through the actions of uncoupling protein 1 ( ucp1 ) ( 2,3 ) . in cells expressing ucp1 , the oxidation of lipids and carbohydrates results in the extraction of high - energy electrons , which flow down the electron transport chain ( etc ) as protons are pumped across the inner mitochondrial membrane ( fig . the resulting electrochemical gradient across the inner mitochondrial membrane , which is usually coupled to atp synthesis via complex v of the etc , is dissipated by a leak of protons back across the inner membrane by ucp1 . thus , much of the chemical energy generated by fuel oxidation in brown fat cells powers a futile proton cycle , which does no work and is instead liberated as heat . this process is typically thought of as being indirectly activated by cold , via the sympathetic nervous system ( sns ) . adaptive thermogenesis can also be activated directly by cold in beige adipocytes and by other stimuli that may signal independently from the -adrenergic receptors . the reducing equivalents generated by the tricarboxylic acid ( tca ) cycle enter the etc . this generates a proton gradient across the inner mitochondrial membrane . instead of linking this gradient to atp synthesis via complex v , it is also worth noting that , from a thermodynamic perspective , there is nothing special about proton cycling , and such a thermogenic machine could , in theory , be built from other components of cellular metabolism . indeed , some deep - diving fish have a heater organ , which is a specialized muscle that entirely lacks a myofibrillar apparatus . this organ , found between the eye and brain in deep - diving marlin and certain tuna , uses a futile cycle of calcium leaking from the sarcoplasmic reticulum and atp - dependent calcium uptake to raise the local temperature near the brain of the fish ( 4 ) . in this case , the physiological goal is not to warm the whole body of the marlin , but to just make it a bit more astute than the other fish around it ! is ucp1 the only thermogenic pathway of importance in mammals ? while no other pathway of thermogenesis except shivering has been convincingly demonstrated , suggestive data from the scientific literature hints that such pathways probably exist . if mice lacking ucp1 are abruptly switched from ambient temperatures to cold ( 4c ) , they develop life - threatening hypothermia ( 5 ) . on the other hand , if they are gradually exposed to lower temperatures , they can survive quite normally at 4c ( 6 ) . this suggests that there are compensatory thermogenic programs that can be activated as long as thermal stress is applied more gradually . in addition , there are examples of experiments in mice where certain mutations have caused very robust increases in energy expenditure and thermogenesis , but where the levels of ucp1 mrna and protein are unchanged . for example , deletion of par-1b / mark2 in mice results in increased energy expenditure and brown fat activation , although ucp1 protein levels were unchanged ( 7 ) .", "ucp1 brown fat has long been known to occur in two distinct anatomical locations in rodents . developmentally formed depots , best typified by the interscapular and perirenal regions , are composed of tightly packed brown fat cells of relatively uniform appearance . these depots exist under most physiological conditions , although they may change in color and lipid content . on the other hand , ucp1 cells can accumulate in small pockets in white fat depots , especially in the subcutaneous adipose tissues , particularly when mice are exposed to long - term cold or stimuli with hormones such as catecholamines and other -adrenergic agonists ( 8) . this pathway of adaptive thermogenesis is robustly activated by cold via an indirect pathway mediated by the sympathetic nervous system . however , fat cells are also able to directly sense cold , though the mechanism is not well understood ( 9 ) . in both anatomical situations , brown fat cells have multiple small lipid droplets , numerous mitochondria , and rich innervation and vascularization . despite these similarities , it is now clear that the classical brown fat cells and the inducible beige fat cells come from different developmental lineages and are , in fact , distinct cell types . the key finding in this regard was the observation that the classical developmentally formed brown fat arises from a myf5 lineage shared with skeletal muscle ( 10 ) . consistent with this , primary cultures of classical brown fat express low levels of certain genes characteristic of skeletal muscle ( 11 ) . subsequent work indicated that the brown fat / skeletal muscle decision occurs between 9.5 and 12.5 days of gestation in mice ( 12 ) . conversely , the ucp1 cells induced in subcutaneous white fat depots by cold or -adrenergic agonists come from a myf5 lineage ( 10 ) . these beige fat cells have now been cloned and express a relatively low level of thermogenic genes , such as ucp1 and type 2 deiodinase in the basal state , but can induce these genes to levels essentially equivalent to classical brown fat cells when given hormonal stimuli ( 13 ) . moreover , the beige and brown fat cells have gene signatures that allow for distinction of these cells types ; however , their comparative physiological properties and overall roles in metabolism are not completely understood . it is now clear that thermogenic adipocytes , taken as a whole , contribute very importantly to metabolic homeostasis in rodents . the partial ablation of ucp1 cells through the transgenic expression of a toxigene led to these animals being more susceptible to obesity and diabetes ( 14 ) . similarly , mice lacking ucp1 through targeted mutation had an increased body weight and fat content , at least when raised at thermoneutrality ( 15 ) . excessive shivering apparently prevented obesity when these experiments were performed at ambient temperatures . the first transgenic model with increased brown and beige fat was the adipose - selective expression of foxc2 , a transcription factor that activated camp metabolism in recipient cells ( 16 ) . these mice , which were resistant to diet - induced obesity and diabetes , had expansions of both the classical brown and beige fat . deciphering the individual roles of brown and beige fat cells prd1-bf-1-riz1 homologous domain - containing protein-16 ( prdm16 ) is an important transcriptional coregulator in both brown and beige fat , but when its expression was elevated through a promoter expressed in all fat depots ( ap2 ) , phenotypic changes were observed in the erstwhile white fat depots , which developed copious pockets of beige fat cells ( 17,18 ) . the classical brown fat , which expresses very high levels of prdm16 in the basal state , showed few or no changes with a further elevation in prdm16 expression . but this occurred only when the transgenic mice also received stimulation with a -adrenergic agonist . while studies in cultured cells had shown that classical brown fat cells require prdm16 to develop and maintain a thermogenic gene program , ablation of prdm16 from all fat cells in vivo was not sufficient to significantly alter the function of the classical brown fat . on the other hand , the development of beige fat cells was severely reduced , at both the histological and molecular level ( 19 ) . these mice developed a moderate obesity , compared with control animals , with an unusual expansion of the subcutaneous fat depots . these beige fat deficient mice also showed a rather profound hepatic insulin resistance that was associated with liver steatosis . one last point of interest is that the subcutaneous fat from the prdm16-ablated mice showed the presence of more inflammatory macrophages and increased the expression of proinflammatory genes . the latter aspect was also observed with cultured subcutaneous cells lacking prdm16 , indicating that this transcriptional coregulator plays a role in the relative resistance of subcutaneous fat to inflammation and is an important determinant of subcutaneous versus visceral fat phenotype . the role of prdm16 in the classical brown fat in vivo is interesting and somewhat more complex . ablation of this factor with a myf5-driven cre recombinase causes a loss of brown fat function and gene expression , but only at 6 months of age ( 20 ) . however , ablation of prdm16 along with its closest homolog , prdm3 , causes an early and severe loss of brown fat thermogenic gene expression and normal histology . thus , it appears that both thermogenic cell types depend on prdm16 , but , at least in early life , the classical brown fat has a second factor that can support development , prdm3 .", "harnessing the therapeutic potential of brown and beige fat requires a detailed understanding of the molecular mechanisms responsible for the determination and maintenance of each cell type . the nuclear hormone receptor peroxisome proliferator activated receptor ( ppar ) is necessary and sufficient for the development of all fat cells ( 21 ) . however , white and brown adipocytes have drastically different phenotypes , indicating that other transcriptional regulators must be involved . a yeast two - hybrid screen identified ppar coactivator-1 ( pgc-1 ) as a cold - inducible binding partner of ppar ( 22 ) . in brown fat pgc-1 is now appreciated to regulate numerous physiological processes in a variety of metabolically important tissues . while brown and beige fat cells lacking pgc-1 have significantly blunted thermogenic gene expression , these cells still retain the molecular signature of brown fat cells ( 23,24 ) . the transcriptional regulator prdm16 was found to be highly enriched in brown fat , whereas it is virtually undetectable in visceral white fat cells . forced expression of prdm16 in cultured white fat cells induces pgc-1 and thermogenic genes , as well as mitochondrial genes and brown fat identity genes ( 17 ) . in addition to activating these pathways , prdm16 also binds ccaat / enhancer binding protein ( c / ebp ) and recruits the corepressor proteins ctbp1 and ctbp2 to repress white fat or muscle gene expression ( 25 ) . importantly , the expression of prdm16 and its binding partner c / ebp in fibroblasts is sufficient to promote differentiation into functional brown fat cells , which can be detected by [ f]-2-fluoro-2-deoxy - d - glucose ( [ f]-fdg ) positron emission tomography ( pet ) when transplanted into mouse models ( 26 ) . an increasing number of transcriptional regulators has been identified as important in brown and beige fat biology . the transcription factor early b - cell factor-2 ( ebf2 ) is enriched in brown relative to beige adipocytes . it functions upstream of prdm16 to promote binding of ppar to the promoters of brown - selective genes ( 27 ) . ehmt1 is a histone lysine methyltransferase that purifies with the prdm16 transcriptional complex in brown fat . ehmt1 is required for brown fat lineage specification and for thermogenesis ( 28 ) . presumably , there are also beige - selective factors upstream of prdm16 and beige - selective epigenetic regulators that modulate the phenotype of these cells . it is able to compete with prdm16 for binding to ppar , and thereby modulates white versus brown / beige phenotype ( 29,30 ) . forced expression of tle3 in adipocytes results in impaired thermogenesis , while deletion enhances thermogenesis in brown and beige fat . a number of other factors have also been shown to play roles in this biology , but a comprehensive review is beyond the scope of this article .", "until fairly recently , brown fat was thought to be present in meaningful amounts only in human infants and small mammals . the combination of insufficient hair , fur , and insulation , along with a high body surface area - to - mass ratio make babies and small mammals particularly susceptible to hypothermia . as a result , they have developed significant interscapular brown fat , which defends body temperature by adaptive thermogenesis . this brown fat was thought to regress by adulthood , unless exposed to catecholamine excess ( as in pheochromocytoma ) or long - term cold exposure ( as in outdoor workers in cold climates ) . in 2009 , these studies made use of [ f]-fdg pet to confirm that adults have glucose - avid tissue with imaging characteristics of adipose in deposits in the supraclavicular and spinal regions . as assayed by [ f]-fdg pet , the amount of active tissue is inversely associated with bmi , and this activity is increased by cold exposure . follow - up studies ( 34 ) have shown that this tissue has the histological appearance of adipose tissue and expresses several molecular markers of brown fat including ucp1 . current efforts are focused on developing optimal imaging modalities to detect this tissue and to quantify its absolute amounts and activity . this is particularly important since brown and beige fat also oxidize lipids , which would not be detectable by [ f]-fdg pet technology . research has increasingly suggested that adult human brown fat shares features of the inducible beige fat described in rodents . brown and beige fat cell lines have now been cloned from mice , allowing for the identification of unique molecular markers of each cell type . multiple studies ( 13,35,36 ) have shown that beige - selective markers are expressed more in human brown fat samples than are markers of the classical brown fat of rodents . a very recent study ( 37 ) characterized a human brown fat cell line and showed that it actually shares more molecular features with beige than brown adipocytes . however , the brown fat in human infants , located in the interscapular region , seems most similar to the interscapular classical brown fat in rodents . in some adult humans , cells with both brown and beige characteristics have been identified , with the depth in the neck seeming to be a determinant of relative brown versus beige character ( 36,38,39 ) . while further studies will clarify this question , this tissue may well contain both brown and beige cells in varying amounts in each individual .", "as a deeper understanding of brown and beige fat has emerged , researchers have turned to strategies to induce the activity of these tissues as a possible therapy for obesity and metabolic diseases . calculations have suggested that maximal cold - induced brown fat thermogenesis would be between 25 and 400 kcal / day in lean healthy volunteers ( 40 ) . given the inducibility of beige fat , activating these cells could result in even more substantial effects . however , the greatest benefits may not relate to increased energy expenditure per se , but rather might result from enhanced glucose and lipid disposal . in addition to taking up and consuming glucose ( 3133 ) , brown adipose tissue takes up free fatty acids from triglyceride - rich lipoproteins . cold exposure in mice upregulates this pathway , resulting in accelerated plasma triglyceride clearance ( 41 ) . while compounds like dinitrophenol can result in impressive weight loss , they have also been associated with complications , including fatal hyperthermia . while this has understandably resulted in caution , it is possible that more specific uncouplers or targeted activators of ucp1 or other futile cycles may provide metabolic benefit with an acceptable safety profile . in that regard , a recent publication ( 42 ) described the development of controlled - release mitochondrial protonophores that can apparently safely uncouple in the liver , and in rats these compounds can improve insulin resistance , diabetes , hypertriglyceridemia , and hepatic steatosis . brown and beige fat activity is robustly induced by thyroid hormone and catecholamines , but side effects would almost certainly limit their use . the 3-selective adrenergic agonist cl 316,243 potently activates brown and beige fat in rodents ( 43 ) . this drug was recently shown to activate human brown fat , which may renew interest in this pathway ( 44 ) . since cold robustly activates brown and beige fat , some investigators have suggested that moderate cold exposure could be used as a therapeutic approach . at least in healthy subjects , daily exposure to 19c for 2 h was sufficient to activate brown fat , resulting in weight loss ( 45 ) . in another small human study ( 46 ) while these studies suggest the potential of cold as a treatment modality , our societal preference for thermal comfort may make this unfeasible as a broad approach . thiazolidinediones ( tzds ) cause browning of the white fat and may do so by stabilizing prdm16 , resulting in its accumulation ( 47 ) . however , these drugs have been associated with weight gain , fluid retention , and cardiovascular events , which have diminished enthusiasm for their use . deacetylation of ppar by sirt1 promotes browning of the white fat , suggesting the possibility of developing compounds that selectively modulate this pathway ( 48 ) . other regulators of interest include bone morphogenetic protein 7 ( bmp7 ) and bmp8b , cyclooxygenase-2 ( cox-2 ) , and natriuretic peptides , though their pleiotropic actions might limit their potential as drug targets . fibroblast growth factor 21 ( fgf21 ) is being examined as a therapeutic agent , but it also has diverse actions and may be associated with bone loss . translating these discoveries into drug candidates will ultimately require a more complete understanding of brown / beige fat biology in humans . mouse studies suggest that prdm16 is a key control point in brown and beige fat phenotype . an increasing understanding of the relevant modifications of prdm16 and the upstream signaling pathways involved may provide new opportunities for therapeutic targeting of brown and beige fat ." ]

[ "in a recent issue of critical care , meyer and colleagues report interesting surveillance data from icus in 30 german hospitals , based on a large amount of microbiology and pharmacy data gathered between 2001 and 2008 . one of the main study findings is a decreasing rate of methicillin - resistant staphylococcus aureus ( mrsa ) and a dramatic increase of third generation cephalosporin ( 3gc ) resistant enterobacteriaceae over the study period . in recent years , most european countries have succeeded in reducing the burden of disease caused by mrsa . this progress has , however , been partly off set by the increase across europe in the prevalence of multiresistant gram - negative pathogens producing extended - spectrum betalactamases ( esbls ) or carbapenemases . the current study confirms this worrisome finding and also shows an increase in carbapenem , 3gc and fluoroquinolone use in german icus with a stable volume of overall antibiotic use . the latter two antibiotic classes have been repeatedly identified as risk factors for carriage of multidrug resistant gram - negatives . carbapenems on the other hand are first - line drugs for the treatment of infections due to esbl - producing bacteria . it is tempting to assume that overuse of fluoroquinolones and 3gc antibiotics contributed to the observed increase in esbl producers , which subsequently increased carbapenem use . the ecological nature of the data , however , makes it difficult to infer clear cause - and - effect relationships , as does the failure to differentiate between hospital- and community - acquired isolates and clinical versus surveillance cultures . in addition , the analysis of trends is hampered by variation in the number of participating icus over time . nevertheless , the increased burden of multidrug - resistant gram - negative bacteria is a real phenomenon . in contrast to mrsa , esbl producers - notably strains of escherichia coli carrying plasmids of the ctx - m family - are mostly imported from the community into the hospital . assuming a relationship with antibiotic use in animals and subsequent transmission of antibiotic resistant e. coli via the food chain is alluring , but strong epidemiological evidence is still lacking . the recently started european union - funded saturn project ( impact of specific antibiotic therapies on the prevalence of human host resistant bacteria ) will gather more information on these risk factors . as mentioned above , antibiotic overuse in humans probably plays a central role in the spread of esbl producers . in the context of this study it is noteworthy that germany has a relatively high level of fluoroquinolone use in the community . as to antibiotic use in hospitals , the overall quantity of antibiotic use in german icus is comparable to that of other countries . although there is important heterogeneity between icus , inappropriate antibiotic use is still common in germany ( as in many other countries ) , where infectious diseases as a stand - alone speciality and antibiotic stewardship programmes are still underdeveloped . compared to other european countries or highly publicised health threats , such as bioterrorism and swine flu , public awareness and political commitment to control multidrug - resistant microorganisms has been slow to rise in germany . only recently ( 2009 ) has a german reference centre for surveillance of gram - negative bacteria been established , which focuses , among other things , on detection of carbapenemase producing bacteria . a first outbreak of carbapenemase producing klebsiella pneumoniae in germany has recently been reported , probably linked to an index patient with previous healthcare contact in greece . the fact that two icus in 2008 reported carbapenem resistant k. pneumoniae in the current study raises the concern that these strains might already be more common in central europe than previously assumed since detection of these strains may be difficult with routine laboratory techniques . with regard to esbl - producers the growing community reservoir makes it unlikely that we will be able to control the spread by conventional measures targeted at nosocomial infection control . the promotion of prudent antibiotic use in the community and animal husbandry should therefore be a key priority . as to carbapenemase - producing enterobacteriaceae , early identification of these strains and aggressive infection control measures seem essential . examin ing novel decolonization strategies for gram - negative enterobacteriacae might be a further strategy worth evaluating . if we manage to enforce all these measures , we will hopefully be able to think positive again - even with regard to gram - negatives .", "3gc : third generation cephalosporin ; esbl : extended - spectrum betalactamase ; mrsa : methicillin - resistant staphylococcus aureus .", "", "preparation of this commentary was supported in part by the 6th and 7th framework programme of the european community in the context of the projects ' changing behaviour of health care professionals and the general public towards a more prudent use of anti - microbial agents ' ( champ , contract sp5a - ct-2007 - 044317 ) and ' impact of specific antibiotic therapies on the prevalence of human host resistant bacteria ' ( acronym saturn , agreement fp7-health-2009-n241796 ) ." ]

[ "primary malignant tumors of the hypopharynx are predominantly squamous cell carcinomas ( sqccs ) , as primary small - cell carcinoma ( smcc ) of the hypopharynx is rare . combined primary smcc and sqcc of the hypopharynx , referred to as composite tumor of the hypopharynx , is even more rare , with only 3 cases previously reported ( 13 ) . we herein report a the case of a patient with primary combined smcc with an sqcc element and investigate the expression of specific proteins for molecular - targeted therapy .", "a 74-year - old man , with a ~50-year history of excessive alcohol consumption and smoking , presented with a 3-month history of throat pain and hoarseness . on hypopharyngoscopy , a tumor was identified in the right anterior wall of the piriform sinus ( fig . a pathologist analyzed the biopsy sample and diagnosed the lesion as sqcc , as no smcc component was identified in the biopsy specimen . the right vocal cord was not fixed . a contrast computed tomography ( ct ) scan of the neck revealed a heterogeneously enhanced tumor sized 251738 mm extending through the right piriform sinus ( fig . moreover , fluorodeoxyglucose positron emission tomography ( fdg - pet ) revealed high - level accumulation in the primary tumor , with a maximum standardized uptake value of 14.6 . there was no evidence of cervical lymph node metastasis , primary lung tumor , or distant metastasis . the patient was diagnosed with hypopharyngeal cancer classified as t2n0m0 , according to the 2009 union for international cancer control staging system ( 4 ) . we performed a tracheostomy , total laryngectomy , right hemithyroidectomy and bilateral lateral neck dissection ( level ii microscopically , two components were identified in the lesion , namely a poorly differentiated sqcc and an smcc containing chromatin - rich nuclei with scanty cytoplasm ( fig . pathologically , multiple metastatic cervical lymph nodes were identifed bilaterally ( right side , levels ii and iii ; and left side , level iii ) . however , after the first course , adjuvant chemotherapy was discontinued due to prolonged bone marrow suppression , although the therapy was effective . however , despite treatment , distant metastases in the thoracic vertebrae ( t5 , 9 and 10 ) were confirmed by ct 6 months later . the patient 's condition started to deteriorate and he succumbed to the disease 7 months after the initial treatment . on gross examination of the laryngectomy specimen , the tumor extended superiorly to involve the right supraglottis and exhibited variable gross morphology , ranging from ulcerated , to nodular , to plaque - like growth . the tumor infiltrated 1.3 cm in depth , but did not involve the thyroid cartilage , vocal cord , or trachea . microscopic sections from the larynx revealed a combined tumor , composed predominantly of smcc localized in the upper side and sqcc localized in the lower side ( figs . 1 and 2a ) . we observed 2 components in the lesion : a poorly differentiated sqcc and a small - sized smcc with finely granular , hyperchromatic nuclei , inconspicuous nucleoli and scanty cytoplasm . the metastatic potential of the lesion was further supported by the smcc component found to infiltrate lymphatic vessels ( fig . diffuse cd56 positivity in the smcc component in the cervical lymph nodes supported the histological diagnosis ( fig . the sqcc component was negative for neuroendocrine markers , including cd56 , synaptophysin and chromogranin a , although it was positive for ck 34e12 ( fig . immunoreactivity to ki-67 was seen in the nuclei of tumor cells . in the smcc component , the ki-67 labeling index was 50.2% , while that in the sqcc component was 47.5% ( fig . to predict the prognosis of this patient , we evaluated the expression of epidermal growth factor receptor ( egfr ) . furthermore , to evaluate the possibility of using molecular - targeted therapy for combined smcc and sqcc , the expression of proteins such as platelet - derived growth factor receptor ( pdgfr ) , vascular endothelial growth factor receptor 2 ( vegfr2 ) and kit was examined . on immunohistochemical analysis , the smcc element was strongly positive for kit and pdgfr ; however , egfr and vegfr2 were not expressed ( fig . the sqcc element was mildly positive for pdgfr and egfr ; however , kit and vegfr2 were not expressed ( fig . genomic dna was extracted from frozen tumor specimens using the qiaamp dna mini kit ( qiagen , hilden , germany ) , according to the manufacturer 's protocol . a molecular genetic analysis of kit ( exons 9 , 11 , 13 and 17 ) and pdgfr ( exons 12 , 14 and 18 ) was performed using the polymerase chain reaction ( pcr ) direct sequencing method , as previously reported ( 58 ) . pcr products were extracted and subjected to a computed automatic dna sequencing ( abi prism 3100 genetic analyzer ; applied biosystems , carlsbad , ca , usa ) . analysis of exon 12 of pdgfr from other normal tonsillar tissues revealed a similar ag change at the 1849 nucleotide position . comparison of this sequence variation with the single - nucleotide polymorphism ( snp ) database revealed the presence of a known snp at codon 567 of pdgfr ( 9 ) . the hpv status was determined using the hpv typing set ( takara bio . , tokyo , japan ) , a pcr primer set specifically designed to identify hpv genotypes 16 , 18 , 31 , 33 , 35 , 52 and 58 in genomic dna . the pcr products were separated by electrophoresis through a 9% polyacrylamide gel and stained with ethidium bromide . our case was negative and not considered as a high - risk hpv status ( fig .", "on gross examination of the laryngectomy specimen , a mass involving the right piriform sinus was palpable . the tumor extended superiorly to involve the right supraglottis and exhibited variable gross morphology , ranging from ulcerated , to nodular , to plaque - like growth . the tumor infiltrated 1.3 cm in depth , but did not involve the thyroid cartilage , vocal cord , or trachea . microscopic sections from the larynx revealed a combined tumor , composed predominantly of smcc localized in the upper side and sqcc localized in the lower side ( figs . 1 and 2a ) . we observed 2 components in the lesion : a poorly differentiated sqcc and a small - sized smcc with finely granular , hyperchromatic nuclei , inconspicuous nucleoli and scanty cytoplasm . the metastatic potential of the lesion was further supported by the smcc component found to infiltrate lymphatic vessels ( fig . diffuse cd56 positivity in the smcc component in the cervical lymph nodes supported the histological diagnosis ( fig . the sqcc component was negative for neuroendocrine markers , including cd56 , synaptophysin and chromogranin a , although it was positive for ck 34e12 ( fig . immunoreactivity to ki-67 was seen in the nuclei of tumor cells . in the smcc component , the ki-67 labeling index was 50.2% , while that in the sqcc component was 47.5% ( fig . furthermore , to evaluate the possibility of using molecular - targeted therapy for combined smcc and sqcc , the expression of proteins such as platelet - derived growth factor receptor ( pdgfr ) , vascular endothelial growth factor receptor 2 ( vegfr2 ) and kit was examined . on immunohistochemical analysis , the smcc element was strongly positive for kit and pdgfr ; however , egfr and vegfr2 were not expressed ( fig . the sqcc element was mildly positive for pdgfr and egfr ; however , kit and vegfr2 were not expressed ( fig . genomic dna was extracted from frozen tumor specimens using the qiaamp dna mini kit ( qiagen , hilden , germany ) , according to the manufacturer 's protocol . a molecular genetic analysis of kit ( exons 9 , 11 , 13 and 17 ) and pdgfr ( exons 12 , 14 and 18 ) was performed using the polymerase chain reaction ( pcr ) direct sequencing method , as previously reported ( 58 ) . pcr products were extracted and subjected to a computed automatic dna sequencing ( abi prism 3100 genetic analyzer ; applied biosystems , carlsbad , ca , usa ) . analysis of exon 12 of pdgfr from other normal tonsillar tissues revealed a similar ag change at the 1849 nucleotide position . comparison of this sequence variation with the single - nucleotide polymorphism ( snp ) database revealed the presence of a known snp at codon 567 of pdgfr ( 9 ) . tokyo , japan ) , a pcr primer set specifically designed to identify hpv genotypes 16 , 18 , 31 , 33 , 35 , 52 and 58 in genomic dna . the pcr products were separated by electrophoresis through a 9% polyacrylamide gel and stained with ethidium bromide . our case was negative and not considered as a high - risk hpv status ( fig .", "on gross examination of the laryngectomy specimen , a mass involving the right piriform sinus was palpable . the tumor extended superiorly to involve the right supraglottis and exhibited variable gross morphology , ranging from ulcerated , to nodular , to plaque - like growth . the tumor infiltrated 1.3 cm in depth , but did not involve the thyroid cartilage , vocal cord , or trachea . microscopic sections from the larynx revealed a combined tumor , composed predominantly of smcc localized in the upper side and sqcc localized in the lower side ( figs . 1 and 2a ) . we observed 2 components in the lesion : a poorly differentiated sqcc and a small - sized smcc with finely granular , hyperchromatic nuclei , inconspicuous nucleoli and scanty cytoplasm . the metastatic potential of the lesion was further supported by the smcc component found to infiltrate lymphatic vessels ( fig . diffuse cd56 positivity in the smcc component in the cervical lymph nodes supported the histological diagnosis ( fig . the sqcc component was negative for neuroendocrine markers , including cd56 , synaptophysin and chromogranin a , although it was positive for ck 34e12 ( fig . immunoreactivity to ki-67 was seen in the nuclei of tumor cells . in the smcc component , the ki-67 labeling index was 50.2% , while that in the sqcc component was 47.5% ( fig . furthermore , to evaluate the possibility of using molecular - targeted therapy for combined smcc and sqcc , the expression of proteins such as platelet - derived growth factor receptor ( pdgfr ) , vascular endothelial growth factor receptor 2 ( vegfr2 ) and kit was examined . on immunohistochemical analysis , the smcc element was strongly positive for kit and pdgfr ; however , egfr and vegfr2 were not expressed ( fig . the sqcc element was mildly positive for pdgfr and egfr ; however , kit and vegfr2 were not expressed ( fig .", "genomic dna was extracted from frozen tumor specimens using the qiaamp dna mini kit ( qiagen , hilden , germany ) , according to the manufacturer 's protocol . a molecular genetic analysis of kit ( exons 9 , 11 , 13 and 17 ) and pdgfr ( exons 12 , 14 and 18 ) was performed using the polymerase chain reaction ( pcr ) direct sequencing method , as previously reported ( 58 ) . pcr products were extracted and subjected to a computed automatic dna sequencing ( abi prism 3100 genetic analyzer ; applied biosystems , carlsbad , ca , usa ) . analysis of exon 12 of pdgfr from other normal tonsillar tissues revealed a similar ag change at the 1849 nucleotide position . comparison of this sequence variation with the single - nucleotide polymorphism ( snp ) database revealed the presence of a known snp at codon 567 of pdgfr ( 9 ) . tokyo , japan ) , a pcr primer set specifically designed to identify hpv genotypes 16 , 18 , 31 , 33 , 35 , 52 and 58 in genomic dna . the pcr products were separated by electrophoresis through a 9% polyacrylamide gel and stained with ethidium bromide . our case was negative and not considered as a high - risk hpv status ( fig .", "in the head and neck , smcc most commonly arises in the larynx , but has also been reported in the sinonasal tract and salivary glands ( 10,11 ) . combined smcc of the larynx has only been reported in 17 cases in the literature to date . moreover , the majority of the patients succumbed to the disease within 2 years of diagnosis , even with adjuvant radiation and chemotherapy ( 1214 ) . despite the rarity of these tumors , the clinical behavior of combined smcc of the larynx appears to be similar to that of the hypopharynx . metastasis from a primary lung smcc must be carefully distinguished from a primary hypopharyngeal or laryngeal smcc by imaging studies of the lung ( 15 ) . in this case , fdg - pet revealed high - level accumulation in the hypopharynx , without evidence of a lung tumor or distant metastasis . a chest contrast ct scan confirmed the absence of a primary lung tumor . according to the world health organization classification of head and neck tumors in 2005 , neuroendocrine carcinomas may be classified as typical carcinoid , atypical carcinoid , and smcc ( 16 ) . neuroendocrine types of smcc associated with an sqcc component are referred to as combined carcinomas . neuroendocrine neoplasms of the hypopharynx and larynx constitute a morphologicaly heterogeneous group of tumors , with considerable differences in clinical behavior and very different treatment strategies ( 12 ) . combined smcc of the hypopharynx is extremely rare and is often difficult to pathologically diagnose prior to surgical treatment . moreover , the prognosis is very poor due to early metastasis , without established treatment regimen ( 3 ) . of the 4 reported cases of combined smcc of the hypopharynx , including our case , 3 succumbed to the disease within 1 year ( table i ) . multi - modality therapy should be performed for combined smcc of the head and neck region . the present case reports new findings : positive expression of kit and pdgfr in the smcc element and positive expression of egfr and pdgfr in the sqcc element . furthermore , to the best of our knowledge , the present study is the first to report a kit and pdgfr mutation analysis in combined hypopharyngeal smcc . kit and pdgfr , both mapped to chromosome 4q12 , have structural similarities with other pdgfr family members . a majority of gastrointestinal stromal tumors ( gists ) display a gain - of - function mutation in the kit proto - oncogene that encodes the kit protein . the majority of mutations occur in kit exon 11 , but mutations may also be found in exon 9 and rarely in exons 13 and 17 . the pdgfr gene is also very similar to the kit gene , and pdgfr mutations have been found in exons 12 , 14 and 18 . terada et al ( 18 ) reported kit protein expression in 100% and pdgfr expression in 65% of small - cell lung carcinoma cases ; however , there were no genetic mutations of kit and pdgfr in small - cell lung carcinoma ( 18 ) . in a previously reported case of esophageal combined smcc , kit and pdgfr were expressed in the smcc component , but not in the sqcc component ( 5 ) . it was found that smcc of the lung and extrapulmonary organs expressed kit and pdgfr , but there were no mutations in these genes within the hot spots of gists . no biomarker has been yet proven to predict response to targeted therapy in either smcc or sqcc . moreover , a predictive biomarker in one cancer type may not be helpful in another cancer type , suggesting that different mechanisms may be involved in combined smcc . to the best of our knowledge , a molecular - targeted therapy for treating combined smcc of the hypopharynx and larynx has not yet been reported . our case is the first to report kit and pdgfr expression in combined smcc in the hypopharynx and larynx ; if similar results are obtained from larger series , these data may have management and prognostic implications through the possible use of targeted biological therapy in these tumors . at present , smcc treatment remains a significant challenge for the oncologists , with several targeted agents under evaluation . however , there is no effective molecular - targeted therapy for smcc . evaluating the expression of the kit and pdgfr genes within hypopharyngeal smcc may facilitate the development of novel agents for the molecular - targeted therapy of this tumor . however , further investigations are necessary regarding these proteins expression and clinical application of molecular targeted therapy for hypophageal combined smcc ." ]

[ "cells and viruses : bovine testicle ( bt ) cells were grown in eagle s \n minimum essential medium ( emem ) ( nissui pharmaceutical , tokyo , japan ) supplemented with \n 0.295% tryptose phosphate broth ( tpb ) ( becton dickinson , san jose , ca , u.s.a . ) and 5% fetal \n bovine serum ( fbs ) ( mitsubishi chemical , tokyo , japan ) . bovine kidney cell line mdbk - hs \n and porcine kidney cell line sk - l were grown in emem supplemented with 0.295% tpb , 10% \n horse serum ( hs ) ( life technologies , carlsbad , ca , u.s.a . ) and 10 mm n , n - bis \n ( 2-hydroxyethyl)-2-aminoethanesulfonic acid ( bes ) ( sigma - aldrich , st . bovine fetal muscle ( bfm ) cells were grown in emem supplemented with 0.295% tpb , 5% fbs , 5% \n hs and 10 mm bes . cells were confirmed to be free from bvdv , and fbs was confirmed to be \n free from both bvdv and anti - bvdv neutralizing antibodies . the bvdv gbk strain is an adventitious bvdv2 isolated from cells of the bovine kidney cell \n line gbk . the \n ndv miyadera strain was propagated in 10-day - old embryonated hens eggs . the new jersey \n serotype strain of vsv was grown in sk - l cells . cloning of a pair of end and end viruses from viral \n stocks : a reverse plaque formation technique was used to obtain a pair of end and end viruses from \n viral stocks as described previously . sequencing : bvdv gbk_e and gbk_e strains , the \n full - length cdna clones and the in vitro - rescued viruses were completely \n sequenced as described previously . in brief , the \n nucleotide sequences of cdna clones and pcr fragments from viral rna were determined using \n the bigdye terminator v3.1 cycle sequencing kit ( life technologies ) and a 3130 genetic \n analyzer or a 3500 genetic analyzer ( life technologies ) according to the manufacturer s \n protocol . sequencing data were analyzed using genetyx version 10 software ( genetyx , tokyo , \n japan ) . plasmid constructs : the cdna fragments from the gbk_e strain , \n obtained by reverse transcription polymerase chain reaction ( rt - pcr ) , were cloned into \n plasmid pcr - blunt ii - topo ( life technologies ) using the zero \n blunt topo cloning kit ( life technologies ) . the cdna sequence was \n flanked by a modified t7 promoter sequence at the 5 end and a pst i \n restriction site at the 3 end . subclones were assembled into a full - length cdna clone , \n termed pgbk_e , by replacing the csfv genome of the full - length cdna clone of the \n csfv alfort187 - 1 strain pa187 - 1 with the genome \n of the gbk_e strain using appropriate restriction enzymes and the in - fusion hd \n cloning kit ( clontech , mountain view , ca , u.s.a . ) according to the manufacturer s protocol . \n the full - length cdna clone \n pgbk_e was transformed and propagated in competent cell stbl3 cells ( life \n technologies ) and purified using the qiagen plasmid plus midi kit ( qiagen , hilden , germany ) \n according to the manufacturer s protocol . g , \n i2623v , d3148 g and d3502y were constructed in the pgbk_e backbone using the \n quickchange lightning multi mutagenesis kit ( agilent technologies , santa clara , ca , u.s.a . ) \n and the in - fusion hd cloning kit . the plasmid pgbk_e / d136 g has a single amino \n acid substitution at position 136 of gbk_e , whereas the plasmids \n pgbk_e / i2623v ; d3148 g ; d3502y and pgbk_e / d136 g ; i2623v ; d3148 g ; \n d3502y have multiple amino acid substitutions at positions 2623 , 3148 and 3502 of \n gbk_e and at positions 136 , 2623 , 3148 and 3502 of gbk_e , \n respectively . full - length pcr amplification , in vitro rna transcription , transfection and viral \n recovery : the cdna - derived viruses were rescued as described previously with some modifications . the full - length genome \n amplification strategy was employed to obtain a \n full - length pcr amplicon for in vitro rna transcription . the cdna clones \n were amplified using primers 5gbke_t7 ( 5-taa tac gac tca cta ta gta tac \n gag att agc taa agt act cg 3 , t7 promoter sequence underlined ) and 3gbke ( 5- \n ggg gct gtt aga ggc atc ctc tag tc 3 ) with accuprime taq dna polymerase high fidelity \n ( life technologies ) . viral rna was transcribed in vitro from the purified \n full - length pcr amplicon using the megascript t7 kit ( life technologies ) , the remaining pcr \n amplicon was digested using dnase ( life technologies ) , and the viral rna was then purified \n with a microspin s-400 column ( ge healthcare , buckinghamshire , u.k . ) . mdbk - hs cells ( 3 \n 10 cells ) were transfected with 10 g of viral rna in a 0.4 \n cm cuvette using the gene pulser xcell electroporation system ( bio - rad , hercules , ca , \n u.s.a . ) the cells were incubated at 37c in 5% \n co2 for 3 days , and the supernatants were then transferred onto naive mdbk - hs \n cells to obtain infectious viruses . the viruses were named according to the plasmid from which they were rescued , replacing the cdna - derived viruses generated in the present study are \n shown in fig . 1.schematic representation of the genomic differences between gbk_e virus \n and gbk_e virus , and mutant viruses derived from cdna clones generated in \n the present study . ( a ) four amino acid substitutions , d136 g , i2623v , d3148 g and \n d3502y , were found in the gbk_e virus in comparison with the \n gbk_e virus . ( b ) three recombinant viruses with combinations of amino \n acid substitutions were generated in the gbk_e backbone by site - directed \n mutagenesis and in - fusion techniques . the white and gray boxes indicate the \n nonstructural and structural proteins , respectively .. schematic representation of the genomic differences between gbk_e virus \n and gbk_e virus , and mutant viruses derived from cdna clones generated in \n the present study . ( a ) four amino acid substitutions , d136 g , i2623v , d3148 g and \n d3502y , were found in the gbk_e virus in comparison with the \n gbk_e virus . ( b ) three recombinant viruses with combinations of amino \n acid substitutions were generated in the gbk_e backbone by site - directed \n mutagenesis and in - fusion techniques . end assay and measurements of type i interferon production : the end assay \n was conducted using bfm cells as described previously . in brief , bfm cells grown in 96-well plates were infected with bvdv and \n incubated for 5 days at 37c in 5% co2 . the end \n phenomenon was regarded as positive , if strong cpe was observed in ndv - infected cells . measurements of type i interferon production in bvdv - infected cells were performed using a \n previously described plaque reduction method with vsv as a challenge virus , with some modifications . in brief , the supernatants \n from bvdv - infected cells were inactivated by exposure to uv light ( 254 nm ) in the uv \n crosslinker ( atto corporation , tokyo , japan ) under the condition of 500 mj / cm . \n viral inactivation of samples was confirmed using indirect fa techniques with anti - bvdv ns3 \n monoclonal antibody # 46/1 after inoculation of \n samples onto mdbk - hs cells and incubation for 2 days at 37c in 5% co2 . then , \n monolayers of sk - l cells in 12-well plates were inoculated with 1 ml of a \n four - fold dilution of uv - inactivated supernatant and incubated for 24 hr at 37c in 5% \n co2 . interferon titers were expressed as reciprocals of dilutions that reduced the number of \n challenge viral plaques by 50% . detection of irf-3 in bvdv - infected cells by sodium dodecyl sulfate - polyacrylamide \n gel electrophoresis ( sds - page ) and western blotting : sds - page and western \n blotting were performed as described previously . \n bfm cells were infected with bvdv ( m.o.i . of 1 ) in six - well plates and incubated for 5 days \n at 37c in 5% co2 . after \n transferring the proteins from the gels to the immunobilion - p transfer membrane ( millipore , \n billerica , ma , u.s.a . ) , the membranes were treated with anti - human irf-3 rabbit polyclonal \n antibodies ( genetex , hsinchu city , taiwan ) , goat anti - rabbit igg - hrp conjugate ( bio - rad ) and \n immunobilion western detection reagents ( millipore ) , in that order . the membranes were read \n using lumi vision pro ( aishin seiki , kariya , japan ) , and specific bands for irf-3 were \n detected .", "cloning of a pair of end/ end viruses from the bvdv gbk \n strain and determining their whole genome sequences : a pair of end \n and end viruses ( gbk_e and gbk_e , respectively ) was \n cloned from the bvdv gbk strain by means of reverse plaque formation techniques and limited \n dilution . the genomes of gbk_e and gbk_e viruses were both 12,284 \n nucleotides in length and encoded 3,897 deduced amino acids . comparison of the complete \n genome sequences of both viruses revealed only six nucleotide and four amino acid \n differences ( d136 g , i2623v , d3148 g and d3502y , the numbers refer to the amino acid position \n in gbk_e ) between the two viruses ( table \n 1table 1.differences of amino acid sequences in the genome of gbk_e and \n gbk_eviral proteinnns4bns5ans5bamino acid position1362,6233,1483,502gbk_ediddgbk_egvgya ) d : aspartic acid , i : isoleucine , g : glycine , v : valine , y : tyrosine . ) . the complete genome sequences of the gbk_e and gbk_e \n strains were deposited in the ddbj / embl / genbank databases under accession numbers ab894423 \n and ab894424 , respectively . a ) d : aspartic acid , i : isoleucine , g : glycine , v : valine , y : tyrosine . generation and characterization of in vitro - rescued viruses : the \n infectious bvdv vgbk_e was successfully rescued by electroporation of mdbk - hs \n cells with viral rna transcribed in vitro from a full - length pcr amplicon \n obtained from a cdna clone pgbk_e . the mutant viruses \n vgbk_e / d136 g , vgbk_e / i2623v ; d3148 g ; d3502y and \n vgbk_e / d136 g ; i2623v ; d3148 g ; d3502y were also recovered from full - length cdna \n clones . sequencing of the complete genomes of these three viruses confirmed the mutations at \n the desired amino acid positions and demonstrated the lack of any other mutations in \n comparison with the parental gbk_e virus . to investigate the biological properties of the mutant viruses , bfm cells were inoculated \n with both the parental gbk_e and the in vitro - rescued \n vgbk_e viruses . the results revealed that both gbk_e and \n vgbk_e were ncp ( data not shown ) and exhibited the end phenomenon \n ( end ) ( fig . bfm cells infected with gbk_e , gbk_e \n or the in vitro - rescued viruses were superinfected with newcastle \n disease virus ( ndv ) . cells infected with gbk_e , vgbk_e or \n vgbk_e / i2623v ; d3148 g ; d3502y exhibited distinguishable cpe after \n superinfection with ndv ( end ) , whereas those infected with \n gbk_e , vgbk_e / d136 g or vgbk_e / d136 g ; i2623v ; \n d3148 g ; d3502y did not exhibit cpe ( end ) in addition , titration of both viruses in mdbk - hs cells revealed that \n vgbk_e exhibited the same growth characteristics as wild - type \n gbk_e ( data not shown ) . moreover , compared with the wild - type \n gbk_e strain , other in vitro - rescued viruses grew equally in \n mdbk - hs cells ( data not shown ) . bfm cells infected with gbk_e , gbk_e \n or the in vitro - rescued viruses were superinfected with newcastle \n disease virus ( ndv ) . cells infected with gbk_e , vgbk_e or \n vgbk_e / i2623v ; d3148 g ; d3502y exhibited distinguishable cpe after \n superinfection with ndv ( end ) , whereas those infected with \n gbk_e , vgbk_e / d136 g or vgbk_e / d136 g ; i2623v ; \n d3148 g ; d3502y did not exhibit cpe ( end ) identification of the amino acid determinants responsible for inhibition of type i \n interferon production : as expected , the mutant viruses vgbk_e / d136 g , \n vgbk_e / i2623v ; d3148 g ; d3502y and vgbk_e / d136 g ; i2623v ; d3148 g ; \n d3502y remained ncp ( data not shown ) . an end assay revealed that the \n vgbk_e / d136 g and vgbk_e / d136 g ; i2623v ; d3148 g ; d3502y viruses were \n changed to end ; bfm cells infected with these viruses did not exhibit cpe after \n ndv superinfection . in contrast , the vgbk_e / i2623v ; d3148 g ; d3502y virus \n remained end ; bfm cells infected with this virus exhibited clear cpe after \n superinfection with ndv , as observed in bfm cells infected with gbk_e ( fig . the amount of type i interferon in supernatants from bfm cells infected with the parent or \n one of the mutant viruses gbk_e , gbk_e , vgbk_e , \n vgbk_e / d136 g , vgbk_e / i2623v ; d3148 g ; d3502y or \n vgbk_e / d136 g ; i2623v ; d3148 g ; d3502y was measured in sk - l cells . secretion of \n type i interferon was inhibited in bfm cells infected with gbk_e , \n vgbk_e or the virus carrying the mutations without npro \n ( vgbk_e / i2623v ; d3148 g ; d3502y ) , which is in consistent with the results \n from the end assay . cells infected with gbk_e or viruses carrying mutations in \n n(vgbk_e / d136 g and vgbk_e / d136 g ; i2623v ; d3148 g ; \n d3502y ) did induce type i interferon ( table \n 2table 2.measurements of type i interferon in cells infected with wild - type and \n in - vitro rescued virusesvirusestype i interferongbk_e<4vgbk_e<4vgbk_e / d136g4vgbk_e / i2623v ; d3148 g ; d3502y<4vgbk_e / d136 g ; i2623v ; d3148 g ; d3502y4gbk_e4 ) . these results clearly indicate that the aspartic acid of n(at position 136 \n of the gbk_e strain ) is the key amino acid residue that determines the capacity \n to inhibit the production of type i interferon and induce the end phenomenon . detection of irf-3 in cells infected with the parental or mutant viruses : \n to investigate the expression levels of irf-3 in bvdv - infected cells , irf-3 in the lysates \n of bfm cells infected with gbk_e , gbk_e , vgbk_e or one \n of the three mutant viruses was detected by western blotting . irf-3 was not detected in the \n lysates of cells infected with gbk_e , vgbk_e or \n vgbk_e / i2623v ; d3148 g ; d3502y , whereas clear bands of irf-3 appeared in the \n lysates of cells infected with the end gbk_e virus or one of the \n n mutant viruses ( vgbk_e / d136 g and vgbk_e / d136 g ; \n i2623v ; d3148 g ; d3502y ) ( fig . 3fig . irf-3 was not \n detected in western blots of the lysates of cells infected with gbk_e , \n vgbk_e or vgbk_e / i2623v ; d3148 g ; d3502y , whereas clear bands \n of irf-3 appeared in western blots of the lysates of cells infected with \n gbk_e , vgbk_e / d136 g or vgbk_e / d136 g ; i2623v ; \n d3148 g ; d3502y . ) . irf-3 was not \n detected in western blots of the lysates of cells infected with gbk_e , \n vgbk_e or vgbk_e / i2623v ; d3148 g ; d3502y , whereas clear bands \n of irf-3 appeared in western blots of the lysates of cells infected with \n gbk_e , vgbk_e / d136 g or vgbk_e / d136 g ; i2623v ; \n d3148 g ; d3502y . comparison of amino acid sequences of zinc - binding trash motif in \n n : the amino acid sequences of the zinc - binding trash motifs in the \n n proteins from various bvdv strains deposited in the ddbj / embl / genbank \n databases were compared with those of the gbk_e and gbk_e strains . \n at least one strain per subgenotype [ 1a1o ( except for 1l ) and 2a2c ] \n [ 11 , 18 ] was \n chosen in the present study . as a result , bvdv strains , except for the g strain , contain \n amino acid residues cys112-cys134-asp136-cys138 in the zinc - binding trash motif of \n n , as observed in gbk_e ( fig . \n 4.comparison of amino acid sequences of the zinc - binding trash motifs in \n n. the amino acid sequences of the zinc - binding trash motifs in the \n n proteins of various bvdv strains deposited in the ddbj / embl / genbank \n databases were compared with those of gbk_e and gbk_e . at \n least one strain per subgenotype [ 1a1o ( except for 1l ) and 2a2c ] \n was chosen . the cysteines ( cs ) at positions 112 , 134 and 138 of the zinc - binding trash \n motif are highlighted in bold and underlined . the accession numbers of strains from the ddbj / embl / genbank are as \n follows : nadl ( m31182 ) , sd-1 ( m96751 ) , osloss ( m96687 ) , cp7 ( u63479 ) , bega ( af049221 ) , \n 721 ( af144463 ) , f ( af287284 ) , 3186v6 ( af287282 ) , w ( af287290 ) , a ( af287283 ) , g \n ( af287285 ) , 23 - 15 ( af287279 ) , ks86 - 1ncp ( ab078950 ) , suwancp ( kc853440 ) , zm-95 \n ( af526381 ) , shitara/02/06 ( ab359930 ) , is25cp/01 ( ab359931 ) , 890 ( u18059 ) , \n hokudai - lab/09 ( ab567658 ) and nrw 14 - 13_dup ( ) ( hg426485 ) . ) . comparison of amino acid sequences of the zinc - binding trash motifs in \n n. the amino acid sequences of the zinc - binding trash motifs in the \n n proteins of various bvdv strains deposited in the ddbj / embl / genbank \n databases were compared with those of gbk_e and gbk_e . at \n least one strain per subgenotype [ 1a1o ( except for 1l ) and 2a2c ] \n was chosen . the cysteines ( cs ) at positions 112 , 134 and 138 of the zinc - binding trash \n motif are highlighted in bold and underlined . the accession numbers of strains from the ddbj / embl / genbank are as \n follows : nadl ( m31182 ) , sd-1 ( m96751 ) , osloss ( m96687 ) , cp7 ( u63479 ) , bega ( af049221 ) , \n 721 ( af144463 ) , f ( af287284 ) , 3186v6 ( af287282 ) , w ( af287290 ) , a ( af287283 ) , g \n ( af287285 ) , 23 - 15 ( af287279 ) , ks86 - 1ncp ( ab078950 ) , suwancp ( kc853440 ) , zm-95 \n ( af526381 ) , shitara/02/06 ( ab359930 ) , is25cp/01 ( ab359931 ) , 890 ( u18059 ) , \n hokudai - lab/09 ( ab567658 ) and nrw 14 - 13_dup ( ) ( hg426485 ) .", "both bvdv and csfv inhibit the production of type i interferon in vitro \n through proteasomal degradation of cellular irf-3 [ 1 , \n 4 , 8 , 28 ] . cells infected with these ( end ) viruses \n exhibit strong cpe after superinfection with ndv or some orbiviruses [ 15 , 20 ] . one of the two domains of \n pestivirus n , the c - terminal domain containing a zinc - binding trash motif \n consisting of cys112-cys134-asp136-cys138 , is required for irf-3 binding and prevention of \n type i interferon production [ 7 , 32 ] . for csfv , it has been reported that mutations at positions 112 , 134 , \n 136 or 138 in n of end viruses abolish the inhibition of type i \n interferon production , while mutations at positions 112 or 136 in n of \n end viruses restore this function [ 24 , \n 32 , 33 ] . for \n bvdv , previous studies have revealed that amino acid substitutions l8p , e22v or h49l in \n n abolish its capacity to interfere with type i interferon production [ 2 , 6 ] . however , to \n the best of our knowledge , there were no studies with bvdv that had approached the basic \n mechanism of the inhibition of type i interferon production using the amino acid differences \n between a pair of end and end viruses as well as viruses genetically \n engineered on the basis of these differences . pestiviruses are quasispecies ; single strains \n consist of populations with various characteristics , such as end and \n end . we cloned a pair of \n end and end viruses ( gbk_e and gbk_e ) from \n the bvdv gbk strain using reverse plaque formation techniques [ 5 , 19 ] . determination of complete \n genome sequences of these viruses revealed that there were only four amino acid differences \n between them ( table 1 ) . to clarify the molecular \n mechanism of the inhibition of type i interferon production and the end phenomenon , we \n generated a full - length cdna clone of gbk_e ( pgbk_e ) as well as \n three other full - length cdna clones with single ( d136 g ) or multiple ( i2623v ; d3148 g ; d3502y \n and d136 g ; i2623v ; d3148 g ; d3502y ) mutations . the ifn bioassay of vgbk_e and three mutant viruses revealed \n that vgbk_e and vgbk_e / i2623v ; d3148 g ; d3502y exhibited the end \n phenomenon and inhibited the production of type i interferon , whereas the n \n mutant viruses vgbk_e / d136 g and vgbk_e / d136 g ; i2623v ; d3148 g ; d3502y \n were end and did not inhibit the production of type i interferon ( fig . this result demonstrates that the single aspartic acid residue in the \n zinc - binding trash motif of n , which occurs at position 136 of the genome of \n the gbk_e virus , is the key to determine viral capacity to inhibit type i \n interferon production and display the end phenomenon . because it has been reported that the \n n proteins of both bvdv and csfv inhibit the production of type i interferon \n by proteasomal degradation of cellular irf-3 [ 1 , 4 , 8 , 28 ] , we assessed whether n of wild - type \n gbk_e , wild - type gbk_e , vgbk_e and the three mutant \n viruses reduced the amount of cellular irf-3 in infected cells by western blotting . an \n apparent reduction in cellular irf-3 was observed in cells infected with wild - type \n gbk_e , vgbk_e or vgbk_e / i2623v ; d3148 g ; d3502y , \n whereas wild - type gbk_e and mutant viruses with mutations in n \n showed no reduction in cellular irf-3 ( fig . the results indicate that the inhibition of type i interferon production and the \n end phenomenon displayed by gbk_e occurs by the degradation of cellular irf-3 \n caused by combining with the zinc - binding trash motif . comparison of \n vgbk_e / d136 g ; i2623v ; d3148 g ; d3502y ( the same amino acid sequence as \n gbk_e ) with vgbk_e / i2623v ; d3148 g ; d3502y revealed that the \n function of n was restored by a single g136d mutation in n of \n the gbk_e virus . in the present study , a comparison of the amino acid sequences of gbk_e , \n gbk_e and viruses from ddbj / embl / genbank databases revealed that amino acid \n residues cys112-cys134-asp136-cys138 in the zinc - binding trash motif of n were \n well conserved among field bvdv isolates ( fig . it is reported that these four residues are required for the inhibition of type i interferon \n production [ 7 , 32 ] . taken together , the above - mentioned results suggest that these field isolates \n inhibit the type i interferon production in infected cells , although there remains a \n possibility that amino acid residues other than those of trash motif contribute to this \n phenomenon . the g strain contains the amino acid residue glu136 in the trash motif of \n n ( fig . 4 ) , and this may affect \n the structure of n and result in the production of type i interferon \n in vitro . it was reported that 35 out of 45 ( 77.8% ) field isolates of \n bvdv in japan contained end virus as the predominant virus population compared \n with end virus and seven isolates ( 15.6% ) contained similar titers of \n end and end viruses , whereas two isolates ( 4.4% ) contained only \n end virus . therefore , further \n studies are needed to investigate how quasispecies of bvdv ( end and \n end ) contribute in vivo . in conclusion , our results indicate that a single mutation in the trash motif at position \n 136 of bvdv n abolishes its interaction with bovine irf-3 and halts the \n degradation of irf-3 . moreover , the mutation of the amino acid residue at position 136 of \n gbk_e restores its function as an interferon antagonist , as shown for csfv . \n however , how n interacts with irf-3 and the nature of the cascade after \n interaction with n and irf-3 are hardly understood . in addition , it is unknown \n how the inhibition of type i interferon production contributes to the viral infection \n strategy when cells are infected with bvdv in vivo . therefore , an \n additional study is also required to reveal the fundamental mechanism by which pestiviruses \n inhibit the production of type i interferon and how this mechanism functions in \n vivo ." ]

[ "in central and northern \n europe as well as in north america a significant \n proportion of patients who suffer from birch pollen allergy develop \n intolerance to certain kinds of fruits and vegetables . such birch pollen - related food allergies are the result \n of initial sensitization to the major birch pollen allergen , bet v \n 1 , and subsequent immunologic cross - reactivity of the bet v 1-specific \n ige antibodies with structurally homologous food proteins . among the \n most frequent triggers of birch pollen - related food allergies are \n apples , with > 70% of all individuals that are sensitized to birch \n pollen developing allergic reactions when consuming apples . symptoms typically occur locally at the site \n of food contact and within minutes after apple consumption , including \n itching and swelling of the lips , tongue , and throat ( oral allergic \n syndromes , oas ) . frequently , allergic \n patients can also exhibit symptoms of food - induced rhinoconjunctivitis \n and dyspnea . in apples ( malus domestica ) , the \n major allergen that is responsible for birch pollen - related food allergies \n is the 17.5 kda protein mal d 1 . mal d \n 1 belongs to group 10 of pathogenesis - related ( pr ) proteins that are \n activated in plants in response to different kinds of stress . the concentration of mal d 1 in apples is highly \n dependent on the cultivar and also influenced by various biotic and \n abiotic factors , storage conditions , and storage duration . typically , 130 g of mal d 1 per gram of fresh apple \n ( accounting for up to 7% of total soluble protein ) is present directly \n after harvest . after storage , these values can \n rise to values exceeding 100 g mal d 1 per gram of apple . although mal d 1 has been found in both the pulp and peel of apples , \n higher concentrations are present in the peel . on \n the basis of this observation , and because mal d 1 appears to be up - regulated \n upon biotic stress , it has been speculated that this protein may play \n a role in plant defense response to pathological situations . mal d 1 is encoded by a multigene family , \n and a number of isoforms \n of mal d 1 have been identified to date , which are clustered into \n four groups on the basis of their dna sequence similarities , that \n is , mal d 1.01 , mal d 1.02 , mal d 1.03 , and mal d 1.04 . pcr screening and mass spectrometric studies \n showed that mal d 1 isoforms are not cultivar specific and that mixtures \n of isoforms are present in apple fruits . along with mal d 1.02 , and depending on the cultivar , isoforms from \n the mal d 1.01 cluster are by far the most abundant isoforms found \n in apples . within the mal d 1.01 cluster , \n immunologic investigations of naturally \n occurring mal d 1 isoforms revealed only small differences of their \n ige binding capacities and it appears that divergent allergenicities \n of apple strains are predominantly determined by different mal d 1 \n expression levels . whereas the \n immunological properties of mal d 1 suggest that this \n protein has a three - dimensional structure and ige binding epitopes \n that are similar to those of bet v 1 and other members of the pr-10 \n protein family , experimental structural data for mal d 1 have not \n been available to date . as a first step toward structural characterization , \n we recently assigned the nmr backbone and side chain h , c , and n chemical shifts of the isoform mal \n d 1.0101 . golden \n delicious , were the first isoforms for which the dna sequence \n was determined and are identical at the amino acid level . here", "the dna of mal d 1.0101 ( genbank nucleotide \n code x83672 , protein code caa58646 ) was cloned into the expression vector pet28b \n by using the restriction sites ncoi and xhoi . construct integrity was ensured by \n dna sequencing ( microsynth ag , balgach , switzerland ) , and the protein \n was expressed in escherichia coli bl21 \n star ( de3 ) . mal d 1.0101 was purified by anion exchange and size exclusion \n chromatography as described in detail elsewhere . the mass and the amino acid sequence of purified mal d \n 1.0101 were confirmed by mass spectrometry using a 7 t fourier transform \n ion cyclotron resonance ( ft - icr ) mass spectrometer ( bruker daltonics , \n bremen , germany ) with an attached electrospray ionization ( esi ) source . protein concentrations for nmr spectroscopic experiments for structure \n determination were 0.5 mm for n / c - labeled \n and 0.8 mm for n - labeled samples in 91% h2o/9% d2o ( v / v ) at ph 6.9 , 10 mm sodium phosphate , and \n 7 or 11.2 mm l - ascorbic acid , respectively . all nmr experiments \n were carried out at 298 k , using either a 500 mhz agilent directdrive \n spectrometer ( agilent technologies , santa clara , ca , usa ) equipped \n with a room temperature probe or a 600 mhz bruker avance ii+ spectrometer \n ( bruker biospin , karlsruhe , germany ) equipped with a prodigy cryoprobe . \n nmr resonance assignments of mal d 1.0101 were made using standard \n triple - resonance methods and were deposited \n at the biological magnetic resonance data bank ( bmrb ) under accession \n no . three - dimensional n and c edited \n noesy - hsqc experiments ( mixing times of 150 ms ) were recorded for \n derivation of distance restraints . nmr data were processed using nmrpipe and analyzed with ccpnmr . for measuring protein translational diffusion , we \n employed a stimulated \n echo pulsed field gradient nmr experiment . experimental details were identical to those reported for bet v \n 1 . for the determination of the hydrodynamic \n radius of mal d 1.0101 , we used dioxane as a standard reference under \n identical buffer conditions , assuming a hydrodynamic radius of 2.12 \n . structure calculations were performed \n with the program xplor - nih 2.42 using a simulated annealing \n protocol . an initial structural model was generated with cs - rosetta using the bmrb cs - rosetta server . a total of 2079 distance restraints were obtained \n from 3d n and c edited noesy - hsqc spectra . \n noe values were converted on the basis of peak intensities into distances \n with upper limits of 3.0 ( strong ) , 4.0 ( medium ) , 5.0 \n ( weak ) , and 6.0 ( very weak ) . dihedral angle restraints \n were predicted using talos+ and cs - rosetta . in all regular secondary structure elements \n hydrogen bonds were included for backbone amide protons , if the n edited noesy - hsqc spectra did not show a water exchange \n cross peak . of 100 generated structures , the 20 lowest energy structures \n were picked and further refined in explicit solvent with the amber14 \n simulation package using pmemd.cuda and the amber force field 99sb - ildn . each structure was soaked into a truncated octahedral \n solvent box of tip3p water molecules with a minimum wall distance \n of 10 . for the refinement , hydrogen atoms and water molecules \n were minimized with fixed heavy atoms . the temperature was increased \n from 0 to 300 k , where the structures were simulated using the noe \n distance restraints , minimized again , and validated using the protein \n structure validation software ( psvs ) suite ( table 1 ) . the coordinates \n of the mal d 1.0101 structures were deposited in the protein data \n bank under the accession code number 5mmu . calculated \n for all residues , using \n sum over r. largest violation among all 20 reported \n structures .", "the three - dimensional \n structure of mal d 1.0101 consists of a curved , \n seven - stranded antiparallel -sheet ( 1-7 ) embracing \n a long helix at the c - terminus of the protein ( 3 ) and two consecutive \n short helices ( 1 , 2 ) ( figure 1 ) . the edges of the -sheet are formed \n by strands 1 and 2 , which are connected by helices 1 \n and 2 that form a v - shaped support for the c - terminal part \n of helix 3 . in total , 35% -sheet \n and ca . 25% helical structure , agreeing well with secondary structure \n estimates from infrared and circular dichroism . as in other \n proteins from the pr-10 family , strands 2 and 3 are \n connected by a glycine - rich loop motif ( gly46-asn47-gly48-gly49-pro50-gly51 ) . \n together , these structural elements create the large internal cavity \n that is typical for the canonical pr-10 fold . from figure 1b it is evident that in our \n nmr structural ensemble of mal d 1 , secondary structure elements are \n very well - defined and conformationally homogeneous in all 20 structural \n models . only slightly elevated levels of conformational heterogeneity \n are observed for some of the solvent - exposed loops that connect secondary \n structure elements and the c - terminus of the protein . nmr solution structure \n of the major apple allergen mal d 1.0101 \n ( pdb accession code 5mmu ) . secondary \n structure elements are labeled 1 ( val2ser11 ) , 2 \n ( gln40glu45 ) , 3 ( ile53thr57 ) , 4 ( tyr66ile74 ) , \n 5 ( ser80gly88 ) , 6 ( glu96val105 ) , 7 \n ( ser111thr121 ) , 1 ( pro15val23 ) , 2 ( ala26ile33 ) , \n 3 ( lys128asp152 ) . ( b ) backbone overlay \n of the ensemble of the 20 lowest energy structures of mal d 1.0101 . \n secondary structure elements are colored from red ( n - terminus ) to \n purple ( c - terminus ) . a peculiar feature of the pr-10 fold is the large internal \n cavity . \n in mal d 1.0101 , the volume of this cavity is ca . 2230 , which is comparable in size to those \n of other pr-10 proteins . as found in the \n birch pollen allergen bet v 1 and other homologous food allergens , \n in mal d 1 the majority of amino acids that form the surface of the \n cavity are hydrophobic ( figure 2 ) . a large proportion of the inner cavity surface is formed \n by amino acid residues in the -sheet whose hydrophobic side \n chains are located at the protein interior ( ile56 ( 3 ) , val67 \n ( 4 ) , ile71 ( 4 ) , tyr81 ( 5 ) , tyr83 ( 5 ) , \n leu85 ( 5 ) , ile98 ( 6 ) , tyr100 ( 6 ) , ile113 ( 7 ) ) \n along with inward - pointing residues in the long amphiphilic helix \n 3 ( val132 , val134 , ala139 , leu142 , phe143 , ile146 ) , the two \n short helices 1 ( phe22 , val23 ) and 3 ( ala26 , ile30 ) , \n and loop regions ( ile38 , phe58 , tyr64 , ala90 ) . in addition , a few \n polar and charged side chains are located at the inside of the molecule \n and form part of the cavity surface , such as asp27 ( 2 ) , his69 \n ( 4 ) , ser115 ( 7 ) , and lys138 ( 3 ) , so that the \n cavity itself is actually amphiphilic , as noted before for the major \n birch pollen allergen , bet v 1 . in crystal \n structures of other pr-10 proteins the cavity is occupied by water , \n amphiphilic ligand molecules , or components of the crystallization \n buffer . for mal d 1 , it is currently not \n known whether ligands bind specifically to the cavity or what the \n biological function of ligand binding could be . ( a ) internal cavity of \n mal d 1.0101 , colored according to the lipophilic \n potential as implemented in moe , where \n hydrophilic regions are colored in blue and lipophilic regions are \n colored in yellow . ( b ) surface representation of the lowest energy \n solution structure of mal d 1.0101 . the two amphiphilic entrances \n to the internal cavity are indicated as 1 ( between the n - terminal \n end of helix 3 and the loops connecting strands 34 \n and 56 ) and 2 ( between the edge of the \n -sheet and the c - terminal end of helix 3 ) . the internal cavity in mal d 1 can be reached by \n two openings ( figure 2 ) . one entrance to \n the protein interior , 1 , is shaped by residues in the n - terminal \n half of helix 3 ( his131 , val134 ) along with the loops connecting \n strands 34 ( gln63 , tyr64 ) and strands 56 \n ( asp89 ) . together , these amino acids create an amphiphilic access \n route to the protein interior . a second amphiphilic entrance , 2 , \n is present at the edge of the -sheet between helix 3 \n ( lys136 , his140 , lys144 , and glu147 ) and strand 1 ( asn7 , phe9 , \n and ser 11 ) . in the nmr solution structures of mal d 1 this access \n route entries \n to the internal cavity at similar locations have also been described \n for other members of the pr-10 protein family . figure 3 shows \n a \n comparison of mal d 1 with bet v 1 and birch pollen - related food allergens \n from the pr-10 family whose structures have been determined so far . \n despite the fact that sequence identities between these proteins are \n only slightly higher than 50% in some cases , their three - dimensional \n structures are generally very similar , with backbone rmsd values for \n secondary structures typically below 2 . in light of the observed immunologic cross - reactivity between \n mal d 1 and the major birch pollen allergen , bet v 1 the backbone \n rmsd between mal d 1.0101 and the hyperallergenic isoform bet v 1.0101 \n ( 61% sequence identity ) of the birch pollen allergen is 2.13 \n ( 1.70 for secondary structure elements ) . of note , mal d 1 and \n bet v 1 differ in length by one amino acid , and divergent presumptions \n have been made about the location of the gap in mal d 1 . on the basis \n of sequence alignments of pr-10 food allergens it has been proposed \n that either the loop right before or right after strand 7 is one residue shorter in mal d 1 . our solution \n structure shows that the loop right before strand 7 is the \n one that is shorter in mal d 1.0101 ( glu96val105 \n in both mal d 1.0101 and bet v 1.0101 ) and 7 ( ser111thr121 \n in mal d 1.0101 and ser112thr122 in bet v 1.0101 ) occupy identical \n positions and have equal hydrogen bonding patterns in the antiparallel \n -sheets of these proteins . they are connected via loops consisting \n of four residues ( cys - gly - ser - gly in mal d 1 ) and five residues ( thr - pro - asp - gly - gly \n in bet v 1 ) , respectively , which produces a small structural difference \n in these loop segments between the two proteins . ( a ) overlay of the lowest energy structure of mal d 1.0101 ( green , \n pdb accession code 5mmu ) with the structures of the major birch pollen allergen bet v 1.0101 \n ( blue , 4a88 ) , the carrot allergen dau c 1.0103 ( orange , 2wql ) , the \n celery allergen api g 1.0101 ( gray , 2bk0 ) , the soybean allergen gly \n m 4.0101 ( yellow , 2k7h ) , the strawberry allergen fra a 1e ( red , 2lpx ) , \n and the cherry allergen pru av 1.0101 ( purple , 1e09 ) . amino acids are marked with asterisks ( identical ) , \n colons ( conserved ) , and dots ( semiconserved ) . mal d 1 is known to have a tendency for cysteine - mediated \n dimerization , \n as shown for the isoform mal d 1.0108 by nonreducing gel electrophoresis \n and size exclusion chromatography . like \n mal d 1.0108 , the isoform mal d 1.0101 contains a single cysteine \n residue , cys107 . in the three - dimensional solution structure of mal \n d 1.0101 cys107 is located at the c - terminal tip of strand 7 , \n with its side chain oriented toward the protein surface . to probe \n the oligomerization state of mal d 1.0101 under the conditions that \n we employed for nmr structure determination ( ph 6.9 , 10 mm sodium \n phosphate , 14 mol equiv of l - ascorbic acid , 298 k ) we performed \n pulsed - field - gradient nmr diffusion experiments . we obtained a value \n of 21.6 0.8 for the hydrodynamic radius of mal d 1.0101 , \n which is comparable to the hydrodynamic radius of monomeric bet v \n 1.0101 ( 20.1 ) under similar experimental conditions . this is consistent with our observation that , \n using the same buffer , mal d 1.0101 elutes from a size exclusion column \n with a retention time that is virtually identical to that of bet v \n 1.0101 . these results were further verified by ft - icr mass spectrometry , \n which shows that mal d 1.0101 does not form dimers or higher order \n aggregates . the nmr solution structure of mal d 1 shows that \n this protein consists \n of a highly curved antiparallel -sheet and three -helices \n forming a large internal cavity , very similar in fashion to other \n pr-10 proteins . this is in agreement with \n the observed immunologic cross - reactivity between mal d 1 and the \n major birch pollen allergen , bet v 1 , as well as other food allergens \n from the pr-10 protein family . in most patients bet \n v 1 is the sensitizing agent , whereas bet v 1-specific ige antibodies \n subsequently cross - react with mal d 1 and elicit an allergic response , \n as reflected by the clinical observation that apple allergy develops \n only after the onset of birch pollinosis . along these lines , cross - inhibition experiments of mal d 1 \n using \n sera from apple - allergic patients showed that mal d 1 shares ige epitopes \n with the major birch pollen allergen , bet v 1 . from \n a structural perspective , limited information about the exact nature \n of binding epitopes of mal d 1 and bet v 1 is available . detailed \n structural information about a sequentially discontinuous ( i.e. , conformational ) \n b - cell epitope in bet v 1 was obtained by cocrystallizing the particular \n isoform bet v 1.0112 with an antigen - binding fragment ( fab ) derived \n from the murine monoclonal igg antibody bv16 . this epitope is formed by the segment between glu42 and thr52 ( including \n the glycine - rich loop motif between strands 2 and 3 ) , \n along with arg70 , asp72 , his76 , ile86 , and lys97 of bet v 1 , covering \n approximately 10% ( 900 ) of the entire protein \n surface . binding of bv16 to this epitope measurably reduces serum \n ige interactions , indicating that ige and monoclonal igg bv16 compete \n for overlapping binding surfaces on bet v 1 . moreover , mutation of a central residue ( glu45ser ) significantly \n reduced the ige binding capacity of bet v 1 , confirming the significance \n of this particular epitope for interactions with ige . figure 4a shows \n the molecular interaction surface that corresponds with the bv16 epitope \n in the apple allergen . in mal d 1 these residues form a contiguous \n surface patch along with a somewhat distal residue ( glu76 ) , similar \n in shape and size to the bv16 epitope of bet v 1 . moreover , the contributing \n amino acids are largely conserved between mal d 1 and bet v 1 . thirteen \n of the 16 amino acids in the bv16 epitope are identical , whereas only \n 3 residues are different in mal d 1.0101 and bet v 1.0101 ( figure 4b ) . these data thus \n provide a structural rationale for the observed allergic cross - reactivity \n between birch pollen and apple allergens . interestingly , mutational \n studies indicate that the ability of mal d 1 to bind serum ige from \n patients with birch pollen allergies can be increased by increasing \n the similarity of the bv16 epitope in mal d 1 to that of bet v 1 , \n indicating that these amino acids are indeed involved in binding of \n bet v 1 specific to mal d 1 . amino acid residues that \n correspond to the molecular interaction surface between monoclonal \n igg bv16 and bet v 1.0112 ( residues glu42thr52 , arg70 , asp72 , \n glu76 , ile86 , and lys97 in mal d 1.0101 ) are colored in blue . amino acid positions that were shown to be crucial \n for ige recognition of mal d 1 in mutational analyses ( thr10 , ile30 , \n thr57 , ser111 , thr112 , and ile113 ) are shown in green ( ile30 and ile113 \n are located in the protein interior and do not contribute to the surface ) . ( b ) amino acid similarities between bet v 1.0101 and mal d 1.0101 \n using a color gradient from lilac ( highly similar ) to teal ( highly \n dissimilar ) . epitope residues that are different between bet v 1.0101 \n and mal d 1.0101 are labeled . similarities were calculated on the \n basis of substitution matrix scores ( blosum62 ) as implemented in moe . it is likely that mal d 1 contains more than a single conformational \n epitope . a number of amino acid positions \n that are relevant for ige recognition have been identified by mutational \n analysis . for a five - point mutant of mal d 1.0108 \n ( thr10pro , ile30val , thr57 asn , thr112cys , \n and ile113val ) a markedly reduced capacity for binding mal \n d 1-specific ige was found in vitro . skin \n prick tests in apple - allergic patients comparing wild - type mal d 1 \n with the five - point mutant further showed a significantly lower ability \n of the mutant protein to induce skin reactions in vivo . further experiments showed that the t - cell recognition \n level of wild - type mal d 1 is conserved in the five - point mutant . because these five amino acids are likely involved \n in ige interactions not only in mal d 1 but also in bet v 1 , they \n could well be part of common cross - reacting epitopes in these two \n allergens . this is corroborated by mutational \n studies , which showed that peptide stretches encompassing these residues \n are indeed involved in immunological cross - reactivity between mal \n d 1 and bet v 1 . in addition , in an independent \n study , ser111 was identified as being essential for ige binding to \n mal d 1 , and a ser111cys mutation resulted in significantly \n reduced affinity for ige in immunoblotting experiments . figure 4a shows \n that these six residues are fairly dispersed on the protein surface \n of mal d 1 and that neither of these amino acids overlaps with the \n bv16 epitope . amino acids thr10 , ser111 , and thr112 form a common \n patch on the protein surface , whereas thr57 is located approximately \n 3739 away and close to the bv16 epitope . considering \n that an epitope of typical size ( 600900 ) and circular shape would have \n an arc length of 2834 on the mal d 1 surface , residues \n thr10 , ser111 , and thr112 are probably too far away from thr57 to \n be part of a common binding epitope . the remaining two residues , ile30 \n and ile113 , do not reach the protein surface in mal d 1 . whereas ile113 \n is close in space to the thr10-ser111-thr112 patch , its hydrophobic \n side - chain is pointing toward the interior of the protein , where it \n participates in a small hydrophobic core located at the inner end \n of the proteins cavity ( between helices 1 and 3 \n and the -sheet ) . residue ile30 is also located in the protein \n interior with its aliphatic side chain forming part of the internal \n cavity and does not contribute to the protein surface . of note , \n because the loop between strands 6 and 7 \n is shorter by one residue in mal d 1 than in bet v 1 , ser111 and thr112 \n of 7 in mal d 1 occupy the 7 positions of ser112 and \n ile113 in bet v 1 . the surface patch formed by thr10 , ser111 , and \n thr112 in mal d 1 thus appears to be less hydrophobic than the corresponding \n surface patch in bet v 1 ( thr10 , ser112 , and ile113 ) . as a matter \n of fact , also the protein surface surrounding these three residues \n displays a considerably lower level of similarity between mal d 1.0101 \n and bet v 1.0101 than other parts of the protein surface , as can be \n seen in figure 4b . \n this might in part be responsible for the different ige binding properties \n of these allergens . it has been noted , on the other hand , that epitope \n coincidence between bet v 1 and mal d 1 may be limited , as exemplified by a recent study describing \n the isolation of human ige binding to bet v 1 but not to mal d 1 . moreover , different mal d 1 isoforms contain \n amino acid substitutions within potential ige interaction surfaces , suggesting that they may influence the immunologic \n reaction . it is clear that high - resolution structural data provide \n the basis \n to determine and compare structural details of ( cross - reactive ) binding \n epitopes in allergenic proteins . in addition , grafting of conformational \n epitopes by transferring stretches of residues between homologous \n allergens has become a valuable experimental tool . epitope grafting \n was used to characterize the role of the bv16 epitope in mal d 1 by \n recreating this epitope on the mal d 1 surface , confirming its importance \n for ige binding and cross - reactivity with bet v 1 . in an orthogonal approach , several mal d 1 stretches encompassing \n residues that are crucial for ige binding were transferred to bet \n v 1 to investigate the role of these structural segments for cross - reactivity , and chimeras of bet v 1 and mal d 1 were created \n to map the epitope of a human monoclonal ige , which was isolated from \n a phage library , to the c - terminus of bet v 1 . in addition , epitope grafting provides access to chimeric \n allergens with fine - tuned antigenic properties , such as reduced ige \n binding capacitites , for molecule - based allergy diagnosis and specific \n immunotherapy . knowledge of the structural \n details of these allergens elements is required to generate correctly \n folded chimeras , because transfer of ( partly ) mismatching stretches \n of secondary structure between different allergens may well be the \n reason for a loss of protein fold and , consequently , reduction of \n ige - binding capacities . the three - dimensional \n structure of mal d 1.0101 presented here provides the biophysical \n basis for elucidating the molecular details of immunological cross - reactivity \n in great detail ." ]

[ "head and neck oncological resections may result in composite oro - mandibular defects involving the oral mucosa ( lining ) , mandibular bone and the skin ( cover ) . anatomically , the fibula bone has a triangular cross section , it has a lateral surface where peroneal muscles are attached , this is a broad flat surface and is ideal for fixing the reconstruction plate during mandibular reconstruction , at the lower end of this peroneal surface is the posterior crural septum through which the peroneal artery perforators supplying the skin paddle run , this septum has variable length and mobility , which determines the movement of the skin paddle . on the posterior and medial facet of this triangle , originates the flexor hallucis longus ( fhl ) muscle and on the supero - medial aspect is the origin of the tibialis posterior muscle and between these two run the vascular pedicle . due to this unique three - dimensional anatomy there is always a possibility of using the bone flap in a more efficient fashion for the reconstructive need . existing literature , however , provides conflicting views about the use of a particular side and orientation of the fibula flap for achieving the optimal outcome . the purpose of this study is to confirm anatomically the effect of bone , soft tissue and vessel orientation on the ease of doing reconstruction . we also propose an algorithm for choosing the correct side of the fibula for a particular reconstructive need and at the same time minimising the donor side morbidity .", "composite fibula flaps of the same dimension were harvested from both legs of a fresh cadaver . the harvested flaps were used to reconstruct the mandibular defect in different orientations , and the best configuration for each reconstructive requirement was assessed . following variables were considered : \n placement of the vascular pedicle the pedicle needs to be placed anteriorly if the microvascular anastomosis is to be done on the opposite side due to vessel paucity in the ipsilateral neck.type of associated soft tissue defect the defect could be a mucosal defect ( lining ) , the skin defect ( cover ) or a combined mucosa and skin defect . \n the pedicle needs to be placed anteriorly if the microvascular anastomosis is to be done on the opposite side due to vessel paucity in the ipsilateral neck . the defect could be a mucosal defect ( lining ) , the skin defect ( cover ) or a combined mucosa and skin defect .", "keeping the peroneal surface for plating , that is , facing outwards , four different configurations of the fibula flap are possible for a given mandibular defect . implications of each one for reconstruction are discussed below . the skin paddle comes out from the lower and outer border of the reconstructed mandible because of the anatomical position of the posterior crural septum [ figure 1 ] . if this skin paddle has to be taken inside the oral cavity , it has to travel over the plated surface of the fibula , which wastes about 3 - 4 cm of skin paddle width . the length of the posterior crural septum being variable also restricts the inside movement of the skin paddle . ipsilateral fibula with pedicle posterior the fhl muscle lies at the submandibular region with the vascular pedicle lying on its superior aspect and therefore pulling the muscle inside for intraoral reconstruction kinks the vascular pedicle . in this situation , the septum location becomes superior and medial in relation to the bone . hence , the skin paddle does not have to go around the bone to come inside and thus we do not lose skin paddle width when we turn it inside . it can thus be used for providing mucosal lining more easily . for moving the skin paddle outside to provide skin cover , it has to travel over the plated surface of the fibula although the wastage of skin paddle width is not much [ figure 2 ] . ipsilateral fibula with pedicle anterior the fhl muscle is supero - medial to the bone and lies within the oral cavity thus it can be easily utilized for intraoral lining . this configuration leads to a tissue orientation similar to that discussed in point 2 ( ipsilateral fibula flap placing the vascular pedicle anterior and thus is suited for intra oral reconstruction [ figure 3 ] . contralateral fibula with pedicle posterior this configuration leads to a tissue orientation similar to that discussed in point 1 ( ipsilateral fibula flap placing vascular pedicle posterior and thus is not particularly suited for intra oral reconstruction and skin paddle is best utilised outside [ figure 4 ] .", "the skin paddle comes out from the lower and outer border of the reconstructed mandible because of the anatomical position of the posterior crural septum [ figure 1 ] . if this skin paddle has to be taken inside the oral cavity , it has to travel over the plated surface of the fibula , which wastes about 3 - 4 cm of skin paddle width . the length of the posterior crural septum being variable also restricts the inside movement of the skin paddle . ipsilateral fibula with pedicle posterior the fhl muscle lies at the submandibular region with the vascular pedicle lying on its superior aspect and therefore pulling the muscle inside for intraoral reconstruction kinks the vascular pedicle .", "in this situation , the septum location becomes superior and medial in relation to the bone . hence , the skin paddle does not have to go around the bone to come inside and thus we do not lose skin paddle width when we turn it inside . it can thus be used for providing mucosal lining more easily . for moving the skin paddle outside to provide skin cover , it has to travel over the plated surface of the fibula although the wastage of skin paddle width is not much [ figure 2 ] . ipsilateral fibula with pedicle anterior the fhl muscle is supero - medial to the bone and lies within the oral cavity thus it can be easily utilized for intraoral lining .", "this configuration leads to a tissue orientation similar to that discussed in point 2 ( ipsilateral fibula flap placing the vascular pedicle anterior and thus is suited for intra oral reconstruction [ figure 3 ] .", "this configuration leads to a tissue orientation similar to that discussed in point 1 ( ipsilateral fibula flap placing vascular pedicle posterior and thus is not particularly suited for intra oral reconstruction and skin paddle is best utilised outside [ figure 4 ] .", "of all the reconstructive options available for the complex mandibular defect , the free fibula osteo - cutaneous flap is the most widely used one . however , there is conflicting literature regarding the ideal donor side of the fibula . hidalgo advocated selection of the donor area based on the quality of the recipient vessels in the neck , that is , ipsilateral fibula for anastomosis with the same sided neck vessels and contralateral otherwise . in all the cases , he prefers the skin paddle orientation of situation 1 and 4 ( the skin paddle should cover the fibula from outside ) . the advantage cited is that the posterior crural septum covers the plate and acts as a second line of defence , preventing the plate exposure in the event of skin breakdown . however in this configuration if one attempts to reconstruct intraoral defects , one has to add at least 3 cm of extra width to the skin paddle because it has to go around the fibula before it could enter the oral cavity . this configuration also has the potential to kink the delicate peroneal perforators as they go around the bone , which could lead to a loss of the skin paddle . as the fhl muscle lies in the submandibular region , it will not be utilised as an intraoral seal . yagi et al . suggested considering the location of the pedicle in the neo - mandible , requirement of a skin paddle and orientation of the fibula to the remnant mandible in determining the ideal choice of side for the mandibular reconstruction , but he has also not emphasised the additional width of the skin paddle required if one does not choose the correct side and orientation of the fibula . put forth that there is no donor side specificity of fibula free flap for complex oro - mandibular reconstruction . however , it has been noted by author that if one takes a very large skin paddle for composite tissue defects , mere need to add 3 cm of width does not make much difference and one could use any side and orientation of fibula for any defect reconstruction . the question of choosing the side and orientation arises only if one considers minimising the donor site deformity by harvesting only the exact amount of skin required . this would be possible only if one plans precisely , and the algorithm , which authors propose [ algorithm 1 ] , helps one to achieve better management of the donor , as well as the recipient site . algorithm for ideal donor side selection the skin paddle of the fibula flap is usually harvested from its lower portion to ensure adequate pedicle length . being the lower end of the leg , only small sized skin defects can be primarily closed . moderate sized defects up to 8 cm in width in the lower leg may be closed using a soleal musculocutaneous perforator based propeller flap . a large skin defect arising from harvesting a big skin paddle needs to be covered with a split skin graft with its potential donor site morbidity . the basis for selection of the side of the donor fibula are the placement of the pedicle ( i.e. , anterior or posterior ) and the reconstructive requirements ( i.e. , bone only , bone and lining , bone and skin cover or bone , lining and cover ) . an anteriorly placed pedicle may be considered in case of a vessel depleted neck or for a ramus reconstruction , when one needs to reconstruct ramus of the mandible and plans to keep the vascular pedicle posteriorly , there is a possibility of acute kink of the vessels if we choose to anastomose the vessels in the neck and if we choose to anastomose the vessels with superficial temporal vessels , one has to grapple with the problem of size mismatch between the donor and recipient vessels with an increased potential of failure . in other situations , based on our results , we can give our recommendations for the different composite defects as under : if the pedicle is positioned anteriorly , such a defect would be adequately reconstructed with an ipsilateral fibula with the fhl muscle ( as described in point 2 ) . the donor area skin can be closed primarily because no skin is harvested . in a posteriorly placed pedicle , when the pedicle is positioned anteriorly , such a defect would need an ipsilateral fibula with optimal sized skin paddle ( as described in point 2 ) . the donor area skin may be closed primarily , with a soleal propeller flap or skin graft . in a posteriorly placed pedicle , buccal mucosal defect extending to the upper alveolus and secondary reconstructions of the mandible would necessitate a contralateral neck anastomosis . these defects are most optimally reconstructed with the ipsilateral fibula if the pedicle has to be brought posteriorly ( point 1 ) or with the contralateral fibula if the pedicle has to be brought out anteriorly ( point 4 ) . the skin paddle may be used to cover the skin defect and the fhl muscle for reconstructing the mucosal defect . however , if the size of the defect is beyond the dimension which could be covered by fibula , such defects are reconstructed with double flaps using the fibula skin paddle for one defect and a second free / regional flap for the other defect . this concept enables us to reduce the donor site defect by choosing the side of fibula flap , which has the skin paddle that will fall into an anatomically correct position to reconstruct the soft tissue defect . closure of smaller defects primarily or with propeller flaps result in superior aesthetic outcome [ figures 5 and 6 ] .", "if the pedicle is positioned anteriorly , such a defect would be adequately reconstructed with an ipsilateral fibula with the fhl muscle ( as described in point 2 ) . the donor area skin can be closed primarily because no skin is harvested . in a posteriorly placed pedicle ,", "when the pedicle is positioned anteriorly , such a defect would need an ipsilateral fibula with optimal sized skin paddle ( as described in point 2 ) . the donor area skin may be closed primarily , with a soleal propeller flap or skin graft . in a posteriorly placed pedicle , buccal mucosal defect extending to the upper alveolus and secondary reconstructions of the mandible would necessitate a contralateral neck anastomosis . these defects are most optimally reconstructed with the ipsilateral fibula if the pedicle has to be brought posteriorly ( point 1 ) or with the contralateral fibula if the pedicle has to be brought out anteriorly ( point 4 ) . the skin paddle may be used to cover the skin defect and the fhl muscle for reconstructing the mucosal defect . however , if the size of the defect is beyond the dimension which could be covered by fibula , such defects are reconstructed with double flaps using the fibula skin paddle for one defect and a second free / regional flap for the other defect . this concept enables us to reduce the donor site defect by choosing the side of fibula flap , which has the skin paddle that will fall into an anatomically correct position to reconstruct the soft tissue defect . closure of smaller defects primarily or with propeller flaps result in superior aesthetic outcome [ figures 5 and 6 ] .", "these defects are most optimally reconstructed with the ipsilateral fibula if the pedicle has to be brought posteriorly ( point 1 ) or with the contralateral fibula if the pedicle has to be brought out anteriorly ( point 4 ) .", "such defects can be reconstructed with a configuration described in points 2 and 3 . the skin paddle may be used to cover the skin defect and the fhl muscle for reconstructing the mucosal defect . however , if the size of the defect is beyond the dimension which could be covered by fibula , such defects are reconstructed with double flaps using the fibula skin paddle for one defect and a second free / regional flap for the other defect . this concept enables us to reduce the donor site defect by choosing the side of fibula flap , which has the skin paddle that will fall into an anatomically correct position to reconstruct the soft tissue defect . closure of smaller defects primarily or with propeller flaps result in superior aesthetic outcome [ figures 5 and 6 ] .", "the algorithm based selection of appropriate sided fibula flap facilitates complex mandibular reconstruction by placing the right kind of tissue at the right place and helps in reducing the donor site morbidity by allowing the surgeon to harvest only the required amount of skin ." ]

[ "mesial temporal lobe epilepsy with hippocampal sclerosis ( mtle - hs ) is the most common type of partial or localization - related seizure disorder in humans . mesial temporal sclerosis ( mts ) with prominent neuronal loss and gliosis in the hippocampus is the most common pathology in temporal lobe epilepsy ( tle ) . although the pathogenesis and epileptogenesis of hippocampal sclerosis ( hs ) has been studied for years , the information available so far still does not fully explain the situation . patients with mtle - hs usually have a history of febrile seizures , status epilepticus , trauma or , in some cases , a mixture of all these symptoms . recently , some studies have implicated inflammatory mechanisms that contribute or predispose to the occurrence of seizures in mtle - hs . cytokines such as interleukins ( il ) are involved in inflammation , immune activation , cell differentiation and apoptosis . in seizures , there is increasing evidence to support the role of il-1 in reducing seizure threshold and epileptogenesis in the pilocarpine status epilepticus model of epilepsy . found a strong association between the homozygotes for allele t at position -511 of the il-1 gene promoter region ( il-1-511 t / t ) and genetic predisposition to the development of hs in japanese tle patients . stated that increased il-1-511 t - allele frequency proved to be a potent determinant of prolonged febrile seizure , thus a discrepancy in prolonged febrile seizure incidence could explain recent conflicting results . three polymorphisms within the il-1 gene promoter region located at positions -1470 , -511 and -31 basepairs from the transcription start site were found to be in strong linkage disequilibrium . additionally , when analyzed in combination , interestingly , however , these two publications have conflicting results in terms of transcriptional activity of il-1 promoter . while chen et al . concluded that the il-1-511 t allele was associated with higher il-1 promoter activity , wen et al . demonstrated that haplotype combinations carrying -511 c allele cause higher levels of secretion of il-1 from the lipopolysaccharide ( lps)-stimulated lymphocytes . in both of these studies , therefore , we intended to analyze the effect of -511 c / t polymorphism on il - i secretion from lps - induced lymphocytes in an attempt to reveal the possible role of this particular polymorhism in the etiopathogenesis of mtle - hs .", "thirty patients with mtle - hs admitted to the akdeniz university hospital neurology department , turkey , were enrolled in this study . a control group of 32 healthy volunteers with matching age , gender ratio and ethnic origins was also included . diagnosis of mtlehs was based on clinical history , electroencephalography , magnetic resonance imaging , and , in some patients , on histopathological findings after the selective amgydalahippocampectomy and anterior temporal lobectomy . the control subjects had no history of seizures or neurological disorders and no central nervous system or other infections at the time of blood sample collection . peripheral venous blood samples ( 3 ml ) were collected from each subject into vacutainer tubes containing edta . dna was extracted from blood samples using the robotic system magnapure ( roche ) and the amount of dna was measured by spectrophotometer . the -511 region of il-1 gene has been genotyped with polymerase chain reaction - restriction fragment length polymorphism ( pcr - rflp ) using 2 mg of genomic dna as template . by using the primers for -511 c / t polymorphism described below , a 304 bp of dna region was amplified by pcr ( total 35 cycles , each cycle 30 s at 95c , 45 s at 50c , and 45 s at 72c ) . each pcr product was then cleaved by ava i enzyme and the digested pcr products were subjected to agarose gel electrophoresis . the gels were stained with ethidium bromide and the dna fragments were visualized by an uv transluminator . pcr products acquired from those with a cc genotype were cleaved into two fragments of 190 and 114 bp by ava i enzyme , whereas the pcr products of people with tt genotype was not digested at all , yielding a product of 304 bp . oligonucleotides used for pcr : forward primer : 5-tggcattgatctggttcatc reverse primer : 5-gtttaggaatcttcccactt peripheral blood of patients and healthy individuals was drawn into heparinized tubes , and lymphocytes were isolated with a density gradient method and pipetted into plates ( 210 cells each well ) and incubated for 24 h ( 5% co2 , 37c ) in an rpmi 1640 culture medium including 10% fcs with ( 0.5 ng / ml ) or without lps . the cells were precipitated by centrifuge at 1600 rpm for 10 min after 4 and 24 h. the supernatants were transferred to another tube and stored at 80c until elisa analyses . il1 levels in cell culture supernatants were studied according to the manufacturer s protocol ( ebioscience , cat . n. 88 - 7010 ) . all statistical analysis was carried out using statistical package for social sciences ( spss ) windows version 15.0 ( spss inc . , tests for differences in allelic and genotypic frequencies were performed using 2 test and fisher s exact test .", "thirty patients with mtle - hs admitted to the akdeniz university hospital neurology department , turkey , were enrolled in this study . a control group of 32 healthy volunteers with matching age , gender ratio and ethnic origins was also included . diagnosis of mtlehs was based on clinical history , electroencephalography , magnetic resonance imaging , and , in some patients , on histopathological findings after the selective amgydalahippocampectomy and anterior temporal lobectomy . the control subjects had no history of seizures or neurological disorders and no central nervous system or other infections at the time of blood sample collection .", "peripheral venous blood samples ( 3 ml ) were collected from each subject into vacutainer tubes containing edta . dna was extracted from blood samples using the robotic system magnapure ( roche ) and the amount of dna was measured by spectrophotometer . the -511 region of il-1 gene has been genotyped with polymerase chain reaction - restriction fragment length polymorphism ( pcr - rflp ) using 2 mg of genomic dna as template . by using the primers for -511 c / t polymorphism described below , a 304 bp of dna region was amplified by pcr ( total 35 cycles , each cycle 30 s at 95c , 45 s at 50c , and 45 s at 72c ) . each pcr product was then cleaved by ava i enzyme and the digested pcr products were subjected to agarose gel electrophoresis . the gels were stained with ethidium bromide and the dna fragments were visualized by an uv transluminator . pcr products acquired from those with a cc genotype were cleaved into two fragments of 190 and 114 bp by ava i enzyme , whereas the pcr products of people with tt genotype was not digested at all , yielding a product of 304 bp .", "peripheral blood of patients and healthy individuals was drawn into heparinized tubes , and lymphocytes were isolated with a density gradient method and pipetted into plates ( 210 cells each well ) and incubated for 24 h ( 5% co2 , 37c ) in an rpmi 1640 culture medium including 10% fcs with ( 0.5 ng / ml ) or without lps . the cells were precipitated by centrifuge at 1600 rpm for 10 min after 4 and 24 h. the supernatants were transferred to another tube and stored at 80c until elisa analyses . il1 levels in cell culture supernatants were studied according to the manufacturer s protocol ( ebioscience , cat . n. 88 - 7010 ) .", "all statistical analysis was carried out using statistical package for social sciences ( spss ) windows version 15.0 ( spss inc . , tests for differences in allelic and genotypic frequencies were performed using 2 test and fisher s exact test .", "there was no significant difference between the mtle - hs patients and controls according to genotype and allele frequencies of il-1-511 . levels of secreted il-1 from the cultured lymphocytes incubated either with or without lps were measured at three different time frames during the culture period ( basal , 4 h and 24 h ) . however , no statistically significant difference was observed between the groups ( table 3 ) . since il-1-511 t allele has been suggested to be associated with increased in vitro production of il-1 and that il-1 is involved in hs pathogenesis , we also analyzed the il-1 secretion between the patients and control individuals who carry the t allele at position -511 of the il-1 gene . however , although spontaneous secretion levels of il-1 were higher in mtle - hs patients , there was no significant difference between the groups ( figure 1 ) . in addition , despite the fact that both spontaneous and stimulated secretion levels of il-1 were higher in mtle - hs patients who carry the t allele with respect to those patients who do not , the difference was not statistically significant ( figure 2 ) .", "in this study , we found that neither the il-1 -511 c / t polymorphism nor the secreted il-1 from the cultured lymphocytes were associated with mtle - hs in a group of turkish patients . in our study , the average age of onset of seizures and gender were similar to those presented in other studies . on the other hand , the incidence of history of febrile seizures was found to be 76.7% , which was higher than the values mentioned in several previous reports . cytokine proteins have both pro - inflammatory and anti - inflammatory forms . among proinflammatory cytokines , three biallelic polymorphisms in il-1 , all of which result from c to t transitions at positions -511 , -31 or + 3954 from the transcriptional start site , have been most frequently evaluated for their association with diverse conditions besides epilepsy . chen et al . demonstrated that the il-1-511 t allele strongly enhanced the transcription of the il-1 gene in the context of the il-1-31 c allele . il-1-511 t single nucleotide polymorphism has been thought to be associated with mtlehs and febrile seizures . previously showed no association between il-1 gene polymorphism and tle with hs in turkish ancestry in a larger group . recently , a meta - analysis and a review was published by kauffman et al . demonstrating a modest association between the il-1-511 t polymorphism and tle with hs . in the current study , we did not find any significant difference between mtlehs patients and control group with respect to the genotype and allele frequencies of il-1-511 ( table 2 ) . in this study , the frequency of t / t genotype was found to be 15.6% whereas , it was reported to be 18% and 20.5% in european and japanese populations , respectively . during the acute phases of status epilepticus , upregulation of il-1 occurred in activated microglia and astrocytes and sustained inflammation was only evident in astrocytes during epileptogenesis . in chronic epileptic tissue , il-1 was still expressed by astrocytes , and it resumed in microglia when rats displayed a high frequency of spontaneous seizures . these findings demonstrated that il-1 is chronically over - expressed in astrocytes after status epilepticus , and il-1 expression in microglia is related to ongoing and severe epileptic activity . together with il-1 , tnf- and il-6 levels increase rapidly after the induction of seizures , then decline to basal levels within 48 - 72 h of the onset of seizures . but il-1 is still up - regulated in the brain 60 days after status epilepticus in rats with spontaneous seizures . the recent data suggested that lipopolysaccharide mimics the actions of an endogenously released protein called high mobility group box 1 that interacts with tolllike receptor 4 to promote seizures . il-1 and high mobility group box 1 activate pathophysiological cascade of inflammatory events in epilepsy through binding to il-1r1 and tolllike receptor 4 , respectively . besides these experimental studies , some clinical studies have measured il-1 secretion from the lymphocytes and serum in febrile seizure and epilepsy patients . showed increased plasma levels of il-6 and levels of il-1 unchanged after seizures in chronic localization - related epilepsy patients . found no difference between febrile seizure patients and controls with respect to serum il-1 levels . reported increased serum levels of il-6 in tle that supported chronic overproduction of cytokines in refractory focal epilepsy . on the other hand , measurement of the secreted il-1 levels from stimulated lymphocytes can differ from serum levels . demonstrated that il-1 secretion from the lymphocytes was significantly higher in febrile seizure patients . in another study ( haspolat , unpublished data , 2008 ) , significantly increased levels of il-1 from lps - stimulated mononuclear cells at 24 h were observed in complicated febrile seizure patients compared to simple ones . although these findings suggest that the increased levels of il-1 in mtle - hs patients may lead to hs and explain epileptogenesis and ictogenesis , in this study we were not able to detect any statistically significant differences in secreted il-1 levels from cultured lymphocytes between the patient and control groups . in addition , comparison of il-1 levels secreted from lymphocytes obtained from individuals carrying a t allele at the -511 position of the il-1 gene to those obtained from individuals who do not carry the t allele did not reveal any significant difference either . interestingly , however , the spontaneous and stimulated levels of il-1 secretion were higher in mtle - hs patients carrying the t allele with respect to the patients who do not . the relatively small number of patients and controls could explain the reason why a statistically significant difference was not observed in this study . in addition , it should be kept in mind that promoter regulation is a complex mechanism involving a large variety of transcription factors acting together . thus , besides ( -511 ) polymorphism , a combination of different haplotypes and many other transcription factors binding to different sites should impinge on the transcriptional activity . in conclusion , in experimental studies , activation of cytokines has been observed especially in areas of onset of seizures and spreading of discharge . in clinical studies , cytokine levels in peripheral blood seem to reflect the central nervous system s production of cytokines , but these are not always correlated ." ]

[ "the presence of high levels of dental anxiety amongst dental care seekers yielded patients with negative attitudes towards dental treatment and rendered dental treatment more difficult to accomplish successfully4,16 . assessment of anxiety in such patients is a crucial factor for the success of their management . avoidance of dental care could be attributed to dental fear and anxiety in many patients7,9,23 . dental anxiety might also affect patient - dentist relationship and obscure proper diagnosis of the actual dental problem8,25 . taani15 ( 2002 ) showed that the levels of dental anxiety were higher among jordanian public school children than those from the private schools . yet , dental fear and anxiety were found among the most reasons that underlie the irregular attendance in two thirds of the public school children and half of those from the private schools . it is a simple , easy to score , short , valid and reliable test for dental visit - associated anxiety5,13,19,22 . humphris , morrison and lindsay21 ( 1995 ) provided a modified scale from the original corah dental anxiety scale . the modified dental anxiety scale was shown to be more comprehensive , highly valid and reliable , with a simpler and more consistent answering system . the modified dental anxiety scale will be , therefore , used to measure dental anxiety in the current study . lack of educational courses specialized in increasing dental awareness amongst non - dental university students in jordan , in addition to shortage of information about correlation between field of study and dental anxiety levels in the dental literature have raised the idea of investigating the level of dental anxiety among the different student populations . this study was therefore designed to investigate the subjective ratings of dental anxiety levels among dental , medical and engineering jordanian university students . in addition , the present study aimed to explore the sources of dental anxiety and the impact of gender on the perceived dental anxiety .", "anxiety related to dental treatment was assessed by means of corah 's dental anxiety scale ( das ) . however , the modified version of das was used21 where an extra item has been included referring to the respondent 's feelings toward local anesthetic injection with especial reference to the site of the injection , because the pain experienced with local anesthetic injections varies according to its location in the mouth14 . moreover , a simplified 5-point scale - answering scheme was devised ranging from not anxious to extremely anxious . the modified dental anxiety scale ( mdas ) contains 5 multiple - choice items including the followings : = if you went to your dentist for treatment tomorrow , how would you feel ? if you were about to have your teeth scaled and polished , how would you feel ? if you were about to have a local anesthetic injection in your gum , how would you feel ? the scores for each of the 5 item responses were summed up to give an estimated value of dental anxiety . the questionnaire was distributed to the third to fifth year undergraduate dental , medical and engineering students at the jordan university of science and technology . students were personally approached in the classrooms by the authors at the end of their class . the students were informed about the study and all the points in the questionnaire were explained and clarified . descriptive statistics were obtained and the means , standard deviation and frequency distribution were calculated . group comparisons were analyzed using twotailed student 's t tests as well as one - way analysis of variance ( anova ) test . statistical significance was based on the probability values of p= 0.05 . furthermore , as a total score of 15 or more almost indicates highly anxious patient19 , the frequencies of subjects with a score of 15 or more in the 3 student groups were also reported .", "the total number of the participants in the current study was therefore 535 , which accounts for a response rate of 89.2 percent . the distribution of the participants according to gender and field of study is presented in table 1 . \n table 2 presents the means and standard deviations of individual items and total scores of the modified corah dental anxiety scale with the results of one - way anova analysis comparing the various groups based on the field of study . out of the several anxiety scale items , the highest anxiety score ( 3.4 ) was given for tooth drilling ( item 3 ) and scored by the engineering students . the next most anxiety - producing item was the local anesthetic injection ( 3.32 ) which was scored by the medical students . however , for the items 1 , 2 , and 5 , the only statistically significant differences were found between the dental and the medical students ( f=7.92 , p = 0.00 ; f=4.69 , p= 0.01 , f=3.39 , p=0.03 , respectively ) . dental students were significantly less anxious about scaling and polishing of teeth ( f = 8.99 , p = 0.00 ) and about tooth drilling ( f = 19.58 , p = 0.00 ) than other groups . however , there were no statistically significant differences between medical and engineering students in relation to these items ( table 2 ) . as expected , dental students scored the lowest total dental anxiety scores ( table 2 ) which were significantly lower than those scored by either medical or engineering students ( f = 14.13 , p = 0.00 ) . generally , in terms of total anxiety scores , women were relatively more anxious than men ( t = -2.21 , p = 0.03 ) ( table 3 ) . women were particularly more anxious than men concerning items related to waiting in the dentist 's sitting room ( t = -2.56 , p = 0.01 ) and local anesthetic injection ( t = -2.62 , p = 0.01 ) . however , such gender variation was not significantly different when items related to going for dental visit tomorrow , having teeth drilled or having teeth scaled or polished were considered . sd = standard deviation the numbers and percentages of subjects who had a total score of 15 or more are shown in table 4 . surprisingly , medical students were found to be the most frequent among those who scored 15 or above .", "this study revealed that dental students do have lower levels of dental anxiety than their engineering and medical counterparts ( p < 0.01 ) . the mean total scores for the mdas showed that severe dental anxiety was mostly associated with drilling and intraoral local anesthetic injection . surprisingly , the medical students showed the highest total anxiety scores and the greatest percentage of subjects scoring 15 or more . the engineering students were already expected to score the highest anxiety scores as they do not receive health or dental awareness education , whilst the medical students are supposed to be more familiar with stress management related to health measures . however , some of the highly anxious patients may avoid showing their anxiety in order not to interfere with the dentist 's procedure . thus , the dentist should be aware of the patient 's possible adverse reaction or distress19,21 . however , when the data are critically appraised , statistically significant difference between groups related to total anxiety scores or the scores of individual items of the anxiety scale could be seriously misleading and possibly do not always reflect an actual clinical significance . this could be attributed to the fact that the sample size is large , which added to the power of the study and revealed even the minor statistical differences between the groups . thus conclusions made on actual differences in numbers and on absolute statistical significance should be drawn with caution . lack of dental health education might result in patients ' fear and anxiety which in turn might end with poor patient compliance and attitudes . this will make it more difficult to manipulate patients and yield difficult patients and thus increase the levels of dental profession - related stress1,12 . assessing the level of patient anxiety before commencing the dental treatment may offer invaluable insight into the probable patient attitudes and behavior towards the dental treatment . this information will be further utilized in developing the best strategies to manage patient anxiety . health - related behavior depends on oral health knowledge14 . in jordan , only students related to the dental field receive adequate dental health education as it is an integral part of their curriculum . jordanian schools and universities pay little attention to the dental health education of their students . students not related to the dental field receive little , if any , dental health education and their curricula lack such courses . since the dental health education is generally ignored in the pre - university stage , medical and engineering students still possess the same ideas about dentistry and dental care at the university . on the other hand , dental field - related students do have the chance to formulate new and better ideas and understanding of the dental health care and for the above mentioned reasons , the dental treatment will still be considered mysterious and stressful for the non - dental students while the dental students will feel better during the dental treatment . this could partly explain the relatively higher scores of dental anxiety among the non - dental students compared to their dental peers . irregular dental attendance may play a major role in increasing the levels of dental anxiety15,23 . if this is added to the lack of dental health education in jordan , the high levels of dental anxiety could be explained and clearly demonstrated among the non - dental students . this might form the bases for explaining the presence of higher levels of dental anxiety among women as well as medical students in this study . female students were found to have higher levels of dental anxiety in all groups and they were more anxious about waiting in the sitting room and taking anesthetic injections . this corroborated the results from previous studies that showed higher levels of dental anxiety among women,11,21,24 . this finding might be explained on the basis that women have higher levels of neuroticism than men and that anxiety is positively associated with neuroticism9,10,20 . however , in the current study , the statistically significant difference between men and women was marginal and the difference in anxiety scores for both genders was minimal ( men 12.29% , women 13.17% ) . therefore , it could be inferred that statistical significance might not be necessarily interpreted as a clinical one . medical students might be prone to have higher levels of neuroticism , due to their courses and stressful field of study2 and thus demonstrate higher levels of dental anxiety than engineering students . on the other hand , although dental students are leading stressful courses and profession they are yet still exposed to better dental health education and knowledge and thus develop favorable dental behavior . presence of suitable dental health education and knowledge seems to be capable , to some extent , of overruling the effect of stress and personality factors on dental anxiety among dental students . the control of dental anxiety might be aided via good dental health education , regular dental visits , good patient - dentist relationship and suitable communication with the patients6 . for a successful dental treatment , a gentle , supportive , professional , sympathetic , quiet and more considerate approach should be followed when managing patients with dental anxiety . on their first visit , patients should be dealt with more sensitively in order to avoid increasing their anxiety and thus avoid their repulsion to the dental care9 . in view of the current available data , it appears that further dental health education measures are required to be applied among the jordanian non - dental university students and the population in general in order to control the levels of dental anxiety and thus improve patient dental attitudes and compliance . suitable standards of dental health knowledge and education could overcome the negative effects of personality and reduce dental anxiety . interception of dental anxiety at early stages will reduce the chance of resistant dental anxiety and fear which are difficult to deal with in the dental clinic . the importance of dental health education can not be overemphasized in the reduction and control of dental anxiety . pre - university as well as non - dental university curricula should include dental health education in order to help reduction of dental anxiety among the population .", "although the current study utilized the modified anxiety scale and investigated the levels of dental anxiety among university students from different fields of study and the sample size was representative and large , further studies are required to investigate the effect of various correlates on dental anxiety ." ]

[ "toxic epidermal necrolysis ( ten ) is a rare life threatening idiosyncratic mucocutaneous drug reaction characterized by widespread epidermal necrosis followed by epidermal detachment . drug - induced ten is the commonest cause and antiepileptics , antibiotics , nonsteroidal anti - inflammatory drugs ( nsaids ) and allopurinol are the most commonly implicated agents . here we report a case of ten which was induced by multiple drugs some of which have been rarely implicated in the causation of ten .", "a 42-year - old woman was referred to medical emergency with extensive epidermal detachment and ocular , genital , and oral lesions of 5 days duration leading to a diagnosis of toxic epidermal necrolysis ( ten ) . seven days prior to this admission , she had presented to a general practitioner with symptoms of vertigo , anxiety , and restlessness . although no provisional or differential diagnosis was found in the documents provided by the patient , she was prescribed antihistamines ( cinnarizine and dimenhydrinate ) , paracetamol / metoclopramide combination and multivitamins . after 8 - 12 hours , she developed fever , malaise , sore throat , and erythematous maculopapular rash in the perioral region . she discontinued all medications and was admitted to a general hospital where she was administered piperacillin / tazobactam and dexamethasone . it is unknown whether paracetamol or antihistamines were restarted in the hospital . her condition deteriorated and she was referred to our hospital . on presentation , she was conscious and afebrile ; her heart rate was 110/min and blood pressure was 190/80 mm hg . cutaneous examination showed involvement of about 70% total body surface area , with skin necrosis , tenderness , and a positive nikolsky 's sign . she had a history of hypertension and was on some antihypertensive medication ( unknown ) for a few years . test reports at three time - points she was administered intravenous fluids , antihypertensive treatment ( amlodipine 5 mg / day ) , injection hydrocortisone ( 50 mg iv twice daily ) , and continued on the same antibiotics . wound care was given by applying topical antiseptics on eroded areas along with nonadherent dressings . biochemical reports showed a decrease in blood urea ( from 68 mg / dl on admission to 57.6 mg / dl on the second day after admission ) and a decrease in serum creatinine ( from 1.1 meq / l on admission to 0.78 parenteral therapy was gradually replaced by oral drugs . on the fourth day , she developed herpetic lesions on the oral mucosa and for that tablet acyclovir was advised thrice a day along with chlorhexidine mouthwash , syrup mucaine gel ( antacid ) , and white petroleum jelly for lips . a tzanck smear , done on fifth day after admission , failed to show any giant cells or acantholytic cells ( acc ) [ table 1 ] . this test was done to rule out pemphigus . the patient showed steady improvement with the therapy given and was discharged , after 8 days , without any sequelae .", "this is most likely a case of drug - induced ten as other causes like upper respiratory tract infection , viral infection , or malignancy were not present . as this was an acute presentation , which occurred right after the drugs were administered , systemic lupus erythematosus ( sle ) and rheumatological disorders were ruled out . ten was diagnosed from the history and typical clinical features and biopsy was not needed . the prodrome was shorter ( 8 - 12 hrs ) than the usual 1 - 3 days . this might have been due to the use of multiple drugs causing an unknown interaction or more likely due to a previous exposure to any or all of these drugs . causality assessment was done for each of the drugs separately by the method of kramer et al . paracetamol and metoclopramide were categorized as probable ( score 5 ) and antihistamines and multivitamins as possible ( score 3 ) causal drugs for this adverse drug reaction . since this drug intake was uneventful for a long time , the chances of this medication causing this episode of ten are minimal . there have been two case reports of ten caused by paracetamol and a case control study showing increased risk of sjs or ten with paracetamol use . there have been no case reports of ten due to a combination of both the drugs , nor have there been any reports regarding ten being caused by antihistamines or multivitamins . however , the probability of other drugs being responsible could not be ruled out completely .", "ten in this patient was probably caused by paracetamol and metoclopramide , but a possibility of an interaction as well as the role of other drugs can not be ruled out ." ]

[ "after hyping deep - etch electron microscopy ( em ) for my whole career ( heuser , 2011 ) , i 'll take this invitation to write an ascb award essay to talk it up some more ! replicas are not only impervious to beam damage in the electron microscope , forever the big problem , because the electron beam heats up the sample so terribly during viewing , but their electron - scattering power is also excellent , so they are simple to image and give super high - contrast . and the key thing to remember is that replicas are utterly faithful to whatever they are replicating they're just surface renderings , copying exactly the contours of the sample and displaying these contours in the electron microscope image . so the whole approach boils down to worrying about how to prepare your biological samples for replication . it takes the right equipment and some practice to make a proper replica , but , once mastered , it 's utterly routine and simple to learn . when mark kirschner first watched me do it while helping me to put it on the map by providing gorgeous cytoskeletons [ heuser and kirschner , 1980]he got bored right away and asked me , ca n't you teach a monkey to do that ? ) anyway , replicas have a glorious history , because in the early days of em , way before thin - sectioning techniques had been developed , they were the only way to go the only way to get any sort of biological sample into the electron microscope . thus the em pioneers in the 1940s used metal replicas to discover viruses and phages and to make the first halting characterizations of macromolecular assemblies like collagen and neurofilaments . what they lacked back then was a way to see inside cells , which keith porter achieved for the first time in 1945 by simply growing cells flat enough to see through in the electron microscope really , really flat and then fixing and staining them properly for em ( his other huge contribution ) . people not familiar with em should be reminded that porter 's 1945 images opened the door to cell biology , and his development of thin - sectioning techniques for cells in the following 10 years really put cell biology on the map . but back to replicas . the whole field of scanning electron microscopy ( sem ) was totally dependent on them because everything had to be coated with metal in order to be seen in the scanning electron microscope . likewise , the exciting field of freeze - fracture em took off after hans moor teamed up with a swiss company that made replicating machines ( balzers of lichtenstein ) and mounted a microtome inside one , so that frozen cells could be fractured open ( not quite thin - sectioned , the microtome was n't that good ) . this made it possible for people to make metal replicas of frozen cells without melting them even a little bit some sort of miracle ! deep - etch em is a variant of what moor introduced ( heuser and salpeter , 1979 ) and deserves special attention only because its purpose has been to avoid all of the fixation and staining and dehydrating procedures that had accompanied previous approaches to em and essentially to get living cells replicated after they were frozen ( figure 1 ) . we found that freeze fracture works just as well or better on unfixed cells and molecules , and therefore made a huge effort to devise a really good way to freeze living cells , tissues , and cell extracts without introducing such artifacts as ice - crystal damage . a platinum replica of the inside surface of a hela cell prepared by unroofing it in culture before quick - freezing and freeze - drying it in the usual way ( heuser , 2000 ) . three - dimensional view was used for the publicity and table cards for our department 's centennial celebration three years ago . clathrin lattices found on all cell membranes and illustrates the various stages in their evolution , from totally flat to fully curved and ready to pinch off during endocytosis . such three - dimensional deep - etch images were the first to illustrate that f - actin filaments ( highlighted in purple ) often become involved in the later stages of such clathrin coated pit formation and stay behind as circular scars after coated vesicles have left the surface ( above the wash in washington university ) . as explained in this essay , the swell opportunity to view such expanses of the plasma membrane at such a high resolution was a lucky outcome of our being able to freeze samples fast enough to avoid ice - crystal formation and then , miraculously , to platinum - replicate such frozen membranes without melting them . the best way to freeze everything turned out to be a spruced - up version of an approach anthonie van harreveld had used in the 1960s at caltech to freeze brains in preparation for classical thin - section em . van harreveld wanted to maintain the proper distribution of electrolytes in the brain and had reason to believe that the classical fixation techniques being used on brain were distorting this distribution . he reasoned that the freeze - substitution technique that ned feder and richard sidman had put on the map in the late 1950s would give him more realistic views . with this technique , a frozen sample is fixed and prepared for embedding in plastic by dissolving the ice out of it at subzero temperatures , using acetone or the like . van reasoned , quite correctly , that this should prevent artifacts from occurring during fixation , because nothing ever melted ; but how he came up with the idea to freeze the brain by slamming it onto an ultracold block of copper remains a mystery to this day . ( it 's fun to mention here that van harreveld did n't start developing this technique until he was already 60 years old ! ) anyway , it sure worked for van , and it also worked for tom reese and me when we copied his slammer , even though we had to spend years ironing out the bugs and making a freezing machine that was mechanically sound and gave reproducible results ( heuser et al . , 1979 ) . the result was our so - called liquid helium cooled cryopress ( renamed to avoid the distressing idea of a delicate piece of tissue being slammed against anything albeit , it 's the abruptness of contact and the superfast extraction of heat from the sample by the copper block that gives such good freezing in the first place ) . fast - forward to today , and we find that freeze substitution is still the backbone of modern efforts to image cells in the electron microscope , and indeed preserves the structure of cells far better than the techniques of fixation and plastic embedding developed by the pioneers of thin - section em . when combined with thicker sections , higher em voltages , and modern tomographic reconstruction techniques , it yields really outstanding images . so why are n't there more than 10 labs in the world using our ( or van harreveld 's ) cryopress to get the quality of freezing our lab has depended on for decades ? the answer lies in part with another advance that hans moor spearheaded in switzerland , again with the same enlightened balzers company producing vacuum evaporators , namely , high - pressure freezing . at the time , phase diagrams of water indicated that water could be frozen into an amorphous glass without the induction of any damaging ice - crystal formation by putting it under extreme pressure ( > 2000 atm ) . today , theories about how water turns into vitreous ( noncrystalline ) ice are much more complex , but moor went ahead and developed ways to put a biological sample under huge pressures and only then freeze it by spraying liquid nitrogen at it rather than slamming it against a liquid nitrogen cooled copper block . ( the rapidity of freezing , he reasoned , should no longer be important if the pressure trick works as apparently it does . ) today , most em labs have a high - pressure freezer , and most of the em papers that are published on freeze - substituted cells have availed themselves of these devices . so why not use our slammer ( or cryopress ) for freezing before freeze substitution , since it 's cheaper , faster , more reliable , and handles larger samples ? frankly , we do n't get it ! not only that , but high - pressure frozen samples can not be freeze - fractured at all at least no one has yet devised a way to do so because the samples end up encased in various sorts of metal pressure chambers , whereas our quick - frozen or cryopressed samples are spread out and open to the world ( mandatory for freeze fracture , but also good for freeze substitution ) . and for that matter , why are n't more labs making good old replicas of quick - frozen , deep - etched molecules ( heuser , 1983 ; goodenough and heuser , 1984 ; hanson et al . , 1997 ) ? that is , of course , the ultimate mystery to us . probably it 's just because people do n't realize that there are still good replicating machines available for purchase , and people do n't realize that these machines are n't so expensive and are easy to operate . well , as i said at the outset , i 've been hyping our technique for decades and ca n't stop now . i believe that an opportunity is being missed and that simplifying techniques so that even a monkey could do it will attract not monkeys to the field , but serious young investigators who want to get their hands on electron microscopes and want to get the most true to life i 'm a photographer at heart and love sharing images , all sorts of images , with people who appreciate them and can learn from them i love that more than anything . what fun it was , to be able to interact on a daily basis with the mark kirschners , tom pollards , ron vales , bernie gilulas , and ira mellmans of cell biology ( and sorry to all those whom i did n't mention you know who you are ! ) . plus , a handful of people really fired me up : tom reese , my boss as a postdoc at the national institutes of health , with whom i became so intertwined for so many years that he and i will never know who did what or who deserves what credit in the original development of quick - freeze , deep - etch em ( heuser and reese , 1973 ; heuser et al . , 1979 ) ; and then nobutaka hirokawa , who came to my lab as a postdoc , and immediately orchestrated a host of collaborations with leading cell biologists around the world that put deep etching on the map ( before leaving for the university of tokyo to become chairman of the department of cell biology , and then dean of the medical school , and now head of the whole human frontier science program ) ; and finally , my ex ursula goodenough , who absorbed my images and simply took off , making huge advances in several fields , thanks to her deep grasp of all aspects of cell biology . finally , i 'd like to simply add this : biological em was terribly interesting for me in the early days , back when it first allowed people to zoom in on the structures that light microscopists had been studying for so long and show what they actually were i used to wait with eager anticipation for each new issue of the journal of cell biology to arrive in the mail and then would devote a whole evening ( maybe with a glass of wine ) to carefully examining every new electron micrograph published that month . but em became even more captivating for me as people began more and more to systematically manipulate cells by physical and pharmacological ( and eventually genetic ) methods and then to look in the microscope to see how this altered the fine structures of their cells . this opened the door to true structure / function correlations at least when the effects of these experimental manipulations of cell physiology and biochemistry were properly determined , along with the microscopy . this era of em was the most fun for me , personally , but as it happened , this heyday was cut short by an overwhelming urge in some quarters to improve the methods of em , in an attempt to make the imaging of cells more this trend particularly captivated the equipment manufacturers and led to an arms race of microscope development that ended up making electron microscopes so very costly that only a few centers could support them anymore . the result was actually a curtailment of general , everyday em as it had been practiced by individual investigators in command of their own microscopes and published every month in the journal of cell biology . and as a consequence , over the past 15 years or so , em has gradually been relegated to a service status , carried out largely by em cores in most major institutions . electron microscopists , and gone also is the use of em for all sorts of fun structure / function correlations . and helping to eclipse the routine em that i enjoyed so much have been all the tremendous advances in light microscopy , coupled with all the advances in digital camera recording of live - cell dynamics ( not to mention the burgeoning field of superresolution light microscopy , crowned this year with the nobel awards ) . these huge advances have captivated nearly everyone still interested in functional correlations of cell structure and have left traditional em sort of out in the cold , an outcome i find most unfortunate . i feel strongly that seeing cell structures at the em level still is the only way to fully grasp their molecular architecture , and that seeing changes in their molecular architecture at this level is the only way to truly understand their function . i 'm permanently stuck with the founding fathers ' view that cell ultrastructure will ultimately display and explain all of cell function ! george palade was my greatest hero , and his fun explanation in his nobel lecture of why he chose to study the pancreatic acinar cell is my favorite quote : perhaps the most important factor in this choice was the appeal of the amazing organization of the pancreatic acinar cell , whose cytoplasm is packed with stacked er cisternae studded with ribosomes . its pictures had for me the effect of the song of a mermaid : irresistible and half transparent . its meaning seemed to be buried under only a few years of work , and reasonable working hypotheses were already suggested by the structural organization itself . irresistible and half transparent , indeed ! thanks , george . and thanks to all of you who cared to look at my images and all the institutions and funding agencies that made it possible for me to generate them !", "every picture i take , i already have an audience for it right as i take it . ( of course , they 're not actually there , they may be continents away , but i 'm imagining them being there and already planning how i will get that picture to them and what i 'll tell them about it as soon as it 's in the computer . ) it 's for showing to someone who immediately comes to mind as soon as that field pops into view in the electron microscope . oh , pietro will love that huge neuromuscular junction ; fulvio will be amazed by that quality of membrane preservation in freeze - substituted yeast ; ursula will be psyched by that run of axonemal dynein ; tom will be impressed with such a clear view of actin branchpoints . only rarely am i lucky enough to have someone actually sitting next to me and to be able to talk to him or her right then , person to person maybe a new postdoc or a close collaborator who really needs to look over my shoulder to see how his or her prep came out . anyway , i want each of my real or imaginary viewers to like that picture , to think it 's a good picture attractive , clear , understandable , useful , illuminating , that is , illuminating something about the subject ( be it a personal portrait or a picture of a cell interior or a molecule ) . i want my audience 's appreciation ! my whole drive of focusing all my work on improving techniques of preparation for em has come from wanting to take better pictures and get more of that appreciation . besides that , there 's just that darn old curiosity : what does it actually look like , what does it look like exactly ? how good a picture of it can i take ? how good - looking can i make it ( or him or her , with my personal portraits ) ? ( nic spitzer once irritably dubbed the latter my thin sections of life as i was clicking away while canoeing with him down a rapids on the allagash river , but not paddling . ) always on my mind is what 's the most expressive or most characteristic or attractive attire or decoration i can outfit it ( them ) with ? osmium or platinum or gold or furs and silks ? capturing that best picture will help me to get to know my subject better , to really see it for what it is . even artifacts can be extremely beautiful and informative , if one knows how one got them and what they say about what the structure was , before it got altered . all these aspects of photography i can appreciate by myself , all alone , but never as much as when there is just one other person with me , with the same inclination and proclivity . sharing , mutual appreciation , communion that has been the whole name of the game for me in my research career . my advisor don fawcett , one of the great masters of em of all times , told me when i graduated from medical school , do n't become an electron microscopist , you 'll become everybody 's slave . actually , i think i can say that it turned out just the opposite : everyone else turned out to be my audience , my source of appreciation and self - worth , my foils , my mentors , and , most important of all , my best source for interesting things to look at in the electron microscope !" ]

[ "in women , cervical cancer is the second most common malignant tumor worldwide and is one of the leading causes of cancer - related death in developing countries . although the incidence of cervical cancer is generally decreasing , it is still a serious public health problem worldwide , especially in developing countries , and the average age of cervical cancer patients is decreasing . human papillomavirus ( hpv ) is considered the major cause of cervical cancer , but viral infection alone is not sufficient for its development . the pathogenesis of cervical cancer is still unclear and probably involves aberrant expression of numerous oncogenes and anti - oncogenes . many distinct advances have been made in the prevention , surgical resection , radiotherapy , and chemotherapy of cervical cancer , but the prognosis of cervical cancer patients remains poor . therefore , it is important to better understand molecular events in the invasion and metastasis of cervical cancer and to develop novel prognostic markers and therapeutic strategies . stromal cell - derived factor-1 ( sdf-1 ) , which is a chemoattractant cytokine , is involved in a variety of physiological and pathological activities . with regard to cancer , substantial evidence indicated that sdf-1 plays crucial roles in the cell growth , apoptosis , invasion , and metastasis of many kinds of cancers [ 79 ] . it is well acknowledged that chronic inflammation is widely connected with the initiation and progression of cervical cancer . inflammatory cytokines such as chemokines , inflammatory proteins , and adhesion molecules are known as the main causes of chronic inflammation . numerous studies have revealed that nf-b , as a transcription factor , is cheifly responsible for the activity of these cytokines and is also implicated in the development of a wide range of cancers . however , to the best of our knowledge , the final role of sdf-1and nf-b in cervical cancer remains unclear . in the present study , we used tissue microarray and the immunohistochemistry method to detect the expression of sdf-1and nf-b in 105 cases of human cervical cancer tissues and their paired adjacent tissues . the purpose of this study was to investigate the association of sdf-1and nf-b expression with clinicopathological parameters of cervical cancer and to evaluate the prognostic value of sdf-1and nf-b expression in cervical cancer patients .", "we analyzed a total of 105 formalin - fixed , paraffin - embedded cervical cancer tissues and the adjacent tissues at the time of operation from january 2002 to november 2013 at east hospital , tongji university . all these cancer tissues were confirmed as cervical cancer by medical examination and hematoxylin and eosin staining after surgical resection . none of the patients received adjuvant chemotherapy , radiation therapy , or other anti - tumor therapies . important related clinicopathological parameters of the patients , such as age , tumor size , figo stage , lymphatic metastasis , stromal invasion , differentiation , and survival time , were obtained from each patient s medical records and are shown in tables 1 and 2 . the survival time was calculated from the date of surgery to the date of death , or the last known follow - up . all cervical cancer tissue samples included in this investigation were obtained with patients written informed consent . a standard immunohistochemistry method was used to study sdf-1and nf-b expression in cervical cancer tissues and the adjacent tissues . briefly , the tumor tissues and adjacent tissues were fixed in 10% formaldehyde and embedded in paraffin and then we cut the paraffin sections into 4-m sections . all the 4-m tissue sections were dewaxed and rehydrated with xylene and graded alcohol , respectively . we washed the sections with buffer solution for 5 min and then added the primary antibody into them at 4c overnight . after that , we washed the sections again and added the second antibody ( r&d systems inc . , minneapolis , mn ; dilution 1:50 ) into the sections . afterwards , we washed the sections with phosphate - buffered saline ( pbs ) and developed them with 3 , 3-diaminobenzidine ( dab ) for 5 min and counterstained them with hematoxylin . results are presented as the percentage of the staining cells ( 0 to 100% ) in tissues . staining under 20% of the tissue cells or no staining was included in the negative group ( ) , while the others belonged to the positive group ( + ) . all the sections were assessed under an optical microscope by 2 independent investigators , and any discrepancy in immunohistochemistry was resolved by consensus . the expression of sdf-1 and nf-b in cervical cancer and adjacent cancer tissues was compared with the paired wilcoxon test . chi - square test and fisher s exact test were used to assess the association between clinical characteristics of cervical cancer patients and sdf-1and nf-b expression . the prognosis of cervical cancer and sdf-1and nf-b expression were determined using kaplan - meier survival analysis and log - rank test for univariate analysis . all statistical analyses were performed using spss version 18.0 software ( spss inc . , chicago , il ) .", "we analyzed a total of 105 formalin - fixed , paraffin - embedded cervical cancer tissues and the adjacent tissues at the time of operation from january 2002 to november 2013 at east hospital , tongji university . all these cancer tissues were confirmed as cervical cancer by medical examination and hematoxylin and eosin staining after surgical resection . none of the patients received adjuvant chemotherapy , radiation therapy , or other anti - tumor therapies . important related clinicopathological parameters of the patients , such as age , tumor size , figo stage , lymphatic metastasis , stromal invasion , differentiation , and survival time , were obtained from each patient s medical records and are shown in tables 1 and 2 . the survival time was calculated from the date of surgery to the date of death , or the last known follow - up . all cervical cancer tissue samples included in this investigation were obtained with patients written informed consent .", "a standard immunohistochemistry method was used to study sdf-1and nf-b expression in cervical cancer tissues and the adjacent tissues . briefly , the tumor tissues and adjacent tissues were fixed in 10% formaldehyde and embedded in paraffin and then we cut the paraffin sections into 4-m sections . all the 4-m tissue sections were dewaxed and rehydrated with xylene and graded alcohol , respectively . we washed the sections with buffer solution for 5 min and then added the primary antibody into them at 4c overnight . after that , we washed the sections again and added the second antibody ( r&d systems inc . , minneapolis , mn ; dilution 1:50 ) into the sections . afterwards , we washed the sections with phosphate - buffered saline ( pbs ) and developed them with 3 , 3-diaminobenzidine ( dab ) for 5 min and counterstained them with hematoxylin . results are presented as the percentage of the staining cells ( 0 to 100% ) in tissues . staining under 20% of the tissue cells or no staining was included in the negative group ( ) , while the others belonged to the positive group ( + ) . all the sections were assessed under an optical microscope by 2 independent investigators , and any discrepancy in immunohistochemistry was resolved by consensus .", "the expression of sdf-1 and nf-b in cervical cancer and adjacent cancer tissues was compared with the paired wilcoxon test . chi - square test and fisher s exact test were used to assess the association between clinical characteristics of cervical cancer patients and sdf-1and nf-b expression . the prognosis of cervical cancer and sdf-1and nf-b expression were determined using kaplan - meier survival analysis and log - rank test for univariate analysis . all statistical analyses were performed using spss version 18.0 software ( spss inc . , chicago , il ) .", "typical immunohistochemistry images of sdf-1 and nf-b in cervical cancer tissues and paired adjacent non - tumor tissues are shown in figures 1 and 2 . the positive percentage of sdf-1 expression in cervical cancer and adjacent non - tumor tissues were 68.6% ( 72/105 ) and 8.6% ( 9/105 ) , respectively . the positive percentage of nf-b expression in cervical cancer and adjacent non - tumor tissues were 60.0% ( 63/105 ) and 5.7% ( 6/105 ) , respectively . the chi - square test was used to confirmed that the difference in the expression level of sdf-1and nf-b between cervical cancer tissues and paired adjacent non - tumor tissues was statistically significant ( p<0.05 ) . the relationship between patients clinical parameters and the expression of sdf-1and nf-b is shown in tables 1 and 2 , respectively , showing that the expression of sdf-1 in the cervical cancer tissues is significantly correlated with tumor size ( =10.794 , p=0.001 ) and figo histology grade ( =7.000 , p=0.008 ) . however , there was no statistical correlation between sdf-1 expression and patient age , lymph node metastasis ( lnm ) status , stromal invasion , or tumor differentiation ( p>0.05 ) . similarly , the expression of nf-b in the cervical cancer tissues was significantly correlated with tumor size ( =7.421 , p=0.006 ) , figo histology grade ( =10.182 , p=0.001 ) , and lymph node metastasis ( lnm ) status ( =4.762 , p=0.029 ) . however , there was no statistical correlation was found between nf-b expression with patient age and stromal invasion and tumor differentiation ( p>0.05 ) . therefore , the results demonstrate that higher sdf-1 and nf-b expression in cervical cancer tissues is positively correlated with tumor metastasis and cancer progression , suggesting that sdf-1and nf-b play important roles in the progression . to further evaluate the relationship between sdf-1and nf-b expression and prognosis of cervical cancer , we performed log - rank survival analysis according to the sdf-1 and nf-b expression level and patient survival data . the survival analysis demonstrates that the cervical survival rate of the patients with negative sdf-1 and nf-b expression is significantly better than that of the patients with positive ones ( p<0.05 , figures 3 , 4 ) . furthermore , a multivariate cox regression analysis demonstrated that sdf-1 expression and lymph node metastasis are independent predictors of the os in cervical cancer patients . the expression of sdf-1 was positively correlated with the expression of nf-b ( r=0.201 , p=0.040 ; table 3 ) .", "typical immunohistochemistry images of sdf-1 and nf-b in cervical cancer tissues and paired adjacent non - tumor tissues are shown in figures 1 and 2 . the positive percentage of sdf-1 expression in cervical cancer and adjacent non - tumor tissues were 68.6% ( 72/105 ) and 8.6% ( 9/105 ) , respectively . the positive percentage of nf-b expression in cervical cancer and adjacent non - tumor tissues were 60.0% ( 63/105 ) and 5.7% ( 6/105 ) , respectively . the chi - square test was used to confirmed that the difference in the expression level of sdf-1and nf-b between cervical cancer tissues and paired adjacent non - tumor tissues was statistically significant ( p<0.05 ) .", "the relationship between patients clinical parameters and the expression of sdf-1and nf-b is shown in tables 1 and 2 , respectively , showing that the expression of sdf-1 in the cervical cancer tissues is significantly correlated with tumor size ( =10.794 , p=0.001 ) and figo histology grade ( =7.000 , p=0.008 ) . however , there was no statistical correlation between sdf-1 expression and patient age , lymph node metastasis ( lnm ) status , stromal invasion , or tumor differentiation ( p>0.05 ) . similarly , the expression of nf-b in the cervical cancer tissues was significantly correlated with tumor size ( =7.421 , p=0.006 ) , figo histology grade ( =10.182 , p=0.001 ) , and lymph node metastasis ( lnm ) status ( =4.762 , p=0.029 ) . however , there was no statistical correlation was found between nf-b expression with patient age and stromal invasion and tumor differentiation ( p>0.05 ) . therefore , the results demonstrate that higher sdf-1 and nf-b expression in cervical cancer tissues is positively correlated with tumor metastasis and cancer progression , suggesting that sdf-1and nf-b play important roles in the progression .", "to further evaluate the relationship between sdf-1and nf-b expression and prognosis of cervical cancer , we performed log - rank survival analysis according to the sdf-1 and nf-b expression level and patient survival data . the survival analysis demonstrates that the cervical survival rate of the patients with negative sdf-1 and nf-b expression is significantly better than that of the patients with positive ones ( p<0.05 , figures 3 , 4 ) . furthermore , a multivariate cox regression analysis demonstrated that sdf-1 expression and lymph node metastasis are independent predictors of the os in cervical cancer patients .", "the expression of sdf-1 was positively correlated with the expression of nf-b ( r=0.201 , p=0.040 ; table 3 ) .", "it has 2 major isoforms : and . both are derived from a single gene due to alternative splicing . it is well acknowledged that sdf-1 mediates a large number of crucial biological processes such as neuronal and cardiac development , angiogenesis , apoptosis , and stem cell motility . substantial evidence indicates that sdf-1 can mediate the activation of sdf-1/crcr4-pi3-mark - nf-b pathway , pi3k / akt , wnt , and erk pathways , and further promotes the invasion and progression of cancers [ 1719 ] . sdf-1 can also improve the expression of mmps and vegf , which are crucial for the aggressive behavior of cancers . many studies have revealed that the over - expression of sdf-1 is found in various malignancies , including lung cancer , prostate cancer , breast cancer , and pancreatic cancer . the nf - kb transcription factor family includes 5 genes : nf-b1 ( p50/p105 ) , nf-b2 ( p52/p100 ) , rela ( p65 ) , c - rel , and relb . normally , nf - kb dimmers are transcriptionally inactive due to their interaction with nf - kb inhibitors ( i kbs ) . activation of nf-b may result from the enhanced expression of epidermal growth factor receptor , insulin growth factor receptor , and tumor necrosis factor receptor families . activation of some other pathways , like pi3k / akt and ras / mapk , also participate in the activation of nf-b . some different kinds of molecular alterations in cancer cells may lead to impaired regulation of nf-b activation . in this situation , nf - kb becomes constantly activated , which leads to aberrant expression of downstream genes of nf-b , including those involved in regulation of cell cycle , proliferation , adhesion , and apoptosis . these pathophysiological changes may finally lead to the initiation , development , and metastasis of cancer . the results of this study indicate that the expression of sdf-1 is increased in cervical cancer tissues and is correrated with tumor size and figo histology grade . we found that the expression of nf-b is increased in cervical cancer tissues and is correrated with tumor size , figo histology grade , and lymph node metastasis . the potential mechanism may be that sdf-1 activates the nf-b pathway by the interaction with cxcr4 . the sdf-1/crcr4-nf-b pathway participates in the regulation of cell prliferation , apoptosis , and angiogenesis in cervical cancer . the increased expression of vegf and mmps caused by sdf-1-cxcr4 interaction and many nf-b - related biological macular - involved cell cycle , apoptosis , and chronic inflammation responses may mediate the initiation and progression of cervical cancer [ 3032 ] . it is well known that cancer pathogenesis is a multi - factor , multi - step , complicated process involved in gene - gene and gene - environment interactions . large and well - designed studies are still needed to elucidate the pathogenesis of cervical cancer .", "results of our study indicate that the expression of sdf-1 is significantly associated with tumor size and figo histology grade . the expression of nf-b is significantly associated with tumor size , figo histology grade , and lymph node metastasis . we also found that positive sdf-1 or nf-b expression is significantly correlated with poor prognosis ." ]

[ "pycnodysostosis was first reported in 1923 by montanari , and he called it as atypical achondroplasia . the main characteristics are short stature , cranial dysplasia , increased bone density and fragility . other clinical features include open cranial sutures , hypoplastic paranasal sinuses , dysplastic lateral clavicle , shortened terminal phalanges , proptosis , blue sclera and frontal or occipital bossing . oral manifestations include obtuse gonial angle , grooved palate , anterior cross - bite , malpositioned teeth associated with increased incidence of dental caries and periodontitis , hypoplastic maxilla , receded chin , delayed eruption of permanent teeth , delayed exfoliation of deciduous teeth and hypoplasia of root - obliterated pulp spaces .", "a 47-year - old man reported to the department with a complaint of deformed lower jaw for the past 10 years . history revealed that the patient had undergone extraction of his teeth that was uneventful , following which there was fracture of jaw at the extraction site . subsequently , there was frequent exfoliation of teeth with fracture at different sites in the lower jaw . his medical history revealed that he had multiple fractures of the upper and lower limbs and a history of diabetes mellitus and hypertension . general examination revealed that the patient 's height was 127 cm and weight was around 49 kg , with proportionate dwarfism [ figure 1 ] . the hand and feet had short digits with overlying cutaneous wrinkles that tapered off with large overriding nails [ figure 2 ] . a 47-year - old man , 127 cm in height shortened digits and cutaneous wrinkles on extraoral examination , there was facial dysmorphia with prominent forehead ( frontal bossing ) , proptosis , beaked nose , deep nasolabial skin folds , micrognathia and obtuse mandibular angle on the right side [ figure 3 ] . mild proptosis and facial dysmorphia intraoral examination revealed multiple clinically missing teeth , chronic periodontitis , narrow and grooved palate , no features of enamel hypoplasia , malposed teeth [ figure 4 ] and evidence of sequestrum in relation to the tooth 46 [ figure 5 ] . based on the history and clinical presentation , a provisional diagnosis of a bone dysplasia , probably pycnodysostosis , was made and differential diagnosis of cleidocranial dysplasia and osteopetrosis was included . grooved palate and clinically missing maxillary anteriors sequestrum in relation to tooth 46 laboratory findings were within normal limits , including hemoglobin conc . , differential count calcium , phosphate , alkaline and acid phophatase level . computed tomography of the bone window of the skull showed open sutures and fontenalles with nonaerated paranasal sinuses , flattening of the mandibular angle on the right side with evidence of fracture and loss of bone architecture on the left side involving the ramus of the body of the mandible and hypoplastic maxilla [ figures 68 ] . orthopantomograph revealed generalized bone loss , multiple missing teeth and obtuse gonial angle with loss of bone structure on the left side of the mandible involving the body and ramus [ figure 9 ] . 3d - reconstructed computed tomography showing open sutures and fontanelles 3d - reconstructed computed tomography showing fractured body of the mandible and obtuse gonial angle 3d - reconstructed computed tomography showing fractured body of the mandible opg showing generalized bone loss , multiple missing teeth , and obtuse gonial angle lateral skull showing open fontanelles with nonaerated paranasal sinus and fractured body of the mandible the patient was surgically managed for osteomyelitis by removal of the sequestrum and curettage , and further mandibular reconstruction was performed .", "the patient presents with characteristic facies , dwarfism , beaked nose , prominent head and generalized increase in the density of bones not sufficient to obliterate medullary canals or cranial orifices . frequent fractures due to trauma can aid in diagnosing this condition . in our case , the patient was negligent about the condition and reported to us with a fractured jaw . intraoral clinical presentation included altered pattern of exfoliation of deciduous teeth and eruption of the permanent dentition . the disease is diagnosed at an early age , wherein the main reasons for consultation are generally short stature and open anterior fontanelles . in later stages , consultation is usually for fracture resulting from slight or moderate trauma , given the severe bone fragility . symptoms include dental abnormalities , with hypoplasia of the enamel , obliterated pulp chambers and hypercementosis . protrusion of the incisors with anterior open bite may be found , and dental crowding associated with extensive caries and periodontitis is frequent . in our case , multiple clinically missing teeth , chronic periodontitis , narrow and grooved palate , features of enamel hypoplasia and malposed teeth these conditions cause the premature loss of dentition that may already be complete by the fourth decade of life , similar to our patients . our patient showed evidence of sequestrum in relation to the tooth 46 as a result of osteomyelitis . orofacial infections are commonly encountered by the dentist and there are wide ranges of modalities that can be implemented in managing them . this is caused by the increased bone volume of the sella turcica that , on compressing the pituitary gland , causes its hypoplasia and a deficient production of the growth hormone . our patient 's height was 127 cm and weight was around 49 kg , with proportionate dwarfism . the hand and feet had short digits with overlying cutaneous wrinkles that tapered off with large overriding nails . diagnosis of pycnodysostosis is based on the clinical presentation , and medical treatment for the condition is symptomatic . the differential diagnosis of pycnodysostosis includes osteopetrosis , acroosteolysis , mandibular acral dysplasia and cleidocranial dysplasia . unlike osteoporosis , hepatosplenomegaly and difference between pycnodysostosis and cleidocranial dysplasia is that dense and brittle bones are found in pycnodysostosis but not in cleidocranial dysplasia . new treatment modalities like gene therapy and bone marrow transplant can be expected to be the mainstay in the future , now that the abnormal expression of cathepsin k and the gene defect has been located .", "now - a - days , symptomatic treatment is provided for patients with pycnodysostosis , with the main intention of prevention of fractures . as pycnodysostosis is associated with inappropriate bone remodeling , it can pose a challenge for a dental health care professional to provide treatment as there can be serious complications , such as osteomyelitis arising as a result of dental infections . tooth extractions in these patients demand certain special care , such as carrying out the surgery atraumatically with proper asepsis . oral hygiene practices and frequent visits to" ]

[ "the accumulation of metals in the environment , stemming from their origination in the earth 's crust , as well as from anthropogenic sources , creates the potential for significant human exposures and subsequent health hazards . deleterious metal - induced health effects , including carcinogenesis and neurodegeneration , have been reported in all body systems , with exposure stemming from multiple sources , including contact with contaminated food , water , air , or soil . the particular metals considered in this review , antimony , arsenic , cadmium , manganese , mercury , silver , and uranium , are among the classes of essential nutrients , as is the case with copper and manganese , as well as the nonessential , naturally occurring metals , such as arsenic , cadmium , and mercury , all of which can induce toxicity depending on the concentration level and exposure duration . over time , organisms have developed protective mechanisms to deal with metal exposure , most of which function in one of three ways : ( 1 ) decreasing the uptake of the metal , ( 2 ) stimulating the expulsion of the metal , or ( 3 ) activating the organism 's general stress response mechanisms . metals can disrupt homeostasis by generating oxidative stress , inhibiting enzyme activity , impairing dna repair , and disrupting protein binding and normal cellular function , including proliferation , cell cycle progression , and apoptosis [ 14 ] . elucidating the mechanism(s ) of metal detoxification has been difficult due to the complexity of mammalian systems and the reductionist approach inherent to cell culture systems . the model organism , caenorhabditis elegans ( c. elegans ) , offers the advantage of an in vivo system that is less complex than the mammalian system while still sharing high homology . c. elegans possess ~60%80% of human genes and contain conserved regulatory proteins [ 68 ] . this soil nematode has been used in a number of toxicity studies due to its well - characterized genetic , physiological , molecular , and developmental stages . some of the advantages afforded by the c. elegans model system are small size ( ~1.5 mm adult ) , short lifespan ( ~3 weeks ) , and rapid lifecycle ( ~3 days ) [ 810 ] . at adulthood , a single c. elegans hermaphrodite is capable of producing ~300 progeny . c. elegans are hermaphrodites , but approximately 1% of c. elegans are male , allowing for genetic experimentation . the nematode 's small genome and relative anatomical simplicity ( less than 1000 cells ) contribute to the appeal of this model system for genetic manipulation . in addition , the use of rna interference ( rnai ) and chromosomal deletion in worms has provided valuable information regarding the increased sensitivity of mutant strains to metal toxicity [ 1214 ] . maintenance of nematode strains is relatively simple ; they grow on bacteria - seeded plates and can be maintained at 20c . strains can also be frozen indefinitely , easily allowing for the accumulation of large stocks of worms . c. elegans provide the researcher with a uniquely powerful model , as the worm 's translucent body allows for the in vivo visualization of fluorescently labeled individual cells and proteins . the in vivo c. elegans model system is especially valuable for the investigation of metal detoxification and is particularly amenable for examining gene - environment interactions , albeit with a few considerations to take into account ( table 1 ) . several toxicity endpoints are readily detected and well documented in the nematode , including mortality , lifespan , reproduction , and feeding [ 1719 ] . acute toxicity can also be assessed in the nematode through behavioral endpoints , such as locomotive behavior , head thrashing , body bending , and other basic movements [ 2024 ] . recently , the role of c. elegans as a biomonitor in environmental risk assessment has also been explored [ 2528 ] . several cellular systems such as the glutathione ( gsh ) , metallothioneins ( mts ) , heat shock proteins ( hsps ) , as well as various pumps and transporters work in concert to detoxify and excrete metals . it remains to be established whether the knockdown or overload of one detoxifying system upregulates other compensatory mechanisms . in this review , we offer a brief summary of the ways in which the c. elegans model has shed novel insights on the various mechanisms of metal detoxification . metals considered herein are antimony ( sb ) , arsenic ( as ) , cadmium ( cd ) , lead ( pb ) , mercury ( hg ) , silver ( ag ) , and uranium ( u ) .", "glutathione ( gsh ) is a cysteine - containing tripeptide , consisting of glutamic acid , cysteine , and glycine and is found in most life forms . gsh possesses antioxidant properties , since the thiol group of cysteine is a reducing agent and can be reversibly oxidized ( gssg ) and reduced ( gsh ) . gsh is maintained in the reduced form by the enzyme , glutathione reductase ( gr ) , and functions by reducing other metabolites and enzyme systems . glutathione peroxidase ( gpx ) catalyzes the oxidation of gsh to gssg in the presence of ros ( figure 1 ) . gssg can be converted back into gsh via gr and the conversion of nadph to nadp+ . proton - translocating mitochondrial nicotinamide nucleotide transhydrogenase ( nnt ) catalyzes the reduction of nadp+ by nadh and is an important source of nadph , as has been demonstrated using nnt-1 deletion mutants . glutathione s - transferases ( gsts ) can catalyze the conversion of gsh to gs , which can then form complexes with various xenobiotics to facilitate excretion . these enzymes are found at particularly high levels in the liver , and gsh is typically the most abundant sulfhydryl - containing compound in cells . additionally , gsh is known to offer protection against metal - generated ros by binding free radicals . gshs have been reported to increase , decrease , or remain constant after exposure to metals [ 5557 ] . the gsh system is found in animals , plants , and microorganisms . c. elegans express approximately 50 gsts . additionally , phytochelatins ( pcs ) , a family of metal - inducible peptides synthesized enzymatically from gsh by pc synthase ( pcs ) in the presence of heavy metal ions , have been identified in c. elegans . although pcs are synthesized from gsh , they are broadly classified as class iii metallothioneins and have been shown to be important in the detoxification of heavy metals . liao and yu investigated the involvement of the gsh system in response to inorganic arsenic exposure . results confirmed that oxidative stress plays a role in arsenic - induced toxicity by mutating glutamylcysteine synthetase ( gcs ) ( gcs-1 ) , the rate - limiting enzyme in gsh synthesis , in worms . the gcs-1 loss - of - function strain demonstrated hypersensitivity to arsenic exposure in lethality testing , as compared to wild - type animals , an effect that was rescued by the addition of gsh to the medium , indicating that these enzymes are crucial for mediating arsenic - induced toxicity . furthermore , helmcke and aschner reported a significant increase in fluorescence in a gst-4::gfp strain following both acute and chronic exposure to mehg . it was also demonstrated that knockout gst-4 worms did not display greater sensitivity to mehg than did n2 wild - type worms . gsh levels were found to be increased in worms subjected to acute exposure , whereas worms subjected to chronic exposure exhibited depleted levels of gsh . in their hormetic model , helmcke and aschner demonstrated an increase in gst-4::gfp expression after low concentration , acute exposure , a finding which indicates that gst-4 may be involved in the hormetic response to mehg . taken together , the data from these studies suggest that gst-4 contributes to the response to mehg exposure , but that knockdown of this gene does not affect with overall lethality . in a study examining the role of pcs in the elimination of cadmium , vatamaniuk and colleagues reported that the c. elegans pcs-1 gene encodes a functional pc synthase critical for heavy metal tolerance . using double stranded rnai against pcs-1 , this group showed that , although the progeny of worms injected with the dsrnai and exposed to cadmium ( 525 m ) managed to reach adulthood , these worms were small , necrotic , sterile , and had a much shorter lifespan than did the wild - type controls . after being exposed to 50 and 100 m concentrations of cadmium , the pcs-1 worms arrested at the l2l4 stage were necrotic and died by day 6 . the results were definitively dependent on the presence of cadmium , because pcs-1-deficient worms , in the absence of cadmium exposure , responded identically to the wild - type controls . hughes and colleagues examined metabolic profiles following cadmium exposure in phytochelation synthase-1 ( pcs-1 ) mutants . results from these studies showed that the primary response to low levels of cadmium is the regulation of the transsulfuration pathway , due to the fact that cadmium exposure caused a decrease in cystathionine concentrations and an increase in phytochelation-2 and -3 . these results were corroborated by additional studies which demonstrated that pcs-1 mutants were an order of magnitude more sensitive to cadmium than were the metallothionein mutants . furthermore , the mt - pcs-1 triple mutant was found to display an additive sensitivity toward cadmium .", "metallothioneins ( mts ) belong to a family of cysteine - rich low - molecular - weight metal - binding proteins ( mw 3,50014,000 da ) involved in metal detoxification and homeostasis . mts bind both metals of physiological importance such as copper and zinc , as well as xenobiotics including arsenic , cadmium , mercury , and silver . the binding of these metals occurs via the interaction of the cysteine residues with thiol groups . binding of metals by mts may be transient , as mts are capable of rapidly releasing metal ions . the protective roles of mts can be ascribed to their three primary functions : ( 1 ) metal homeostasis , ( 2 ) heavy metal detoxification , and ( 3 ) protection from oxidative stress . additionally , these proteins have been identified as contributors to the hormetic response [ 39 , 61 ] . mammals express four known metallothionein isoforms ( mt - i , mt - ii , mt - iii , mt - iv ) . mt - i and mt - ii are expressed in almost all tissues and have been best characterized with regard to their protection of the brain . mt - iii is especially enriched in the central nervous system , although its role has not yet been clearly defined . mt - iv is most abundantly expressed in the stratified squamous epithelia [ 6466 ] . mt expression has been shown to be induced under stressful cellular conditions such as exposure to cytokines , glucocorticoids , reactive oxygen species ( ros ) , and metal ions . mts can bind directly and sequester the toxicant ; they also can provide protection by acting as antioxidants ( figure 1 ) . further , mts can limit apoptosis and promote the survival of mitochondrial dysfunctional cells by serving as highly efficient reducing elements against reactive oxygen species ( ros ) . c. elegans contain two distinct isoforms of mts , known as mtl-1 and mtl-2 , which can be induced in response to exposure to various metals . jiang and colleagues examined the effects of mts on depleted uranium ( du ) in c. elegans . results from their study showed that mtl-1 was an important factor in uranium accumulation in c. elegans as knockouts displayed increased cellular accumulation . in a study investigating lead and methylmercury toxicity , ye and colleagues demonstrated the involvement of mts in affording a protective cross - adaptation response to neurobehavioral toxicity . this endpoint was assessed by observing behavioral alterations ( head thrashing and body bending ) in worms that were exposed during the l2 phase to either pb or mehg . the study was conducted in conjunction with mild heat shock , wherein pretreatment of the larva with heat shock prevented the neurobehavioral deficits and the stress response at lower concentrations ( 50100 m ) but not at higher concentrations ( 200 m ) . additionally , mild heat shock coupled with exposure to a low concentration of either metal was found to induce mtl-1 and mtl-2 promoter activity and gfp gene expression , results that were not observed in either the metal - exposed or heat - shocked cohort alone . finally , the overexpression of mtl-1 or mtl-2 at the l2 stage was shown to significantly repress neurobehavioral toxicity , suggesting that the accumulation of mt protein is necessary to confer the protective response to the toxicant . similarly , helmcke and aschner reported that mtl knockouts displayed increased lethality upon exposure to mehg . this group also demonstrated increases in mtl-1::gfp fluorescence in response to acute mehg exposure at the l1 stage ; however , chronic mehg exposure produced no change in florescence . their results indicate that mtl-1 is important in mediating mehg toxicity and that the effects occur in a concentration- and time - dependent manner . meyer and colleagues examined the aggregation of silver nanoparticles ( agnps ) in wild type and mtl-2 c. elegans . results from these studies showed that the mtl-2 strain displayed greater agnp sensitivity than did the wild - type controls . these data indicate that there may be a differential preference for mtl-1 over mtl-2 depending on the particular metal to which an organism is exposed . in a 2004 study by swain and colleagues , using gfp - expressing transgenes , mt - null alleles , and the rnai knockdown of mts , this group demonstrated that cadmium but not copper or zinc was able to influence a concentration - dependent , temporal transcription response . cadmium exposure caused a reduction in body size , generation time , brood size , and lifespan , effects that were magnified in the mt knockdown worms . hughes and colleagues studied metabolic profiles using proton nmr spectroscopy and uplc - ms following cadmium exposure in single and double mtl knockouts . results showed that the metallothionein status did not influence the metabolic profile in cadmium - exposed or unexposed worms . the primary response to low levels of cadmium was the regulation of the transsulfuration pathway , due to the fact that cadmium exposure resulted in a decrease in cystathionine concentrations and an increase in phytochelation-2 and -3 . these results were corroborated by data showing that pcs-1 mutants ( phytochelation synthase-1 ) were an order of magnitude more sensitive to cadmium than were mt mutants . further , an additive sensitivity toward cadmium was observed in the mt - pcs-1 triple mutant . a study by bofill and colleagues examined zinc and cadmium toxicity ; results indicated differential metal binding behavior for mt-1 as compared to mt-2 . specifically , the mt-1 isoform showed optimal behavior when binding zn , and mt-2 showed optimal behavior when binding cd . accordingly , it was hypothesized that , due to its induction following cd exposure , mt2 is primarily responsible for detoxification , whereas mt1 possesses some degree of constitutive expression and is , therefore , primarily involved in physiological metal metabolism ( e.g. zinc ) . these findings were corroborated by additional studies which showed that mt - knockout worms exhibited significantly decreased levels of overall fitness after the knockout of mt1 than after mt2 knockout . further , both mt isoforms displayed a clear preference for divalent metal ion binding as opposed to copper coordination , likely due to the presence of histidines in the mts . using both in vitro and in vivo models , zeitoun - ghandour and colleagues examined zinc and cadmium exposures and showed different roles for mtl-1 and mtl-2 . both isoforms their affinities and stoichiometries were measured , and both isoforms displayed equal zinc- binding ability ; however , mtl-2 had a higher affinity for cd than did mtl-1 . these experiments were repeated in vivo in mtl-1 , mtl-2 , and double knockouts following exposure to 340 m zn or 25 m cd . zinc levels were found to be significantly increased in all knockout strains , but mtl-1 knockout worms demonstrated the most acute level of sensitivity . however , cadmium accumulation was found to be the highest in the mtl-2 knockout and double mutant strains . additional studies assessed metal speciation , and results indicated that o - donating ligands play an important role in maintaining zinc levels , independent of metallothioneins status . further , cadmium was shown to interact with thiol groups , and cd speciation was significantly different in the mtl-1 strain when compared with both the mtl-2 strain and the double knockout strain , suggesting that the two mt isoforms have distinct in vivo roles . the authors suggested that mts are not functioning as metal storage proteins but , rather , are mediating the accumulation and excretion of metals . a follow - up study , showed in vitro evidence for the partitioning of zinc and cadmium with different metallothionein isoforms . employing electrospray ionization mass spectrometry ( esi - ms ) to directly observe zinc and cadmium binding preferences , more cadmium ions were found to be preferentially bound to mt-2 than to mt-1 ; however , cd was shown to be capable of inducing both isoforms . finally , partitioning was also demonstrated to be more effective at lower cd : zn ratios . using daf-2 ( insulin receptor - like protein ) and age-1 ( phosphatidylinositol-3-oh kinase catalytic subunit ) mutants , barsyte et al . examined the expression of mt genes under noninducing conditions and after exposure to cadmium and copper . they reported that mt-1 mrna levels were significantly higher in daf-2 mutants compared to both age-1 mutants and wild - type worms under basal conditions . exposure to cadmium treatment resulted in a three - fold induction of mt-1 and a two - fold induction of mt-2 mrna in daf-2 mutants as compared to wild - type controls . copper did not induce mt-1 or mt-2 mrna expression in any of the strains tested . collectively , these studies show differential metal preferences for one mt isoform over another depending on the metal to which an organism is exposed . most significantly , these studies indicate that the mts play crucial roles in metal detoxification ( table 2 ) . indeed , mts have been associated with a protective effect in cells under numerous states of disease and stress . interestingly , serum mt levels of cancer patients are three times higher than those of control patients . a study conducted in denmark revealed the increased expression of mt-1 and mt-2 mrna and protein in many human cancers such as breast , kidney , lung , nasopharynx , ovary , prostate , salivary gland , testes , urinary bladder , cervical endometrial skin carcinoma , melanoma , acute lymphoblastic leukemia , and pancreatic cancers . this information is of particular import given the use of metals for the treatment of certain cancers , for example , arsenic as treatment for promyelocytic leukemia . it is interesting to postulate that higher levels of mts may enhance the efficacy of metal therapeutic agents or , conversely , may lead to resistance to such therapies . understanding the factors that modulate mt expression will allow for the improved understanding of metalloid toxicity and will provide more effective therapeutic approaches to metalloid - based chemotherapy .", "there are a number of pumps and transporters that have been implicated in metal detoxification . these include atp - binding cassette ( abc ) transporters , such as the multidrug resistance - associated protein ( mrp ) as well as two members of the p - glycoprotein subfamily ( pgp-1 and pgp-3 ) , which have been shown to contribute to heavy metal tolerance through the use of c. elegans deletion mutants . in c. elegans , there are approximately 60 genes encoding abc transporters , and these genes make up the largest family of transporters . c. elegans have four mrp homologues and fifteen pgp homologues . the pgps are ubiquitously expressed and are most abundantly found in the apical membranes of the gut and in the excretory organs of the worm . pgp-2 is expressed in the intestine and is required for the acidification of lysosomes and lipid storage ; pgp-1 and -3 contribute to heavy metal and drug resistance . tseng and colleagues investigated the arsa protein - mediated detoxification of the metalloids , as(iii ) and sb(iii ) . bacterial arsa atpase is the catalytic component of an oxyanion pump that is responsible for resistance to arsenite and antimonite . in this study , wild - type and asna-1-mutant nematodes were evaluated for as and sb response and toxicity . the asna-1 gene of c. elegans was found to be stimulated by as(iii ) ; further , sb(iii ) was determined to be crucial for establishing tolerance . although these results occurred in response to as and sb exposure , the ubiquity of the arsa atpase - dependent pathway has not been observed in other species or in response to other metals . the role of multidrug resistance - associated protein ( mrp ) in arsenite and cadmium toxicity was explored in a study by broeks and colleagues . the targeted inactivation of mrp-1 rendered the arsenite and cd exposed worms incapable of recovering from temporary exposure to high arsenic and cadmium , whereas the wild - type controls were able to recover . additionally , worms were also shown to be hypersensitive to arsenite and cd exposures when both mrp-1 and pgp-1 ( p - glycoprotein-1 ) were deleted . lastly , no increased sensitivity in response to exposure to antimony was observed in mrp-1-deletion mutants as compared to wild - type controls . vatamaniuk and colleagues characterized the half - molecule abc transporter of the heavy metal tolerance family-1 ( hmt-1 ) subfamily in response to cadmium exposure . the suppression of hmt-1 expression by rnai was shown to produce punctuate refractive inclusions within the vicinity of the nucleus of the intestinal epithelial cells upon exposure to toxic levels of cadmium . similarly , schwartz and colleagues described the c. elegans hmt-1 following exposure to arsenic , copper , and cadmium . hmt-1 conferred tolerance in response to exposure to all three metals as shown by lethality testing following the knockdown of hmt-1 . kurz and colleagues demonstrated the three - fold induction of pgp-5 following cadmium exposure . results of this study showed that strong fluorescence was induced in the intestinal cells of pgp-5::gfp worms , where the gfp - encoding gene is under the control of the upstream pgp-5 promoter . copper and zinc were also found to be capable of inducing pgp-5 expression in these worms . accordingly , it was concluded that pgp-5 is required for establishing full resistance to cadmium and copper . in addition , the rnai knockdown of tir-1 , an upstream component of the p38 mapk pathway in the pgp-5 transgenic reporter strain , was shown to significantly reduce pgp-5 induction following exposure to cadmium . however , the double - stranded rna knockdown of erk ( mpk-1 ) and jnk ( med-1 and kgb-1 ) did not affect the induction of pgp-5 in response to cadmium exposure . au and colleagues studied the divalent - metal transporter ( dmt1 ) following exposure to manganese . the dmt1-like family of proteins has been shown to regulate manganese and iron in the cell . the deletion of the three worm dmt1-like genes resulted in differential effects on manganese toxicity . the deletion of smf-1 and smf-3 increased mn tolerance , whereas the deletion of smf-2 increased mn sensitivity .", "heat shock proteins ( hsps ) are cytosolic molecular chaperones . hsps promote the refolding and repair of denatured proteins and facilitate protein synthesis upon activation by cellular stress [ 75 , 76 ] . hsps , particularly those in the hsp70 family , have also been shown to participate in the hormetic response . hsp70s are atp - binding proteins that convert atp to adp and bind to peptides , thereby , inactivating them and preventing aggregation ( figure 1 ) . oxidative stress can cause a reduction in cellular atp levels . decreased levels of atp result in the continued prevention of the aggregation of damaged proteins . the functions of the hsp70 products are mediated by the conserved n - terminal atpase and the c - terminal peptide - binding region . the human and c. elegans hsp70 genes have a high degree of homology and share a conserved core the hsp16 family of stress proteins is produced in c. elegans only under stress conditions [ 8082 ] . in a study examining the effects of mehg exposure , hsp-4::gfp was measured immediately following the treatment of l1 worms for 30 minutes and l4 worms for 15 hours with this toxicant . after 30 minutes of acute exposure to mehg , however , in l4 worms chronically exposed to mehg for 15 hours , hsp-4::gfp was induced . at the same time point in the chronic treatment paradigm , a four - fold increase in gst-4 fluorescence was detected , but there were no changes in either mtl-1 or mtl-2::gfp expression . jones and candido exposed nematodes to cadmium or mercury and measured feeding behavior . for these studies , transgenic lines containing the promoter sequence for hsp16 genes which regulate the production of e. coli-galactosidase were used . accordingly , to measure stress , levels of this protein were assessed . results showed that cadmium inhibited feeding behavior significantly but not completely , as a minimal rate of feeding continued at high cadmium concentrations . further , exposure to cadmium ( 1 ppm ) induced a detectable production of -galactosidase without inhibiting feeding behavior . the stress response was induced at a concentration of cadmium that was ten times lower than the lc50 . mercury also was shown to inhibit feeding at concentrations similar to those necessary for the induction of a stress response ; however , the difference in this instance was less than two fold .", "the use of c. elegans as an experimental model has produced considerable insight and valuable information regarding the multiple and varied processes of metal detoxification . conclusive biochemical evidence has indicated that different metals are not handled in the same capacity . the selectivity and sensitivity of each of these proteins is highlighted in the large body of accumulated research on different metal toxicities as well as various systems of metal detoxification . however , the overall mechanisms , temporal activation , and interplay between different cell detoxification systems remain elusive . future studies are necessary in order to enhance our understanding of the complex interplay of multiple - cell detoxification systems in response to exposure to different metals . the c. elegans model system will be critical for these investigations , as knockouts are easily generated and provide a wealth of information about metal detoxification in a genetically retractable , inexpensive , and in vivo model ." ]

[ "obesity is a complex heterogeneous disease that is caused by genes , environmental factors , and the interaction between the two . obesity is also a multifactorial condition , and many endocrine and inflammatory pathways are involved in its development and in obesity - related diseases . excess weight in obesity may come from muscles , bone , fat , and/or body water , but obesity specifically refers to having an abnormally high proportion of total body fat . the world health organization defines overweight as a body mass index ( bmi ) of 25 or more and obesity as a bmi of 30 or more . the prevalence of obesity has been stated as being near epidemic size [ 13 , 57 ] , and obesity has been associated with type ii diabetes , hypertension , coronary artery disease , stroke , and many forms of cancer [ 8 , 9 ] . therefore , it is important that the underlying pathophysiology of obesity - related diseases is understood . obesity results from the combined effects of genes , lifestyle , and the interactions of these factors , and both familial and nonfamilial factors play an important role in its development . a genetic predisposition to obesity has been reported as a major risk factor for individuals . with the increasing prevalence of obesity , studies on candidate genes for obesity most obesity - predisposing genes encode the molecular components of physiological systems related to energy balance . leptin is a protein product of the ob gene and is expressed and secreted by adipose tissue in amounts proportional to the body weight content ; studies on its receptor have greatly advanced the comprehension of the mechanism for regulating body weight and energy homeostasis . the lipostat system , mediated by leptin and its hypothalamic receptor , reduces food intake and increases thermogenesis [ 10 , 12 ] . the leptin ( lep ) and leptin receptor ( lepr ) genes have been evaluated for polymorphisms that could potentially be related to the pathophysiology of obesity and its complications . although the polymorphisms in these genes have been evaluated [ 1315 ] , the association of these polymorphisms with obesity is still controversial . therefore , we investigated whether the lep gene g2548a polymorphism and lepr gene 668a / g ( q223r ) polymorphism might be involved in the pathogenesis of obesity .", "this study included 127 obesity patients ( 93 women , 34 men ) and 105 controls ( 62 women , 43 men ) provided from the department of internal medicine , gazi osmanpaa university in tokat , turkey . informed consent was in accordance with the study protocol , and all patients and controls signed a written consent form . all patients received a complete clinical evaluation , and all individuals in the control group were healthy and were selected by excluding the diagnosis of obesity . genomic dna was isolated from white blood cells by a kit procedure ( invitrogen life technologies , carlsbad , ca , usa ) and stored at 20c . lep g2548a and lepr 668a / g polymorphisms were analyzed by polymerase chain reaction based restriction fragment length polymorphism ( pcr - based rflp ) methods . the pcr protocol consisted of an initial melting step of 2 min at 94c , followed by 35 cycles of 30 s at 94c , 30 s at 55c ( for lep ) , 30 s at 60c ( for lepr ) , and 30 s at 72c , and a final elongation step of 5 min at 72c . amplification was carried out using primers forward 5-ttt cct gta att ttc ccg tga g-3 and reverse 5-aaa gca aag aca ggc ata aaa a-3 for the lep gene and forward 5-tcc tct tta aag cct atc cag tat tt-3 and reverse 5-agc tag caa ata ttt ttg taa gca at-3 for the lepr gene . pcr was performed with a 25 l reaction mixture containing 2550 ng/l dna , 1 l of 10 pmol/l of each primer , 1 l of dntp mixture ( 5 mm dntp , 1 l 2.5 mm mgcl2 , 1 u taq dna polymerase ) , 2.5 l 10x pcr buffer ( mg free , invitrogen life technologies , carlsbad , ca , usa ) , and dh2o . amplified products were digested with hhai at 37c for lep and mspi at 37c for lepr , and the resulting fragments were separated by 2% agarose gel electrophoresis . the fragments were stained with ethidium bromide and visualized through a vilber - lourmat gel quantification and documentation system ( quantum - st4 ; vilber lourmat bp 66 , torcy , france ) . analysis of the data was performed using spss 16.0 ( spss , chicago , il , usa ) and openepi info ( http://www.openepi.com ) . the frequencies of the alleles and genotypes ( hardy - weinberg equilibrium ) in patients and controls were compared with analysis , and 95% confidence intervals were calculated . a p value less than 0.05 ( two - tailed ) was regarded as statistically significant .", "the mean age and bmi were 44.86 1.51 , 35.45 4.56 and 34.25 15.43 , 21.57 1.89 in patients and control groups , respectively . the mean age of obesity patients with bmi > 35 kg / m and bmi < 35 kg / m was 45.01 1.38 and 44.7 1.65 , respectively , while the mean bmi of these patients was 38.68 3.11 ( bmi > 35 kg / m ) and 32.06 1.56 ( bmi < 35 kg / m ) . patients and controls were genotyped for both the g2548a polymorphism in the lep gene promoter and the 668a / g polymorphism in the lepr gene . the distribution of the lep g2548a and lepr 668a / g polymorphisms of the patients and control groups are presented in tables 2 and 3 . we found no statistically significant difference in the genotype frequencies of the lep gene polymorphism in patients and control groups ( p > 0.05 ) . the lepr genotypes differed between the obesity patients and controls , but this was not statistically significant after the bonferroni correction ( p = 0.05 ) . allele frequencies in the lepr gene showed no statistically significant association ( p > 0.05 ) ( table 3 ) . the lepr gene a allele was 61.4% in patients and 69.5% in the control group , while the g allele frequency was 38.5% in patients and 30.4% in the control group . in the combined analysis of the lep and lepr genes , the lep / lepr gg / gg combined genotype was found to increase the risk of obesity compared to the controls ( p < 0.05 ) ( table 4 ) . in the combined genotype analysis based on the mean bmi of obesity patients , there was no association of the lep / lepr combined genotype and obesity between patients with a bmi 35 kg / m and patients with a bmi near 30 kg / m ( p > 0.05 ) ( table 4 ) .", "human obesity is a complex trait determined by the interaction of multiple genes and environmental factors . obesity may arise as a result of increased energy intake , decreased energy expenditure , or increased partitioning of nutrients into fat , either alone or in combination . the prevalence of obesity and being overweight continues to increase worldwide , not only causing serious personal health problems but also imposing a substantial economic burden on societies . genetic influences are difficult to elucidate , and identification of the involved genes is not easily achieved . in the present study , we analyzed the frequencies of lep g2548a and lepr 668a / g polymorphisms in obesity patients in a turkish population . there was no statistically significant difference between the groups with respect to the lep genotype distribution ( p > 0.05 ) and allele frequencies ( p > 0.05 ) . suggested that the lep g2548a variant may influence gene expression of leptin and leptin secretion by adipose tissue . noted that the lep g2548a polymorphism may influence a bmi increase by means of its effects on leptin secretion ; however , they identified a significant and independent association between the lep 2548gg carrier status and higher leptin levels . an association of the lep g2548a polymorphism and increased bmi was reported in overweight europeans and in taiwanese subjects with obesity and the combined lep 759c / t and lep g2548a genotype may be a determinant of obesity . the results of our study do not support the results of these studies but do support those of other studies that showed no association between the lep g2548a polymorphism and obesity - related phenotypes [ 11 , 14 , 15 , 21 ] . we found that lepr genotypes show a difference , but not statistically significant , between obesity patients and controls . we attribute this lack of significance to the low number of patients included in our study , but finding obese patients that have no other disease is difficult . some researchers have proposed that the polymorphisms of the leptin receptor gene ( especially lepr 668a / g polymorphism ) may contribute to common forms of human obesity [ 11 , 14 , 2224 ] . our results with respect to the lepr polymorphism are in agreement with the results of these studies . our results showed a statistically significant difference between groups with respect to the distribution of the lep / lepr gg / gg combined genotype . obesity results from both gene - gene and gene - environment interactions , and in our study we examined the gene - gene interactions of the lep / lepr genes and their link to obesity . demonstrated that the haplotype association of the lep g2548a and lepr q223r variants was related to a 58% increase in obesity risk , and they considered the interactions between lep and lepr gene polymorphisms to intensively influence modulation of energy homeostasis . in agreement with the findings of our study , boumaiza et al . reported that the lep g2548a and lepr q223r polymorphisms and haplotype combination were associated with a metabolic syndrome and obesity risk in tunisian subjects . the g2548a and 3hvr variants of the lep gene have been noted as being in linkage disequilibrium , and i / g combined genotypes are associated with obesity . in addition , the interactions between the polymorphisms of the lep and lepr genes have been shown to increase the risk of non - hodgkin 's lymphoma and influence insulin plasma concentrations and blood pressure levels . our findings indicate that the lep g2548a polymorphism is not a relevant obesity marker and that the lepr 668a / g polymorphism may be related to obesity in a turkish population . additionally , the lep / lepr gg / gg combined genotype was found to increase the risk of obesity in patients compared to controls . however , the association of these polymorphisms with obesity is still controversial , and further research with larger patient populations is necessary ." ]

[ "susceptibility weighted imaging ( swi ) is a relatively new magnetic resonance ( mr ) technique that provides innovative sources of contrast enhancement visualising the changes in magnetic susceptibility that are caused by different substances like iron , haemorrhage or calcium . the basic concept of this technique is maintaining phase information into the final image , discarding phase artefacts and keeping just the local phase of interest . sensitivity to susceptibility effects increases , progressing from fast spin - echo ( se ) to conventional se to gradient - echo ( ge ) sequences , from t2-weighting to t2 * -weighting , from short to long echo times and from lower to higher field strengths . before the clinical implementation of swi , susceptibility imaging relied only on ge sequences . swi differs significantly from a t2 * -weighted ge sequence : it is based on a long echo time ( te ) high - resolution , flow - compensated , three - dimensional ( 3d ) ge imaging technique with filtered phase information in each voxel . the combination of magnitude and phase data produces an enhanced contrast magnitude image that is particularly sensitive to haemorrhage , calcium , iron storage and slow venous blood , thus allowing a significant improvement compared with t2 * ge sequences . after imaging acquisition , the phase mask is then multiplied with the magnitude data to enhance the visualisation of vessels or foci with susceptibility effects . swi is therefore especially helpful in the detection of calcifications and microhaemorrhages , which are both characterised by low signal . the evaluation of the corrected phase images allows the differentiation between the two substances , as calcifications appear bright because of a positive phase shift and haemorrhages appear dark because of a negative phase shift . a supplementary source of information in swi swi represents a technical improvement in high - resolution blood oxygenation level - dependent venography ( hrbv ) , originally developed by reichenbach et al . , which was based on 3d long te , flow - independent ge sequences and manipulation of the images with the phase data . the paramagnetic properties of deoxyhaemoglobin [ bold ( blood oxygen level dependent ) effect ] and the prolonged t2 * of venous blood were used as an intrinsic contrast agent , leading to a phase difference between vessels containing deoxygenated blood and surrounding brain tissue , resulting in signal intensity cancellation . thus , deoxyhaemoglobin can behave like a contrast agent with long tes for differentiating arteries from small veins , which can be as small as 100200 m and therefore difficult to detect with conventional mr angiography techniques , such as time of flight ( tof ) or phase contrast ( pc ) . for this reason , the phase - added information that is usually not available in the conventional magnitude image makes swi well suited for the visualisation of very small vessels such as the caput medusae of venous angiomas and telangiectasias as a result of a combination of slow flow with changes in deoxyhaemoglobin concentration . latest advances have allowed the technique to be refined , thereby expanding its clinical applicability to brain imaging as a complementary source of information to conventional t1-weighted and t2-weighted imaging sequences .", "at our institution , mr imaging ( mri ) is performed by using a 1.5-t system ( magnetom avanto ; siemens , erlangen , germany ) with a 12-channel head coil . swi is obtained with a long - te , fully flow - compensated 3d ge sequence with the following parameters : repetition time ( tr)/te , 49/40 ms ; flip angle , 15 ; rectangular field of view ( fov ) , 7/8 ; matrix size , 280 320 ; slice thickness , 1.6 mm ( 80 slices in a single slab matrix size ) ; ipat factor , 2 ; acquisition time , 5 min . the swi sequences are reconstructed with the minimum intensity projection algorithm ( minip ) and multiplanar reformation ( mpr ) techniques to obtain images with thickness ( 310 mm ) and position comparable to those of conventional sequences . the minip algorithm has the characteristic to enhance the visualisation of veins while attenuating the signal from the brain tissue ( fig . 1 ) . \n b swi , minimal intensity projection ( minip ; 10 mm ) image : cortical and subependymal veins are well displayed . c swi , phase map : the distinction between the pars reticulata ( arrow ) and compacta ( asterisk ) of the substantia nigra is enhanced compared with conventional fast se and ge sequences . the outer margins of the red nucleus are also better displayed ( see d for comparison ) . b swi , minimal intensity projection ( minip ; 10 mm ) image : cortical and subependymal veins are well displayed . c swi , phase map : the distinction between the pars reticulata ( arrow ) and compacta ( asterisk ) of the substantia nigra is enhanced compared with conventional fast se and ge sequences . the outer margins of the red nucleus are also better displayed ( see d for comparison ) . d t2-weighted axial image swi sequences have some intrinsic disadvantages , which are mainly represented by artefacts caused by undesirable sources of magnetic susceptibility that occur at air tissue interfaces , therefore limiting the investigation of areas next to paranasal sinuses and temporal bone . the blooming artefact , useful in most cases , might also not be needed in some situations , producing normal tissue signal cancellation and loss of anatomical borders . the sequence acquisition time on a 1.5-t system ranges from 5 to 8 min , depending on the spatial resolution and the coverage of the brain needed , leading to an increased incidence of movement artefacts . imaging at a high field strength has some advantages over 1.5 t in the delineation of even smaller vessels belonging to the venous network , with shorter imaging times because of the higher signal - to - noise ratio , higher spatial resolution and increased susceptibility effects . tissue interfaces or other sources of local field heterogeneity are much more severe at higher fields , thus reducing swi s usefulness in the evaluation of the posterior fossa and skull base . swi has been investigated at 3 t with excellent results , especially concerning brain tumours and detection of cerebral microbleeds [ 6 , 7 ] .", "sporadic cerebral amyloid angiopathy ( caa ) is a common small - vessel disease associated with ageing , dementia and alzheimer s disease , that can only be diagnosed histopathologically following biopsy or at post - mortem examination . it consists of deposition of amyloid protein within the small and medium - sized cerebral arteries , which is likely responsible for increased vessel fragility and it is one of the major causes of lobar intraparenchymal haemorrhages in the elderly . computed tomography ( ct ) and conventional mr techniques are usually unable to show cerebral microbleeds ( cmbs ) , which can be frequently observed on t2 * -weighted gradient - echo mri and have a typical lobar distribution . recent findings indicate that cmbs in the general elderly population are relatively common and are even more frequently observed in patients with ad . swi is much more sensitive than ge sequences in demonstrating cmbs in and around the arteriole vessel wall , which appear as black dots better displayed by minip images ( fig . 2 ) . histologically , they represent perivascular clusters of haemosiderin - laden macrophages resulting from leakage from cerebral small vessels . higher field strengths , as expected , can improve cmb detectability , increasing the lesion s contrast enhancement . \n fig . 2recent onset of altered mental status and cognitive impairment in a 67-year - old man . a fluid attenuated inversion recovery ( flair ) mri : diffuse hyperintensity of the white matter , more pronounced in the right parietal region . b diffusion - weighted imaging ( dwi ) , apparent diffusion coefficient ( adc ) map : the white - matter hyperintensity represents vasogenic oedema . c ge t2 * -weighted image : small punctate hypointense foci in the right parietal cortex ( open arrow ) . d swi minip : increased visualisation of markedly hypointense foci surrounding the white matter abnormalities which correspond to multiple cerebral microbleeds . on the basis of swi imaging findings , caa with diffuse inflammatory changes was suspected and steroid therapy was administered . e follow - up mri at 3 months showed significant reduction of the vasogenic oedema recent onset of altered mental status and cognitive impairment in a 67-year - old man . a fluid attenuated inversion recovery ( flair ) mri : diffuse hyperintensity of the white matter , more pronounced in the right parietal region . b diffusion - weighted imaging ( dwi ) , apparent diffusion coefficient ( adc ) map : the white - matter hyperintensity represents vasogenic oedema . c ge t2 * -weighted image : small punctate hypointense foci in the right parietal cortex ( open arrow ) . d swi minip : increased visualisation of markedly hypointense foci surrounding the white matter abnormalities which correspond to multiple cerebral microbleeds . on the basis of swi imaging findings , caa with diffuse inflammatory changes was suspected and steroid therapy was administered . e follow - up mri at 3 months showed significant reduction of the vasogenic oedema caa is also characterised by white matter hyperintensities on conventional mri sequences , which have been associated with cognitive impairment . vascular amyloid deposition is believed to be involved in the pathophysiological mechanisms that determine white matter hypoperfusion through either vessel stenosis or vascular dysfunction . caa should also be suspected in elderly patients with clinical signs of a progressive encephalopathy syndrome with seizures , in which extensive white matter abnormalities are discovered in brain mri together with multiple cmbs with lobar and subcortical distribution ( fig . 2 ) . it has been recently reported that this combination of mr findings should be interpreted as caa - related inflammation , which can be treated with steroid therapy with a prompt resolution of the symptoms . in those cases , the demonstration of an apoe 44 genotype can definitely support the neuroradiological diagnosis . hypertensive encephalopathy and especially posterior reversible encephalopathy syndrome ( pres ) , when the signal abnormalities of the white matter are more symmetrical and located in parieto - occipital regions , should be taken into consideration for differential diagnosis . hypertensive encephalopathy is characterised by multiple cmbs which are usually silent and can be discovered when the patient is investigated with mri in order to understand the cause of an intraparenchymal haemorrhage located outside the basal ganglia . swi is more sensitive to cmbs than t2 * -weighted ge sequences in blood pressure ( bp)-related small vessels disease . cmbs are usually discovered both in deep basal ganglia and subcortical white matter ( fig . histologically they represent focal accumulations of haemosiderin - containing macrophages in the perivascular space of small brain vessels , indicating previous extravasation of blood , and are often associated with the presence of a symptomatic haemorrhage in the corresponding area . the number of cmbs , which remain detectable for years , is significantly associated with bp levels . moreover , the presence of deep cmbs can be a useful marker of bp - related small vessels disease , helping in the differential diagnosis with caa . \n a t2-weighted image shows a thin hypointense band in the right thalamus , which represents haemosiderin deposition ( arrow ) . b ge t2 * -weighted image shows multiple microbleeds , the largest one in the right thalamus ( arrow ) . c swi minip : the number of identifiable microbleeds is increased compared with ge images . they can be recognised bilaterally in the basal ganglia and in the subcortical temporal white matter ( arrows ) patient with long - standing hypertension . a t2-weighted image shows a thin hypointense band in the right thalamus , which represents haemosiderin deposition ( arrow ) . b ge t2 * -weighted image shows multiple microbleeds , the largest one in the right thalamus ( arrow ) . c swi minip : the number of identifiable microbleeds is increased compared with ge images . they can be recognised bilaterally in the basal ganglia and in the subcortical temporal white matter ( arrows ) iron deposition increases in the brain as a function of age , primarily in the form of ferritin and particularly in oligodendrocytes , but also in neurons and microglia . typical sites of iron deposition include the globus pallidum , substantia nigra , and red and dentate nuclei . swi filtered - phase images are particularly suitable for showing increased iron content in the brain ( . abnormally elevated iron levels are evident in many neurodegenerative disorders , including parkinson s disease , alzheimer s disease , huntington s disease and amyotrophic lateral sclerosis . the ability to measure the amount of ferritin in the brain can be used for a better understanding of the progression of the disease and is also helpful in predicting the treatment outcome . phase images allow a better distinction between the pars compacta and the pars reticulata of the substantia nigra , which contains iron ( fig . 1 ) . as in patients with idiopathic parkinson s disease , there is evidence of increased iron in the substantia nigra , swi sequences can be proposed as a useful imaging tool to identify iron deposition as a biomarker for disease progression , although longitudinal studies are required to support the usefulness of this specific application . accurate localisation of the subthalamic nucleus can also be achieved in the swi phase maps at 3 t , allowing safe direct targeting for placement of electrodes in the treatment of parkinson s disease . in 7098% of patients with alzheimer s disease recent evidence has shown that patients with multiple cmbs have more white matter hyperintensities and perform worse on mini mental state examination compared with patients with no cmbs , despite similar disease duration . a significant correlation has also been reported recently between patients with at least one hypointensity in gre - t2 * imaging and those homozygous for the apolipoprotein e 44 gene , a well - known risk factor for alzheimer s disease ( fig 4 ) . \n b coronal reformatted swi section shows a cerebral microbleed in the right frontal white matter ( arrow ) alzheimer s disease . b coronal reformatted swi section shows a cerebral microbleed in the right frontal white matter ( arrow ) swi has been demonstrated to be very useful in the acute phase of stroke for several reasons . first of all , it is very sensitive to the presence of cmbs , whose early identification is believed to predict the probability of potential haemorrhagic transformation after thrombolytic treatment . it has also been hypothesised that cmbs can represent a link between cerebral haemorrhage and ischaemia . swi is also capable of identifying the acute intravascular clot in the main and distal branches of the cerebral arteries . the evaluation of the intravascular content of deoxyhaemoglobin in hypoperfused brain is one of the most interesting applications of swi . this technique has been proved to be useful for the assessment of tissue viability in patients with hyperacute cerebral ischaemia , as it provides functional information about the ischaemic penumbra . in acute arterial stroke , flow reduction or absence causes an increase in oxygen extraction , leading to a growth in the amount of deoxyhaemoglobin in the hypoperfused brain tissue . deoxyhaemoglobin represents a supplementary source of contrast enhancement in swi because of its paramagnetic properties , which determine a reduction in t2 * as well as a phase difference between the vessel and its surrounding parenchyma . as the t1 and t2 properties of blood depend on the oxygen saturation of the blood , there are differences between arterial and venous vessels that can not be discovered with conventional sequences . the visualisation of draining veins within areas of impaired perfusion allows the identification of penumbral brain tissue in a different way from the current perfusion - weighted imaging ( pwi ) techniques ( fig . 5left internal carotid artery ( ica ) dissection with acute watershed infarct in a 42-year - old man with sudden onset of speech disturbance . a mr angiography , single partition at the level of the skull base showing dissection of the left ica , which is characteristically enlarged with high - signal - intensity methaemoglobin ( arrowheads ) representing the intramural thrombus and a small eccentric lumen ( arrow ) . b mr angiography , coronal mip : narrowing of the ica in its vertical segment just below the skull base ( arrow ) . d swi minip : improved visualisation of the veins of the left cerebral hemisphere , which is related to increased oxygen extraction in the ischaemic penumbra and corresponds to the hypoperfusion deficit shown by the mean transit time ( mtt ) map . e perfusion mri , mtt map , showing delayed transit time in the left middle cerebral artery ( mca ) territory . f intra - arterial digital subtraction angiography ( dsa ) , left carotid angiogram , confirming the ica stenosis ( arrow ) . g intra - arterial dsa : the patient was treated using stent placement , which resulted in partial restoration of the vessel lumen . h perfusion mri , mtt map 24 h after stent placement : complete resolution of the perfusion deficit . i swi minip , normalisation of the venous drainage of the left hemisphere left internal carotid artery ( ica ) dissection with acute watershed infarct in a 42-year - old man with sudden onset of speech disturbance . a mr angiography , single partition at the level of the skull base showing dissection of the left ica , which is characteristically enlarged with high - signal - intensity methaemoglobin ( arrowheads ) representing the intramural thrombus and a small eccentric lumen ( arrow ) . b mr angiography , coronal mip : narrowing of the ica in its vertical segment just below the skull base ( arrow ) . c dwi , axial image : acute watershed infarct in the left centrum semiovale . d swi minip : improved visualisation of the veins of the left cerebral hemisphere , which is related to increased oxygen extraction in the ischaemic penumbra and corresponds to the hypoperfusion deficit shown by the mean transit time ( mtt ) map . e perfusion mri , mtt map , showing delayed transit time in the left middle cerebral artery ( mca ) territory . f intra - arterial digital subtraction angiography ( dsa ) , left carotid angiogram , confirming the ica stenosis ( arrow ) . g intra - arterial dsa : the patient was treated using stent placement , which resulted in partial restoration of the vessel lumen . h perfusion mri , mtt map 24 h after stent placement : complete resolution of the perfusion deficit . i swi minip , normalisation of the venous drainage of the left hemisphere swi has become a functional method for evaluating cerebral venous sinus thrombosis ( cvst ) by demonstrating engorgement of the venous system as a result of venous hypertension and collateral slow flow . dural sinus thrombosis causes an increase in deoxyhaemoglobin concentration in the veins involved that appears as a prominent area of hypointense signal intensity on swi ; if cvst is treated successfully , this effect will disappear . swi is also capable of identifying the thrombosed pial vein , which is responsible for the development of oedema into the white matter , due to its high sensitivity in the demonstration of an intravascular clot . swi can depict at the same time the features of parenchymal or extra - axial haemorrhages that can occur in the case of infarction , with higher sensitivity compared with conventional ge sequences ( fig . a sagittal t1-weighted image demonstrates hyperintensity of the posterior third of the sagittal sinus ( open arrow ) . b mr venography confirms the thrombosis of the posterior third of the sagittal sinus ending at the torcular ( open arrow ) . c dwi , b = 1,000 image demonstrates a right occipital acute ischaemic lesion . d swi minip shows a right thalamic haemorrhagic lesion in the vascular territory of the right deep internal cerebral vein ( arrow ) epileptic seizures and sagittal sinus thrombosis in a 3-year - old child . a sagittal t1-weighted image demonstrates hyperintensity of the posterior third of the sagittal sinus ( open arrow ) . b mr venography confirms the thrombosis of the posterior third of the sagittal sinus ending at the torcular ( open arrow ) . c dwi , b = 1,000 image demonstrates a right occipital acute ischaemic lesion . d swi minip shows a right thalamic haemorrhagic lesion in the vascular territory of the right deep internal cerebral vein ( arrow ) cerebral arteriovenous malformations ( avms ) are easily displayed by conventional mri and mr angiography because of their characteristic high flow , whereas venous malformations such as cavernomas , developmental venous anomalies and capillary telangiectasias can not be adequately visualised without contrast medium administration and ge sequences , as they mainly consist of slow - flow small vessels . mr angiography techniques are often inadequate for visualising small vessels with slow flow . on the contrary , swi sequences are well suited for the visualisation of very small vessels , such as the caput medusae of venous angiomas and telangiectasias as a result of a combination of slow flow with changes in deoxyhaemoglobin concentration . the combined information derived from both phase and magnitude images is responsible for enhanced visualisation of such lesions . swi plays a substantial role in the identification and characterisation of cerebral vascular malformations , first of all improving their detection rate . on the other hand , allowing simultaneous visualisation of the different compartments of cerebral avms and of the relationship with one another and with the brain parenchyma , it is particularly valuable for therapeutic planning . the clinical usefulness of a similar technique , bold mr venography , was first demonstrated by essig et al . . phase changes caused by the susceptibility differences between oxygenated and deoxygenated red blood cells and the different relaxation rates between venous and arterial blood provide a natural separation of arteries and veins . the 3d dataset of swi can therefore be used as an alternative to mr angiography in order to better differentiate afferent arteries from draining veins . this advantage becomes relevant with small avms with recent bleeding , in which it is mandatory to identify the nidus in relation to eloquent areas in order to plan the best therapeutic approach , endovascular or surgical or both ( fig . veins appear dark due to t2 * loss and processing of the phase image , while arteries show bright signal from time - of - flight ( tof ) inflow enhancement . the use of higher flip angles and shorter tr make it possible to increase the contrast enhancement in the arteries without overly degrading the venography . for the evaluation of high - flow vascular malformations ( i.e. avms and dural arteriovenous fistulas ) the use of both minip and mip post - processing techniques can improve the image quality . the identification of small niduses , the exact location of the fistulous point and the depiction of the venous drainage patterns are the most relevant information that should be obtained from this technique . the elevated diagnostic accuracy of swi compared with digital subtraction angiography ( dsa ) has been recently reported for the detection of arteriovenous shunting ( avs ) in brain avms . \n b the swi minip image clearly depicts the brainstem haemorrhage , which is markedly hypointense . c , d swi , axial and reformatted sagittal images , processed with a maximum intensity projection algorithm ( mip ) , show flow - related enhancement within the haemorrhagic lesion ( arrow ) and a hypertrophied right posterior inferior cerebellar artery ( pica ; open arrow ) which are consistent with a small ruptured brainstem avm . e cerebral angiography , selective injection of the right vertebral artery : brainstem avm ( arrow ) fed by branches of the right pica and early venous drainage into a cerebellar vein ( arrowheads ) brainstem haemorrhage in a 43-year - old patient . b the swi minip image clearly depicts the brainstem haemorrhage , which is markedly hypointense . c , d swi , axial and reformatted sagittal images , processed with a maximum intensity projection algorithm ( mip ) , show flow - related enhancement within the haemorrhagic lesion ( arrow ) and a hypertrophied right posterior inferior cerebellar artery ( pica ; open arrow ) which are consistent with a small ruptured brainstem avm . e cerebral angiography , selective injection of the right vertebral artery : brainstem avm ( arrow ) fed by branches of the right pica and early venous drainage into a cerebellar vein ( arrowheads ) dural av fistulas ( davfs ) are usually more difficult to identify than avms on conventional mri , especially when venous ectasia is absent , as in type i and ii davfs ; the patients have vague symptoms ( i.e. headache or tinnitus ) and there are no localising signs . swi is capable of better identifying the extent of the abnormal venous drainage compared with mr angiography . the conspicuity of the venous structures on swi can be explained by the combination of a prolonged cerebral circulation time that favours increased oxygen extraction resulting in increased concentration of deoxyhaemoglobin , with the typical venous engorgement ( fig . b , c swi axial and coronal reformatted minip images show dilated right temporal veins ( arrows ) , which arouse suspicion of a tentorial dural av fistula . d cerebral angiography , selective injection of the right external carotid artery : dural av fistula of the right tentorium , fed by the right middle meningeal artery ( open arrow ) , with early venous drainage into the tentorial and temporal veins ( arrows ) patient with headache and pulsatile tinnitus of the right ear . a t2-weighted image shows a dilated right temporal vein ( arrows ) . b , c swi axial and coronal reformatted minip images show dilated right temporal veins ( arrows ) , which arouse suspicion of a tentorial dural av fistula . d cerebral angiography , selective injection of the right external carotid artery : dural av fistula of the right tentorium , fed by the right middle meningeal artery ( open arrow ) , with early venous drainage into the tentorial and temporal veins ( arrows ) concerning slow - flow vascular malformations ( i.e. cavernomas , developmental venous anomalies and capillary telangiectasias ) the phase information contained in swi is responsible for an artefactual enhancement that dramatically improves mri sensitivity to these pathological conditions . without swi individuals with cavernous malformations can present with epilepsy and focal neurological deficits or acute intracranial haemorrhage , although these vascular malformations are often discovered as incidental findings . cavernomas that have previously bled are usually detectable on routine mri because of the prominent signal intensity of haemorrhagic products . however , these lesions tend to be barely visible when they have not bled , except for faint enhancement after contrast medium administration that is often non - specific . patients with multiple lesions can be diagnosed with familial cavernous angiomatosis and should be referred for genetic evaluation and counselling ( fig . individuals with symptomatic , growing , or haemorrhagic malformations should be considered for surgical resection . close follow - up after diagnosis and treatment is helpful to identify lesion progression or recurrence . \n a t2-weighted image demonstrates a left frontal cavernous malformation surrounded by a thick ring of haemosiderin . b ge t2 * -weighted image shows multiple punctate hypointense foci located in both hemispheres , corresponding to small cavernous malformations ( arrows ) . c swi minip : there are supplemental cavernous malformations that are not detected by ge sequences ( arrows ) . d swi minip , reformatted sagittal section : multiple small cavernous malformations located in the brainstem ( arrows ) multiple cavernous malformations in a 32-year - old man investigated for intraparenchymal haemorrhage . a t2-weighted image demonstrates a left frontal cavernous malformation surrounded by a thick ring of haemosiderin . b ge t2 * -weighted image shows multiple punctate hypointense foci located in both hemispheres , corresponding to small cavernous malformations ( arrows ) . c swi minip : there are supplemental cavernous malformations that are not detected by ge sequences ( arrows ) . d swi minip , reformatted sagittal section : multiple small cavernous malformations located in the brainstem ( arrows ) developmental venous anomalies ( dva ) consist of a radially arranged venous complex converging on a centrally located venous trunk , which drains the normal brain parenchyma . they are better displayed with swi than with t1-weighted contrast - enhanced images ( fig . 10incidental discovery of a cerebellar developmental venous anomaly in a 48-year - old man investigated for right - sided sensorineural hearing loss . a t2-weighted image shows thin hypointense bands of flow void , in the right cerebellar hemisphere ( open arrow ) . b swi minip demonstrates multiple thin medullary veins ( arrows ) that converge into a dilated collector vein ( open arrow ) , consistent with a cerebellar venous angioma , better defined as developmental venous anomaly . c post - contrast t1-weighted axial image shows the enhancing dilated veins ( open arrow ) incidental discovery of a cerebellar developmental venous anomaly in a 48-year - old man investigated for right - sided sensorineural hearing loss . a t2-weighted image shows thin hypointense bands of flow void , in the right cerebellar hemisphere ( open arrow ) . b swi minip demonstrates multiple thin medullary veins ( arrows ) that converge into a dilated collector vein ( open arrow ) , consistent with a cerebellar venous angioma , better defined as developmental venous anomaly . c post - contrast t1-weighted axial image shows the enhancing dilated veins ( open arrow ) capillary telangiectasias are asymptomatic venous vascular malformations , which are smaller and less common than cavernomas and can sometimes occur in mixed cavernoma / telangiectasia lesions . these may occur sporadically or may be infrequently associated with syndromes , like hereditary haemorrhagic telangiectasia or ataxia telangiectasia . they may also manifest as a result of endothelial injury , such as radiation - induced vascular injury , particularly in children who have received cranial irradiation . they are characterised by poor contrast enhancement and can therefore be missed on conventional t1-weighted and t2-weighted sequences ( fig . 11incidental discovery of a pontine telangiectasia in a 34-year - old woman investigated for migraine . swi minip axial ( b ) and sagittal reformatted section ( c ) show a markedly hypointense lesion with regular margins , located in the pons . . the capillary telangiectasia is characterised by mild enhancement with an arbor - like pattern incidental discovery of a pontine telangiectasia in a 34-year - old woman investigated for migraine . swi minip axial ( b ) and sagittal reformatted section ( c ) show a markedly hypointense lesion with regular margins , located in the pons . . the capillary telangiectasia is characterised by mild enhancement with an arbor - like pattern ataxia telangiectasia is a rare autosomal recessive disorder characterised by an early - onset of progressive cerebellar ataxia , immunodeficiency , ocular and cutaneous telangiectasia and is associated with an elevated risk of intracranial tumours . swi can discover brain gliovascular nodules with perivascular haemorrhage and small haemosiderin deposits presumably only in older patients , during the later stages of the disease ; however , in our experience this technique can also be a useful screening tool in young patients ( fig . a 31-year - old woman with a diagnosis of ataxia telangiectasia by the age of 7 , investigated for dizziness and headache . c swi minip : additional hypointense black spots can be identified , corresponding to haemosiderin deposits presumably due to gliovascular nodules with perivascular haemorrhage ataxia telangiectasia . a 31-year - old woman with a diagnosis of ataxia telangiectasia by the age of 7 , investigated for dizziness and headache . c swi minip : additional hypointense black spots can be identified , corresponding to haemosiderin deposits presumably due to gliovascular nodules with perivascular haemorrhage mri with ge sequences has been extensively used in the past for investigating young patients with traumatic brain injuries ( tbi ) , either in the acute phase , when the clinical picture of a severe coma is not explained by ct , or months after trauma , in order to understand the causes of an unsuccessful recovery . in both cases , swi has been widely used in the identification of smaller haemorrhages due to a shear - strain mechanism of injury , thus refining the prediction of outcome . haemorrhagic dai lesions on swi can be six - times better detected than t2 * -weighted conventional ge sequences and the recognisable volume of haemorrhage is approximately twofold greater ( fig . reformatted swi images in the sagittal plane are particularly helpful in the assessment of corpus callosum haemorrhages . fig . 13diffuse axonal injury in a 14-year - old boy who had severe tbi following a motorcycle crash . brain mr study was performed three days after the trauma , when the patient was in a severely comatose state ( gcs 5 ) . a flair image identifies multiple hyperintense lesions in the splenium of the corpus callosum and in frontal subcortical and periventricular white matter . b ge t2 * -weighted image : haemorrhagic shearing injuries are barely visible in the right fronto - opercular and parieto - occipital regions ( arrows ) . c swi minip : additional microhaemorrhages are recognisable at the grey matter - white matter junction of the frontal lobes and in the right parieto - occipital white matter ( black arrows ) . d swi minip , reformatted sagittal section shows microhaemorrhages in the corpus callosum ( white arrows ) diffuse axonal injury in a 14-year - old boy who had severe tbi following a motorcycle crash . brain mr study was performed three days after the trauma , when the patient was in a severely comatose state ( gcs 5 ) . a flair image identifies multiple hyperintense lesions in the splenium of the corpus callosum and in frontal subcortical and periventricular white matter . b ge t2 * -weighted image : haemorrhagic shearing injuries are barely visible in the right fronto - opercular and parieto - occipital regions ( arrows ) . c swi minip : additional microhaemorrhages are recognisable at the grey matter - white matter junction of the frontal lobes and in the right parieto - occipital white matter ( black arrows ) . d swi minip , reformatted sagittal section shows microhaemorrhages in the corpus callosum ( white arrows ) brain mri predictors of tumour grade include contrast enhancement , oedema , mass effect , cyst formation or necrosis , haemorrhage , metabolic activity and cerebral blood volume . it is well known that the growth of solid tumours , such as gliomas , is dependent on the angiogenesis of pathological vessels . swi can provide a thorough assessment of the internal angioarchitecture of brain tumours ( increased microvascularity inside and beyond the tumour margins ) , together with the identification of foci of haemorrhage and calcification , thus representing an additional tool in the neuroradiological grading of cerebral neoplasms . the administration of a contrast agent ( ce - swi ) allows discrimination among those three entities , as only blood vessels will change their signal intensity , while calcifications and regions of inactive haemorrhage ( which can be differentiated from each other by the evaluation of phase images , as described above ) will not . the clinical potential of contrast - enhanced bold mr venography at 3 t and 1.5 t for the study of brain tumours was first reported by barth et al . , who demonstrated variable venous patterns in various types of tumours and in different parts of the lesions ( oedema , contrast - enhancing areas , necrosis ) , which might represent increased blood supply and particular vascular patterns around fast - growing malignant tumours . kim et al . recently assessed the added value provided by swi in the differential diagnosis of solitary enhancing brain lesions compared with the use of conventional mri alone . ce - swi has been found to be equivalent or even superior to ce - t1 images in the evaluation of most tumours with necrotic areas ( figs . 14 and 15 ) : the particular contrast combination within swi images permits the simultaneous visualisation of the information otherwise obtained by a multimodal imaging approach including ct , ce - t1 se , flair and t2 * conventional ge sequences . \n 14characterisation and grading of a glial tumour . a 30-year - old man with dizziness , long - standing behavioural changes and subsequent diagnosis of intra - axial cerebral tumour . b swi , unprocessed image : absence of intratumoural susceptibility signals ( due either to calcifications , haemorrhages or venous vasculature ) . c contrast - enhanced swi : detailed visualisation of the margins of a large anaplastic area ( asterisk ) , which is in good correlation with enhancement characteristics ( d ) , hypervolaemia on pwi relative cerebral blood volume ( rcbv ) map ( e ) and mr spectroscopy ( f ) . e pwi , rcbv map : area of increased cerebral blood volume ( open arrow ) . f single - voxel mr spectroscopy , showing inversion of the cho / naa ratio and the presence of a lactate peak . targeted stereotactic biopsy in the supposed necrotic area confirmed the hypothesis of gliomatosis cerebri who grade iii with several necrotic focifig . b t2-weighted image : the mass is irregularly hyperintense with cystic changes and small hypointensities representing small vessels and calcifications . c post - contrast t1-weighted image shows patchy enhancement of the lateral part of the lesion and some vascular structures . d ce - swi minip : calcifications appear as punctate hypointensities , unchanged after gadolinium injection ( white arrows ) . post - processing with minip algorithm allows the simultaneous visualisation of arteries ( hyperintense , black arrows ) and veins ( linear hypointensities , open arrow ) around and inside the tumour . ce - swi is superior to t1-weighted post - gadolinium sequences in demonstrating a diffuse bbb rupture in the lateral necrotic area ( bright signal , asterisk ) . histopathology revealed a primary neuroectodermal tumour ( pnet ) with extensive cystic and necrotic changes and increased vascularity characterisation and grading of a glial tumour . a 30-year - old man with dizziness , long - standing behavioural changes and subsequent diagnosis of intra - axial cerebral tumour . b swi , unprocessed image : absence of intratumoural susceptibility signals ( due either to calcifications , haemorrhages or venous vasculature ) . c contrast - enhanced swi : detailed visualisation of the margins of a large anaplastic area ( asterisk ) , which is in good correlation with enhancement characteristics ( d ) , hypervolaemia on pwi relative cerebral blood volume ( rcbv ) map ( e ) and mr spectroscopy ( f ) . e pwi , rcbv map : area of increased cerebral blood volume ( open arrow ) . f single - voxel mr spectroscopy , showing inversion of the cho / naa ratio and the presence of a lactate peak . targeted stereotactic biopsy in the supposed necrotic area confirmed the hypothesis of gliomatosis cerebri who grade iii with several necrotic foci internal architecture of a high - grade tumour . a 2-year - old child with headache , vomiting and right hemiparesis . b t2-weighted image : the mass is irregularly hyperintense with cystic changes and small hypointensities representing small vessels and calcifications . c post - contrast t1-weighted image shows patchy enhancement of the lateral part of the lesion and some vascular structures . d ce - swi minip : calcifications appear as punctate hypointensities , unchanged after gadolinium injection ( white arrows ) . post - processing with minip algorithm allows the simultaneous visualisation of arteries ( hyperintense , black arrows ) and veins ( linear hypointensities , open arrow ) around and inside the tumour . ce - swi is superior to t1-weighted post - gadolinium sequences in demonstrating a diffuse bbb rupture in the lateral necrotic area ( bright signal , asterisk ) . histopathology revealed a primary neuroectodermal tumour ( pnet ) with extensive cystic and necrotic changes and increased vascularity pinker et al . demonstrated a correlation between intratumoural susceptibility effects , positron emission tomography ( pet ) results and histopathological grading . swi has been proposed in the evaluation of clinical response to anti - angiogenetic drugs and in the differential diagnosis with pseudo - progression after chemo- and radiotherapy . a correlation with mr pwi has also been attempted . however , larger comparative studies of pwi and swi are still needed to determine a more precise role of the new techniques in the grading of cerebral neoplasms .", "sporadic cerebral amyloid angiopathy ( caa ) is a common small - vessel disease associated with ageing , dementia and alzheimer s disease , that can only be diagnosed histopathologically following biopsy or at post - mortem examination . it consists of deposition of amyloid protein within the small and medium - sized cerebral arteries , which is likely responsible for increased vessel fragility and it is one of the major causes of lobar intraparenchymal haemorrhages in the elderly . computed tomography ( ct ) and conventional mr techniques are usually unable to show cerebral microbleeds ( cmbs ) , which can be frequently observed on t2 * -weighted gradient - echo mri and have a typical lobar distribution . recent findings indicate that cmbs in the general elderly population are relatively common and are even more frequently observed in patients with ad . swi is much more sensitive than ge sequences in demonstrating cmbs in and around the arteriole vessel wall , which appear as black dots better displayed by minip images ( fig . 2 ) . histologically , they represent perivascular clusters of haemosiderin - laden macrophages resulting from leakage from cerebral small vessels . higher field strengths , as expected , can improve cmb detectability , increasing the lesion s contrast enhancement . \n fig . 2recent onset of altered mental status and cognitive impairment in a 67-year - old man . a fluid attenuated inversion recovery ( flair ) mri : diffuse hyperintensity of the white matter , more pronounced in the right parietal region . b diffusion - weighted imaging ( dwi ) , apparent diffusion coefficient ( adc ) map : the white - matter hyperintensity represents vasogenic oedema . c ge t2 * -weighted image : small punctate hypointense foci in the right parietal cortex ( open arrow ) . d swi minip : increased visualisation of markedly hypointense foci surrounding the white matter abnormalities which correspond to multiple cerebral microbleeds . on the basis of swi imaging findings , caa with diffuse inflammatory changes was suspected and steroid therapy was administered . e follow - up mri at 3 months showed significant reduction of the vasogenic oedema recent onset of altered mental status and cognitive impairment in a 67-year - old man . a fluid attenuated inversion recovery ( flair ) mri : diffuse hyperintensity of the white matter , more pronounced in the right parietal region . b diffusion - weighted imaging ( dwi ) , apparent diffusion coefficient ( adc ) map : the white - matter hyperintensity represents vasogenic oedema . c ge t2 * -weighted image : small punctate hypointense foci in the right parietal cortex ( open arrow ) . d swi minip : increased visualisation of markedly hypointense foci surrounding the white matter abnormalities which correspond to multiple cerebral microbleeds . on the basis of swi imaging findings , caa with diffuse inflammatory changes was suspected and steroid therapy was administered . e follow - up mri at 3 months showed significant reduction of the vasogenic oedema caa is also characterised by white matter hyperintensities on conventional mri sequences , which have been associated with cognitive impairment . vascular amyloid deposition is believed to be involved in the pathophysiological mechanisms that determine white matter hypoperfusion through either vessel stenosis or vascular dysfunction . caa should also be suspected in elderly patients with clinical signs of a progressive encephalopathy syndrome with seizures , in which extensive white matter abnormalities are discovered in brain mri together with multiple cmbs with lobar and subcortical distribution ( fig . 2 ) . it has been recently reported that this combination of mr findings should be interpreted as caa - related inflammation , which can be treated with steroid therapy with a prompt resolution of the symptoms . in those cases , the demonstration of an apoe 44 genotype can definitely support the neuroradiological diagnosis . hypertensive encephalopathy and especially posterior reversible encephalopathy syndrome ( pres ) , when the signal abnormalities of the white matter are more symmetrical and located in parieto - occipital regions , should be taken into consideration for differential diagnosis .", "hypertensive encephalopathy is characterised by multiple cmbs which are usually silent and can be discovered when the patient is investigated with mri in order to understand the cause of an intraparenchymal haemorrhage located outside the basal ganglia . swi is more sensitive to cmbs than t2 * -weighted ge sequences in blood pressure ( bp)-related small vessels disease . cmbs are usually discovered both in deep basal ganglia and subcortical white matter ( fig . histologically they represent focal accumulations of haemosiderin - containing macrophages in the perivascular space of small brain vessels , indicating previous extravasation of blood , and are often associated with the presence of a symptomatic haemorrhage in the corresponding area . the number of cmbs , which remain detectable for years , is significantly associated with bp levels . moreover , the presence of deep cmbs can be a useful marker of bp - related small vessels disease , helping in the differential diagnosis with caa . \n a t2-weighted image shows a thin hypointense band in the right thalamus , which represents haemosiderin deposition ( arrow ) . b ge t2 * -weighted image shows multiple microbleeds , the largest one in the right thalamus ( arrow ) . c swi minip : the number of identifiable microbleeds is increased compared with ge images . they can be recognised bilaterally in the basal ganglia and in the subcortical temporal white matter ( arrows ) patient with long - standing hypertension . a t2-weighted image shows a thin hypointense band in the right thalamus , which represents haemosiderin deposition ( arrow ) . b ge t2 * -weighted image shows multiple microbleeds , the largest one in the right thalamus ( arrow ) . c swi minip : the number of identifiable microbleeds is increased compared with ge images . they can be recognised bilaterally in the basal ganglia and in the subcortical temporal white matter ( arrows )", "iron deposition increases in the brain as a function of age , primarily in the form of ferritin and particularly in oligodendrocytes , but also in neurons and microglia . typical sites of iron deposition include the globus pallidum , substantia nigra , and red and dentate nuclei . swi filtered - phase images are particularly suitable for showing increased iron content in the brain ( . abnormally elevated iron levels are evident in many neurodegenerative disorders , including parkinson s disease , alzheimer s disease , huntington s disease and amyotrophic lateral sclerosis . the ability to measure the amount of ferritin in the brain can be used for a better understanding of the progression of the disease and is also helpful in predicting the treatment outcome . phase images allow a better distinction between the pars compacta and the pars reticulata of the substantia nigra , which contains iron ( fig . 1 ) . as in patients with idiopathic parkinson s disease , there is evidence of increased iron in the substantia nigra , swi sequences can be proposed as a useful imaging tool to identify iron deposition as a biomarker for disease progression , although longitudinal studies are required to support the usefulness of this specific application . accurate localisation of the subthalamic nucleus can also be achieved in the swi phase maps at 3 t , allowing safe direct targeting for placement of electrodes in the treatment of parkinson s disease . in 7098% of patients with alzheimer s disease , recent evidence has shown that patients with multiple cmbs have more white matter hyperintensities and perform worse on mini mental state examination compared with patients with no cmbs , despite similar disease duration . a significant correlation has also been reported recently between patients with at least one hypointensity in gre - t2 * imaging and those homozygous for the apolipoprotein e 44 gene , a well - known risk factor for alzheimer s disease ( fig 4 ) . \n b coronal reformatted swi section shows a cerebral microbleed in the right frontal white matter ( arrow ) alzheimer s disease . b coronal reformatted swi section shows a cerebral microbleed in the right frontal white matter ( arrow )", "swi has been demonstrated to be very useful in the acute phase of stroke for several reasons . first of all , it is very sensitive to the presence of cmbs , whose early identification is believed to predict the probability of potential haemorrhagic transformation after thrombolytic treatment . it has also been hypothesised that cmbs can represent a link between cerebral haemorrhage and ischaemia . swi is also capable of identifying the acute intravascular clot in the main and distal branches of the cerebral arteries . the evaluation of the intravascular content of deoxyhaemoglobin in hypoperfused brain is one of the most interesting applications of swi . this technique has been proved to be useful for the assessment of tissue viability in patients with hyperacute cerebral ischaemia , as it provides functional information about the ischaemic penumbra . in acute arterial stroke , flow reduction or absence causes an increase in oxygen extraction , leading to a growth in the amount of deoxyhaemoglobin in the hypoperfused brain tissue . deoxyhaemoglobin represents a supplementary source of contrast enhancement in swi because of its paramagnetic properties , which determine a reduction in t2 * as well as a phase difference between the vessel and its surrounding parenchyma . as the t1 and t2 properties of blood depend on the oxygen saturation of the blood , there are differences between arterial and venous vessels that can not be discovered with conventional sequences . the visualisation of draining veins within areas of impaired perfusion allows the identification of penumbral brain tissue in a different way from the current perfusion - weighted imaging ( pwi ) techniques ( fig . 5 ) . \n 5left internal carotid artery ( ica ) dissection with acute watershed infarct in a 42-year - old man with sudden onset of speech disturbance . a mr angiography , single partition at the level of the skull base showing dissection of the left ica , which is characteristically enlarged with high - signal - intensity methaemoglobin ( arrowheads ) representing the intramural thrombus and a small eccentric lumen ( arrow ) . b mr angiography , coronal mip : narrowing of the ica in its vertical segment just below the skull base ( arrow ) . d swi minip : improved visualisation of the veins of the left cerebral hemisphere , which is related to increased oxygen extraction in the ischaemic penumbra and corresponds to the hypoperfusion deficit shown by the mean transit time ( mtt ) map . e perfusion mri , mtt map , showing delayed transit time in the left middle cerebral artery ( mca ) territory . f intra - arterial digital subtraction angiography ( dsa ) , left carotid angiogram , confirming the ica stenosis ( arrow ) . g intra - arterial dsa : the patient was treated using stent placement , which resulted in partial restoration of the vessel lumen . h perfusion mri , mtt map 24 h after stent placement : complete resolution of the perfusion deficit . i swi minip , normalisation of the venous drainage of the left hemisphere left internal carotid artery ( ica ) dissection with acute watershed infarct in a 42-year - old man with sudden onset of speech disturbance . a mr angiography , single partition at the level of the skull base showing dissection of the left ica , which is characteristically enlarged with high - signal - intensity methaemoglobin ( arrowheads ) representing the intramural thrombus and a small eccentric lumen ( arrow ) . b mr angiography , coronal mip : narrowing of the ica in its vertical segment just below the skull base ( arrow ) . d swi minip : improved visualisation of the veins of the left cerebral hemisphere , which is related to increased oxygen extraction in the ischaemic penumbra and corresponds to the hypoperfusion deficit shown by the mean transit time ( mtt ) map . e perfusion mri , mtt map , showing delayed transit time in the left middle cerebral artery ( mca ) territory . f intra - arterial digital subtraction angiography ( dsa ) , left carotid angiogram , confirming the ica stenosis ( arrow ) . g intra - arterial dsa : the patient was treated using stent placement , which resulted in partial restoration of the vessel lumen . h perfusion mri , mtt map 24 h after stent placement : complete resolution of the perfusion deficit .", "swi has become a functional method for evaluating cerebral venous sinus thrombosis ( cvst ) by demonstrating engorgement of the venous system as a result of venous hypertension and collateral slow flow . dural sinus thrombosis causes an increase in deoxyhaemoglobin concentration in the veins involved that appears as a prominent area of hypointense signal intensity on swi ; if cvst is treated successfully , this effect will disappear . swi is also capable of identifying the thrombosed pial vein , which is responsible for the development of oedema into the white matter , due to its high sensitivity in the demonstration of an intravascular clot . swi can depict at the same time the features of parenchymal or extra - axial haemorrhages that can occur in the case of infarction , with higher sensitivity compared with conventional ge sequences ( fig . a sagittal t1-weighted image demonstrates hyperintensity of the posterior third of the sagittal sinus ( open arrow ) . b mr venography confirms the thrombosis of the posterior third of the sagittal sinus ending at the torcular ( open arrow ) . c dwi , b = 1,000 image demonstrates a right occipital acute ischaemic lesion . d swi minip shows a right thalamic haemorrhagic lesion in the vascular territory of the right deep internal cerebral vein ( arrow ) epileptic seizures and sagittal sinus thrombosis in a 3-year - old child . a sagittal t1-weighted image demonstrates hyperintensity of the posterior third of the sagittal sinus ( open arrow ) . b mr venography confirms the thrombosis of the posterior third of the sagittal sinus ending at the torcular ( open arrow ) . c dwi , b = 1,000 image demonstrates a right occipital acute ischaemic lesion . d swi minip shows a right thalamic haemorrhagic lesion in the vascular territory of the right deep internal cerebral vein ( arrow )", "cerebral arteriovenous malformations ( avms ) are easily displayed by conventional mri and mr angiography because of their characteristic high flow , whereas venous malformations such as cavernomas , developmental venous anomalies and capillary telangiectasias can not be adequately visualised without contrast medium administration and ge sequences , as they mainly consist of slow - flow small vessels . mr angiography techniques are often inadequate for visualising small vessels with slow flow . on the contrary , swi sequences are well suited for the visualisation of very small vessels , such as the caput medusae of venous angiomas and telangiectasias as a result of a combination of slow flow with changes in deoxyhaemoglobin concentration . the combined information derived from both phase and magnitude images is responsible for enhanced visualisation of such lesions . swi plays a substantial role in the identification and characterisation of cerebral vascular malformations , first of all improving their detection rate . on the other hand , allowing simultaneous visualisation of the different compartments of cerebral avms and of the relationship with one another and with the brain parenchyma , it is particularly valuable for therapeutic planning . the clinical usefulness of a similar technique , bold mr venography , was first demonstrated by essig et al . . phase changes caused by the susceptibility differences between oxygenated and deoxygenated red blood cells and the different relaxation rates between venous and arterial blood provide a natural separation of arteries and veins . the 3d dataset of swi can therefore be used as an alternative to mr angiography in order to better differentiate afferent arteries from draining veins . this advantage becomes relevant with small avms with recent bleeding , in which it is mandatory to identify the nidus in relation to eloquent areas in order to plan the best therapeutic approach , endovascular or surgical or both ( fig . veins appear dark due to t2 * loss and processing of the phase image , while arteries show bright signal from time - of - flight ( tof ) inflow enhancement . the use of higher flip angles and shorter tr make it possible to increase the contrast enhancement in the arteries without overly degrading the venography . for the evaluation of high - flow vascular malformations ( i.e. avms and dural arteriovenous fistulas ) the use of both minip and mip post - processing techniques can improve the image quality . the identification of small niduses , the exact location of the fistulous point and the depiction of the venous drainage patterns are the most relevant information that should be obtained from this technique . the elevated diagnostic accuracy of swi compared with digital subtraction angiography ( dsa ) has been recently reported for the detection of arteriovenous shunting ( avs ) in brain avms . \n b the swi minip image clearly depicts the brainstem haemorrhage , which is markedly hypointense . c , d swi , axial and reformatted sagittal images , processed with a maximum intensity projection algorithm ( mip ) , show flow - related enhancement within the haemorrhagic lesion ( arrow ) and a hypertrophied right posterior inferior cerebellar artery ( pica ; open arrow ) which are consistent with a small ruptured brainstem avm . e cerebral angiography , selective injection of the right vertebral artery : brainstem avm ( arrow ) fed by branches of the right pica and early venous drainage into a cerebellar vein ( arrowheads ) brainstem haemorrhage in a 43-year - old patient . b the swi minip image clearly depicts the brainstem haemorrhage , which is markedly hypointense . c , d swi , axial and reformatted sagittal images , processed with a maximum intensity projection algorithm ( mip ) , show flow - related enhancement within the haemorrhagic lesion ( arrow ) and a hypertrophied right posterior inferior cerebellar artery ( pica ; open arrow ) which are consistent with a small ruptured brainstem avm . e cerebral angiography , selective injection of the right vertebral artery : brainstem avm ( arrow ) fed by branches of the right pica and early venous drainage into a cerebellar vein ( arrowheads ) dural av fistulas ( davfs ) are usually more difficult to identify than avms on conventional mri , especially when venous ectasia is absent , as in type i and ii davfs ; the patients have vague symptoms ( i.e. headache or tinnitus ) and there are no localising signs . swi is capable of better identifying the extent of the abnormal venous drainage compared with mr angiography . the conspicuity of the venous structures on swi can be explained by the combination of a prolonged cerebral circulation time that favours increased oxygen extraction resulting in increased concentration of deoxyhaemoglobin , with the typical venous engorgement ( fig . b , c swi axial and coronal reformatted minip images show dilated right temporal veins ( arrows ) , which arouse suspicion of a tentorial dural av fistula . d cerebral angiography , selective injection of the right external carotid artery : dural av fistula of the right tentorium , fed by the right middle meningeal artery ( open arrow ) , with early venous drainage into the tentorial and temporal veins ( arrows ) patient with headache and pulsatile tinnitus of the right ear . swi axial and coronal reformatted minip images show dilated right temporal veins ( arrows ) , which arouse suspicion of a tentorial dural av fistula . d cerebral angiography , selective injection of the right external carotid artery : dural av fistula of the right tentorium , fed by the right middle meningeal artery ( open arrow ) , with early venous drainage into the tentorial and temporal veins ( arrows ) concerning slow - flow vascular malformations ( i.e. cavernomas , developmental venous anomalies and capillary telangiectasias ) the phase information contained in swi is responsible for an artefactual enhancement that dramatically improves mri sensitivity to these pathological conditions . without swi individuals with cavernous malformations can present with epilepsy and focal neurological deficits or acute intracranial haemorrhage , although these vascular malformations are often discovered as incidental findings . cavernomas that have previously bled are usually detectable on routine mri because of the prominent signal intensity of haemorrhagic products . however , these lesions tend to be barely visible when they have not bled , except for faint enhancement after contrast medium administration that is often non - specific . patients with multiple lesions can be diagnosed with familial cavernous angiomatosis and should be referred for genetic evaluation and counselling ( fig . . individuals with symptomatic , growing , or haemorrhagic malformations should be considered for surgical resection . close follow - up after diagnosis and treatment is helpful to identify lesion progression or recurrence . \n a t2-weighted image demonstrates a left frontal cavernous malformation surrounded by a thick ring of haemosiderin . b ge t2 * -weighted image shows multiple punctate hypointense foci located in both hemispheres , corresponding to small cavernous malformations ( arrows ) . c swi minip : there are supplemental cavernous malformations that are not detected by ge sequences ( arrows ) . d swi minip , reformatted sagittal section : multiple small cavernous malformations located in the brainstem ( arrows ) multiple cavernous malformations in a 32-year - old man investigated for intraparenchymal haemorrhage . a t2-weighted image demonstrates a left frontal cavernous malformation surrounded by a thick ring of haemosiderin . b ge t2 * -weighted image shows multiple punctate hypointense foci located in both hemispheres , corresponding to small cavernous malformations ( arrows ) . c swi minip : there are supplemental cavernous malformations that are not detected by ge sequences ( arrows ) . d swi minip , reformatted sagittal section : multiple small cavernous malformations located in the brainstem ( arrows ) developmental venous anomalies ( dva ) consist of a radially arranged venous complex converging on a centrally located venous trunk , which drains the normal brain parenchyma . they are better displayed with swi than with t1-weighted contrast - enhanced images ( fig . 10incidental discovery of a cerebellar developmental venous anomaly in a 48-year - old man investigated for right - sided sensorineural hearing loss . a t2-weighted image shows thin hypointense bands of flow void , in the right cerebellar hemisphere ( open arrow ) . b swi minip demonstrates multiple thin medullary veins ( arrows ) that converge into a dilated collector vein ( open arrow ) , consistent with a cerebellar venous angioma , better defined as developmental venous anomaly . c post - contrast t1-weighted axial image shows the enhancing dilated veins ( open arrow ) incidental discovery of a cerebellar developmental venous anomaly in a 48-year - old man investigated for right - sided sensorineural hearing loss . a t2-weighted image shows thin hypointense bands of flow void , in the right cerebellar hemisphere ( open arrow ) . b swi minip demonstrates multiple thin medullary veins ( arrows ) that converge into a dilated collector vein ( open arrow ) , consistent with a cerebellar venous angioma , better defined as developmental venous anomaly . c post - contrast t1-weighted axial image shows the enhancing dilated veins ( open arrow ) capillary telangiectasias are asymptomatic venous vascular malformations , which are smaller and less common than cavernomas and can sometimes occur in mixed cavernoma / telangiectasia lesions . these may occur sporadically or may be infrequently associated with syndromes , like hereditary haemorrhagic telangiectasia or ataxia telangiectasia . they may also manifest as a result of endothelial injury , such as radiation - induced vascular injury , particularly in children who have received cranial irradiation . they are characterised by poor contrast enhancement and can therefore be missed on conventional t1-weighted and t2-weighted sequences ( fig . 11incidental discovery of a pontine telangiectasia in a 34-year - old woman investigated for migraine . swi minip axial ( b ) and sagittal reformatted section ( c ) show a markedly hypointense lesion with regular margins , located in the pons . the capillary telangiectasia is characterised by mild enhancement with an arbor - like pattern incidental discovery of a pontine telangiectasia in a 34-year - old woman investigated for migraine . swi minip axial ( b ) and sagittal reformatted section ( c ) show a markedly hypointense lesion with regular margins , located in the pons . . the capillary telangiectasia is characterised by mild enhancement with an arbor - like pattern ataxia telangiectasia is a rare autosomal recessive disorder characterised by an early - onset of progressive cerebellar ataxia , immunodeficiency , ocular and cutaneous telangiectasia and is associated with an elevated risk of intracranial tumours . swi can discover brain gliovascular nodules with perivascular haemorrhage and small haemosiderin deposits presumably only in older patients , during the later stages of the disease ; however , in our experience this technique can also be a useful screening tool in young patients ( fig . a 31-year - old woman with a diagnosis of ataxia telangiectasia by the age of 7 , investigated for dizziness and headache . c swi minip : additional hypointense black spots can be identified , corresponding to haemosiderin deposits presumably due to gliovascular nodules with perivascular haemorrhage ataxia telangiectasia . a 31-year - old woman with a diagnosis of ataxia telangiectasia by the age of 7 , investigated for dizziness and headache . c swi minip : additional hypointense black spots can be identified , corresponding to haemosiderin deposits presumably due to gliovascular nodules with perivascular haemorrhage", "mri with ge sequences has been extensively used in the past for investigating young patients with traumatic brain injuries ( tbi ) , either in the acute phase , when the clinical picture of a severe coma is not explained by ct , or months after trauma , in order to understand the causes of an unsuccessful recovery . in both cases , swi has been widely used in the identification of smaller haemorrhages due to a shear - strain mechanism of injury , thus refining the prediction of outcome . haemorrhagic dai lesions on swi can be six - times better detected than t2 * -weighted conventional ge sequences and the recognisable volume of haemorrhage is approximately twofold greater ( fig . reformatted swi images in the sagittal plane are particularly helpful in the assessment of corpus callosum haemorrhages . both number and volume of haemorrhagic lesions correlate with neuropsychological deficits . \n fig . 13diffuse axonal injury in a 14-year - old boy who had severe tbi following a motorcycle crash . brain mr study was performed three days after the trauma , when the patient was in a severely comatose state ( gcs 5 ) . a flair image identifies multiple hyperintense lesions in the splenium of the corpus callosum and in frontal subcortical and periventricular white matter . b ge t2 * -weighted image : haemorrhagic shearing injuries are barely visible in the right fronto - opercular and parieto - occipital regions ( arrows ) . c swi minip : additional microhaemorrhages are recognisable at the grey matter - white matter junction of the frontal lobes and in the right parieto - occipital white matter ( black arrows ) . d swi minip , reformatted sagittal section shows microhaemorrhages in the corpus callosum ( white arrows ) diffuse axonal injury in a 14-year - old boy who had severe tbi following a motorcycle crash . brain mr study was performed three days after the trauma , when the patient was in a severely comatose state ( gcs 5 ) . a flair image identifies multiple hyperintense lesions in the splenium of the corpus callosum and in frontal subcortical and periventricular white matter . b ge t2 * -weighted image : haemorrhagic shearing injuries are barely visible in the right fronto - opercular and parieto - occipital regions ( arrows ) . c swi minip : additional microhaemorrhages are recognisable at the grey matter - white matter junction of the frontal lobes and in the right parieto - occipital white matter ( black arrows ) . d swi minip , reformatted sagittal section shows microhaemorrhages in the corpus callosum ( white arrows )", "brain mri predictors of tumour grade include contrast enhancement , oedema , mass effect , cyst formation or necrosis , haemorrhage , metabolic activity and cerebral blood volume . it is well known that the growth of solid tumours , such as gliomas , is dependent on the angiogenesis of pathological vessels . swi can provide a thorough assessment of the internal angioarchitecture of brain tumours ( increased microvascularity inside and beyond the tumour margins ) , together with the identification of foci of haemorrhage and calcification , thus representing an additional tool in the neuroradiological grading of cerebral neoplasms . the administration of a contrast agent ( ce - swi ) allows discrimination among those three entities , as only blood vessels will change their signal intensity , while calcifications and regions of inactive haemorrhage ( which can be differentiated from each other by the evaluation of phase images , as described above ) will not . the clinical potential of contrast - enhanced bold mr venography at 3 t and 1.5 t for the study of brain tumours was first reported by barth et al . , who demonstrated variable venous patterns in various types of tumours and in different parts of the lesions ( oedema , contrast - enhancing areas , necrosis ) , which might represent increased blood supply and particular vascular patterns around fast - growing malignant tumours . kim et al . recently assessed the added value provided by swi in the differential diagnosis of solitary enhancing brain lesions compared with the use of conventional mri alone . ce - swi has been found to be equivalent or even superior to ce - t1 images in the evaluation of most tumours with necrotic areas ( figs . 14 and 15 ) : the particular contrast combination within swi images permits the simultaneous visualisation of the information otherwise obtained by a multimodal imaging approach including ct , ce - t1 se , flair and t2 * conventional ge sequences . \n 14characterisation and grading of a glial tumour . a 30-year - old man with dizziness , long - standing behavioural changes and subsequent diagnosis of intra - axial cerebral tumour . b swi , unprocessed image : absence of intratumoural susceptibility signals ( due either to calcifications , haemorrhages or venous vasculature ) . c contrast - enhanced swi : detailed visualisation of the margins of a large anaplastic area ( asterisk ) , which is in good correlation with enhancement characteristics ( d ) , hypervolaemia on pwi relative cerebral blood volume ( rcbv ) map ( e ) and mr spectroscopy ( f ) . e pwi , rcbv map : area of increased cerebral blood volume ( open arrow ) . f single - voxel mr spectroscopy , showing inversion of the cho / naa ratio and the presence of a lactate peak . targeted stereotactic biopsy in the supposed necrotic area confirmed the hypothesis of gliomatosis cerebri who grade iii with several necrotic focifig . b t2-weighted image : the mass is irregularly hyperintense with cystic changes and small hypointensities representing small vessels and calcifications . c post - contrast t1-weighted image shows patchy enhancement of the lateral part of the lesion and some vascular structures . d ce - swi minip : calcifications appear as punctate hypointensities , unchanged after gadolinium injection ( white arrows ) . post - processing with minip algorithm allows the simultaneous visualisation of arteries ( hyperintense , black arrows ) and veins ( linear hypointensities , open arrow ) around and inside the tumour . ce - swi is superior to t1-weighted post - gadolinium sequences in demonstrating a diffuse bbb rupture in the lateral necrotic area ( bright signal , asterisk ) . histopathology revealed a primary neuroectodermal tumour ( pnet ) with extensive cystic and necrotic changes and increased vascularity characterisation and grading of a glial tumour . a 30-year - old man with dizziness , long - standing behavioural changes and subsequent diagnosis of intra - axial cerebral tumour . b swi , unprocessed image : absence of intratumoural susceptibility signals ( due either to calcifications , haemorrhages or venous vasculature ) . c contrast - enhanced swi : detailed visualisation of the margins of a large anaplastic area ( asterisk ) , which is in good correlation with enhancement characteristics ( d ) , hypervolaemia on pwi relative cerebral blood volume ( rcbv ) map ( e ) and mr spectroscopy ( f ) . e pwi , rcbv map : area of increased cerebral blood volume ( open arrow ) . f single - voxel mr spectroscopy , showing inversion of the cho / naa ratio and the presence of a lactate peak . targeted stereotactic biopsy in the supposed necrotic area confirmed the hypothesis of gliomatosis cerebri who grade iii with several necrotic foci internal architecture of a high - grade tumour . b t2-weighted image : the mass is irregularly hyperintense with cystic changes and small hypointensities representing small vessels and calcifications . c post - contrast t1-weighted image shows patchy enhancement of the lateral part of the lesion and some vascular structures . d ce - swi minip : calcifications appear as punctate hypointensities , unchanged after gadolinium injection ( white arrows ) . post - processing with minip algorithm allows the simultaneous visualisation of arteries ( hyperintense , black arrows ) and veins ( linear hypointensities , open arrow ) around and inside the tumour . ce - swi is superior to t1-weighted post - gadolinium sequences in demonstrating a diffuse bbb rupture in the lateral necrotic area ( bright signal , asterisk ) . histopathology revealed a primary neuroectodermal tumour ( pnet ) with extensive cystic and necrotic changes and increased vascularity pinker et al . demonstrated a correlation between intratumoural susceptibility effects , positron emission tomography ( pet ) results and histopathological grading . swi has been proposed in the evaluation of clinical response to anti - angiogenetic drugs and in the differential diagnosis with pseudo - progression after chemo- and radiotherapy . a correlation with mr pwi has also been attempted . however , larger comparative studies of pwi and swi are still needed to determine a more precise role of the new techniques in the grading of cerebral neoplasms .", "swi , which is a combination of ge techniques with phase information , represents a useful tool for the identification and characterisation of vascular malformations and for a better understanding of cerebrovascular diseases . despite some inherent limitations , swi has increasing indications for neuroradiology and should be included in the routine imaging protocols of trauma and vascular abnormalities . further investigation is still needed into its extensive clinical application in neurodegenerative diseases and tumoural pathological conditions ." ]

[ "shallots ( wild garlic / osghordion ) with the scientific name of allium hertifolium , is one of the most famous plants from the alliaceae family . for a long time , shallots have been used as a source of food and medicine in iran . the active ingredients of the plant could be referred to agapentagenin , allicin , omega-3 , omega-6 , and minerals such as potassium , sodium , magnesium , iron , copper , zinc , and manganese . this study was conducted to compare shallots in the traditional and modern medicine in order to make a better use of this precious plant .", "to collect appropriate data , resources and articles in trustworthy databases ( e.g. cochrane library , pubmed , google scholar ) and traditional literature ( e.g. makhzan - ul - adwiah , canon , zakhireh - ye khwarazmshahi ) were studied . subsequently , the findings were reviewed , classified , and reported in a tabular format .", "shallots are rich in fatty acids and minerals with many pharmacological effects such as its effect on the respiratory and nervous system and blood dilution , as reflected in the modern medicine . however , certain effects as mentioned in traditional medicine ( e.g. anti - warts , anti - lipoma , anti - kidney stone , and its diuretic effects ) are not covered in research studies of the modern medicine .", "depending on its natural habitats , shallots have different pharmacological effects for which many usages are mentioned in traditional medicine . some of these effects have been investigated in modern medicine ; however , further evaluation of its safety and dosages for clinical use is necessary . furthermore , some cases have not been studied in modern medicine , which could be the basis for future research ." ]

[ "multiple sclerosis ( ms ) is a chronic idiopathic disorder of the central nervous system ( cns ) sustained by a multifocal inflammatory process predominantly affecting myelin - sheathed axons . although traditionally viewed as a white matter ( wm ) demyelinating disorder , ms is characterized by acute and chronic axonal and neuronal loss , as shown for long by pathological and neuroimaging studies [ 1 , 2 ] . acute inflammation causes the development of plaques , characterized by blood - brain barrier ( bbb ) breakdown , perivascular cellular infiltration , demyelination , and axonal degeneration . notably , axonal damage occurs not only in the acute phase but also in inactive ms lesions [ 3 , 4 ] . plaques represent the underlying pathological substrate of clinical events , with occurrence of focal / multifocal neurological symptoms and signs that eventually subside in many cases as inflammation ceases . lesions may also involve the cortical gray matter ( gm ) in which case they are characterized by myelin / axonal injury and microglial activation but not bbb disruption and less cellular infiltration compared to wm lesions [ 6 , 7 ] . it is increasingly perceived that the severity of ms clinical outcome does not simply result from the extent of wm damage , but it rather represents a complex balance among wm and gm tissue damage , tissue repair , and cortical reorganisation [ 810 ] . the evidence that axonal loss highly correlates with neurological disability and disease progression has spurred the search for reliable markers of axonal degeneration . although ms aetiology still remains undetermined , genetic and environmental risk factors have been identified or are suspected ( i.e. , female gender , hla - drb1 allele , genome - wide association studies candidate genes , epstein - barr virus infection , low vitamin d levels , cigarette smoking , etc . ) mainly influencing immune system modulation and although much less evidently myelin and axonal repair mechanisms [ 1115 ] . the complex and unique interplay between genetic background and environmental exposure in each case likely determines the clinical heterogeneity of ms both between and within subjects varying from benign or even subclinical types to highly disabling forms and making it a challenge to predict the clinical course at the individual level . given that ms is mostly diagnosed in subjects in the third and fourth decade of life , the availability of reliable predictors of long - term prognosis is extremely important . the objective of this paper is to review the current literature and to discuss evidence on clinical , paraclinical , magnetic resonance imaging ( mri ) , and cerebrospinal fluid ( csf ) markers as predictors of disability progression in ms .", "the typical clinical course of ms is relapsing - remitting ( rr ) , characterized by an initial event of acute or subacute neurological disturbance , generally indicated as clinically isolated syndrome ( cis ) followed by recurrence of symptoms over time . cis is the type of onset in around 85% of ms cases , while the remaining 15% of patients have a progressive disease from onset ( primary progressive ( pp ) ms ) . progressive onset is an unfavourable prognostic predictor per se , since motor , sphincter control , and cognitive impairment are prominent features of the clinical picture , neurological disability continues to worsen over time , and no effective treatment exists . conversely , ciss generally recover well and may remain monophasic for a long time interval before conversion to clinically definite ms occurs . since cis represents the earlier clinical manifestation of rr ms , this patient population is of great value for identification of predictive and prognostic disease markers compared to definite ms cases who are necessarily in a more advanced stage . typical cis presentation includes acute partial myelitis ( 3050% of cases ) , brainstem / cerebellum syndromes ( 2530% ) unilateral optic neuritis ( 2025% ) , and cerebral hemisphere syndromes ( 5% ) ; more than 20% of ciss present with symptoms and/or signs of more than one anatomical location ( multifocal presentation ) [ 1820 ] . the percentage of cis patients who develop clinically definite ms in prospective observational studies ranges from 16% at 1 year to 80% at 25 years [ 21 , 22 ] . however , these figures date back to studies conducted before the introduction of the most recent revision of ms diagnostic criteria according to which patients previously classified as cis already have ms at the time of initial symptoms if mri demonstrates space and time dissemination of demyelinating lesions . after ms develops , irreversible disability may be the result of accumulation of fixed sequelae after each attack or may be due to transition to a secondary progressive ( sp ) phase , in which insidious neurological deterioration substitutes the preceding rr stage of the disease ( 3258% of cases in major prospective studies ) . clinical predictors of long - term disability in ms include male gender , older age , multifocal symptoms , efferent systems involvement , incomplete remission of the initial event , a short interval to the second event , and high relapse rate in the first 25 years after onset , although not all studies replicated the same findings ( table 1 ) [ 2427 , 30 , 31 , 48 , 73 ] . one single study reported a shorter time to secondary progressive ms in patients with family history of ms . the relevance of age as a prognostic factor is subject to interpretation depending on the temporal frame in which disability levels are captured . indeed , while older age at onset is associated with a more rapid disability progression likely due to prevalence of the primary progressive disease course , age - dependent degenerative processes , and dysfunction of repair mechanisms in older subjects early onset ms patients reach disability milestones at a younger age compared to late onset ms cases , even though in a longer time interval [ 26 , 75 ] . in addition , it has been shown that the progressive phase of ms is an age- rather than a disease duration - dependent process , since age at pp and sp ms onset overlaps significantly in large observational studies and subsequent disability progresses along a common age - driven trajectory independent of onset epoch and previous clinical course . in this perspective , older age at onset may be viewed as a favourable prognostic factor , meaning a longer disease - free interval before ms symptoms occurrence in life and an older age at which significant disability milestones are reached , compared to early onset . also the influence of relapses on later disability progression is debated . according to the authors who found a positive correlation between relapses and long - term disability , the association is stronger in younger patients ( < 25 years old at ms onset ) , it diminishes significantly after the first 25 years of disease , and it is minimal after the progressive phase has begun [ 32 , 78 ] . a sizable proportion of ms patients does not accumulate clinically relevant disability during the entire natural history of the disease . this type of course is known as benign ms , although there is no general agreement on its definition and consequently , on its prevalence in the ms population . while initial definitions predominantly stressed the absence of significant ambulatory disability ( expanded disability status scale ( edss ) score < 3.5 or 2.5 ) after a reasonable time interval from initial symptoms ( 10 or 15 years ) [ 81 , 82 ] , more recent studies highlighted the importance of carefully considering cognitive status and quality of life when defining benign ms . indeed , edss , which is the most largely used disability scale in ms clinical practice , is clearly unbalanced towards ambulation impairment . scores range from 0 meaning no disability to 10 meaning death due to ms : from score 1 to 3.5 , there can be a wide range of neurological deficit but ambulation is unrestricted ; from 4 to 5.5 , independent ambulation is below 500 meters ; from 6 to 7.5 , ambulation is only possible with support ; and from 8 on , the patient is wheelchair - bound or bedridden . whatever the definition , it has been shown that benign status at 10 years after ms onset persist at 20 or more years in 5269% of patients , leading to the conclusion that benign ms is a transient condition for a considerable proportion of cases [ 82 , 8486 ] . however , studies addressing this topic are generally limited by the clinic - based design in which ms patients with mild disease who are not seen on a regular basis in the neurology practices are not included in the analysis falsely reducing the proportion of benign cases . there are no diagnostic tools or validated markers to identify ms patients who will have a favourable clinical course ; however , female gender , younger age , and absence of motor symptoms at onset have been associated with a benign disease form . neurophysiological assessment with visual , somatosensory , motor , and brainstem auditory evoked potentials is traditionally used as a paraclinical tool for ms evaluation , although its diagnostic relevance has progressively decreased after mri became largely available as a more sensitive technique . however , evoked potentials still maintain a prognostic significance likely because they reflect the functional integrity of specific anatomical pathways and consequently tend to better correlate with neurological disability than conventional mri , which provide purely morphological information . several cross - sectional and longitudinal studies established that the degree of evoked potentials abnormalities is significantly associated with the edss score at the time of neurophysiological evaluation and up to 14 years later in patients with ms [ 8893 ] . a recent study found that cis patients with at least three abnormal evoked potentials at baseline have an increased risk of reaching moderate disability over a mean follow - up period of six years , independent of initial mri features . in recent years optical coherence tomography ( oct ) has emerged as a powerful tool to detect retinal nerve fiber layer ( rnfl ) thinning in ms patients with and without optic neuritis history . rnfl thickness decrease results from axonal loss in optic nerve , possibly reflecting diffuse neuroaxonal injury in the cns , and correlates with markers of ms activity such as relapses , new / gadolinium - enhancing lesions , and parenchymal atrophy on brain mri [ 96 , 97 ] . the extent of rnfl thinning in optic neuritis patients predicts visual recovery and exhibits a modest correlation with overall neurological disability in ms patients . it has been recently suggested that thinning of inner and outer nuclear layers of the retina identifies a subset of ms patients with primary retinal neuronal pathology and more aggressive disease course [ 98 , 99 ] .", "given its increased availability and its sensitivity in detecting ms lesions , conventional mri has become the main imaging tool in the ms diagnostic work up as well as in monitoring treatment response to disease - modifying drugs . its diagnostic sensitivity reflects the ability to identify clinically silent lesions , thus , favouring the early demonstration of dissemination in space and time of the lesions ( figure 1 ) according to the recent revision of diagnostic criteria . however , while it may seem obvious that patients who develop new wm lesions are worse off than those without new lesions , conventional mri has been shown to have a prognostic value only in patients at disease onset : high t2-weighted lesion load in patients with a cis has been associated with an increased risk of subsequent conversion to clinically definite ms and long - term disability accumulation [ 101 , 102 ] . by contrast , in a more advanced phase of the disease , the strength of relationship between conventional mri measures and subsequent disability progression is rather weak [ 33 , 34 ] . in a recent study including 548 placebo - treated rr ms patients , the multivariate analysis indicated just edss score and t2 lesion load as factors independently predicting the clinical progression . nevertheless , these two variables taken together were able to account for only 3% of the probability to have an edss increase over follow - up time , thus , confirming the limited value of these metrics in predicting short - term disability changes in rr ms . such result is in line with those of several previous cross - sectional and longitudinal studies conducted on smaller groups of patients with different clinical characteristics , which have shown only modest correlation between t2- and t1-weighted brain mri activity and subsequent changes in disability [ 3638 ] . although edss is not without limitations in terms of reliability and responsiveness to disease changes , the lack of a strong correlation between wm lesion load and clinical disability had prompted investigations of the so - called normal - appearing brain tissue . for this purpose , unconventional and quantitative mri techniques , having increased sensitivity and specificity to irreversible tissue damage , have been consistently applied to monitor and predict ms evolution . given these premises , several studies have been focused on brain atrophy showing its relevant clinical impact not only in the diagnostic phase but also in predicting subsequent disability progression both in rr ms and in pp ms ( figure 2 ) . a recent study published by the magnims group , included 261 ms patients who had mr imaging at baseline and after 1 - 2 years and edss scoring at baseline and after 10 years ; in the whole patient group , after correction for imaging protocol , whole brain and central atrophy were good predictors of edss at 10 years ( r = 0.74 ) . despite the good sensitivity of brain atrophy , even better results in predicting disability progression have been further achieved by the regional analysis of brain atrophy . jasperse and colleagues , for example , suggested that atrophy of central brain regions was related to decline in ambulatory function , whereas atrophy of both central and peripheral brain regions was associated to decline in neurologically more complex tasks for coordinated hand function . the best results , however , have been obtained by the study of gm and wm atrophy separately . indeed several voxel - based and surface - based studies , both in rrms and in ppms , revealed strong relationship between gm , but not wm , atrophy and disability progression [ 4345 ] . even when a very long followup , a very large sample size , or more sophisticated disability scales ( i.e. , ms functional composite ) were considered , gm atrophy reflected disease subtype and disability progression to a greater extent than wm atrophy or lesions [ 4547 ] . a further step forward in the comprehension of the pathological mechanisms underlying the accumulation of irreversible disability in ms was obtained by the regional analysis of gm atrophy ; since the first studies , indeed , it was clear that some cortical and deep gm structures were more prone to inflammatory and degenerative damage [ 49 , 50 ] than others and that , when damaged , some cortical areas had a greater impact on the accumulation of physical [ 8 , 51 ] and cognitive disability [ 52 , 53 ] than others . in particular thalamus and cerebellum were consistently related to clinical disability and its progression over time . thalamus was found to be one of the earliest structures involved by the neuropathological process taking place in the gm and the rate of thalamic atrophy in ms subjects was correlated with changes in edss . moreover , in a longitudinal study , baseline thalamic fraction ( odds ratio = 0.62 ) was identified as independent predictor of worsening disability at 8 years . cerebellum has been indicated as a preferred site of demyelination , especially in patients with progressive ms , whose cerebellar cortex was found to be affected by ms - related pathology in up to 92% of its extension [ 105 , 106 ] . in a recent 5-year longitudinal study cerebellar cortical atrophy , together with age and cortical lesion load , was indicated among the predictive parameters of progression in those rr ms patients who convert to the sp phase . beyond diffuse gm damage , the relevance of cortical damage in determining disability has been pointed out by the strong correlation observed between focal gm damage as visible by double inversion recovery ( dir ) sequence ( i.e. , cortical lesions ; figure 3 ) and clinical progression . indeed , high number of cortical lesions has been demonstrated to characterize patients with the poorest prognosis and having early and severe cortical atrophy and cognitive impairment . in a 5-year longitudinal study on more than 300 ms patients with different clinical phenotypes , cortical lesion volume and gm atrophy were found to be associated to each other and to physical and cognitive disability progressions . patients having high cortical lesion load at baseline showed the worse clinical evolution and a significant progression of cortical atrophy after 5 years . of course a complete and accurate evaluation of the risk of clinical progression should not disregard the evaluation of spinal cord damage that has suggested as a major determinant of disability in patients with ms . in line with what has been happened for the brain damage , the application of quantitative mri techniques to the spinal cord damage has convincingly demonstrated that cord area , rather than t2 lesion load , might have a role in predicting the accumulation of disability [ 56 , 57 , 108 ] . in the last 10 years , finally , other non - conventional sequences have received considerable attention since their high sensitivity for the most disabling pathological features of ms ( i.e. , irreversible demyelination and neuroaxonal injury ) and their ability to detect occult changes occurring in the normal - appearing brain tissue . among these unconventional techniques magnetization transfer and diffusion tensor imaging gave the most interesting results . in 73 patients , who were followed prospectively with clinical visits for a median period of 8 years , a multivariable model identified baseline gm magnetization transfer ratio histogram peak height and average lesion magnetization transfer ratio percentage change after 12 months as independent predictors of disability worsening at 8 years ( r = 0.28 ) . in a longitudinal study on 54 primary progressive ms patients , lower level of disability and gm damage evaluated at study entry on the base of average gm mean diffusivity identified patients with high risk of disease progression over the following 5 years . in a more recent prospective study fractional anisotropy of normal appearing gm and t2 lesion load were independent predictors of edss score , while change in fractional anisotropy of normal appearing gm ( b = 0.523 ) and disease duration ( b = 0.342 ) were independent predictors of edss change . finally , the application of diffusion tensor imaging to the spinal cord damage revealed that baseline cord cross - sectional area and its fractional anisotropy correlated with increase in disability at follow - up . all together these studies confirmed that neurological and neuropsychological disability in ms are likely the consequence of both visible and invisible wm and gm damage . the strength of correlation between gm tissue loss and progression of disability exceeds that related to wm lesions or atrophy ( table 2 ) . unfortunately , gm damage is poorly evaluated by conventional mri and to achieve more accurate estimates of such a damage it requires multiparametric mri approach including unconventional and quantitative mri techniques , many of which are not yet available or practicable in routine diagnostics .", "the examination of csf represents a valuable procedure in investigating a number of inflammatory and degenerative neurological disorders . in addition to the classical biochemical and electrophoretic approaches , the proteome complexity of csf can be tackled today by a number of methods , hence , indicating that scientists involved in this frontier are fishing in the right pond . however , in a disorder with a complex pathogenesis , such as ms , individual biomarkers , taken singly , are likely to reflect only isolated components of ongoing neuroinflammation and neurodegeneration , hence , lacking prognostic significance . moreover , most of the investigated ms biomarkers , while of invaluable diagnostic help , are currently unsuitable for predicting disease progression . according to their biological role , molecules of potential prognostic significance for ms may be classified as follows : ( i ) markers of immune activation ( e.g. , cytokines , chemokines , antibodies , complement factors , adhesion molecules , etc . ) ; ( ii ) markers of blood - brain barrier disruption ( e.g. , matrix metalloproteinases ) ; ( iii ) markers of demyelination ( e.g. , myelin basic protein , myelin oligodendrocyte glycoprotein , proteolytic enzymes , proteases inhibitors , etc . ) ; ( iv ) markers of oxidative stress and cytotoxicity ( e.g. , advanced oxidation protein products , total thiol , hydroxyl radicals , divalent iron , etc . ) ; ( v ) markers of axonal / neuronal damage and gliosis ( e.g. , neurofilaments , tau , 14 - 3 - 3 protein , glial fibrillary acidic protein , etc . ) ; and ( vi ) markers of remyelination / neural repair ( e.g. , nerve growth factor , brain - derived growth factor , nogo - a , etc . ) . a correlation with ms disability progression over time has been suggested for several csf markers , including but not limited to 14 - 3 - 3 protein ; tau ; neurofilament heavy chain [ 66 , 67 ] ; chitinase 3-like 1 ; and cystatin c . csf igg oligoclonal bands , which have a recognized relevance for the diagnosis of ms and predict conversion from cis to ms , do not influence the long - term risk of disability , although a contrasting observation has been described . conversely , csf oligoclonal igm , particularly if directed against myelin lipids , have been associated with a poor ms outcome in terms of frequency of relapses and disability progression [ 68 , 69 ] . several csf markers of inflammation have been investigated for potential prognostic value in cis and early ms patients . some studies have identified novel biological predictors of conversion from cis to ms , for instance measles - rubella - varicella zoster virus igg antibody reaction ( mrzr ) and high levels of c - x - c motif ligand 13 ( cxcl13 ) and in the csf . however , no predictive value for progression of disability has been shown for such molecular candidates [ 111 , 112 ] . since neurodegeneration is regarded as the biological determinant of irreversible neurological disability in demyelinating disorders , csf markers of neuroaxonal injury ( e.g. , tau , 14 - 3 - 3 protein , and neurofilaments ) are the most promising candidates for predicting disease progression [ 113 , 114 ] . csf tau concentration in ms patients with both relapsing and progressive forms of the disease has been reported to be higher compared to controls in several studies [ 110 , 115119 ] , although other researchers did not replicate this finding [ 120122 ] . a correlation between csf tau and progression of disability in ms patients has been shown only in one 3-year follow - up study . in a small group of patients with cis and clinically definite ms , investigated either at the acute attack ( i.e. , within 30 days ) or several weeks or months later , our group found values of tau within normal limits , a finding that we later confirmed in a larger mostly independent cohort of cis patients . interestingly , it has been shown that csf tau levels decrease during the course of ms , as a likely effect of progressive parenchymal brain loss , hence , showing a negative correlation with clinical severity . the latter findings are consistent with studies showing progressive brain atrophy in ms patients , regardless of disease subtype . taken together , while the determination of csf tau concentration in ms deserves further scrutiny , it is possible that in a subset of ms patients , this protein represent a reliable marker of axonal injury . conversely , available evidence shows that determination of p - tau has no value as a biomarker . 14 - 3 - 3 protein has also been detected in the csf of subjects with cis / ms by several [ 61 , 62 , 116 , 126 , 127 ] but not all research groups . . showed that a positive csf 14 - 3 - 3 assay at the first neurological event suggestive of ms predicted the development of significant neurological disability over a median follow - up period of 32 months , while colucci et al . found that 14 - 3 - 3 positive ms patients had a higher rate of edss progression over 10 months compared to 14 - 3 - 3 negative cases . fiorini et al . found variable upregulation of csf 14 - 3 - 3 / in cis / ms patients investigated at active or inactive disease stages ( as observed in other inflammatory / demyelinating conditions ) but not overexpression of 14 - 3 - 3 and , the isoforms typical of disorders characterized by ongoing axonal and neuronal degeneration , such as sporadic creutzfeldt - jakob disease ( scjd ) and motor neuron disease . these findings encourage an in - dept - analysis in larger cohorts of patients , before ruling out the usefulness of this biomarker . among csf biomarkers of neurodegeneration that have been tested in ms , concentrations are increased in the csf of ms patients compared to age - matched normal controls . furthermore , csf nfl levels seem to better correlate with ms acute inflammatory activity ( higher levels in cis patients who convert to ms and during relapse compared to remission phase ) , while csf nfh concentrations appear to be related to irreversible neuroaxonal injury as indicated by the correlation with confirmed edss score progression and brain atrophy both in cross - sectional and longitudinal studies [ 64 , 65 , 122 , 128130 ] . \n table 3 shows csf markers for which a correlation with ms disability progression has been reported in longitudinal studies .", "which is not straightforward in all cases , clinicians have to be prepared for the challenge of prognostic predictions in order to give adequate responses to patients concern about their future life with ms . among the determinants of ms burden , development of irreversible neurological disability , particularly when affecting motor and cognitive functions , has the highest impact on patients quality of life and health system costs . therefore , prognostic markers of long - term disability progression are strongly needed in ms . a prognostic marker is a specific parameter or a combination of parameters that can be measured in a subject with a given condition and that is significantly correlated with a relevant clinical outcome of that condition . ideally , reliable prognostic marker studies should fulfil the following methodological requirements : ( 1 ) prospective or longitudinal design ; ( 2 ) long - term followup ; ( 3 ) adequate marker and outcome measurement ; ( 4 ) clinical significance of the marker ( i.e. , good correlation and consistency with relevant clinical outcomes ) ; and ( 5 ) reproducibility . clinical prognostic markers that are associated with an increased risk of disability progression in the longterm ( e.g. , male gender , older age , progressive onset , etc . ) have been identified in several ms natural history studies . however , besides identifying subjects who are more likely to experience a severe disease course , such markers do not offer real advantages in terms of prediction potential , since they are not modifiable risk factors , do not directly reflect biological processes , and do not generally distinguish between responders and nonresponders to available ms treatments . conversely , mri and csf parameters , which can be classified as biomarkers as they express more closely biological mechanisms underlying the disease pathophysiology , have a good potential of quantitative assessment as well as variation according to disease stage . considering the complex pathogenesis of ms however , families of biomarkers representative of specific pathogenetic pathways particularly those related to axonal / neuronal damage may correlate with irreversible neurological dysfunction and be used as prognostic indicators to identify patients at risk of a more aggressive disease course . furthermore , such a biomarker might be helpful for identifying patients who could benefit from therapy in case it showed a reliable correlation with the response to a given treatment . unfortunately , no conventional mri measure has shown strong correlation with long - term disability progression in ms , while unconventional mri techniques particularly those assessing gm damage are currently being investigated with promising results , although they are still difficult to apply in clinical settings . on the other hand , research on csf biomarkers has gathered convincing preliminary evidence only for nfh and nfl as predictors of disability progression . so far , biomarkers studies have mainly focused on selected candidates and have generally recruited relatively small sample of cases with a cross - sectional design , often showing conflicting results . it is likely that discrepancies across studies are at least in part explained by differences in selection of patients , marker measurement , and outcome assessment . although a considerable level of international agreement has been reached on methodological requirements of mri studies in ms , an effort is being made by the csf markers research community in order to standardize collection and biobanking of samples from well clinically characterized ms patients to develop reproducible laboratory assays for csf analysis and to find common definitions of healthy and diseased controls [ 132 , 133 ] . to identify reliable prognostic markers , future ms research will need to focus on large longitudinal observational studies and clinical trials exploring the correlation of unconventional mri measures and selected csf proteins with the development of irreversible neurological disability ." ]

[ "we assumed a monopolar spherical electrode in an infinite homogeneous extracellular medium with a resistivity of e=300 cm . under quasi - static conditions the extracellular potentials ve along the rectified neuron was calculated by ve=eiel/4r , where r is the distance from a compartment to the electrode radiating a monophasic stimulating current pulse with amplitude iel . the current to the center of the n - th compartment of the model neuron consists of the following components : capacitive current , ion currents across the membrane and ohmic currents to the left and right neighbors . applying kirchhoff 's law for compartment n results in(1)d(vi , nve , n)dtcm , n+iion , n+vi , nvi , n1rn/2+rn1/2+vi , nvi , n+1rn/2+rn+1/2=0 with vi , r and cm denoting the intracellular potential , axial resistance and membrane capacity , respectively . intracellular resistivity was 150 cm , membrane capacity 1 f / cm . the following system of differential equations is deduced by introducing the transmembrane voltage v = vive to compute the time courses of vn in every compartment ( rattay , 1999):(2)dvndt=[iion , n+vn1vnrn1/2+rn/2+vn+1vnrn+1/2+rn/2+ve , n1ve , nrn1/2+rn/2+ve , n+1ve , nrn+1/2+rn/2]/cm , n the direct stimulating influence of the extracellular potential on compartment n is defined by the activating function(3)fn=[ve , n1ve , nrn1/2+rn/2+ve , n+1ve , nrn+1/2+rn/2]/cm , n the physical dimension of fn is [ v / s ] . if the neuron is in the resting state before a stimulating current impulse is applied , fn is the slope of membrane voltage vn at stimulus onset ( fig . as r decreases with increasing compartment diameters , f increases with diameter and consequently processes with larger diameters are generally easier to excite . the excitability of the rectified model neuron was initially tested with the original model data obtained from the neuron modeldb corresponding to the model of a cortical pyramidal cell by hu et al . this model incorporates tapering diameters , uneven channel distribution within compartments of one type as well as a reduced membrane capacity of 0.02 f / cm in order to simulate myelination . high threshold sodium nav1.2 and low threshold nav1.6 , fast voltage - gated k , slow non - inactivating potassium current , high - voltage activated ca and calcium dependent k currents are non - uniformly integrated and combined with a linear leakage current throughout the cell . during single pulse experiments for a reduced model we obtained quite similar threshold characteristics in spite of either assuming constant channel densities within every main segment or including only nav1.2 , nav1.6 , fast voltage - gated k channels and a linear leakage current with eleak=70 mv and gleak=0.033 ms / cm throughout the whole neuron . in detail , the reduced model distinguishes soma and dendrites compartment , both with maximum conductances of gnav1.2=8 , gnav1.6=0 , gkv=10 ms / cm , the axon hillock with gnav1.2=320 , gnav1.6=0 , gkv=100 ms / cm , the ais with gnav1.2=100 , gnav1.6=320 , gkv=100 ms / cm , the unmyelinated axon with gnav1.2=0 , gnav1.6=300 , gkv=150 ms / cm and the nodes of ranvier with gnav1.2=0 , gnav1.6=160 , gkv=20 ms / cm . sodium current kinetics are calculated via inav1.j = gnav1.j mh ( v ena ) with j equals either 2 or 6 and a reversal potential of ena=60 mv . details on the differential equations of the different variables have been presented for example by mainen et al . the values for the half ( in)activation voltages v1/2 , the slopes k and the coefficients a were obtained from a previously published model in the neuron model db ( hu et al . , 2009 ) after subtracting the corresponding value for the shift of voltage dependence of the kinetics . therefore the sodium currents inav1.j have the same values for a , that is a(m)=0.182 , a(m)=0.124 , a(h)=0.024 , a(h)=0.0091 , and the slope of inactivation , that is k(h)=5 and k(h)=6.2 , in contrast to altered slope of activation , that is k(m)=k(m)=7 for nav1.2 but k(m)=k(m)=6 for nav1.6 . to account for the reduced threshold of nav1.6 channels v1/2 ( m ) is decreased to 41 mv compared to the calculated value of 28 mv for activation of nav1.2 channels . the corresponding values in mv for the inactivation of nav1.2/nav1.6 channels are v1/2(h)=35/41 , v1/2 ( h)=60/73 , v1/2 ( h)=57/70 . the potassium currents are determined by ik = gk n ( v ek ) with ek=90 mv . to be consistent the corresponding values of a()=0.02 , a()=0.002 , v1/2()=v1/2()=25 mv and k()=k()=9 which were used for the reduced model were also obtained from the neuron model db ( hu et al . , 2009 ) . since the presented results are simulated for 37 c , a temperature coefficient of 3.209 has to be applied when calculating the conductances and the channel kinetics to account for the original model temperature of 23 c and a q10 of 2.3 . the internodes are simulated with 17 sheets of membrane with a conductance of 1 ms / cm and c=1 f / cm per sheet ( rattay , 1999 ) .", "dendritic excitation with spike propagation across the soma into the axon was analyzed with a simple compartment model with a straight axis ( fig . the model neuron consists of a single non - branching dendrite ( 500 m , d=5 m ) , soma ( 20 m ) , axon hillock ( 10 m , d=3.1 m ) , ais ( 50 m , d=1.22 m ) , unmyelinated axon ( 200 m , d=1 m ) , myelinated axon ( 500 m , d=1 m ) and unmyelinated terminal ( 50 m , d=1 m ) . also assumptions for ion channel distribution are quite similar as modeled by hu et al . ( 2009 ) : the same constant nav1.2 channel density for dendrite and soma , but 40 times higher sodium channel density in hillock and ais with a change to the low threshold type nav1.6 in the axon ( fig . 1b ) . in a distance of 80 m from the dendrite axis a propagating spike is generated by a 100 s cathodic pulse from the tip of an electrode which is modeled as point source in an infinite homogeneous medium ( fig . according to the activating function concept ( rattay , 1986 , 1999 ) the slopes of membrane voltage at the beginning of the stimulus pulse reflect the first response of the applied electric field and these slopes are proportional to fictive current pulses injected individually in each of the compartments . therefore spike initiation is expected in a region with positive activating function , that is , where the slopes at stimulus onset are positive ( fig . assuming a homogeneous external medium and a straight long fiber with constant diameter , the activated region is limited by an angle of 70 ( fig . this initial excitation pattern is independent from ion channel composition as well as internal and external resistivities . in order to obtain a propagating spike , inward sodium current has to be large enough to supply the neighboring region with enough intracellular axial current flow to reach threshold . this fact demands for a minimum sodium channel density which is not achieved on the assumptions mentioned above with a ratio of 1:1500 concerning dendrites and ais . moving the electrode closer to the dendrite reduces the length of the activated region as a consequence of the 70 limit . the rather low maximum sodium conductance gna=8 ms / cm is not enough for nearby stimulation and therefore direct cathodic dendritic spike excitation fails for electrode distances below a limit which is about 56 m for the 5 m dendrite1 . excitation is however possible when the inward dendritic sodium current shifts the ap initiation site by axial current into the low threshold ais ( fig . maximum membrane voltage increases with stimulus strength in the activated 70 region , still passive excitation is prevented by the hyperpolarized regions on both sides ( fig . for an infinite long fiber , reduction of fiber diameter by a factor four causes quite the same numerical values in model evaluation when fiber - electrode distance , compartment length as well as stimulus current is divided by 2 ( quadratic relation ) . this means that thinner dendritic regions are directly excitable for shorter electrode - fiber distances , for example the limit is 28 m for 1.25 m dendrite diameter . in this case , propagating spikes are generated in the small operating window from 25 to 41 a . higher stimuli cause cathodic block by strong hyperpolarized side lobes that hinder spike propagation ( ranck , 1975 ; rattay , 1986 ) in a similar way as shown in fig . the geometry of the last example is used to compare dendritic and axonal excitability : moving the electrode from the dendrite ( 1.25 m diameter ) to the distal end of the ais ( xelectrode=70 m ) with constant axial distance ( zelectrode=28 m ) reduces cathodic threshold current from 25 to 8.1 a . in this example the ais and the dendrite have quite similar diameters but the ais threshold is less than one - third of the dendrite 's value . furthermore , the electrode with the same axial distance is positioned above the third node of ranvier . in spite of the reduction of diameter ( from 1.22 to 1 m ) as well as the decrease in the maximum sodium conductance gnav1.6 ( from 320 to 160 ms / cm ) for compartments of type nr compared to those of type ais , the firing threshold is further reduced by 5% to 7.7 a and cathodic block threshold appears again at 24 a . above the center of the second internode ( xelectrode=518 m , zelectrode=27.6 m ) threshold current increases for propagating spikes to 21.8 a and blockade threshold to 45 a . when the electrode is shifted in constant distance along the axis of the neuron , several phenomena ( 1)the distal end of the axonal initial segment with high nav1.6 channel density is very sensitive to cathodic stimulation and this is the site of spike initiation in many cases.(2)however , the neuron is even more excitable at the third and fourth node of ranvier , in spite of essentially lower ion channel densities . this is a consequence of high activating function values in nodes of ranvier that are favored by the properties of the internodes ( coburn , 1989 ; rattay , 1986 , 1989 ; rattay et al . , 2000).(3)moving the electrode along the thick dendrite in a distance of 100 m from the neuron axis needs about twice the threshold current in comparison to the positions along the axon.moving the electrode with 50 m distance along the neuron shows the following discrepancies ( 4 and 5 ) to a quadratic distance threshold current relation as expected from literature ( stoney et al . , 1968 ; tehovnik , 1996 ; tehovnik et al . , 2006).(4)the thresholds are higher as expected ( prediction according to the quadratic rule is shown as dashed blue line in fig . 3a).(5)electrodes above the center of an internode need doubled threshold currents compared to positions above a node of ranvier . this node - internode threshold relation known from peripheral nerve stimulation becomes more and more pronounced when electrode - fiber distance is reduced ( rattay , 1987).(6)within a dendritic region ( 149 m < xelectrode<37 m ) anodic threshold is lower than cathodic . this is in agreement with experiments ( ranck , 1975 ) and theory ( rattay , 1999).(7)anodic threshold current of the 5 m diameter dendrite example increases in an exponential way with distance from the soma ( comp . the rather linear relation of the left part of the green line in the logarithmic current scaling of fig . the thin dendrite has another threshold characteristic as explained below.(8)the extreme node - internode threshold fluctuations seen for cathodic pulses are lost when pulse polarity is reversed.(9)in general thin dendrites have higher thresholds ( comp . thin and thick green and blue lines in fig . the distal end of the axonal initial segment with high nav1.6 channel density is very sensitive to cathodic stimulation and this is the site of spike initiation in many cases . however , the neuron is even more excitable at the third and fourth node of ranvier , in spite of essentially lower ion channel densities . this is a consequence of high activating function values in nodes of ranvier that are favored by the properties of the internodes ( coburn , 1989 ; rattay , 1986 , 1989 ; rattay et al . , 2000 ) . moving the electrode along the thick dendrite in a distance of 100 m from the neuron axis needs about twice the threshold current in comparison to the positions along the axon . moving the electrode with 50 m distance along the neuron the thresholds are higher as expected ( prediction according to the quadratic rule is shown as dashed blue line in fig . electrodes above the center of an internode need doubled threshold currents compared to positions above a node of ranvier . this node - internode threshold relation known from peripheral nerve stimulation becomes more and more pronounced when electrode - fiber distance is reduced ( rattay , 1987 ) . within a dendritic region ( 149 m this is in agreement with experiments ( ranck , 1975 ) and theory ( rattay , 1999 ) . anodic threshold current of the 5 m diameter dendrite example increases in an exponential way with distance from the soma ( comp . the rather linear relation of the left part of the green line in the logarithmic current scaling of fig . the extreme node - internode threshold fluctuations seen for cathodic pulses are lost when pulse polarity is reversed . in general thin dendrites have higher thresholds ( comp . thin and thick green and blue lines in fig . threshold currents are also computed in radial direction for three cell regions : middendritric , soma and ais ( fig . thresholds in table 1 are calculated with charge - balanced triphasic pulses with an interpulse time of 100 s and 100 s per phase . this way they can be compared with our previous data as well as with symmetric biphasic pulse widths of 200 s per phase which are often applied in cortical experiments . as for such pulse durations , the charge per phase is essential for threshold current ( nowak and bullier , 1998 ) , half of our reported threshold values are expected when 200 s pulses are applied . for low current stimulation ( left part of table 1 ) deviations between charge - balanced and monophasic pulses note that the spike initiation site or the excitation mechanisms ( dendritic spike ais spike anodal break , etc . ) suddenly may change when the electrode is slightly moved . a continuous trend from linear ( doubled threshold for doubled distance ) to quadratic relationship ( fourfold threshold for doubled distance ) 3b , table 1 cover also the range above 100 m in order to demonstrate that a quadratic current distance rule ithreshold = kr ( with constant k and electrode - neuron distance r ) as reported by tehovnik ( 1996 ) is a good fit for this area . this behavior changes continuously to a rather linear relation when the electrode enters the region below 50 m where low current stimulation is possible . in contrast to experimental recording , computer simulation allows snapshots of membrane voltages as functions of neuron 's length coordinate ( fig . 4 ) . we analyze cathodic and anodic stimulation for three electrode positions , always with zel=50 m to be comparable with fig . 3a : ( i ) above the second node of ranvier ( xel=370 m , fig . 4 right traces ) , ( ii ) above the distal ais end ( xel=70 m , fig . 4 central traces ) , ( iii ) above the dendrite , symmetrical to position 1 ( xel=370 m , fig . starting at rest , first compartment responses are proportional to the activating function which is independent of the ion channel composition ( rattay , 1986 , 1999 ) . they appear because of irregularities of the electric field and due to changes of the neural geometry . with our assumptions irregularities of the electric field in other cases inhomogeneity of extracellular medium and curvature of fibers essentially contribute to high activating function values . this first response is smoothed by intracellular current flow ( compare red lines with the activating functions in fig . c ) of cathodic stimulation the maxima of the red lines appear at the electrode position which is marked by dashed red lines in fig . 4 and consequently spikes are initiated in the neuron compartment closest to the electrode in cases fig . 4b , but as explained above , the low dendritic sodium channel densities hinder spiking also for higher stimuli , because of the hyperpolarized regions predicted by the negative side lobes of the activating function ( comp . the stimulus polarity causes these side lobes of the activating function to become stimulating ( comp . 4 g , h. for a fiber with constant diameter the activating function values of the side lobes converge asymptotically to zero when the x distance to the electrode position is increased . in case ( fig . 4 g ) , the rather small activating function values in the ais are , however , still more powerful than the higher values in the dendrite with its low sodium channel density resulting in distant spike initiation , that is more than 400 m away from the electrode . this phenomenon explains the almost exponential increase for anodic threshold current when the electrode is moved distally along the dendrite ( left part of green curve in fig . a similar effect , but not as spectacular in its shifting size , is seen for anodic axon stimulation : in case ( fig . 4e ) causes a mirrored pattern similar to the dendritic excitation of case ( fig . red lines ) but the low dendritic sodium channel density together with its higher nav1.2 threshold voltage are the reasons why the spike is initiated within the unmyelinated axon distal to ais . putting the electrode above the second internode results in an asymmetric activating function with a larger right side lobe value ( fig . 4f ) . note that this rather small difference in activating function minima favors nodal excitation in spite of half nodal nav1.6 channel densities when compared to the other activating function side lobe in the unmyelinated axon . increasing the cathodic stimulus of the subthreshold case of fig . 4a from 50 to 57 a causes a delayed spike development for the 5 m diameter dendrite at its distal end ( fig . , the primarily excited region is too short for direct spiking but the axial current flow in both directions prefers to stimulate the left side in spite of the rather large hyperpolarization at the end of the pulse ( red bottom line in fig . is assumed to be sealed , the axial current is also restricted to flow backwards which escalates the increase of membrane voltage in that particular area . this effect is also reflected by the asymmetry of the excitation process of the neighboring compartments of the cyan line in fig . 1c with differing response on left side compared to the behavior on the right . when the electrode is slightly moved further away the left hyperpolarized side lobe of the red line in fig . this explains the decrease of threshold as can be seen at the left end of the corresponding thick blue curve in fig . however the effect vanishes when the positive segment of the red curve is reduced , that is before the electrode position reaches the distal end ( comp . blue curve in fig . 3 ) . reduction of fiber diameter to one - fourth allows direct dendritic spiking for this dendrite changing the polarity reduces the length for direct anodic dendritic spiking ( constant threshold values of thin green curve in fig .", "the aim of the study was to study the effects of external low current stimulation on a cortical model neuron with respect to changing microelectrode position and configuration . in order to enlighten the neural elements which are activated , the electrode has been shifted in the axial direction , showing a varying , non - uniform excitation pattern along the neural axis . a model based on recently reported nav1.2 and nav1.6 channel distribution ( hu et al . , 2009 ) has been used to calculate thresholds for different pulses and varying distances to the electrode by radial offset . during simulations we identified the spike initiation sites with special emphasis on spikes generated in the dendrite by direct stimulation . the performed simulations indicate that the nodes of ranvier and the distal end of the ais are the most excitable structures ( fig . 3a ) showing the lowest threshold for stimulation which is in accordance with experiments ( nowak and bullier , 1998 ; gustafsson and jonkowska , 1976 ) . in comparison to the sensitive ais and electrode positions close to nodes of ranvier , about 23 times higher currents are needed to excite thick dendrites and even higher thresholds are expected for thin branches ( fig . this trend seems to be applicable to various cell types , since gustafsson and jonkowska ( 1976 ) revealed similar results with their experiments on spinal motor neurons and spinal border cells . the excitability essentially differs along the neural axis displaying the varying electrical properties of different functional elements of the neuron . therefore parameter values shall be collected carefully , for example it should be pointed out that different modeling of myelination considering the presence of nav1.2 channels , but with decreased capacitance as formerly described by hu et al . ( 2009 ) , also leads to a zig - zag behavior of the threshold function as mentioned previously , but not as pronounced as in fig . 3a . since non - branching long straight excitable fibers with constant diameters show an anodic cathodic threshold ratio in the order of 4:1 ( ranck , 1975 ; rattay , 1986 ) , we focused our simulations on phenomena occurring for the cathodic excitation . nonetheless , note that only within a small region in the vicinity of the soma dendrites are easier to stimulate with anodic pulses and that the extreme node - internode threshold fluctuations are lost when pulse polarity is reversed . charge - balanced triphasic pulses with an interpulse time of 100 s and 100 s per phase have also been tested for both polarities , that is anodic first and cathodic first stimuli . when the electrode is placed close to the neuron the thresholds are similar to monophasic cathodic stimulation . nonetheless we observed an alternative activation of the cell when the electrode is placed in the vicinity of the dendrites . shortening the electrode - fiber distance allows indirect spike generation in the ais ( fig . 2a ) , but still this type of stimulation demands for electrode positions rather close to the soma . in order to reach their threshold value the neighboring compartments profit from high intracellular resistance . lower resistances caused by branching or suddenly increased diameters , for example at the border to the soma , not only reduce the possibility of spike development but are also capable of preventing the conduction into the axon . 5b demonstrates a reduction in amplitude to 42% before the thin dendrite spike enters the soma , whereas for the 5 m dendrite the dendrite - soma diameter ratio of 1:4 is secure and the spike enters the soma with full amplitude ( fig . our results indicate that a reasonable hypothesis to explain the sparely distributed population of pyramidal cell bodies when stimulated at low intensities ( histed et al . , 2009 ) is to assume a rather dense concentration of collaterals of many neurons in the close surrounding of the electrode tip . these thin axons and dendrites are capable of generating an ongoing nerve impulse or activate the distant ais via intracellular current flow . therefore the cell bodies of stimulated pyramidal cells can be situated within a distance of several hundred micrometers to the electrode tip , whereas most of the cell bodies closer to the electrode wo n't be activated . the performed simulations further approve the ability of dendrites to generate and successfully transmit an ongoing action potential when stimulated extracellularly . ( 2009 ) report that the current needed to activate at least one cell was 10 a or less . according to their fig . , 2009 ) , axon and dendrite diameters are estimated with 0.2 m . with our assumptions such tiny dendrites can be directly activated in the range from 10 to 16 a . our results demonstrate that , by applying a 100 s pulse with an amplitude between 25 and 41 a , a 1.25 m diameter dendrite that passes the electrode tip in 28 m distance is able to evoke a spike , whereas the 5 m thick dendrites needs a minimum distance of 56 m for a local spike and much higher currents . starting at the resting state , we have demonstrated that regular dendritic spikes need an excited region of minimum size to obtain enough intracellular current for the neighboring segments to reach threshold . in case of external point source stimulation the polarized and depolarized regions are separated by a 70 angle , that is the length of the activated area can be calculated by 1.4 distance to electrode . this area does not depend on ion channel compositions , however the minimum electrode fiber distance for a local spike depends essentially on the sodium and potassium channel densities and the intracellular resistance . the inverse recruitment order for peripheral nerve stimulation which is known since 1933 ( blair and erlanger , 1933 ) states that large diameter fibers have smaller threshold currents . this behavior is also predicted by the activating function both for myelinated and unmyelinated fibers ( see methods , rattay , 1989 , 1999 ) . moreover , the strength of dendritic spike propagation is vulnerable to modulation by the extracellular ion concentrations or any other circumstances that change the dendritic voltage - gated channel profile ( gasparini et al . , 2004 ) . although most of these components are beyond the scope of this article , the threshold behavior has also been tested with a more complex model as used by hu et al . generally , the obtained results were quite similar to the reported ones , demonstrating that dendritic na and k channels play a key role in setting the threshold and determining the shape and forward - propagation of dendritic spikes ( gasparini et al . , 2004 ) . ( 2008 ) with a simple model of reduced geometrical complexity and an even distribution of only traditional hodgkin huxley style na and k currents which is able to explain properties like rapid spike onset and its high threshold variability . another obvious reason for the missing effect of the addition of currents ica , ikca and ikm to somatodendritic compartments is their relatively low conductances of gca=0.03 , gkca=0.3 , gca=0.03 ms / cm ( hu et al . , 2009 ) . since royeck et al . ( 2008 ) among other authors report that these ion currents amplify spike after depolarizations and cause the generation of spike bursts , their absence does not influence the initial respond of neurons in the resting start during the onset of microstimulation . mainen and sejnowski ( 1996 ) suggest to include these complex ion channels to expand from single spikes to spike trains , that is repitative firing , which is beyond the purpose of this article . in order to concentrate the analysis on phenomena already observed in a non - branching rectified neuron , we avoided to include the anatomical diversity of pyramidal cells concerning their diverging pattern of dendrites and axons . one should be aware that many more parameters influence the recruitment of extracellular microelectrodes , such as synaptic activities ( spruston , 2008 ; sjstrm et al . , 2008 ) , refractory behavior ( miocinovic and grill , 2004 ) , inhomogeneity in ion channel densities ( migliore and shepherd , 2002 ; schaefer et al . , 2007 ; keren et al . , 2009 ) as well as branching ( manita and ross , 2009 ) , curvature ( rattay et al . , 2000 ; iles , 2005 ) or irregularities in diameters of cell processes ( rattay , 1999 ) . more details on the influence of 3d structures and ion channel distributions will be reported in a forthcoming paper ." ]

[ "peptide- and protein- hydration is the dominant factor in the stabilization of spatial molecular structure , in the process of protein folding by gating hydrophobic residues , and in the mechanisms of peptide and protein mediated reactions [ 14 ] . water molecules , therefore , can be considered as an integral component of biomolecular systems with dynamic , functional , and structural roles [ 47 ] . investigation of the structural and functional role of water molecules , bound to proteins and peptides , requires a sufficient understanding of the hydration process of their building blocks [ 1 , 2 ] . the hydration of amino acids and their derivatives at a molecular level , therefore , is of great importance and has been extensively studied with x - ray crystallography [ 1 , 3 ] and a variety of spectroscopic techniques including multinuclear magnetic resonance spectroscopy [ 2 , 813 ] , ir and raman spectroscopy [ 1416 ] , icr mass spectrometry , and laser ablation in combination with microwave spectroscopy . we present here , for the first time in the international literature , a comparative investigation of literature d , c , n , and o nmr and crystallographic data in order to provide a coherent hydration model of amino acids and selected derivatives at different ionization states in aqueous solution and in the crystal state .", "\n o nmr has received little attention in amino acid and peptide research [ 2 , 12 , 13 , 19 , 20 ] . this neglect is due to the fact that of the three naturally occurring oxygen isotopes , only o possesses a nuclear spin ( i = 5/2 ) . owing to its electric quadrupole moment ( qe = 2.6 10 em ) and , thus , broad line widths , and its low absolute sensitivity compared with that of h ( ~1.1 10 ) , the o- isotope is one of the more difficult to observe by nmr spectroscopy [ 12 , 13 , 21 , 22 ] . o nmr studies , therefore , of compounds at natural abundance require high concentrations ( > 0.1 m ) and extensive signal averaging . recording of spectra can be greatly facilitated by the use of o enriched samples [ 2327 ] . figure 1(a ) illustrates the natural abundance o nmr spectrum of glutamic acid , 0.1 m in o - depleted water at 40c . despite the extensive signal averaging ( number of scans ( ns ) =3 10 ) and the total experimental time of 4.2 hours , the achievable signal - to - noise ( s / n ) ratio is very poor and practically prohibitive for the accurate determination of chemical shifts and line widths . figure 1(b ) illustrates the clear advantages of working with o - labelled glutamic acid ( o enrichment 1 at.% ) . \n o shieldings of various chemical functional groups are very sensitive for studying hydrogen bonding interactions because of the large chemical shift range of the o nucleus [ 12 , 13 ] . the effect of solvent - induced hydrogen bonding interactions on (o ) of the carboxyl groups is , however , rather small compared with the substantial sensitivity of over 80 ppm to hydrogen bonding interaction of (o ) of amide and carbonyl oxygens [ 12 , 13 ] . only a single o resonance absorption is observed for the carboxylic group since the shifts of the individual resonance absorptions (c = o ) and (oh ) are averaged out by rapid intermolecular proton transfer with protic solvents , traces of h2o , and/or through hydrogen bonding aggregates of the cooh groups in organic solvents [ 12 , 13 , 23 , 24 , 26 , 28 ] . reuben from dilution studies of acetic acid in 1,2-dichloroethane estimated a deshielding effect of ~12 ppm due to breaking of a hydrogen bond involving the carbonyl oxygen of the acid and a shielding effect of 6 ppm due to breaking of a oho hydrogen bond . therefore , a total shift of only + 6 ppm is expected for the monomeric acetic acid in apolar media ( dichloroethane ) compared with the dimeric form . despite the relatively low sensitivity of the o shieldings of the carboxyl group to hydrogen bond interactions , spisni and collaborators attempted to estimate the solvation state of the -carboxyl group of amino acids in the different ionization states . figures 2(a ) and 2(b ) show the dependence of (o ) of l - alanine and l - proline as a function of molar fraction of dmso in the ph range 7 - 8 and 12 - 13 . since dmso can not form a hydrogen bond interaction with the carboxylate group , contrary to the case of h2o , the shielding difference of 1017 ppm between the two solvents was interpreted with the hypothesis that the carboxylate group of these amino acids is hydrated by two water molecules in aqueous solution with one hydrogen bond per carboxylate oxygen . in the acidic ph range ( figures 2(c ) , 2(d ) ) , a nonlinear behaviour of the chemical shift at high dmso molar fractions was observed . for dmso molar fractions up to 0.6 , a linear dependence of the chemical shift was observed which , on extrapolation to 100% dmso , results in a shielding of 1517 ppm , the same as in the neutral ph . this was interpreted with the hypothesis that two hydrogen bonds ( one to each oxygen ) are being ruptured . when the dmso molar fraction is between 0.6 and 0.8 , it was suggested that a third molecule of water , which is hydrogen bonded to the hydroxyl hydrogen , is dissociated due to the interaction with dmso . this might explain the deflection from linearity and the plateau - like dependence of the o shielding . the protonated form , therefore , of the carboxyl group of the amino acids is more hydrated with an access of a bound molecule of h2o than the deprotonated form . this conclusion is in qualitative agreement with multinuclear nmr relaxation data ( see below ) . for quadrupolar nuclei , such as d , n , and o , the longitudinal ( t1 ) and transverse ( t2 ) relaxation times are essentially due to quadrupolar interaction \n ( 1)1t1=1t2(1+23)2f(,d ) , \n where is the nuclear quadrupole coupling constant . the asymmetry parameter varies from 0 to 1 and describes the deviation of the electric field gradient from axial symmetry , and f( , d ) is the correlation function , which depends on the rotational diffusion constant d and its relative orientation with respect to the principal axes of the field gradient tensor [ 12 , 13 ] . when isotropic reorientation is assumed , f( , d ) reduces to a single overall correlation time mol which is given by the stokes - debye formula \n ( 2a)mol = vmkbt , \n where vm is the molecular volume , the viscosity of the solution , kb the boltzman constant , and t the absolute temperature . vm can be estimated as \n ( 2b)vm=0.74mwn0 , \n where n0 is the avogadro 's number and mw and are the molecular weight and the density of the solute ( amino acid ) , respectively . \n the removal , therefore , of these impurities is necessary in studies of t2 and t1 relaxation times . figure 3 illustrates the ph dependence of the o line widths of 0.1 m glycine in h2o . this o line width minimum has been previously explained by a decrease of the molecular tumbling time attributable to a reduction in hydration and , thus , intermolecular association of glycine in the zwitterionic form . in the high ph region , a broad maximum at ph 11 was observed . addition of 2 mm ethylenediamine - n , n , n,n-tetraacetate ( edta ) to the original solution resulted in no line width variation in the neutral and high ph region . it can , therefore , be concluded that this broad minimum at ph 11 should be attributed to the effect of paramagnetic impurities and not to a hydration change of glycine in the neutral and high ph region . \n d t1 relaxation times of cd2 of glycine at acidic ph were shown to be shorter relative to those at neutral ph . this shortening in t1 implies an increase in mol and , thus , in the effective molecular weight mw ( ( 1 ) , ( 2a ) , and ( 2b ) ) , which was interpreted with an increase in the hydration state in the cationic form . \n tritt goc and fiat investigated in detail the viscosity and temperature dependence of the o nmr line width of glycine , alanine , proline , leucine , histidine , and phenylalanine at ph 2 , 7 , and 12.5 . the experimentally observed viscosity / temperature ( /t ) dependence of the reorientation correlation time was compared with various hydrodynamic models . a model of the hydration state in the primary solvation sphere of the carboxylic group of amino acids in their cationic state was suggested in which two water molecules are hydrogen bonded to the oxygens and one to the hydrogen of the oh group . in the zwitterionic and anionic states , the hydration model of the carboxylate group can be presented by a structure in which one water molecule is hydrogen bonded to each of the oxygens . the o [ 10 , 11 ] and n nmr line widths of several protein amino acids were measured in aqueous solution to investigate the effect of molecular weight on the line widths ( table 1 ) . the n and o line widths , under composite proton decoupling , increase with the bulk of the amino acid , and increase at low ph . assuming an isotropic molecular reorientation of a rigid sphere and , thus , a single correlation time from overall molecular reorientation ( mol ) , then , the line width 1/2 can be expressed in the following form : \n ( 3)1/2=1t2=0+1mw , \n where mw is the molecular weight , 1 is the contribution to the line width of the quadrupolar coupling constant , density and temperature , ( 1 ) , ( 2a ) , and ( 2b ) , and 0 is the solvent viscosity - independent contributions to the line width due to the primary hydration sphere of the amino acids . the linear correlation between 1/2 and mw at ph 6 for both n and o nuclei ( figure 4 ) is in agreement with the hydrodynamic model of ( 3 ) . furthermore , the (o ) of the amino acid is independent of both the ionization and the degree of hydration of the carboxyl group . the increase in the o line widths at acidic ph ( ~100 31 hz ) , relative to those at neutral ph , was interpreted by a change in the rotational correlation time and , thus , effective mw of the amino acids , ( 3 ) . this implies that the cationic form of the amino acids is more hydrated by an access of 1.3 to 2.5 molecules of water relative to that in the zwitterionic form with lifetimes that are longer than the overall molecular rotational correlation time , presumably 210 ps . in the case of a stochastic diffusion of the amino and carboxyl groups comprising contributions from internal ( int ) and overall ( mol ) motions , the correlation time c for n or o is given by ( 4a)c=mol[a+(b+c)(12/r)intmol+(12/r)int ] \n with \n ( 4b)a=34(3cos21)2 , b=3sin2cos2 , c=34sin4,where is the angle between the rotation axis and the main field gradient ( r denotes an r - fold jump mechanism ) . since the sum of a , b , and c is equal to 1 , ( 4a ) can be rewritten as(5a)c=molamol+imol+i , \n where \n ( 5b)i = ( 12r)int.equations ( 5a ) and ( 5b ) can be rewritten as \n ( 6)c=(1a)i+amol(1a)i2mol+i . \n since a and i can be assumed to be constant for all the amino acids , ( 4a ) and ( 4b ) can be written as \n ( 7)1/2=0+1mw+2mw+3 , \n where 03 are constants . the minimization of ( 7 ) on the basis of the o experimental data gave the mean difference of 35.8 17.3 in mw between ph 0.5 and 6.0 for three different 1/2 values : 250 , 350 ( figure 4(b ) ) , and 500 hz . the difference in the n line widths at the two ionization states ( figure 4(a ) ) should be attributed to differences in the correlation times and to a decrease in the (n ) on deprotonation of the carboxyl group . in the case of the linear model , the influence of variations of values of the (n ) to the line width , 1/2 , is less for small molecular weights . therefore , for 1/2 = 70 hz ( figure 4(a ) ) , the difference in mw will be a reasonable approximation of the difference in hydration in the two states . the calculated value was found to be 45.2 7.4 , which corresponds to an excess of 2 - 3 water molecules in the cationic form compared to that in the zwitterionic form , in reasonable agreement with the o nmr data . more recently , takis et al . investigated the cc longitudinal relaxation times ( t1 ) and n line widths ( 1/2 ) of amino acids and acetyl - amino acids in aqueous solutions at acid and neutral ph . both c and n values indicate that amino acids and acetyl - amino acids at acid ph interact with an access of one water molecule with respect to their deprotonated form at neutral ph . on the contrary , c and n values of betaines ( r3nch(r)coo ) crystal structure databases provide a rich source of information to extract details on the architectures and interactions of molecules . this kind of search provides the opportunity to examine the formation of intramolecular and intermolecular hydrogen bond in small molecule crystal structures [ 33 , 34 ] . propensities for the hydration of the -carboxylate group of amino acids and their derivatives were derived on the basis of exhaustive searches in the cambridge crystallographic database ( csd ) . since intermolecular hydrogen bonds are preferred when five- or six - membered conjugated rings are formed , particular attention has been given to the hydrogen bond patterns in the vicinity of the carboxylate group that involves two simultaneous hydrogen acceptors . the concept of five- and six - membered conjugated rings , along with three - center ( bifurcated ) and 4-center ( trifurcated ) hydrogen bonds , has been acknowledged and accepted widely as an important factor in determining the structure and function of molecules ranging from inorganic to organic and biological molecules [ 1 , 3539 ] . furthermore , port and pullman studied theoretically the formate ion - water interaction as a prototype of the carboxylate group . three energetically favourable hydration sites were obtained , two equivalent sites on the carboxylate oxygens at the exterior of the ion and one water bridging the two oxygen atoms . the conquest 1.13 program was used for all the statistical analysis described in this paper . specifically , the csd version 5.32 ( november 2010 ) for small molecules was searched , with the following general search flags : r > 0.5 , , and only organic . in order to extract the number of entries present in the current database that form six - membered conjugated rings between the two oxygens of the -carboxylate and the carboxylic group with a molecule of water in the vicinity , the following geometric cut - offs were used : upper limits d = 3 for ( ow)ho = c and ( ow)ho c , and d = 3.5 for owo = c and owo c 44 hits were obtained for the carboxylate state ( figure 5 ) , whereas only one was derived for the protonated form . figure 5 demonstrates that the oxygen of water , ow , is reasonably close to the carboxylate oxygens and displays a significant preference for the o1c there is a general correlation between hydrogen bond lengths and hydrogen bond angles ( figure 6 ) similar to that observed by jeffrey and maluszynska in the case of water molecules in the hydrates of small biological molecules . furthermore , crystallographic database searches were performed to identify the propensity for the formation of intramolecular hydrogen bond interaction in the carboxylate ( nho ) and the carboxylic acid ( nho = c ) state . interestingly , 946 and 118 hits were retrieved for the carboxylate and 621 and 6 hits for the carboxylic form in the absence and presence of two molecules of water , respectively . it is evident from figure 7 that in the presence of two bound water molecules there is a significant reduction in the number of structures with intramolecular hydrogen bond interaction for the carboxylate group and , concurrently , a significant increase in the distance ( nho ) . it is important to note that no intramolecular nh3ooc hydrogen bonds were observed for 82 amino acid carboxylates with sp - hybridized c - atoms in agreement with an early survey of amino acid structures determined by neutron diffraction .", "\n o shielding changes of amino acids as a function of molar fraction of dmso / h2o , the decrease in the longitudinal relaxation times ( t1 ) of c d and c , and the increase in line widths of n and o at acidic ph relative to those at neutral ph may be interpreted with the hypothesis that the cationic form of amino acids is more hydrated by 1 to 3 molecules of water than the zwitterionic form . similar behaviour was also observed for acetylated derivatives of amino acids , but not for betaines , between the protonated and deprotonated carboxyl group . although the precise hydration differences observed for various nuclei deviate somehow , it may be concluded that these hydrated complexes have lifetimes that are shorter than the nmr chemical shift time scale , but presumably longer than the overall molecular rotational correlation time of 210 ps . an exhaustive search in the cambridge crystallographic database ( csd ) demonstrates a strong tendency of the two oxygens of the deprotonated carboxylate group to form hydrogen bonds with a single molecule of water . even though statistical analysis of structural parameters in crystals can not be used in a straightforward way to derive quantitative structural models in solution , it is of interest to note that this mode of six - membered conjugated ring , which is absent in the case of the carboxylic group , might result in a more compact and , thus , less hydrated structure in aqueous solution , in accordance with the nmr data ( figure 8) . furthermore , it may be concluded that the bound molecules of water alleviate the nho interaction and very probably this effect is even more pronounced in aqueous solution . from the above , it is evident that the reduced hydration of the carboxylate group , relative to the carboxylic group , should be attributed mainly to the strong tendency of the carboxylate group to form a six - membered conjugated ring with a single molecule of water . constructively , the tentative models illustrated in figure 8 should be further validated by in silico and experimental approaches . computational methods complement the experimental results by providing information on the microscope and physicochemical details on the interplay between water and the biomolecule of interest [ 4447 ] . for example , introduction of solvent effects into molecular dynamics can provide an atomic description of the folding and unfolding of a protein . furthermore , there is an array of theoretical approaches that have been utilized for treating nmr shieldings in solution , that can be classified as continuum models [ 49 , 50 ] and molecular dynamics simulations . experimental approaches could involve o nmr both in powders and in the crystal state with varying degrees of hydration ." ]

[ "fabrication of the passive electrode arrays began with solid state phosphorus doping ( ph-1000n source , saint gobain , usa , 1000 c for 10 minutes ) of p - type device si on a si on insulator ( soi , top si ~300 nm , soitec , france ) wafer . removing the buried oxide layer of the soi by wet etching with concentrated hf released the device si as a si nm , retrieved with a slab of the elastomer poly(dimethylsiloxane ) ( pdms ) and transfer - printed to a spin - cast bilayer of poly(methylmethacrylate ) ( pmma , ~800 nm thick ) and polyimide ( pi , ~300 nm thick ) on a si wafer . photolithography and reactive ion etching ( rie ) defined a pattern of electrodes and interconnects in the si nms . plasma enhanced chemical vapor deposition ( pecvd ) formed a layer of sio2 ( thickness 100 nm ) as encapsulation . patterned etching with buffered oxide etchant removed the sio2 from the electrode regions . spin casting and patterning a top coating of pi ( ~300 nm thick ) placed the si nm electrodes and interconnects near the neutral mechanical plane . patterning a mesh structure across the multilayer ( i.e. pi , sio2 , pi and pmma ) by rie followed by immersion in buffered oxide etchant exposed the base layer of pmma to allow its dissolution in acetone . retrieval onto a slab of pdms enabled removal of the bottom exposed layer of pi by rie . transfer onto a film of plga ( ~30 m thick ) , facilitated by heating to temperatures close to the glass transition of the plga ( 55~60 c , lactide / glycolide ratio of 75:25 composition ) , followed by elimination of the top layer of pi by rie completed the fabrication . bonding an acf cable to the terminal regions of the si nm interconnects yielded connection points for interfaces to external data acquisition ( daq ) systems . the fabrication began with growth of 200 nm of thermal oxide on a p - type soi wafer ( top si ~320 nm , soitec ) , photolithography and immersion in buffered oxide etchant to create a mask for solid state phosphorus diffusion ( 1000 c for 6 minutes ) to define the source and drain contacts . releasing , retrieving and transferring the doped si nms onto a temporary substrate , consisting of si wafer with a bilayer coating of pi / pmma , followed procedures similar to those described for passive electrode arrays . photolithography and rie etching patterned the si nms into geometries for an 88 array of unit cells , each consisting of two transistors connected in series for purpose of actively multiplexed readout . a thin layer of sio2 ( pecvd at 220 c , thickness ~100 nm ) served as the gate dielectric . buffered oxide etching through a photolithographically patterned mask formed openings through the sio2 to expose the source and drain contact regions . photolithography and lift off in acetone defined a patterned layer of mo ( sputter deposited , thickness ~300 nm ) for the gate electrodes and metal interconnects . deposition of a trilayer of sio2 ( ~300 nm)/si3n4 ( ~400 nm)/sio2 ( ~300 nm ) by pecvd formed the interlayer dielectric . photolithography and buffered oxide etching created vertical interconnect access ( via ) holes for electrical connections between layers . an additional layer of mo ( thickness ~300 nm ) patterned by photolithography and liftoff defined column select lines . another trilayer of sio2 ( ~300 nm)/si3n4 ( ~400 nm)/sio2 ( ~300 nm ) served as encapsulation , with openings at the locations of the sensing electrodes and peripheral contact pads for interfacing to an external daq system . selective rie and buffered oxide etching through these multilayer stacks ( diluted pi / trilayers of inorganic materials / trilayers of inorganic materials / diluted pi ) formed mesh structures that enabled release of active layers from the temporary substrate by dissolving the pmma layer in acetone . transfer printing steps followed , according to procedures similar to those for the passive electrode fabrication . . 2 is representative of four different acute experiments , each of which lasted 5 - 6 hours . the procedures , which were approved by the institutional care and use committee of the university of pennsylvania , involved an anaesthetized rat with its head fixed in a stereotaxic apparatus . the animal was anesthetized with initially ketamine / xylazine and then isoflurane throughout the craniotomy and neural recordings . a craniotomy exposed a 48 mm region of cortex in either left or tight or in both hemispheres . all recordings were taken in reference to a distant stainless steel bone screw inserted through the skull during the surgery . a commercial stainless steel microwire electrode ( ~100 um stainless steel wire from california fine wire ) placed at 0.5 mm depth from the cortical surface in close proximity to the bioresorbable electrodes served as a control during acute recordings . neural data was acquired by a fhc multi - channel neural amplifier ( fhc inc , bowdoin , me , usa ) and an acquisition system ( 16 bit axon instruments digidata 1322a , axon instruments , foster city , ca ) . neural recording data were analyzed offline using clampfit software ( axon instruments ) and custom matlab software for neural signal analysis . the following procedures were approved by the institutional care and use committee of the university of pennsylvania . one 150-g , sprague - dawley rat was anesthetized with a ketamine ( 60 mg / kg ) , dexdomitor ( 0.25 mg / kg ) solution and placed in a stereotaxic frame . a skull screw was placed in the left frontal bone to serve as the reference electrode for the recordings . a pair of needle stimulating electrode were inserted into the left mystacial pad at various locations . brief electrical currents ( ~250 - 600 a , 1 ms / phase , biphasic pulse ) were passed between the electrodes to activate the intrinsic muscles of the vibrissae , causing a visible protraction of the whiskers . current amplitude and electrode spacing was adjusted for focal activation , usually 1 - 4 whiskers . an adult long evans rat was anesthetized with isoflurane and placed in a stereotactic frame ( david kopf instruments tujunga , ca ) . body temperature was maintained with a heating blanket and the eyes were covered with ointment to prevent drying . the skull was exposed and a large craniotomy ( 4 8 mm ) was made between bregma and lambda and laterally to the midline . the electrode was placed on the exposed dura and a slurry of gel foam and saline was layered on top of the electrode . a screw electrode was placed contralaterally to the experimental array , with another such electrode placed posterior to lambda as a ground and reference . the skull and electrodes were then covered with dental cement and the connecting plug was secured on top . the rat was given meloxicam for postoperative pain and allowed to recover on a heating pad . after 1 week the animal was placed in a cage for video / eeg recording . eeg signals were collected continuously from 3 channels on the array and from the screw . the signals were amplified and low pass filtered at 600 hz ( multichannel systems , reutlingen , germany ) and sampled at 2000 hz with a 16 bit digitizer ( national instruments , austin , tx ) . rats ( n=14 ) were anesthetized and transcardiac perfusion was performed using phosphate buffered saline ( pbs 10x , cat . # bm-220 , boston bioproducts , ashland , ma ) , followed by 4% paraformaldehyde ( pfa , cat . whole brains were then removed and post - fixed overnight at 4c in the same 4% pfa solution . # 57 - 50 - 1 , sigma - aldrich , saint louis , mo ) at 4c and coronal sections were cut at 20 m using a leica cm3050 s cryostat ( leica biosystems inc . ) . serial sections , spanning the entire craniotomy site , were mounted on charged slides and stored at 20c until use . for immunostaining , slides were first immersed in an antigen retrieval solution ( 0.1 m citrate buffer , ph 6.0 , cat # ab64214 , abcam , cambridge , ma ) and placed in a water bath at 95c for 10 minute . after cooling , sections were rinsed in distilled water , incubated in a blocking solution containing 0.1% triton x-100 ( cat . # 9002 - 93 - 1 , sigma - aldrich ) and 5% normal goat serum ( cat . kerrville , tx ) for one hour at room temperature ( rt ) and then incubated overnight at 4 c with the following primary antibodies : anti- glial fibrillary acidic protein ( gfap , 1:1000 , cat . # smi-22r , covance , princeton , nj ) , and anti - ionized calcium binding adapter molecule 1 ( iba-1 , 1:1000 , cat . after 320-minute washes in pbs , sections were incubated with the corresponding fluorescent secondary antibodies ( alexa fluor 488 goat anti - mouse igg2b , 1:1000 , cat . # a-21141 , and alexa fluor 568 goat anti - rabbit igg , 1:1000 , cat . after the final washes ( 3 20 min in pbs ) , the slides were cover - slipped with an anti - fade medium containing the nuclear stain dapi ( fluoromount - g+dapi , cat . control sections were incubated with omission of one or both primary antibodies , adding only the secondary antibodies to exclude false - positive labeling . slides were examined on an epifluorescence microscope ( zeiss axioscope , germany ) and images were acquired with a 20 objective and a spot rt3 digital camera , using the spot software 5.1 ( diagnostic instruments , sterling heights , mi ) . digital images were processed using adobe photoshop 12.0 ( adobe systems , san jose , ca ) .", "fabrication of the passive electrode arrays began with solid state phosphorus doping ( ph-1000n source , saint gobain , usa , 1000 c for 10 minutes ) of p - type device si on a si on insulator ( soi , top si ~300 nm , soitec , france ) wafer . removing the buried oxide layer of the soi by wet etching with concentrated hf released the device si as a si nm , retrieved with a slab of the elastomer poly(dimethylsiloxane ) ( pdms ) and transfer - printed to a spin - cast bilayer of poly(methylmethacrylate ) ( pmma , ~800 nm thick ) and polyimide ( pi , ~300 nm thick ) on a si wafer . photolithography and reactive ion etching ( rie ) defined a pattern of electrodes and interconnects in the si nms . plasma enhanced chemical vapor deposition ( pecvd ) formed a layer of sio2 ( thickness 100 nm ) as encapsulation . patterned etching with buffered oxide etchant removed the sio2 from the electrode regions . spin casting and patterning a top coating of pi ( ~300 nm thick ) placed the si nm electrodes and interconnects near the neutral mechanical plane . patterning a mesh structure across the multilayer ( i.e. pi , sio2 , pi and pmma ) by rie followed by immersion in buffered oxide etchant exposed the base layer of pmma to allow its dissolution in acetone . retrieval onto a slab of pdms enabled removal of the bottom exposed layer of pi by rie . transfer onto a film of plga ( ~30 m thick ) , facilitated by heating to temperatures close to the glass transition of the plga ( 55~60 c , lactide / glycolide ratio of 75:25 composition ) , followed by elimination of the top layer of pi by rie completed the fabrication . bonding an acf cable to the terminal regions of the si nm interconnects yielded connection points for interfaces to external data acquisition ( daq ) systems .", "the fabrication began with growth of 200 nm of thermal oxide on a p - type soi wafer ( top si ~320 nm , soitec ) , photolithography and immersion in buffered oxide etchant to create a mask for solid state phosphorus diffusion ( 1000 c for 6 minutes ) to define the source and drain contacts . releasing , retrieving and transferring the doped si nms onto a temporary substrate , consisting of si wafer with a bilayer coating of pi / pmma , followed procedures similar to those described for passive electrode arrays . photolithography and rie etching patterned the si nms into geometries for an 88 array of unit cells , each consisting of two transistors connected in series for purpose of actively multiplexed readout . a thin layer of sio2 ( pecvd at 220 c , thickness ~100 nm ) served as the gate dielectric . buffered oxide etching through a photolithographically patterned mask formed openings through the sio2 to expose the source and drain contact regions . photolithography and lift off in acetone defined a patterned layer of mo ( sputter deposited , thickness ~300 nm ) for the gate electrodes and metal interconnects . deposition of a trilayer of sio2 ( ~300 nm)/si3n4 ( ~400 nm)/sio2 ( ~300 nm ) by pecvd formed the interlayer dielectric . photolithography and buffered oxide etching created vertical interconnect access ( via ) holes for electrical connections between layers . an additional layer of mo ( thickness ~300 nm ) patterned by photolithography and liftoff defined column select lines . another trilayer of sio2 ( ~300 nm)/si3n4 ( ~400 nm)/sio2 ( ~300 nm ) served as encapsulation , with openings at the locations of the sensing electrodes and peripheral contact pads for interfacing to an external daq system . selective rie and buffered oxide etching through these multilayer stacks ( diluted pi / trilayers of inorganic materials / trilayers of inorganic materials / diluted pi ) formed mesh structures that enabled release of active layers from the temporary substrate by dissolving the pmma layer in acetone . transfer printing steps followed , according to procedures similar to those for the passive electrode fabrication .", "2 is representative of four different acute experiments , each of which lasted 5 - 6 hours . the procedures , which were approved by the institutional care and use committee of the university of pennsylvania , involved an anaesthetized rat with its head fixed in a stereotaxic apparatus . the animal was anesthetized with initially ketamine / xylazine and then isoflurane throughout the craniotomy and neural recordings . a craniotomy exposed a 48 mm region of cortex in either left or tight or in both hemispheres . all recordings were taken in reference to a distant stainless steel bone screw inserted through the skull during the surgery . a commercial stainless steel microwire electrode ( ~100 um stainless steel wire from california fine wire ) placed at 0.5 mm depth from the cortical surface in close proximity to the bioresorbable electrodes served as a control during acute recordings . neural data was acquired by a fhc multi - channel neural amplifier ( fhc inc , bowdoin , me , usa ) and an acquisition system ( 16 bit axon instruments digidata 1322a , axon instruments , foster city , ca ) . neural recording data were analyzed offline using clampfit software ( axon instruments ) and custom matlab software for neural signal analysis .", "the following procedures were approved by the institutional care and use committee of the university of pennsylvania . one 150-g , sprague - dawley rat was anesthetized with a ketamine ( 60 mg / kg ) , dexdomitor ( 0.25 mg / kg ) solution and placed in a stereotaxic frame . a skull screw was placed in the left frontal bone to serve as the reference electrode for the recordings . a pair of needle stimulating electrode were inserted into the left mystacial pad at various locations . brief electrical currents ( ~250 - 600 a , 1 ms / phase , biphasic pulse ) were passed between the electrodes to activate the intrinsic muscles of the vibrissae , causing a visible protraction of the whiskers . current amplitude and electrode spacing was adjusted for focal activation , usually 1 - 4 whiskers .", "an adult long evans rat was anesthetized with isoflurane and placed in a stereotactic frame ( david kopf instruments tujunga , ca ) . body temperature was maintained with a heating blanket and the eyes were covered with ointment to prevent drying . the skull was exposed and a large craniotomy ( 4 8 mm ) was made between bregma and lambda and laterally to the midline . the electrode was placed on the exposed dura and a slurry of gel foam and saline was layered on top of the electrode . a screw electrode was placed contralaterally to the experimental array , with another such electrode placed posterior to lambda as a ground and reference . the skull and electrodes were then covered with dental cement and the connecting plug was secured on top . the rat was given meloxicam for postoperative pain and allowed to recover on a heating pad . after 1 week the animal was placed in a cage for video / eeg recording . eeg signals were collected continuously from 3 channels on the array and from the screw . the signals were amplified and low pass filtered at 600 hz ( multichannel systems , reutlingen , germany ) and sampled at 2000 hz with a 16 bit digitizer ( national instruments , austin , tx ) .", "rats ( n=14 ) were anesthetized and transcardiac perfusion was performed using phosphate buffered saline ( pbs 10x , cat . # bm-220 , boston bioproducts , ashland , ma ) , followed by 4% paraformaldehyde ( pfa , cat . whole brains were then removed and post - fixed overnight at 4c in the same 4% pfa solution . # 57 - 50 - 1 , sigma - aldrich , saint louis , mo ) at 4c and coronal sections were cut at 20 m using a leica cm3050 s cryostat ( leica biosystems inc . ) . serial sections , spanning the entire craniotomy site , were mounted on charged slides and stored at 20c until use . for immunostaining , slides were first immersed in an antigen retrieval solution ( 0.1 m citrate buffer , ph 6.0 , cat # ab64214 , abcam , cambridge , ma ) and placed in a water bath at 95c for 10 minute . after cooling , sections were rinsed in distilled water , incubated in a blocking solution containing 0.1% triton x-100 ( cat . # 9002 - 93 - 1 , sigma - aldrich ) and 5% normal goat serum ( cat . # gs-0500 , equitech - bio inc . , kerrville , tx ) for one hour at room temperature ( rt ) and then incubated overnight at 4 c with the following primary antibodies : anti- glial fibrillary acidic protein ( gfap , 1:1000 , cat . # smi-22r , covance , princeton , nj ) , and anti - ionized calcium binding adapter molecule 1 ( iba-1 , 1:1000 , cat . after 320-minute washes in pbs , sections were incubated with the corresponding fluorescent secondary antibodies ( alexa fluor 488 goat anti - mouse igg2b , 1:1000 , cat . # a-21141 , and alexa fluor 568 goat anti - rabbit igg , 1:1000 , cat . # a-11011 , invitrogen by life technologies , grand island , ny ) . after the final washes ( 3 20 min in pbs ) , the slides were cover - slipped with an anti - fade medium containing the nuclear stain dapi ( fluoromount - g+dapi , cat . control sections were incubated with omission of one or both primary antibodies , adding only the secondary antibodies to exclude false - positive labeling . slides were examined on an epifluorescence microscope ( zeiss axioscope , germany ) and images were acquired with a 20 objective and a spot rt3 digital camera , using the spot software 5.1 ( diagnostic instruments , sterling heights , mi ) . digital images were processed using adobe photoshop 12.0 ( adobe systems , san jose , ca ) .", "" ]

[ "integrated care is a common concern for health and social care systems throughout the world . the definition that is used in this study describes integrated care as the .co - ordinated set of services which are planned , managed and delivered to individual service users across a range of organisations and by a range of co - operating professionals and informal carers [ 2 , p. 14 ] . this definition suggests that there is a need for both inter- and intra - organisational cooperation since multiple organisations and professionals must cooperate and coordinate their services in order to provide care to an individual . when discussing integrated care , elderly care is often in focus [ 25 ] . the challenges concerning the complicated elderly care situation involve demographic changes , elderly with multiple care demands and the endeavour to accommodate the care needs of elderly at home for as long as possible [ 6 , 7 ] . what is more , the trend towards deinstitutionalisation in combination with the move towards advanced palliative care , which previously was provided by the hospital , into the patients home , increases the number of home care interventions [ 810 ] . all these factors increase the need for coordinating home care work between all parties involved . integrated elderly care at home has been studied from various perspectives that have taken different strategies , structures and processes into account , as argued by wijngaarden et al . . previous studies have focused on policy approaches to integrated care [ 4 , 12 ] , system models [ 13 , 14 ] and organisational structures . in addition , information technology ( it ) is often suggested as a way to improve cooperation and coordination and support integrated care [ 10 , 15 , 16 ] . however , research into this field has not explored fully how the daily work is actually carried out in situ . one approach that can be used to explore this perspective is an ethnographic workplace study . in this paper , the aim is to explore how home care workers coordinate their daily work , identify coordination issues in situ and discuss possible actions for supporting seamless and integrated elderly care at home .", "the concept of cooperation has a long history within the fields of social sciences and sociology . in recent years , cooperation has also been a focus within the field of computer supported cooperative work ( cscw ) . it involves several research disciplines such as computer science , sociology , anthropology , organizational theory and design [ 17 , 18 ] . the main endeavour with cscw research is to understand the nature and requirements of cooperative work with the objective of designing computer - based technologies for cooperative work arrangement [ 19 , p. 5 ] . while some of this research focuses on the first part of the cscw acronym , other centres focus mainly on the social aspect in different kinds of cooperative work arrangements [ 21 , 22 ] . the interest for cscw has grown also in other research areas and in the industry . one of the reasons may be the demands of industry for improved tools that support coordination and help control group activities . furthermore , a growing interest for cscw can also be found in research areas such as medical and health informatics . at the same time , these medical settings provide a rich domain for studying cooperative work from a cscw perspective . within cscw , cooperative work is not defined by formal organizational boundaries or structures , but by actual cooperative behaviour . according to schmidt , cooperative work emerges in response to the requirements and constraints of the transformation process and the social environment on one hand and the limitations of the technical and human resources available on the other [ 18 , p. 352 ] . bannon argue , people engage in cooperative work when they are mutually dependent in their work and , therefore , are required to cooperate in order to get the work done [ 19 , p. 7 , a cooperative work setting furthermore raises the need for coordination of the individual inter - related distributed activities . coordination in this context implies the need to mesh , allocate , relate , schedule , etc . activities , actors , and resources with respect to each other [ 25 , p. 689 ] . cscw research has highlighted several important aspects of the nature and requirements of cooperative work . in general , it has been shown that supporting cooperative work is a complex issue that requires more than the improvement of information access , communication and coordination [ 26 , 27 ] . for example , cscw has highlighted the importance of supporting awareness ( awareness is here understood as the way in which cooperative actors make sense of and act upon their joint endeavours ) . in addition , it has been shown that human actions are both situated and flexible according to the social and physical conditions that are in place . as a response to the latter finding , cooperative work is most often explored through workplace studies in situ . these workplace studies often use an ethnographic approach that focus on the work , actions , interactions and technologies typical of complex organizational settings . this methodology was also employed in the present study as described in the next section . furthermore , in this work , the analysis of the cooperative work focuses on the coordination activities that take place in order to provide integrated care .", "the present study was carried out in an inter - organisational home care setting in a county in southern sweden during 20022004 . at the studied county , home health care the study was part of a broader project with the general aim to support the cooperative work in home care with mobile it tools . this implies that analysis is conducted through every stage of the research procedure , in the process sharpening the focus of the data collection . the actual methods used to collect data may vary in ethnographic studies . in the present study , data were gathered through observational studies , interviews and group discussions . to gain a general overview of the work domain , some 15 semi - structured interviews were carried out with managers from both the home help service and home health care . this was followed by observational studies that were focused on the cooperation activities taking place in the daily work . during these studies , observation of 30 work shifts taking place during the day , the evening and the night were included in the study . during these studies , field notes were taken and transcribed the day after the observations . in order to obtain a more comprehensive understanding of the views and perceptions of the home care workers , group discussions were conducted . participants in the group discussions were three district nurses , one assistant nurse , one home help service manager and two home help service workers . the group discussions focused on four themes : problematic issues concerning the inter - organisational division of labour and the division of responsibility between home help service and home health care , problematic issues concerning work activities , problematic issues concerning cooperative activities and , finally , general issues concerning information needs and tools . the aim when analysing data was to identify and categorise common themes , activities and/or issues which can explain how cooperative work and coordination is accomplished in the examined work domain . therefore , the collected data were indexed according to which theme , activity or issue they illustrated . for this paper , two coordination activities are identified as particularly important for providing integrated care : planning the process and coordination during home care . the work , actions , interactions and relevant technology used in these two activities are described in the result section .", "coordination of the home care process can be divided into two main activities ; planning the process and coordination during home care . in this paper , these two activities are considered as crucial when providing integrated elderly care at home . the first activity is the starting point for the cooperation between all parties involved while the second describes how the home care workers share information and communicate their efforts during the home care process . during the observations , some interesting situations occurred , which in the group discussions with the care workers were found relevant when improving cooperation and coordination in this setting . the observed situations are described below . in order to deliver integrated elderly care at home , the municipalities and the county councils are required to follow the requirements from the national board of health and welfare . one of these requirements concerns information sharing between care providers while the other deals with cooperative care planning before discharge from hospital . therefore , when an individual is scheduled to receive care at home after hospital discharge , a care plan meeting with all parties involved is mandatory . during this meeting , the care providers and the care receiver with relatives discuss the home care process . the following care providers attend these meetings : the nurse at the hospital who initiates the meeting , personnel from home health care , the care administrator and physiotherapists from both the hospital and from primary care if needed . the care interventions provided by the home help service and by home health care are different . the home health care provides care interventions defined as health care , such as binding up wounds , giving insulin , taking samples for testing , inserting pharmaceuticals into medical dispenser units and dispensing medicine and eye drops . the home help service provides care interventions defined as care along with social care such as help with food , getting dressed , cleaning , care assistance , practical services and they also respond to alarms . furthermore , some care interventions are defined by home health care as self - treatment and are , therefore , not provided by the home health care personnel . an elderly patient at the hospital is about to be discharged and is in need of home care . five persons attend this meeting ; the patient himself , a relative , the hospital nurse , the district nurse and the care administrator . the nurse describes the patient s visit to the hospital and what care interventions are needed when the patient returns to his home . the patient needs help with treating a wound with a cream twice a day , a procedure that takes some time . the district nurse responds that this kind of care intervention is classified as self - treatment and should not be conducted by the home health care workers . the hospital nurse informs the home care workers that the patient can not perform the treatment himself due to his injuries . the care administrator responds that this kind of task is something that certainly should be done by the home health care workers since this task is not something a care receiver could apply for help for and thus can not be classified as part of the home help service , .besides the home help service unit has no resources for these kinds of care interventions that take so much time . the meeting ends without a final decision about who should take care of the wound . during the group discussion , the home health care workers argued that if the patient is not mentally capable , even simple treatments may be considered as health care interventions . however , some patients are physically incapable to conduct the self - treatment themselves , as in the above observation , and the question then is which organisation is responsible for this person . to determine responsibility in these cases , the question is often asked if the person s health will deteriorate until a health care intervention is needed unless given the treatment in question . another way of determining responsibility is to decide which organisation would have had responsibility for the patient if he or she had not applied for home help service . in addition to this , it is important to note that for the care receiver , home help service may be more costly than home health care . during the group discussions , the care workers argued for clearer rules or agreements about what organisation should be responsible for which care intervention . the care workers also stated that resources are wasted when both the home help service and home health care are at the care receiver s home at the same time : when one person is making a sandwich while another person gives insulin at the same time , this does not feel as an efficient way to conduct a home visit . however , the care workers also stated that sometimes the unit chiefs or care administrators bend the rules in the best interest of the care receiver and sometimes both the home help service and home health care workers actually conduct care interventions beyond those agreed upon during the care plan meeting . the home help service groups from all shifts store information about the care receiver and the approved care interventions separately in non - digital form . the home health care personnel use a computerised patient record system , which is used within all primary care . however , while working in the patients homes , it is impossible to access this patient record system . to manage cooperation and coordination between the organisations , svop is an acronym for coordinated health care and care planning ( in swedish : samordnad vrd- och omsorgs planering ) . the binder is placed in the care receiver s home and consists of several documents and material which provide the care workers with information for administering home care interventions , for supporting involved individuals in daily work situations and for facilitating communication between the care workers involved . the binder and its use has been described and analysed in more detail in . particularly important for the present study is the fact that although the current events document facilitates communication , it was rarely used since it was available only in the care receiver s home . this is unfortunate , since some of the information is needed for coordination purposes and important information may be received late , which in turn may affect the care receiver s health condition . to circumvent this problem , workers who add information to the current events document also try to reach the day - shift personnel who need the information by phone . this is further complicated by the fact that the home help service workers during dayshifts ( 10 persons ) share only two mobile phones , making it difficult for district nurses to quickly contact a specific home help service worker . for the night - shift personnel , the notes in the svop binder are the only way to stay informed . to continue , care receivers in need of multiple care interventions can trigger alarms if the need arises . the home help service responds to these alarms and is , therefore , equipped with keys . not having these keys , the home health care personnel must coordinate their visits according to the home help service s schedule . however , information about the scheduled home help service visits is placed in the svop binder , accessible only during home visits . the following observation shows one consequence of this situation : a district nurse is about to make a home visit . she knows that the home help service usually makes its home visit at a certain time and she , therefore , plans the home visit according to this . when she arrives to the patient , the door is locked and she realises that the home help service worker has not arrived yet . she makes another home visit and attempts to visit the patient later when the home help service has arrived . in addition to this , the information in the svop binder is not always up - to - date for several reasons . in particular , pharmaceutical information , updated by external primary care workers and taken from the patient s medical record , tends to be inaccurate . the following observed situation illustrates an actual consequence of this problem : a district nurse is conducting a home visit in order to insert pharmaceuticals into medical dispenser units . she takes the pharmaceuticals from the packages and in order to insert the right dosage she looks at the prescribed pharmaceuticals document in the svop binder . during this home visit the district nurse consults the prescribed pharmaceuticals document and notices that the dosage of the new pharmaceutical is inconsistent with the prescribed pharmaceuticals document . she , therefore , drives back to the reception to search for the new document . to summarise , the empirical findings clearly show that the tools used for information and communication do not fully support coordination during the home care process , thus forcing care workers to make additional coordination efforts .", "in order to deliver integrated elderly care at home , the municipalities and the county councils are required to follow the requirements from the national board of health and welfare . one of these requirements concerns information sharing between care providers while the other deals with cooperative care planning before discharge from hospital . therefore , when an individual is scheduled to receive care at home after hospital discharge , a care plan meeting with all parties involved is mandatory . during this meeting , the care providers and the care receiver with relatives discuss the home care process . the following care providers attend these meetings : the nurse at the hospital who initiates the meeting , personnel from home health care , the care administrator and physiotherapists from both the hospital and from primary care if needed . the care interventions provided by the home help service and by home health care are different . the home health care provides care interventions defined as health care , such as binding up wounds , giving insulin , taking samples for testing , inserting pharmaceuticals into medical dispenser units and dispensing medicine and eye drops . the home help service provides care interventions defined as care along with social care such as help with food , getting dressed , cleaning , care assistance , practical services and they also respond to alarms . furthermore , some care interventions are defined by home health care as self - treatment and are , therefore , not provided by the home health care personnel . an elderly patient at the hospital is about to be discharged and is in need of home care . five persons attend this meeting ; the patient himself , a relative , the hospital nurse , the district nurse and the care administrator . the nurse describes the patient s visit to the hospital and what care interventions are needed when the patient returns to his home . the patient needs help with treating a wound with a cream twice a day , a procedure that takes some time . the district nurse responds that this kind of care intervention is classified as self - treatment and should not be conducted by the home health care workers . the hospital nurse informs the home care workers that the patient can not perform the treatment himself due to his injuries . the care administrator responds that this kind of task is something that certainly should be done by the home health care workers since this task is not something a care receiver could apply for help for and thus can not be classified as part of the home help service , .besides the home help service unit has no resources for these kinds of care interventions that take so much time . the meeting ends without a final decision about who should take care of the wound . during the group discussion if the patient is not mentally capable , even simple treatments may be considered as health care interventions . however , some patients are physically incapable to conduct the self - treatment themselves , as in the above observation , and the question then is which organisation is responsible for this person . to determine responsibility in these cases , the question is often asked if the person s health will deteriorate until a health care intervention is needed unless given the treatment in question . another way of determining responsibility is to decide which organisation would have had responsibility for the patient if he or she had not applied for home help service . in addition to this , it is important to note that for the care receiver , home help service may be more costly than home health care . during the group discussions , the care workers argued for clearer rules or agreements about what organisation should be responsible for which care intervention . the care workers also stated that resources are wasted when both the home help service and home health care are at the care receiver s home at the same time : when one person is making a sandwich while another person gives insulin at the same time , this does not feel as an efficient way to conduct a home visit . however , the care workers also stated that sometimes the unit chiefs or care administrators bend the rules in the best interest of the care receiver and sometimes both the home help service and home health care workers actually conduct care interventions beyond those agreed upon during the care plan meeting .", "the home help service groups from all shifts store information about the care receiver and the approved care interventions separately in non - digital form . the home health care personnel use a computerised patient record system , which is used within all primary care . however , while working in the patients homes , it is impossible to access this patient record system . to manage cooperation and coordination between the organisations , svop is an acronym for coordinated health care and care planning ( in swedish : samordnad vrd- och omsorgs planering ) . the binder is placed in the care receiver s home and consists of several documents and material which provide the care workers with information for administering home care interventions , for supporting involved individuals in daily work situations and for facilitating communication between the care workers involved . particularly important for the present study is the fact that although the current events document facilitates communication , it was rarely used since it was available only in the care receiver s home . this is unfortunate , since some of the information is needed for coordination purposes and important information may be received late , which in turn may affect the care receiver s health condition . to circumvent this problem , workers who add information to the current events document also try to reach the day - shift personnel who need the information by phone . this is further complicated by the fact that the home help service workers during dayshifts ( 10 persons ) share only two mobile phones , making it difficult for district nurses to quickly contact a specific home help service worker . for the night - shift personnel , the notes in the svop binder are the only way to stay informed . to continue , care receivers in need of multiple care interventions can trigger alarms if the need arises . the home help service responds to these alarms and is , therefore , equipped with keys . not having these keys , the home health care personnel must coordinate their visits according to the home help service s schedule . however , information about the scheduled home help service visits is placed in the svop binder , accessible only during home visits . the following observation shows one consequence of this situation : a district nurse is about to make a home visit . she knows that the home help service usually makes its home visit at a certain time and she , therefore , plans the home visit according to this . when she arrives to the patient , the door is locked and she realises that the home help service worker has not arrived yet . she makes another home visit and attempts to visit the patient later when the home help service has arrived . in addition to this , the information in the svop binder is not always up - to - date for several reasons . in particular , pharmaceutical information , updated by external primary care workers and taken from the patient s medical record , tends to be inaccurate . the following observed situation illustrates an actual consequence of this problem : a district nurse is conducting a home visit in order to insert pharmaceuticals into medical dispenser units . she takes the pharmaceuticals from the packages and in order to insert the right dosage she looks at the prescribed pharmaceuticals document in the svop binder . during this home visit the district nurse consults the prescribed pharmaceuticals document and notices that the dosage of the new pharmaceutical is inconsistent with the prescribed pharmaceuticals document . she , therefore , drives back to the reception to search for the new document . to summarise , the empirical findings clearly show that the tools used for information and communication do not fully support coordination during the home care process , thus forcing care workers to make additional coordination efforts .", "if seamless and integrated care is to be supplied , the daily work situation needs to be improved . the first issue considers the difficulties that derive from the fact that home help service and home health care are separated between two organisations . the second issue is not related to organisational boundaries , but rather to the fact that the work needs to be coordinated while the care workers are distributed across time and/or space , regardless of organisational belonging . coordinating home care interventions where home care is divided between two organisations is not a straightforward process . the present study has identified the planning activity , specifically during the care plan meeting , as a particularly problematic subject . the most crucial issue here is , who should assume responsibility for care interventions defined as self - treatment by home health care . this shows that it is difficult to demarcate between treatments considered as health care interventions and treatments that are considered as possible for the patient himself / herself to conduct . there is no established definition of self - treatment and , consequently , each case is judged separately . it is quite clear that this is primarily a policy issue that needs to be resoled at an organisational level . in 2006 , the national board of health and welfare initiated an investigation on how to define self - treatment in relation to health care and during 2008/2009 directions will be issued on how to handle this issue . another possible solution for improving the quality of home care that has been discussed is the merger of the home help service and home health care into one organisation . in 2004 , the ministry of health and social affairs investigated how medical and social care including rehabilitation and aids for disabled should be organised to ensure integrated care and service for the elderly [ 36 , p. 14 ] . the main argument of this bill was that the responsibility for the home help service and home health care should be provided by the municipalities , which would entail a merger of the home help service and home health care . the bill also stated that advanced home health care should remain the responsibility of the county council . furthermore , it is possible that other services appear that also need to cooperate and coordinate with a merged home help service / home health care team . for example , the county council has recently introduced a trial service called the mobile doctor consisting of a physician from primary care who makes home visits during daytime . thus , even if the home help service and home health care would merge , there will still be different care providers visiting elderly in their homes . this means that there always will be a need for inter - organisational cooperation and coordination . furthermore , integrating care by merely merging home health care and the home help service may not be the ultimate solution , as discussed below . the second issue considers the care workers need to coordinate their efforts not only between organisations , but also within their own organisation across space and between work shifts . however , the empirical findings show that the information in the binder is equally important for the intra - organisational coordination taking place between dayshift and nightshift . in this light , the binder supports the core aim of the cooperation and coordination between home care workers , which is the care of the care receiver . unfortunately , the present study suggests , like , that the svop binder fails to fully serve the need for effective dissemination of information and coordination during the home care process . additionally , the empirical findings also show that home help service workers lack the mobile phones necessary for communicating effectively with other care providers . providing home help service workers with mobile phones , thus , coordination during the home care process is clearly not only an organisational matter , but primarily a technical challenge . from this perspective , the most direct way of facilitating information and communication between care workers may be the introduction of it tools that improve information access and asynchronous communication . certainly , this may improve the care workers possibility to coordinate during the home care process . following the cscw research , further aspects need to be considered , explored and analysed in order to develop it tools that effectively support coordination . the ethnographic workplace study was conducted in order to understand how coordination is carried out in the daily work , to identify coordination issues in situ and possible actions that may be taken to improve coordination in the studied setting . therefore , the analysis of the common themes , activities and issues is an important activity during the research process . as in all qualitative research , the data may be influenced by the researcher s point of view a problematic issue that needs to be considered during the research process . this is even more the case when conducting ethnographic studies , since one of the objectives is to capture the members point of view . to evaluate the researcher s interpretation of the empirical findings , common themes , activities and issues have been discussed with the care workers and managers throughout the project , thus confirming the reliability of the data . in cscw , some criticism has been presented in regard to workplace studies intended to inform systems design [ 38 , 39 ] . it has been argued that the understanding of a particular workplace is a valuable contribution in its own right . it is believed that such an understanding can inform cscw design through raising awareness of important conceptual issues and questioning taken - for - granted assumptions about work activities and how they should be supported [ 38 , p. 321 the assumption in this paper is that this kind of understanding is equally important in the research area of integrated care . the need for coordination in this particular case shows that formal organisational boundaries do not describe the actual behavioural patterns and division of daily work among health care providers . the core aim in the studied setting is to provide the care receiver with good - quality care , and the actors must coordinate their efforts regardless of their organisational belonging to achieve this aim . bend the boundaries regarding the issue of self - treatment in the best interest of the care receiver . with this in mind , care workers have invented additional tools and routines to support cooperation and coordination in the workplace . integrated care is clearly much more than organisational boundaries , policies , strategies , structures and processes . in their daily work , people cooperate and coordinate their activities to get the work done , regardless of their organisational belonging . again , it tools may help to resolve some of the organizational problems faced by home care providers . in care settings , efforts have been made to develop electronic patient records , medical records and health records . these technologies were developed for a particular process or organization , and take the boundaries and structures of this process or organization into consideration . however , as shown in the present study , home care coordination often needs to transcend organizational boundaries . this further emphasises the findings of , which show that the context of elderly care at home involves coordination also with other care providers as well as the inclusion of the care receiver himself / herself and relatives . furthermore , the present study also stresses the fact that new care providers might visit the care receiver at home in the future .", "coordinating home care interventions where home care is divided between two organisations is not a straightforward process . the present study has identified the planning activity , specifically during the care plan meeting , as a particularly problematic subject . the most crucial issue here is , who should assume responsibility for care interventions defined as self - treatment by home health care . this shows that it is difficult to demarcate between treatments considered as health care interventions and treatments that are considered as possible for the patient himself / herself to conduct . there is no established definition of self - treatment and , consequently , each case is judged separately . it is quite clear that this is primarily a policy issue that needs to be resoled at an organisational level . in 2006 , the national board of health and welfare initiated an investigation on how to define self - treatment in relation to health care and during 2008/2009 directions will be issued on how to handle this issue . another possible solution for improving the quality of home care that has been discussed is the merger of the home help service and home health care into one organisation . in 2004 , the ministry of health and social affairs investigated how medical and social care including rehabilitation and aids for disabled should be organised to ensure integrated care and service for the elderly [ 36 , p. 14 the main argument of this bill was that the responsibility for the home help service and home health care should be provided by the municipalities , which would entail a merger of the home help service and home health care . the bill also stated that advanced home health care should remain the responsibility of the county council . furthermore , it is possible that other services appear that also need to cooperate and coordinate with a merged home help service / home health care team . for example , the county council has recently introduced a trial service called the mobile doctor consisting of a physician from primary care who makes home visits during daytime . thus , even if the home help service and home health care would merge , there will still be different care providers visiting elderly in their homes . this means that there always will be a need for inter - organisational cooperation and coordination . furthermore , integrating care by merely merging home health care and the home help service may not be the ultimate solution , as discussed below .", "the second issue considers the care workers need to coordinate their efforts not only between organisations , but also within their own organisation across space and between work shifts . however , the empirical findings show that the information in the binder is equally important for the intra - organisational coordination taking place between dayshift and nightshift . in this light , the binder supports the core aim of the cooperation and coordination between home care workers , which is the care of the care receiver . unfortunately , the present study suggests , like , that the svop binder fails to fully serve the need for effective dissemination of information and coordination during the home care process . additionally , the empirical findings also show that home help service workers lack the mobile phones necessary for communicating effectively with other care providers . providing home help service workers with mobile phones , thus , coordination during the home care process is clearly not only an organisational matter , but primarily a technical challenge . from this perspective , the most direct way of facilitating information and communication between care workers may be the introduction of it tools that improve information access and asynchronous communication . certainly , this may improve the care workers possibility to coordinate during the home care process . following the cscw research , further aspects need to be considered , explored and analysed in order to develop it tools that effectively support coordination .", "the ethnographic workplace study was conducted in order to understand how coordination is carried out in the daily work , to identify coordination issues in situ and possible actions that may be taken to improve coordination in the studied setting . therefore , the analysis of the common themes , activities and issues is an important activity during the research process . as in all qualitative research , the data may be influenced by the researcher s point of view a problematic issue that needs to be considered during the research process . this is even more the case when conducting ethnographic studies , since one of the objectives is to capture the members point of view . to evaluate the researcher s interpretation of the empirical findings , common themes , activities and issues have been discussed with the care workers and managers throughout the project , thus confirming the reliability of the data . in cscw , some criticism has been presented in regard to workplace studies intended to inform systems design [ 38 , 39 ] . it has been argued that the understanding of a particular workplace is a valuable contribution in its own right . it is believed that such an understanding can inform cscw design through raising awareness of important conceptual issues and questioning taken - for - granted assumptions about work activities and how they should be supported [ 38 , p. 321 the assumption in this paper is that this kind of understanding is equally important in the research area of integrated care . the need for coordination in this particular case shows that formal organisational boundaries do not describe the actual behavioural patterns and division of daily work among health care providers . the core aim in the studied setting is to provide the care receiver with good - quality care , and the actors must coordinate their efforts regardless of their organisational belonging to achieve this aim . bend the boundaries regarding the issue of self - treatment in the best interest of the care receiver . with this in mind , care workers have invented additional tools and routines to support cooperation and coordination in the workplace . integrated care is clearly much more than organisational boundaries , policies , strategies , structures and processes . in their daily work , people cooperate and coordinate their activities to get the work done , regardless of their organisational belonging . again , it tools may help to resolve some of the organizational problems faced by home care providers . in care settings , efforts have been made to develop electronic patient records , medical records and health records . these technologies were developed for a particular process or organization , and take the boundaries and structures of this process or organization into consideration . however , as shown in the present study , home care coordination often needs to transcend organizational boundaries . this further emphasises the findings of , which show that the context of elderly care at home involves coordination also with other care providers as well as the inclusion of the care receiver himself / herself and relatives . furthermore , the present study also stresses the fact that new care providers might visit the care receiver at home in the future .", "two important issues of the studied home care setting must be addressed to support seamless and integrated care : the division of care interventions that are defined as self - treatment and the coordination difficulties that arise from the fact that workers are distributed in time and/or space . the first issue concerns the fact that care is provided by two organisations and can be resolved through policymaking or through the merger of the home help service and home health care into one organisation . however , since informing and communicating between time shifts within an organisation was shown to be more difficult than between organisations , this may not help resolve the second issue . a more effective solution to this issue may be the introduction of it tools that support distributed access to information and communication between all care workers involved , regardless of organisational belonging . the findings of this paper thus suggest that integrated care needs to be developed not only between organisations but also within each organisation . in the daily work , cooperation and coordination cooperative work goes beyond organisational boundaries and integrated care must be developed with this situation in mind , beyond organisational boundaries . in view of this , a core subject for future practice and research is to develop it tools that reach beyond formal organisational boundaries and processes while remaining adaptable in view of future structure changes .", "yves couturier , mss , phd , researcher at the geriatric institute of the sherbrooke university and at the research centre for research in youth , science teaching and learning , department of social services , university of sherbrooke , sherbrooke , canada alison petch , dr . director research in practice for adults , blacklers , dartington hall trust , totnes , uk isabella scandurra , phd , medical informatics , chief science officer , apri | ehealth , sjlevad , sweden and maria hgglund , phd , medical informatics , karolinska institutet , health informatics centre , stockholm , sweden" ]

[ "\n aspergillus species are ascomycetes that are classified in the form subdivision deuteromycotina , as many of them do not show a sexual reproductive phase . generally , they are common ubiquitous saprophytes in soil and on dead organic substrates . being classic opportunistic pathogens , invasive infections by aspergillus species almost exclusively develop in immunocompromised patients , while localized infections and allergic bronchopulmonary aspergillosis occur in individuals without immunosuppression . generally , the species aspergillus fumigatus represents the most common inducer of invasive and allergic manifestations , followed by a. terreus , a. flavus , and a. niger [ 1 , 2 ] . invasive aspergillosis ( ia ) considerably contributes to the morbidity and mortality among immunocompromised individuals , including patients with haematological malignancies , recipients of haematological stem cell and solid organ transplants , aids patients , and patients treated with immunosuppressive regimens due to autoimmune diseases . the most important single risk factor is prolonged and profound neutropenia ( < 500 neutrophils/l for more than 10 days ) [ 1 , 46 ] . over the last decades , invasive fungal infections , particularly aspergillosis , have become more frequent due to a higher number of immunocompromised patients ( new chemotherapy regimens , increasing number of solid organ transplant recipients , and immunosuppressive regimens ) and extended survival time in hiv patients ( haart therapy ) [ 1 , 79 ] . on the side of the pathogen , several characteristics and various putative virulence factors that may facilitate the infection have been described for a. fumigatus . it differs from nonpathogenic species by its growth at 37c ; furthermore , it is rapidly growing and has very small conidiospores ( 35 m ) . these include melanin and a hydrophobic protein - coat layer on the surface of conidia that may help to protect them against recognition , ingestion and/or elimination by complement and phagocytes [ 1014 ] . various proteases that may help to pass tissue barriers and to degrade proteins of the immune response are secreted by the fungus [ 1517 ] , and mycotoxins like gliotoxin might also contribute to undermine the host defence [ 1820 ] . the most important path of aspergillus infections is via inhalation of the conidia into the respiratory tract . as conidia of pathogenic aspergillus species are very small , they can be inhaled deeply into the lung and even into the pulmonary alveoli . in immunocompetent individuals , conidia are effectively phagocytosed and eliminated by alveolar macrophages and infiltrating neutrophils [ 12 , 21 , 22 ] , but in the case of immunologic deficits , they are able to germinate and to penetrate the lung tissue , thus causing an invasive pulmonary aspergillosis . infections of the lung are the by far most frequent type of ia . by penetration of blood vessels , aspergillus can disseminate and invade other organs , including the heart , the liver , and the central nervous system ( cns ) . cerebral aspergillosis occurs in 10%20% of all cases of ia and thus is the most common extrapulmonary form . neuropathologic features include hemorrhagic infarcts and/or necrosis , vascular thrombosis , meningitis , granuloma , and formation of solitary as well as multiple abscesses [ 6 , 2325 ] . according to the division of bacterial and mycotic diseases ( dbmd ) , the incidence of aspergillosis is 1 - 2 per 100,000 per year . incidence rates of ia in high - risk populations depend on the respective group and rise up to 24% in patients with prolonged and profound neutropenia . furthermore , ia is the most expensive opportunistic infection in immunosuppressed patients , with annual treating costs in europe of approximately 200 million . in - hospital stays complicated by ia cause additional costs of 75,000 per patient . despite antimycotic therapy and surgical interventions , the fatality of ia is high and depends on the degree of immunosuppression and on the affected organs . without treatment , the mortality is nearly 100% , while under treatment the overall case - fatality rate is nearby 60% and rises to more than 90% in cases of cns aspergillosis [ 1 , 6 , 26 ] .", "complement consists of approximately 30 fluid - phase and membrane - bound proteins that cooperate to form the cascade . regulatory factors control and modulate its activity , and cellular receptors mediate the interaction between complement factors and immune cells . representing a potent component of the innate host defence and an interface to adaptive immunity the most outstanding roles are the direct and indirect defence against infections , the stimulation and regulation of b- and t - cell response , and the disposal of debris [ 2731 ] . hepatocytes are the main producers of complement factors ; however , several other cell types participate in the synthesis . activation of the complement system is triggered by a multiplicity of danger signals , such as pathogen - associated molecular patterns ( pamps ) , antigen / antibody complexes , and the presence of transformed cells , apoptotic cells , or cell debris . three different activation pathways start the complement cascade , all of them resulting in the cleavage of the central complement factor c3 by proteolytic enzyme complexes ( c3 convertases ) , and subsequently leading to the common terminal pathway ( figure 1 ) . in the classical pathway , binding of complement factor c1q to immunoglobulin class g or m ( igg , igm ) of antigen - antibody complexes represents the initial step . alternatively , the globular heads of c1q can interact with microbial surfaces that had been covered by pentraxins , a class of soluble pattern recognition molecules . the thereby induced conformational changes of c1q subsequently activate the associated proteases c1r and c1s , which cleave the factors c4 and c2 . the resulting fragments form the c3 convertase c4b2a . in the lectin pathway , foreign carbohydrate molecules on the surface of pathogens are recognized by mannose - binding lectin ( mbl ) or the related ficolins . mbl - associated serine proteases ( masps ) cleave c4 and c2 , and the fragments build up the c3 convertase c4b2a , which is identically equal to the one of the classical pathway . ficolin-2 can also interact with pentraxin - covered microbes , thus starting the lectin pathway in an alternative manner . interestingly , mbl was recently described to support c3 cleavage by a c2 bypass mechanism , which results in activation of the alternative pathway . the alternative pathway is triggered via activating foreign surfaces and creates an amplification loop by spontaneous reaction of c3 with h2o ( c3(h2o ) ) ; alternatively , c3b generated by the other pathways represents the starting trigger . surface - bound c3b associates with factor b , which is then cleaved by the plasma serine protease factor d. these steps result in the formation of the c3 convertase c3bbb [ 27 , 36 ] . proteolytic cleavage of c3 by one of the c3 convertases is the common and central step of all three activation pathways . this split generates the fragments c3a and c3b , which are two important components that mediate a multitude of complement functions ( see below ) . the product c3b associates with the c3 convertases , thus forming the c5 convertases , which cleave factor c5 into c5a and c5b . this step initiates a chain of assembly processes of the proteins c6 , c7 , c8 , and c9 . the bound and polymerized c9 units create the terminal complement complex ( tcc ) that can form a pore in the target lipid bilayer , called membrane attack complex ( mac ) . targeted cells , bacteria and viruses die or are inactivated by efficient disruption of the membrane integrity [ 31 , 37 ] . beneath the mac formation and direct pathogen destruction , complement displays several additional antimicrobial mechanisms aiming to neutralize invading microbes and to restore body homeostasis . surface - bound c3b undergoes internal cleavage steps ; the derived products ic3b , c3d , and further , coat and label the pathogens for phagocytosis ( opsonization ) . effector cells with specific membrane - bound complement receptors ( crs ) recognize the opsonizing complement fragments , ingest the labeled pathogens , and eliminate them . furthermore , the interaction of the opsonized particles with cr - bearing immune cells results in their activation and their increased proliferation . the receptor cr3 , a heterodimer of cd11b and cd18 , is regarded to be the most important mediator for complement - driven phagocytosis . being expressed on phagocytes like dendritic cells , neutrophils , macrophages , and microglia , it interacts with ic3b on the pathogen [ 27 , 38 ] . the complement receptors cr3 and cr4 allow adhesion of cells to cells of the same and other cell types , respectively , ( homotypic and heterotypic adhesion ) . immune cells can bind via these receptors to their ligands on endothelium of the blood vessels , a prerequisite for penetration through the vessel wall into the tissue and migration to the site of infection and inflammation . surface - bound ic3b can be further cleaved proteolytically to generate the opsonizing fragment c3d . binding of c3d - opsonised pathogens to the corresponding complement receptor cr2 ( cd35 ) on b cells induces cross - linkage with the b cell receptor complex , a process that lowers the threshold for b cell activation by the specific antigen by several orders of magnitude . cleavage of c3 and c5 generate the potent anaphylatoxins c3a and c5a , respectively , which exert several biological functions by binding to their corresponding cellular receptors c3ar , c5ar ( cd88 ) , and c5l2 . they provoke chemotactic attraction of immune cells to the site of infection and an increase of vascular permeability [ 40 , 41 ] . furthermore , c3a and c5a trigger an efficient proinflammatory response by stimulating cytokine synthesis and secretion [ 40 , 41 ] . various cell types harboring the corresponding anaphylatoxin receptors on their surface react on ligand binding with cell activation , stimulation of cell specific signaling pathways , or of oxidative burst [ 27 , 42 ] . the complement cascade needs a tight control to prevent host damage by cell / tissue lysis and excessive inflammation . a variety of both soluble and membrane - bound regulators can influence all steps of the complement cascade , with the c3/c5 convertases as main control targets [ 27 , 37 , 4345 ] . under normal conditions , these regulators should protect all body cells against auto - attack by the complement system . the serine protease factor i cleaves both c4b and c3b and is thereby supported by various cofactor molecules . c4 binding protein ( c4 bp ) and factor h ( fh ) are fluid - phase proteins that enable the cleavage of c4b and c3b , respectively , moreover , the membrane - anchored molecules complement receptor 1 ( cr1 , cd35 ) and membrane cofactor protein ( mcp , cd46 ) support the degradation of both c3b and c4b . in addition , fh and c4 bp accelerate the decay of assembled c3 convertase ; cr1 affects both c3 and c5 convertases . decay accelerating factor ( daf , cd55 ) is another notable membrane - bound regulator that efficiently prevents the assembly and promotes the disintegration of both c3 and c5 convertases [ 27 , 4345 ] . in the terminal pathway , the membrane - anchored cd59 ( protectin ) binds to c8 in the c5b-8 complex and thus inhibits further incorporation and polymerization of c9 units to form the mac [ 27 , 4345 ] . the potency of complement represents a valuable tool to attack invading pathogens and to defend the host against penetration and dissemination . one particular advantage of complement lies in the fact that activation can start within seconds after contact with the microbe and ends with a multifaceted spectrum of antimicrobial reactions . however , the fact that microbial infections occur in a considerable proportion , already implicates that the pathogens have developed appropriate counterstrategies to avoid elimination , thus starting a vicious circle of reaction and counterreaction . furthermore , the antimicrobial effector mechanisms of the complement system might also harbour harmful consequences for the affected host . as known from several infectious and noninfectious diseases , chronic or exceeding complement - mediated inflammation putative mechanisms for such complement - induced tissue damage may include a fulminant inflammatory reaction and opsonization of surrounding bystander cells with subsequent lysis .", "\n aspergillus conidia and hyphae activate the complement system via all three pathways [ 4648 ] ( figure 1 ) . initiation of the complement cascade by resting conidia is mediated predominantly by the alternative pathway . however , when the conidia begin to swell and transform into hyphae , there is a progressive involvement of the classical pathway . these differences in the activation pathways are reflected by different kinetics ; the slowest initiation is seen with resting conidia . furthermore , mbl as a pattern recognition molecule of the lectin pathway is able to bind to carbohydrate structures on the surface of aspergillus and promotes complement activation via the lectin pathway , which results in the deposition of c4 . as mentioned above , mbl can support c3 cleavage by a c2 bypass mechanism after contact with a. fumigatus conidia , resulting in activation of the alternative pathway and avoiding formation of the classical pathway c3 convertase . this mechanism is not restricted to a. fumigatus , but can also take place in the presence of a. terreus , a. niger , and a. flavus . mbl generally seems to be a molecule of high significance for innate defence against a range of pathogens . in several studies , it was shown to bind to various sugars on the surfaces of viruses , bacteria , yeasts , fungi , and protozoa [ 4851 ] . further evidence for the crucial role of mbl arises from findings in patients with chronic necrotizing pulmonary aspergillosis and mouse models of pulmonary aspergillosis [ 52 , 53 ] ; these facts suggest mbl as a promising molecule for prophylaxis and therapeutical treatment ( see below ) . a further mechanism for complement activation driven by aspergillus involves the interaction with the pattern recognition molecule pentraxin-3 ( ptx-3 ) . when a. fumigatus is opsonized with ptx-3 , the complement cascade can be activated either by interaction between ptx-3 and c1q via the classical pathway , or by interaction between ptx-3 and ficolin-2 via the lectin pathway . after seroconversion , anti - aspergillus antibodies in the serum can trigger the start of the classical complement pathway .", "the thesis that complement represents a central tool in antifungal host defence is supported by several findings . furthermore , recognition by the complement system and activation of the cascade seems to interfere with efficient dissemination in the host . this conclusion is strongly indicated by the fact that the level of complement deposition on different aspergillus species correlates inversely with their pathogenicity : highly virulent species like a. fumigatus and a. flavus bind less c3 on their surface than nonpathogenic species like a. glaucus or a. nidulans . the antimicrobial potency of the complement cascade appears to be independent from direct killing via formation of a mac ; presumably , the thick fungal cell wall block the formation of a pore by the c9 polymers and the subsequent lysis of the cells . opsonization of the fungal surface with c3-derived fragments are presumably the most relevant complement - associated weapon , stimulating efficient phagocytosis or release of damaging compounds , oxidative burst and killing by monocytes , bronchoalveolar macrophages , and polymorphonuclear cells [ 46 , 47 , 57 ] . the capacity to opsonize pathogens and to exert antifungal effects strictly depends on the available complement levels in the respective compartment of the body . the complement concentrations in the central nervous system ( cns ) are low and thus only allow a rather weak deposition . consequently , the complement amounts in the cerebrospinal fluid are unable to induce a significant oxidative burst in immune cells and to result in reduced fungal viability , thus making the cns a highly vulnerable organ . however , the brain cells react to the fungal presence with an upregulation of complement synthesis to enable better opsonization and therefore a more efficient clearance of the fungus . mice deficient in complement factor c5 can exert the early opsonization processes with c3 fragments , but are unable to fulfil the complete cascade . when infected with a. fumigatus , these mice show decreased resistance and lower 50% lethal conidia dosage for a disseminated infection [ 59 , 60 ] . since this enhanced susceptibility is unlikely to be due to absent mac formation in the fungal cell membrane , it might be supposed that the inability to form the anaphylatoxin c5a could be the relevant deficit in these mice [ 59 , 60 ] . c5a exerts a wide range of proinflammatory effects ; by binding to its receptor c5ar , c5a recruits inflammatory cells to the site of infection , enhances cellular adhesion , and stimulates oxidative metabolism . in addition , c5a can trigger the release of lysosomal enzymes and of inflammatory mediators such as tumor necrosis factor - alpha ( tnf- ) and interleukin-6 ( il-6 ) [ 61 , 62 ] . furthermore , a higher susceptibility to aspergillosis in c5-deficient mice might be attributed to missing tcc . low doses of this soluble complex were shown to bind to the membrane of a range of cell types , thereby triggering various effects like activation , rescue from apoptosis , and secretion of prostaglandins , which are important regulators of the immune response [ 6367 ] .", "the potency of aspergillus to cope with the complement system and to undermine its mechanisms for elimination determines how successful the fungus can establish an infection . aspergillus has developed a complex repertoire of effector mechanisms for this purpose ( table 1 ) . single or multiple abscess formation is a characteristic feature of aspergillosis , particularly in the central nervous system ( cns ) . the fungal hyphae are found in brain blood , vessels with invasion through vascular walls into adjacent parenchymal tissue . fungal brain abscesses may arise from these sites of localized parenchymal infection . in this case , white blood cells collect in the affected part of the brain , and fibrous tissue forms around this area , creating a mass . cns abscesses typically present with headache , focal neurological abnormalities , and/or seizure , which is the consequence of local destruction or compression of adjacent brain tissue . mature fungal abscesses exhibit a central necrotic area with fungal hyphae , surrounded by a capsule of newly formed fibrous tissue . the formation of abscesses represents a host mechanism to inhibit further spreading of invading pathogens . however , this sealing off not only inhibits fungal dissemination , but also forms some kind of protection shields against the complement attack . immunohistochemical staining revealed that effect : whereas the fibrous surrounding tissue was intensely stained for complement proteins , the central necrotic area contained only minor complement concentrations . no deposition of complement factors on the fungal surface in the abscess was visible , implying that the encapsulation protects the fungus within the abscess from any efficient complement attack . putative complement recognition sites on the conidial surface of a. fumigatus are optimally masked to minimize the stimulus for complement activation . experiments aiming to identify the relevant fungal structure indicated that melanin could play a substantial role for masking ; for this purpose , knock out mutants lacking enzymes of the melanin biosynthesis pathway were used [ 10 , 11 , 76 ] . disruption of the gene alb1 , which encodes a polyketide synthase in the synthesis of melanin , results in increased opsonization of the conidia with c3 and in a better ingestion by human neutrophils . deposition of pigments on the conidial surface might mask the c3 binding sites , and disruption of the alb1 gene might expose these sites and thus allow enhanced c3 binding . a mouse model confirms this function of alb1 in fungal pathogenesis , since the alb1-deficient mutant of a. fumigatus turned out to be less virulent than the wild - type fungus [ 11 , 76 ] . inactivation of the gene for the pigmentation protein arp1 similarly increased the deposition of c3 on conidia . this pigment seems to be a central element of aspergillus against the host defence , as it is also involved in scavenging reactive oxygen species ( ros ) and inhibits the acidification of phagolysosomes of alveolar macrophages , monocyte - derived macrophages , and human neutrophil granulocytes after ingestion of conidia [ 12 , 13 , 77 ] . as mentioned above , the activity of the complement cascade is strictly limited by several fluid - phase inhibitors . immunofluorescence analysis , adsorption assays and flow cytometry studies showed that aspergillus acquires fh , factor h - like protein 1 ( fhl-1 ) , factor h - related protein 1 ( fhr-1 ) , and c4 bp from the host [ 70 , 71 ] . fhl-1 is a splicing product of the fh gene , and fhr-1 is a related protein belonging to the fh family . bound to the conidial surface , fh maintained its regulatory activity and could act as a cofactor for the factor i - mediated cleavage of c3b . as a consequence of covering the fungal surface with these complement inhibitors , all three pathways might be downmodulated . the attachment molecules on aspergillus are not yet known , whereas the corresponding binding regions within fh were recently described . one of them was identified within n - terminal short consensus repeats ( scrs ) 1 to 7 and a second one within c - terminal scr 20 . \n a. fumigatus not only acquires complement inhibitors from the host , but also produces and releases its own soluble factor that inhibits complement activation and opsonization of the fungus [ 72 , 73 ] . this complement inhibitor ( ci ) , which is also synthesized by a. flavus , selectively abolishes activation of the alternative pathway and interferes with c3b - dependent phagocytosis and killing . the exact chemical composition of ci is as yet unknown ; it contains 15% protein and 5% polysaccharide . recent own results raise the possibility that this described ci or a closely related activity also contributes to the pathogenesis in cerebral aspergillosis , since immunohistochemical studies show deposition of c1q and c4 , but not of c3 , on the fungal hyphae in the cns . studies by sturtevant revealed the synthesis of a proteolytic enzyme that is able to degrade c3 ( , reviewed in : ) . this is confirmed by own experiments showing complement - degrading proteolysis in the supernatant of aspergillus when grown in cerebrospinal fluid ( csf ) . the fungus - induced degradation of complement in csf evoked a drastic reduction of the opsonization of the fungal hyphae . in parallel , the fungal serine protease alp1 might participate in complement degradation and thus be partly responsible for the complement evasion [ 15 , 17 ] . to date , degradation of c1q , c3 , c4 , c5 , mbl , and factor d were shown [ 15 , 17 ] .", "the high morbidity and lethality of invasive aspergillosis strongly demands for an expansion of the current treatment options . the antimycotic therapy might be completed by new approaches aiming to strengthen the host immune response against the fungus . putative approaches could be to increase the available complement concentrations or to improve the efficiency of complement attack by undermining the fungal evasion strategies . appropriate strategies that target the complement system may aim to the following . our own studies about cerebral aspergillosis showed a clear correlation between the complement levels in the csf and the capacity of csf to opsonize fungal hyphae and designate them for phagocytic killing . a therapeutic increase of mbl concentrations in invasive aspergillosis might be an appropriate approach , since patients with chronic necrotizing pulmonary aspergillosis show more frequently mbl haplotypes that encode for low levels of the protein than healthy control persons . further support comes from a murine model of invasive pulmonary aspergillosis : those mice with externally administered recombinant mbl reveal better survival , compared to untreated animals . detailed studies confirmed that mbl - treated mice show a significant increase in the levels of the proinflammatory cytokines tnf- and il-1 , together with a marked decrease of anti - inflammatory il-10 and of fungal hyphae in the lung . blocking of the fungal surface pigments by specific antibodies or peptides might be a hypothetical approach that could help to expose the c3 binding sites and thus improve complement deposition and ingestion of conidia by phagocytes . another approach might target the acquisition of the negative complement regulators fh , fhl-1 , and c4 bp to the fungal surface . for candida albicans , some molecules that bind c4b and fh have recently been identified [ 78 , 79 ] , while the attachment sites on aspergillus are still unknown but might include related molecules . blocking antibodies , designed peptides or other inhibitors against these complement regulator binding molecules might help to make the fungus more vulnerable towards complement attack . a similar approach might be developed for the aspergillus - derived complement inhibitor described by washburn [ 72 , 73 ] . \n our own results open the possibility to neutralize the fungal protease(s ) that is / are secreted by aspergillus to degrade complement proteins . two different strategies were tested by first in vitro experiments : the neutralization of the protease by specific inhibitors or interference with the production of this proteolytic enzyme . in our studies , we could prevent the complement degrading activity by serine protease inhibitors . however , a therapeutically used protease inhibitor must be highly specific , since a general block of serine proteases might be fatal for the host . alternatively , our experiments exhibited that the secretion of complement - degrading enzymes strictly depends on the availability of nitrogen sources . thus , the supply of amino acids in the infected host might downmodulate the secretion of the relevant fungal protease that cleaves the complement factors of the host .", "despite new antifungal drugs and improved medical treatment , invasive aspergillosis remains a dangerous threat for immunocompromised patients , as the innate immune defence is the most crucial weapon against this infection . the complement system is of particular importance , as it harbours multiple effects against infectious diseases , bridges the elements of the human defence network by a multitude of factors , and helps to preserve the homeostasis of the body . the presence of fungal pathogens is detected by different pattern recognition molecules ; three pathways guarantee the activation of the complement cascade by resting , swollen , and germinating conidia as well as by hyphae of aspergillus . direct lysis of fungal cells by the membrane attach complex ( mac ) appears to be of minor importance for the antifungal defence . presumably , attraction and activation of immune cells ( monocytes , pulmonary macrophages , and polymorphonuclear neutrophils ) are the most essential mechanisms . anaphylatoxins ( c3a , c5a ) chemotactically recruit immune cells to the site of the infection and induce further inflammatory reactions . opsonization of conidia and hyphae with complement fragments like c3b and ic3b mediate phagocytosis , oxidative burst , and release of damaging compounds by binding to corresponding receptors on immune cells . however , highly virulent aspergillus species have evolved mechanisms to evade the attack by complement . they hide from recognition , acquire complement regulatory molecules from the host , and secrete proteases to degrade complement factors . the multifaceted interactions between complement and aspergillus represent promising approaches for future therapeutic strategies that may help to improve the outcome of invasive aspergillosis ." ]

[ "isolation of rat brain mitochondria all procedures involving \n animals were approved by the children 's hospital of pittsburgh ( animal \n protocol number chp upmc arcc 1805 ) and were in compliance with \n principles of laboratory animal care and current laws of the united states . \n rat brain mitochondria were isolated from the cortex of adult sprague - dawley \n rats . after removal , tissue was minced and homogenized in ice - cold isolation \n buffer i , which contained 225 mm mannitol , 75 mm \n sucrose , 5 mm hepes buffer ( ph adjusted to 7.3 with koh ) , 0.1 mg / ml \n fatty acid - free bovine serum albumin , 1 mm tetrapotassium edta , and \n 12% percoll . the homogenate thus obtained was carefully layered on the top of \n a discontinuous gradient of percoll ( 24 and 42% ) prepared using the same \n buffer . the preparation was then centrifuged at 16,000 g for \n 10 min . the fraction containing the mitochondria located between 42 and 24% \n percoll was carefully withdrawn by a syringe and washed from percoll twice by \n pelleting in isolation buffer i. the resulting mitochondrial suspension was \n diluted in isolation medium ii , which was similar to isolation buffer i , \n except for the concentration of edta ( 0.1 mm ) and lack of albumin , \n and spun down at 12,000 g for 10 min . the deposit of \n mitochondria was homogenized in isolation buffer ii at a final protein \n concentration of 20 mg / ml and stored on ice until use . the protein \n concentration in the mitochondrial samples was determined using a protein \n assay kit . mitochondria prepared in this way were active for at least \n 56 h , as determined by their ability to maintain a stable transmembrane \n potential in the presence of oxidizable substrates . fluorescence measurements measurements were performed in a \n stirred cuvette mounted in a shimatzu rf-5301 spectrofluorimeter maintained at \n 37 c . mitochondria ( 0.2 mg / ml protein ) were added to 1.5 ml of the basic \n incubation medium , which contained 125 mm kcl , 5 mm \n mgcl2 , 10 mm tris , 10 mm hepes ( ph adjusted \n to a particular value between 6 and 8 with koh or hcl ) , 10 m \n egta , 5 mm succinate or 5 mm glutamate , and 5 \n mm malate as oxidizable substrates . due to a tendency of \n precipitation at alkaline ph in the presence of inorganic phosphate , \n concentration of mgcl2 was reduced to 0.5 mm . hydrogen peroxide hydrogen peroxide was measured using a2 \n m concentration of the fluorescent amplex red dye in the \n presence of 1 unit / ml horseradish peroxidase as previously described \n ( 26 ) . measurements were \n carried out at excitation / emission wavelengths of 560 nm ( slit 1.5 nm)/590 nm \n ( slit 3 nm ) , respectively . h2o2 generation was \n calibrated by constructing calibration curves using known \n h2o2 concentrations in the standard incubation buffer \n together with amplex red and horseradish peroxidase but without mitochondria \n at all ph values 68 . at acidic ph , the correction coefficients calculated from the \n titration curves for different medium ph were as follows : ph 6.0 , 0.68 ; ph \n 6.5 , 0.90 ; ph 7.0 , 1.00 ; ph 7.5 , 1.08 ; ph 8.0 , 1.10 . these correction \n coefficients were applied to compensate for a difference in \n h2o2 reporting system response when determining rates of \n ros release at a given ph . mitochondrial \n transmembrane potential ( m ) was estimated \n using fluorescence quenching of the cationic dye safranin o that is \n accumulated and quenched inside energized mitochondria . the excitation \n wavelength was 495 nm ( slit 3 nm ) and emission 586 nm ( slit 5 nm ) , and the dye \n concentration used was 2.5 m \n ( 11 ) . respiration of mitochondria respiration of mitochondria was \n measured by a clark type oxygen electrode ( hansatech co. ; oxygen electrode \n unit dw1 ) . the water - jacketed chamber was maintained at 37 c and \n constantly stirred with a magnetic stirring bar . statistical analysis was performed using student 's \n t test for comparison of two groups or one - way analysis of variance \n with bonferroni 's correction for multiple comparison implemented in prism 4.0 \n software ( graphpad software , san diego , ca ) .", "to study the ph - dependent component in the mechanism of reactive oxygen \n species generation in mitochondria , we used three types of incubation media \n with different impacts on the ph and electrical components of proton motive \n force through the inner membrane : condition 1 , the medium without permeant \n anions , such as pi ; condition 2 , the medium of condition 1 \n supplemented with pi ; condition 3 , the medium of condition 2 \n supplemented with nigericin . rat brain mitochondria respiratory characteristics measured in condition 2 \n and ph variations from 6 to 8 with succinate or glutamate plus malate as \n substrates are presented in fig . \n 1 . these data show that our mitochondrial preparations are good in \n coupling between respiration and phosphorylation in physiological range of ph . \n at ph 7 , the addition of oligomycin inhibits respiration , whereas the addition \n of adp ( state 3 ( st 3 ) ) essentially stimulates it , so that the ratio \n of respiration rates ( state 3/state oligomycin ( st 3/st \n oligo ) ) was 5.6 0.6 ( n = 3 ) for succinate and 6.5 \n 0.3 ( n = 6 ) for glutamate plus malate . at alkaline ph , adp \n stimulated respiration to a smaller degree , possibly because the lack of \n protons in the medium limits the process . the first condition provides the maximal value of ph ( maximal ph in \n mitochondrial matrix ( phin ) at a given ph of incubation medium \n ( phout ) and minimal value of . pi co - transported with h ( or in exchange to \n oh ) decreases ph ( and respectively increases ) . \n therefore , the second condition stabilizes phin at a value less \n alkaline compared with the first condition ; it provides the \n h distribution between electrical and chemical \n components similar to that in mitochondria of living cells . figure 1.ph dependence of mitochondrial respiration in state 3 \n ( triangles ) , state 4 ( squares ) , and state 4 with added \n oligomycin ( inverse triangles ) , with succinate ( a ) and \n glutamate and malate ( b ) as substrates . basic incubation medium \n ( see experimental procedures ) supplemented with 1 mm \n pi was used . statistical analysis was as follows . a , rates \n of respiration in state 3 taken at ph 8 were significantly different from that \n at ph 67 ( n = 34 ; , p < 0.05 , \n t test ) . b , rates of respiration in state 3 taken at ph 8 \n were significantly different from that at ph 67.5 ; rates at ph 7 and \n 7.5 were significantly different from each other ( n = 6 ; \n , p < 0.05 ; , p < 0.001 , \n t test ) . ph dependence of mitochondrial respiration in state 3 \n ( triangles ) , state 4 ( squares ) , and state 4 with added \n oligomycin ( inverse triangles ) , with succinate ( a ) and \n glutamate and malate ( b ) as substrates . basic incubation medium \n ( see experimental procedures ) supplemented with 1 mm \n pi was used . a , rates \n of respiration in state 3 taken at ph 8 were significantly different from that \n at ph 67 ( n = 34 ; , p < 0.05 , \n t test ) . b , rates of respiration in state 3 taken at ph 8 \n were significantly different from that at ph 67.5 ; rates at ph 7 and \n 7.5 were significantly different from each other ( n = 6 ; \n , p < 0.05 ; , p < 0.001 , \n t test ) . nigericin as an h / k exchanger adjusts ph \n according to the k gradient across the inner membrane . since \n k is the major osmogenic component , mitochondrial volume normally \n is stabilized when k concentrations inside and outside are the \n same and therefore under the third condition phin = \n phout . since the chemical component of h is \n close to zero , could reach a higher value than under the other \n conditions ( fig . 3a \n ( squares ) shows that the rate of ros production under condition 1 is \n 3 times increased when the ph of media changed from 6 to 7 , whereas the levels \n of membrane potential decreased ( fig . \n usually , high is considered to be a factor \n promoting ros production ( factor iii in the introduction ) . here the situation \n is controversial ; ros production is increased when is decreased . \n obviously , substrate and oxygen concentration ( factors ii and i ) remain the \n same for all conditions ; therefore , medium ph is the only factor correlating \n with increase of ros . at more alkaline ph ( 7.5 and 8) , 4 \n demonstrates , the loss of membrane potential can be delayed by the presence in \n the medium of the agents known to inhibit permeability transition pore \n opening ; cyclosporine a together with adp and oligomycin . this fact indicates \n that the loss of membrane potential in this condition is due to permeability \n transition pore opening . additions at the time points indicated \n by the arrows were as follows : nigericin , 100 nm ; fccp , \n 200 nm . nigericin increases membrane potential of mitochondria oxidizing \n succinate at ph 68 . additions at the time points indicated \n by the arrows were as follows : nigericin , 100 nm ; fccp , \n 200 nm . in the presence of 1 mm of pi ( condition 2 ) , which \n makes the conditions more physiological , the behavior of the analyzed system \n is similar to that under condition 1 . as \n fig . 3a \n ( triangles ) shows , the rate of ros production steeply increased upon \n increase of medium ph , reaching a maximum at ph 7.5 , while membrane potential \n ( fig . the decrease of ros production at ph 8 \n correlates with a slight decrease of respiration rate \n ( fig . 3c , \n triangles ) . when pi and nigericin were present in the medium ( condition 3 ) , \n the rates of ros production also increased along with the medium ph increase \n from 6 to 8 ( fig . 3c , \n circles ) repeated the pattern when only pi was present \n ( condition 2 ) , reaching maximum at ph 7.5 . if ph = 0 , the whole \n pattern remains qualitatively similar to that observed in the presence of \n pi ( condition 2 ) . the ph dependences presented in fig . \n 3 clearly show that ph itself , and not ph , is an important \n determinant of ros production . as can be seen , at ph ranging from 6 to 7 , when \n mitochondria do not undergo permeability transition , ros production under \n condition 1 is higher than under conditions 2 or 3 , which could indicate an \n important role of matrix ph . the difference in ros production can be seen when \n the conditions change in the course of the experiment , producing qualitatively \n the same outcome ( i.e. the addition of pi or nigericin \n always resulted in decrease of the ros generation rate despite the increase in \n ( fig . 5 , a and \n b ) because of trading the ph component of proton \n motive force to the electric one ) . the difference in ros production between the three conditions presented in \n fig . in addition to the use of \n pi and nigericin , application of the ionophores fccp and \n valinomycin can be instrumental in manipulating matrix ph to study its role in \n mitochondrial ros generation . both of these ionophores can decrease membrane \n potential ; however , protonophore fccp acidifies the matrix by increasing \n proton leak , whereas valinomycin causes increase of matrix ph by carrying \n k into the matrix , decreasing and , thus , facilitating \n the proton ejection by the electron transport chain . to stress the \n qualitatively different effects of these ionophores , we applied a small \n concentration of fccp that did not substantially change and a \n concentration of valinomycin that essentially depolarized mitochondria \n ( fig . 6b shows , fccp \n induced a dramatic decrease of ros production , wheras valinomycin temporarily \n increased it . figure 3.effect of medium ph on rate of ros release ( a ) , level of \n membrane potential ( b ) , and rates of respiration of mitochondria \n oxidizing succinate ( c ) . basic medium ( see experimental \n procedures ) was used in all experiments with different \n supplementations : condition 1 , basic medium alone ( squares ) ; \n condition 2 , basic medium with 1 mm pi \n ( triangles ) ; condition 3 , basic medium with 1 mm \n pi and 100 nm nigericin ( circles ) . statistical \n analysis was as follows . a , ros rates taken at all conditions and all \n ph values were significantly different from each other ( n = \n 310 , p < 0.05 ) , with an exception for conditions 2 and 3 at \n ph 8 ( one - way analysis of variance test ) . within the same condition , ros rates \n taken at each ph were significantly different from that at the adjacent ph \n value ( n = 310 , p < 0.05 ) , with the exceptions of \n the rates taken at ph 7.5 and 8 in condition 3 ( t test ) . b , \n the levels of membrane potential measured at ph 7.08.0 for condition 1 \n were significantly different from that in condition 2 or 3 ( n = \n 38 ; , p < 0.05 , analysis of variance test ) and \n also from the membrane potential at ph 6.06.5 for the same condition \n ( n = 3 ; + , p < 0.05 , t test ) . c , at ph \n 8.0 , rates of respiration measured in conditions 2 and 3 were significantly \n different from that in condition 1 ( n = 38 ; + , p < \n 0.05 , analysis of variance test ) . in condition 1 , rates at ph 8.0 were \n significantly different from that at all other ph values ( n = \n 78 ; , p < 0.0001 , t test ) ; in \n condition 3 , rates at ph 7.5 were significantly different from that at ph 6.0 \n and 6.5 ( n = 34 ; # , p < 0.05 , t \n test ) . effect of medium ph on rate of ros release ( a ) , level of \n membrane potential ( b ) , and rates of respiration of mitochondria \n oxidizing succinate ( c ) . basic medium ( see experimental \n procedures ) was used in all experiments with different \n supplementations : condition 1 , basic medium alone ( squares ) ; \n condition 2 , basic medium with 1 mm pi \n ( triangles ) ; condition 3 , basic medium with 1 mm \n pi and 100 nm nigericin ( circles ) . statistical \n analysis was as follows . a , ros rates taken at all conditions and all \n ph values were significantly different from each other ( n = \n 310 , p < 0.05 ) , with an exception for conditions 2 and 3 at \n ph 8 ( one - way analysis of variance test ) . within the same condition , ros rates \n taken at each ph were significantly different from that at the adjacent ph \n value ( n = 310 , p < 0.05 ) , with the exceptions of \n the rates taken at ph 7.5 and 8 in condition 3 ( t test ) . b , \n the levels of membrane potential measured at ph 7.08.0 for condition 1 \n were significantly different from that in condition 2 or 3 ( n = \n 38 ; , p < 0.05 , analysis of variance test ) and \n also from the membrane potential at ph 6.06.5 for the same condition \n ( n = 3 ; + , p < 0.05 , t test ) . c , at ph \n 8.0 , rates of respiration measured in conditions 2 and 3 were significantly \n different from that in condition 1 ( n = 38 ; + , p < \n 0.05 , analysis of variance test ) . in condition 1 , rates at ph 8.0 were \n significantly different from that at all other ph values ( n = \n 78 ; , p < 0.0001 , t test ) ; in \n condition 3 , rates at ph 7.5 were significantly different from that at ph 6.0 \n and 6.5 ( n = 34 ; # , p < 0.05 , t \n test ) . in the experiments described above , succinate was applied as a substrate . \n used in much higher concentrations than normally produced in krebs cycle , it \n stimulates high ros production rate , and this was convenient to study the \n process , although the ros production rates were much higher than could be \n observed under physiological conditions . this high concentration of succinate \n could be observed , however , at pathologies like ischemia \n ( 27 , \n 28 ) . more physiological \n substrates , glutamate and malate , produce much smaller amounts of ros \n ( 11 , \n 29 ) . however , as \n fig . 7a illustrates , \n the rates of ros release in this case also significantly increased with the \n increase of medium ph . these experiments were performed in the presence of \n both pi and nigericin to ensure the conversion of the ph \n component of proton motive force into the electric one ( condition 3 ) . \n fig . 7 , b and \n c , shows that , indeed , the addition of inorganic \n phosphate and nigericin did increase membrane potential the same way as it was \n observed with succinate as substrate . figure 4.spontaneous depolarization of mitochondria takes place in the medium \n without pi ( condition 1 ) at ph 8 . a , membrane \n potential 2 m csa , \n 200 m adp , and 2 m oligomycin were present in \n the medium before the addition of mitochondria ( curve 2 ) . spontaneous depolarization of mitochondria takes place in the medium \n without pi ( condition 1 ) at ph 8 . a , membrane \n potential ; b , ros . 2 m csa , \n 200 m adp , and 2 m oligomycin were present in \n the medium before the addition of mitochondria ( curve 2 ) . 7b ) \n and dramatically stimulated ros generation \n ( 11 , \n 29 ) . \n 8 shows that upon \n application of rotenone , regardless of depolarization , the rate of ros \n generation increased with the increase of medium ph . thus , all presented data \n show the same pattern of ros production increase in conjunction with the \n alkalization of medium .", "in a study performed recently by lambert and brand \n ( 30 ) , the authors came to the \n conclusion that ph gradient through the inner mitochondrial membrane \n determines ros production . our results clearly show that even in the absence \n of ph gradient ( figs . \n 3a ( circles ) , \n 7a , and \n 8) medium ph is an essential \n factor defining ros production by the respiratory chain , and ph increase \n itself induces the increase in ros generation these data are in agreement with \n an early study presented by turrens and boveris \n ( 31 ) , where a simpler model of \n submitochondrial particles was used . the authors have shown that the rate of \n superoxide generation increased in conjunction with an increase of medium ph \n from 7 to 9.2 in the presence of nadh or succinate as substrates and \n inhibitors rotenone or antimycin a , respectively . figure 5.inorganic phosphate ( a ) and nigericin ( b ) increase the \n value of membrane potential but decrease the rate of ros release . the same \n relative scale of fluorescence was used for both dyes . a , membrane \n potential ( traces 13 ) and ros ( traces 46 ) . \n basic incubation medium , ph 7 , was used ( see experimental \n procedures ) ; 1 mm pi was present ( traces \n 1 and 4 ) , or no pi was present ( traces 2 , \n 3 , 5 , and 6 ) . the additions of mitochondria \n ( mt ) ( all traces ) , 1 mm pi ( traces \n 3 and 6 ) and 200 nm fccp ( traces 13 ) \n were made at the time points indicated by arrows . b , basic \n incubation medium without pi was used ; ph was adjusted to 6.5 . \n trace 1 , ros ; trace 2 , membrane potential . at the time \n points indicated by arrows , the following additions were made : \n mitochondria ( all traces ) , 100 nm nigericin ( all \n traces ) , and 200 nm fccp ( trace 2 ) . inorganic phosphate ( a ) and nigericin ( b ) increase the \n value of membrane potential but decrease the rate of ros release . a , membrane \n potential ( traces 13 ) and ros ( traces 46 ) . \n basic incubation medium , ph 7 , was used ( see experimental \n procedures ) ; 1 mm pi was present ( traces \n 1 and 4 ) , or no pi was present ( traces 2 , \n 3 , 5 , and 6 ) . the additions of mitochondria \n ( mt ) ( all traces ) , 1 mm pi ( traces \n 3 and 6 ) and 200 nm fccp ( traces 13 ) \n were made at the time points indicated by arrows . b , basic \n incubation medium without pi was used ; ph was adjusted to 6.5 . \n trace 1 , ros ; trace 2 , membrane potential . at the time \n points indicated by arrows , the following additions were made : \n mitochondria ( all traces ) , 100 nm nigericin ( all \n traces ) , and 200 nm fccp ( trace 2 ) . the ph dependence of ros production could be explained mechanistically , as \n outlined in the introduction . proton binding could be a limiting step in \n semiquinone radical - ubiquinol transformation ; in this case , the decrease of \n proton concentration must stabilize semiquinone radical . ohnishi and trumpower \n ( 32 ) directly measured that \n semiquinone radical concentration in respiratory chain steeply increased with \n the increase of ph . this supports the proposed role of medium ph in \n mitochondrial ros generation by respiratory chain . figure 6.ionophores valinomycin and fccp have opposite effect on the rate of ros \n release by brain mitochondria . a , recordings of membrane \n potential basic medium without \n pi ( ph 6 ) was used . at the time points indicated by \n arrows , the following additions were made : mitochondria ( mt ) \n ( all traces ) , 20 pm valinomycin ( traces 1a and \n 1b ) , and 2.5 nm fccp ( traces 2a and 2b ) . \n concentration of k ions in the medium was 125 mm . to \n achieve a complete depolarization , ionophores valinomycin and fccp have opposite effect on the rate of ros \n release by brain mitochondria . a , recordings of membrane \n potential basic medium without \n pi ( ph 6 ) was used . at the time points indicated by \n arrows , the following additions were made : mitochondria ( mt ) \n ( all traces ) , 20 pm valinomycin ( traces 1a and \n 1b ) , and 2.5 nm fccp ( traces 2a and 2b ) . \n concentration of k ions in the medium was 125 mm . to \n achieve a complete depolarization , the experimental results of \n ( 30 ) are in line with the ones \n presented here , and we believe that they could be better interpreted in terms \n of absolute value of matrix ph . indeed , ph = phin - \n phout could be increased by two ways ( either by increasing \n phin or by decreasing phout ) with a different outcome . \n higher phin would stabilize semiquinone radical on the matrix side \n of the membrane , thus stimulating ros production , and this was observed in the \n work ( 30 ) as well as in the \n present study . however , lowering outside ph ( increase of outside proton \n concentration ) would counteract the dissociation of protons from ubiquinol and \n formation of semiquinone radical ( reaction \n 2 ) . in the latter case 3a indicates that in the same medium ph , ros production \n is different , apparently because the matrix ph is different as defined by the \n respective conditions of incubation . the decrease of matrix ph induced by the \n addition of pi and nigericin ( switch from condition 1 to condition \n 2 or 3 ) is accompanied by an increase of \n ( fig . 3b ) , which is \n expected to stimulate ros production . however , the ros generation rate \n decreased ( figs . 3a \n ( triangles and circles ) and \n 5 ) , thus indicating that in \n these circumstances the effect of ph dominates over the opposite effect of \n . figure 7.effect of medium ph on rate of ros release ( a ) and level of \n membrane potential of mitochondria oxidizing complex i substrates glutamate \n and malate ( b and c ) . basic incubation medium was \n supplemented with 1 mm pi ( a and c ) \n and 100 nm nigericin ( a ) . for traces of membrane \n potential ( b and c ) , ph was adjusted to the values indicated \n by each trace . the additions of mitochondria ( mt ) , 1 mm \n pi , 1 m rotenone , 100 nm nigericin , and \n 200 nm fccp were made at the time points indicated by the \n arrows . all data sets for ros \n rates were statistically different from each other ( n = 4 ; \n , p < 0.05 , t test ) . effect of medium ph on rate of ros release ( a ) and level of \n membrane potential of mitochondria oxidizing complex i substrates glutamate \n and malate ( b and c ) . basic incubation medium was \n supplemented with 1 mm pi ( a and c ) \n and 100 nm nigericin ( a ) . for traces of membrane \n potential ( b and c ) , the additions of mitochondria ( mt ) , 1 mm \n pi , 1 m rotenone , 100 nm nigericin , and \n 200 nm fccp were made at the time points indicated by the \n arrows . all data sets for ros \n rates were statistically different from each other ( n = 4 ; \n , p < 0.05 , t test ) . the mentioned above comparison of the three conditions could give an \n approximate quantitative estimation of ph gradient and the role of matrix ph \n in ros production . in the presence of nigericin , matrix ph is the same as \n external ph ; therefore , subtracting the ros production rate under condition 3 \n from that under two other conditions ( as indicated in \n fig . 9 ) , we can deduce the role \n of matrix ph difference in ros production . as \n fig . 9 shows , at ph 6 , the \n difference between ros production rate under the condition when pi \n and nigericin are absent ( condition 1 ) and when they are present ( condition 3 ) \n is 650 pmol / min / mg protein . the reason for this difference is different matrix \n ph , which is higher in the absence of pi and nigericin ( condition \n 1 ) . the same difference in ros production ( 650 pmol / min / mg prot ) could be \n induced by a 0.5-unit ph shift from ph 6 to 6.5 in the presence of both \n pi and nigericin ( under condition 3 ) \n ( fig . therefore , if under condition 1 matrix ph defines this \n difference in ros production , it must be not less than 6.5 when ph of the \n medium is 6 . figure 8.acidic ph inhibits rotenone - induced ros generation by rbm in the \n presence of glutamate and malate . basic incubation medium supplemented \n with 1 mm pi was used ; concentration of rotenone was 1 \n m . data sets taken at ph \n 6.0 , 6.5 , and 7.0 were significantly different from that at ph 7.5 and 8.0 \n ( n = 58 ; , p < 0.01 ; , \n p < 0.001 , t test ) . acidic ph inhibits rotenone - induced ros generation by rbm in the \n presence of glutamate and malate . basic incubation medium supplemented \n with 1 mm pi was used ; concentration of rotenone was 1 \n m . data sets taken at ph \n 6.0 , 6.5 , and 7.0 were significantly different from that at ph 7.5 and 8.0 \n ( n = 58 ; , p < 0.01 ; , \n p < 0.001 , t test ) . figure 9.difference between the rates of ros release calculated from the data \n presented in fig . \n 3 . results of subtraction of ros release rates are \n plotted versus corresponding ph of the medium . circles , \n condition 1 to condition 3 ; squares , condition 2 to condition 3 . \n values of difference between condition 1 \n and condition 3 ( circles ) at ph 6.5 and 7 were significantly \n different from that at ph 6 ( n = 310 ; , \n p < 0.01 , t test ) ; values of difference between condition \n 2 and condition 3 ( squares ) at ph 7 were significantly different from \n that at ph 8 ( n = 310 ; , p < 0.05 , \n t test ) . difference between the rates of ros release calculated from the data \n presented in fig . \n results of subtraction of ros release rates are \n plotted versus corresponding ph of the medium . circles , \n condition 1 to condition 3 ; squares , condition 2 to condition 3 . \n values of difference between condition 1 \n and condition 3 ( circles ) at ph 6.5 and 7 were significantly \n different from that at ph 6 ( n = 310 ; , \n p < 0.01 , t test ) ; values of difference between condition \n 2 and condition 3 ( squares ) at ph 7 were significantly different from \n that at ph 8 ( n = 310 ; , p < 0.05 , \n t test ) . at ph 7 , the difference between ros production under conditions 1 and 3 is \n even higher ( 1300 pmol / min / mg protein ( fig . \n 9 , circles ) ) . using the same reasoning as above , we can \n deduce that under condition 1 at ph 7 , an even higher difference between \n external and internal ph should be expected . thus , high ph must result \n in decrease , and it was measured as \n fig . moreover , so high a ph difference assumes extremely \n low intramitochondrial proton concentrations ; this could be a limiting factor \n restricting respiration rate when mitochondria are in state 3 , as demonstrated \n by fig . this indirect estimation of ph gradient by ros production is in agreement with \n measurements made by conventional methods , 0.51.4 ph units \n ( 30 , \n 33 , \n 34 ) . redox reactions in electron transport chain and proton \n translocation are fed by glycolysis and the krebs cycle , which provide nadh , \n complex i substrate , and succinate , complex ii substrate . respiratory complex \n i accepts electrons from nadh , oxidizing it to nad , and delivers \n these electrons to ubiquinone ( q ) . proton transport in complex iii is coupled \n with electron transport in accordance with the generally accepted \n ubiquinone / ubiquinol ( q / qh2 ) cycle mechanism , which is shown in \n more detail . the overall reaction performed by complex iii is as follows , \n qh2 + 2cyt cox + 2hn q + 2cyt \n cred + 4hp(i.e . it oxidizes ubiquinol , \n reducing cytochrome c and releasing two h to the \n cytosolic side ( positive or p - side ; this is reflected in the index of released \n h ) ) . in addition , it translocates two protons from matrix \n ( negative or n - side ) to cytosol . ubiquinol ( qh2 ) delivers its first \n electron to fe , releasing two protons at the p - side of the inner \n mitochondrial membrane and producing semiquinone radical ( sq ) . \n then the latter gives its unpaired electron to cytochrome \n bl , and produced ubiquinone ( q ) dissociates from the \n complex . free q binds at the n - side and receives two electrons from cytochrome \n bh , resulting from oxidation of two qh2 \n molecules , thus producing subsequently sq and qh2 , \n taking protons from the n - side . redox reactions in electron transport chain and proton \n translocation are fed by glycolysis and the krebs cycle , which provide nadh , \n complex i substrate , and succinate , complex ii substrate . respiratory complex \n i accepts electrons from nadh , oxidizing it to nad , and delivers \n these electrons to ubiquinone ( q ) . proton transport in complex iii is coupled \n with electron transport in accordance with the generally accepted \n ubiquinone / ubiquinol ( q / qh2 ) cycle mechanism , which is shown in \n more detail . the overall reaction performed by complex iii is as follows , \n qh2 + 2cyt cox + 2hn q + 2cyt \n cred + 4hp(i.e . it oxidizes ubiquinol , \n reducing cytochrome c and releasing two h to the \n cytosolic side ( positive or p - side ; this is reflected in the index of released \n h ) ) . in addition , it translocates two protons from matrix \n ( negative or n - side ) to cytosol . ubiquinol ( qh2 ) delivers its first \n electron to fe , releasing two protons at the p - side of the inner \n mitochondrial membrane and producing semiquinone radical ( sq ) . \n then the latter gives its unpaired electron to cytochrome \n bl , and produced ubiquinone ( q ) dissociates from the \n complex . free q binds at the n - side and receives two electrons from cytochrome \n bh , resulting from oxidation of two qh2 \n molecules , thus producing subsequently sq and qh2 , \n taking protons from the n - side . ros levels are physiologically important as a metabolic signal \n ( 35 , \n 36 ) . ros overproduction is one \n of the main factors inducing apoptosis \n ( 37 ) . here we show that \n mitochondrial matrix ph is one of the factors controlling ros signaling . higher matrix ph in the absence of pi and nigericin ( condition \n 1 ) at medium ph more than 7 resulted in spontaneous depolarization , probably \n due to permeability transition as figs . \n 3b ( squares ) \n and 4a illustrate . in \n these conditions , mitochondria experience dramatic perturbations with the loss \n of all gradients across the inner membrane . this resulted in a substantial \n decrease of ros release , which requires functional integrity of the \n mitochondrial membrane ( figs . \n 10 that \n sq is not produced if the electron efflux from the fes center is \n blocked . on the other hand , matrix ph was shown to be an essential factor in \n induction of permeability transition \n ( 38 ) . evidence from the \n literature indicates that the induction of permeability transition may occur \n in the presence of inorganic phosphate and/or ca \n ( 39 ) . since these compounds \n were not present in the medium of condition 1 , our results demonstrate that ph \n itself could regulate permeability transition ; this proves the conclusion of \n our previous theoretical study \n ( 40 ) . the permeability transition was not observed under conditions 2 and 3 ; \n however , the alkalization - induced increase of respiration rate when \n remained constant ( figs . \n 3c , triangles \n and circles ) indicates that ros affects membrane permeability , \n increasing leaks . in the presence of pi and nigericin ( under \n condition 3 ) , respiration increased more steeply with ph increase despite \n slower ros production ( fig . \n this could be due to the fact that \n in the presence of both pi and nigericin ( condition 3 ) \n is higher and that the leak strongly depends on . at ph 8 , \n respiration decreases ( remains unchanged ) , apparently due to the \n decrease in proton leak , since external h concentration decreases . \n steep inhibition of respiration observed in condition 1 at ph 7.5 and 8 , in \n all likelihood , is the consequence of the loss of cytochrome c in the \n course of permeability transition . the data discussed above leave no doubts that change in matrix ph is vital ; \n it controls such physiologically important processes as ros production and \n permeability transition . proton translocation coupled with \n electron transport itself can not change ph extensively , because a small amount \n of translocated protons results in high increase of electric potential , which \n stops the process . however , if proton translocation is coupled with an influx \n of some cations , such as ca or k , it could result in \n a considerable alkalization of matrix . mitochondria are also permeable for ca , and it is \n possible that ca overload of cells and , consequently , \n mitochondria could take place under some stress conditions . although proton \n leak normally is compensated by respiratory proton translocation , it could set \n a dynamic steady state with matrix less alkaline . application of uncoupler \n fccp shown in fig . 6 modulates \n this situation . in cells , the activation of ucps can down - regulate the level of ros by controlling the \n current of protons into the matrix without considerable compromise of \n mitochondrial atp production . to demonstrate the phenomenon of ph regulation of ros production by the \n respiratory chain , mitochondria were studied in the conditions of artificially \n high succinate levels . in this case , they produce ros with a higher rate than \n when respiring on physiological substrates glutamate and malate ( although with \n similar ph dependence , as figs . \n ph , \n evidently , is not the only factor defining ros production rate . if the levels \n of ubiquinol are high and , according to mass conservation , the levels of \n ubiquinone are low , the lack of electron acceptor ubiquinone results in a \n block of electron efflux from cytochrome bh and , \n subsequently , bl ( see \n fig . the levels of cytosolic side semiquinone radical and , consequently , the rate \n of ros production must be high . oxidation of succinate , available in excess , \n produces much higher levels of ubiquinol than would be produced by complex i , \n since ubiquinone reduction by respiratory complex ii is not related with \n proton translocation ( as in complex i ) and is not restricted by the proton \n electrochemical gradient . we suggest that this is the main reason why \n mitochondria , when respiring on succinate , produce much more ros than when \n they respire on complex i substrates . it should be noted that the increase of matrix ph stabilizes not only \n matrix side but also cytosolic side semiquinone . the stabilization of matrix \n side semiquinone slows down the electron transport in the chain upstream , thus \n increasing the life - time of semiquinone radical bound to cytosolic side . this \n rationale is in accordance with the finding that complex iii of mitochondria \n respiring on succinate produces substantial amounts of ros on the cytosolic \n side of the membrane ( 42 , \n 43 ) . the whole set of experiments presented here supports the proposed \n ph - dependent mechanism of ros generation in mitochondrial respiratory chain \n and its link with mitochondrial permeability transition . it allows us to \n understand the mechanism by which ucps down - regulate the level of ros in \n cells . the specific properties and quantitative data featuring mitochondrial \n ros generation could be a basis for a more detailed study aimed at exploration \n of the mechanism of ros production ." ]

[ "myositis is an acute , sub - acute , or chronic inflammation of skeletal muscles .", "a previously healthy 12-year - old boy was admitted to driscoll children s hospital in corpus christi , tx , usa , with a 9 day history of fever , and 7 day history of pain and weakness in the lower extremities . three days prior to admission , he was seen by a nurse practitioner and diagnosed with otitis media and pharyngitis . his temperature was 36.8c and the physical examination was significant for antalgic , broad - based gait and tender gastrocnemius soleus muscles . sensation and deep tendon reflexes were normal with absent romberg s and babinski s sign . the white blood cell count was 8300 cells per cubic mm with 60% neutrophils and 40% lymphocytes , hemoglobin was 13.5 g / dl , and the platelet count was 172,000 cells per cubic mm . creatinine phosphokinase ( cpk ) was elevated at 2394 /l , aspartate aminotransferase ( ast ) was 222 /l and , alanine aminotransferase ( alt ) was 143 /l . a diagnosis of myositis was made and the patient was given analgesics and oral prednisone as dermatomyositis and polymyositis were amongst the differential diagnosis . rhabdomyolysis was considered in the differential diagnosis and the patient was started on one and a half maintenance intravenous fluids . our patient s clinical course was not severe and did not develop the complications usually associated with rhabdomyolysis . on day 2 of his hospital stay , he was able to stand up with support and cpk levels came down to 1713 cerebrospinal fluid analysis showed a protein concentration of 62 mg / dl and white blood cell count of 4 . a multiplex respiratory viral pcr was positive for rhinovirus and negative for influenza a and b , parainfluenza , metapneumovirus and adenovirus . arboviral titers for west nile virus was negative and so was the rocky mountain spotted fever titers . on the day 3 of his hospitalization , serum was evaluated by use of an indirect immunofluorescence antibody ( ifa ) test kit for immunoglobulin g ( igg ) to rickettsia typhi and rickettsia rickettsii ( focus diagnostics , cypress , ca , usa ) . the complete recovery of the patient took about 48 hours which included 100 mg of doxycycline given twice daily by mouth . he was discharged home on doxycycline to complete a 10 day course and prednisone was discontinued .", "myositis can be caused by strenuous activity , muscle trauma , illicit drug injections , genetic diseases , connective tissue disorders , diabetes and infections . infectious myositis can involve single or multiple muscle groups in the limbs with the proximal muscles being predominantly affected the exception being trichinosis which commonly involves orbital muscles . signs and symptoms of myositis include fever , malaise , muscle swelling , weakness and pain that is worse with movement . with this presentation , other diseases that should be considered are polymyositis , dermatomyositis , rhabdomyolysis , guillain barre syndrome , cellulitis , osteomyelitis , deep vein thrombosis , metabolic and other inflammatory myopathies ( table 1 ) . rhabdomyolysis was considered as one of the top differential diagnosis for our patient and given intravenous fluids . it presents 1 - 2 weeks after the bite of an infected flea although a history of such exposure is rarely elicited . . the rash may not be present in 50% of the patients with murine typhus . doxycycline is the treatment of choice , which our patient was prescribed at the time of discharge . it has been known that murine typhus resolves even without treatment . to our knowledge , this is the first pediatric case reported of murine typhus associated with myositis and paraparesis .", "physicians practicing in or near rickettsia typhi - endemic areas need to consider this in the differential diagnosis of children with evidence of myositis . making this diagnosis may have a significant impact on the course of the disease because of the availability of antimicrobial therapy against rickettsial organism ." ]

[ "microarrays , once a selectively used expensive tool , have become increasingly common due to their falling costs and increased credibility over the past 10 years . in contrast to the bulk of dna sequencing , which has been taken over by large centers that automatically submit sequencing reads to centralized databases ( e.g. genbank ) , the majority of microarray expression data is still generated by smaller laboratories addressing particular biological questions . given the diversity of expression possibilities in the cell and the stochastic nature of transcription and of microarrays themselves , previous studies have found computational analysis of large sets of microarrays ( compendia ) to be a powerful means of identifying strong biological signals between genes and across conditions ( 14 ) . historically these compendia have been generated as large internally controlled projects from a single laboratory , often excluding smaller datasets from independent laboratories . yet , the many small microarray datasets generated worldwide represent a large and underutilized resource for genome - scale analyses such as compound mode of action identification ( 5 ) and network inference ( 6,7 ) . the creators of the geo database at ncbi ( 8) and the arrayexpress database at ebi ( 9 ) have sought to address this opportunity by providing a central repository of expression data for large and small laboratories . while valuable first initiatives , the geo and arrayexpress databases are not yet structured in a way that facilitates efficient exploration or analysis of the data . four main obstacles exist : first , submitting microarray datasets to repositories is more difficult than submitting sequences to genbank the data itself is more complicated , requiring submission formats that are beyond the means of many non - computational researchers . this problem has been addressed to some degree as more journals require submission of microarray data to geo or arrayexpress . the second obstacle is the presence of platform - specific biases in expression data due to the use of many different microarray platforms in a compendium . these biases obfuscate the interpretation of the integrated dataset . for dual - channel arrays , the situation is often further complicated by the lack of a single physiological reference condition used across all arrays in the platform . hence , the first step in the analysis of an array compendium is often to segregate data into sets with a uniform reference condition and a consistent array type . this time - consuming step often reduces the compendium to a far less expansive dataset . the third obstacle is the lack of uniformity in the format of expression data , even within a single expression platform . the data deposited in geo and arrayexpress does not necessarily employ a uniform preprocessing approach , nor is the raw intensity data always provided with the deposits . the fourth obstacle is the incompleteness and inconsistency in the curation of metadata describing the details of each experimental condition . each expression profile run for a given species can have a different genetic background , media , growth conditions and any number of chemicals , which might have an effect on the cell 's expression . such data is fundamental to the meaningful interpretation of expression data . even when provided , this metadata is found as unstructured prose in the database deposit or in the methods sections of each publication . ideally , this metadata would be collected in a computable format with uniform units across all laboratories . although standards like miame ( 10 ) promote the human interpretation of experimental conditions , the standard is unevenly applied and it does not facilitate computational analysis . to address the latter three of these problems , we have constructed the many microbe microarrays database ( m ) . m currently contains over 1000 microarrays for escherichia coli ( 507 ) , saccharomyces cerevisiae ( 530 ) and shewanella oneidensis ( 14 ) , all of which were collected and combined from individual investigators , geo ( 8) , arrayexpress ( 9 ) and asap ( 11 ) . to avoid problems with platform - specific biases , the expression data is uniformly normalized to enable web - based or offline ( via a database dump ) analysis without further user - dependent normalization . this facilitates analysis of the data across all laboratories and conditions , even by non - expert users . a set of web - based browsing and analysis tools is provided to facilitate efficient interrogation of the dataset without extensive computational skills . importantly , experimental metadata in m is human curated from each microarray publication converting each chemical and growth attribute into a structured and computable set of experimental features with consistent naming conventions and units . finally , all versions of the database builds are maintained and accessible in perpetuity on the website to facilitate the future interpretation and comparison of results published on m data . the various attributes of m comprehensive data and metadata , uniform normalization , access to raw data dumps , a computable structure , versioning of the database and web - based analysis tools facilitate both efficient human interrogation of the dataset and machine - based computational analysis . moreover , the consistency and uniformity of the dataset facilitates downstream comparison of results and findings based on the dataset .", "large microarray depositories like geo and arrayexpress focus on the archiving of expression data as used in specific publications . these archives play an essential role in biological science by allowing transparent replication of microarray analyses by other researchers . experimenters using the same array platform often use different normalization methods for their analyses , so that data downloaded from different projects on geo or arrayexpress are unlikely to be directly comparable . a geo dataset contains a collection of biologically and statistically comparable microarray samples processed using the same platform . unfortunately , there is a significant delay between when a sample is submitted to geo and when it is available as a geo dataset . only one - fifth of the number of samples in m were available from geo datasets ( figure 1a and b ) . \n figure 1.all of the available e. coli affymetrix antisense2 expression data for the transcription factor lexa and its known target reca were downloaded from ncbi geo profiles ( a ) and from m compendium e_coli_v3_build_1 ( b and c ) . ncbi geo profile data is derived from ncbi geo datasets that contain only a subset of the data in geo , therefore many more samples were available for plotting from m ( 445 ) than from geo ( 85 ) . the correlation between lexa and its known target was higher when the raw data was uniformly normalized with rma ( c ) rather than normalizing each microarray individually with mas5 ( a and b ) . all of the available e. coli affymetrix antisense2 expression data for the transcription factor lexa and its known target reca were downloaded from ncbi geo profiles ( a ) and from m compendium e_coli_v3_build_1 ( b and c ) . ncbi geo profile data is derived from ncbi geo datasets that contain only a subset of the data in geo , therefore many more samples were available for plotting from m ( 445 ) than from geo ( 85 ) . the correlation between lexa and its known target was higher when the raw data was uniformly normalized with rma ( c ) rather than normalizing each microarray individually with mas5 ( a and b ) . we have initially chosen to include only single - channel affymetrix microarrays in m. the photolithography process used by affymetrix allows all laboratories to start with a very consistent substrate for hybridization . in addition , the single - channel design eliminates the need for a common reference condition for all arrays . thus , in contrast to two - color array designs , data from different laboratories and projects can be integrated without artifacts due to an inconsistent reference condition . the remaining systematic biases in the affymetrix platform are due to researcher - specific differences in the rna preparation and hybridization protocols . however , when the raw probe - level microarray data ( cel files ) are normalized as a group with rma ( 12 ) , we find that these systematic researcher biases are small relative to the biological changes that occur across experimental conditions ( 7 ) . in addition , the rma normalized data tends to have higher correlation between the expression of transcription factors and their known targets ( figure 1b and c ) . to employ the rma normalization approach in m , all expression profiles for a particular array design ( e.g. the e. coli antisense 2 array ) are collected , uniformly normalized and deposited as a build. periodically , we add new expression profiles for a particular array design , renormalize all data , and release a new build. this ensures that all experiments in any build are uniformly normalized and comparable across conditions . the renormalization process may result in small changes in the expression values of all profiles . thus , all builds are labeled with a version number that references the underlying mysql schema of the database and a build number that denotes the particular set of microarray data ( e.g. e_coli_v3_build_2 uses mysql schema version 3 and is the second compendium built for e. coli ) . this system , like the build system used by the human genome assembly , allows computational researchers to specify the exact dataset used for a particular analysis .", "the experimental condition information underlying each microarray sample is the most under - utilized aspect of compendia collected from multiple disparate sources . in scientific language there are typically multiple units that can be used to describe a particular aspect of an experiment . for example , the amount of glucose added to a media can be described in weight / volume , as a percent solution , or using molarity . to promote large - scale analyses of the relationship between experimental conditions and the expression values of each gene , we provide the quantitative and qualitative features of each experimental condition cataloged in a consistent framework suitable for computation . we use human curation to convert the condition metadata in each publication into consistent units and naming conventions , and we use computer validation to provide data integrity .", "to facilitate large - scale computational analyses of compendium data , we provide bulk downloads of the normalized expression data in m. for each build , we provide separate files containing normalized data for all genes , all genes + intergenic regions , and all genes + intergenic regions + control probes . we also provide flat files containing the gene names , probe set names and curated experimental condition information . in addition , we provide the raw cel files as a tar archive for researchers interested in using or developing other normalization methods .", "for more targeted analysis and data exploration , m allows the flexible construction , visualization and download of custom datasets . users can select any subset of the experiments in m using checkboxes or by selecting projects that represent larger groups of experiments ( typically a project is the set of microarrays available in a single publication ) . similarly , users can choose a subset of genes by typing or uploading a list of gene and/or probe names . genes can also be selected by differential expression as measured by t - test , z - test or fold change ( e.g. choose all genes with a significant expression change between experiments a , c , e versus b , d , f , g as measured by a t - test with a user - chosen significance threshold ) . once a user selects a set of genes and experiments , the data can be downloaded or visualized . although , there are many existing general plotting tools and a few software visualization products dedicated to microarrays , it is often convenient to be able to choose a few conditions of interest , type in a few genes and see a quick plot of the data . m currently provides heat plots ( with and without clustering ) , expression histograms ( for individual genes and groups of genes ) , scatter plots and a genome browser for visualization of expression in a genome context ( figure 2 ) ( 13 ) . \n figure 2.custom datasets constructed on m can be visualized with scatterplots ( a ) , histograms of individual genes ( b ) , heatplots ( c ) , histograms of collections of genes ( d ) and in their genome context using a genome browser ( e ) . custom datasets constructed on m can be visualized with scatterplots ( a ) , histograms of individual genes ( b ) , heatplots ( c ) , histograms of collections of genes ( d ) and in their genome context using a genome browser ( e ) . all browsing , analytical and download features of m3d are accessed from the same page , the analysis page , on the website . this page guides the user step - by - step through the process of database selection , experiment selection , gene selection , visualization , analysis and download . at each step , a user 's selections are saved in a cookie , enabling the user to return to and modify any prior selection without losing any other selections . context - specific help is provided on each page by mousing over the [ ? ] ' symbol .", "the power of the internet resides in its interconnectivity . biological databases like ncbi ( 14 ) , ecocyc ( 15 ) and regulondb ( 16 ) provide easy linking mechanisms so that other databases can automatically generate hyperlinks to their content . m provides a simple mechanism for generating links to m genepages , which contain a basic set of plots for each gene . in addition all of the plots on m are generated using a simple url syntax so that websites can easily include plots generated by m on their own sites . for example , strong correlation is often found between the expression of a transcription factor and its targets , a website cataloging known or predicted regulatory interactions might find it useful to provide a scatter plot of the transcription factor 's expression versus its target gene 's expression to allow users to see if expression data currently supports the regulatory interaction . the url syntax for this and other plots can be found by clicking the help tab on the main menu of m.", "raw cel files are periodically collected from the arrayexpress and geo microarray databases . upon accumulation of approximately 50 new chips for a particular species , all of the old and new microarrays are normalized together into a new compendium build . for researchers preferring to submit cel files directly to m , we can generate a template submission to geo , which the researcher can then edit as desired ." ]

[ "pharmacists embedded in primary care medical practices , such as those modeled on the patient - centered medical home , are playing increasing roles in interdisciplinary primary healthcare teams.13 however , with limited pharmacist resources and high patient to pharmacist ratios , it is important to identify which patients would benefit most from consultation with a primary care pharmacist . we have previously completed a similar assessment of patient requirements for dietician services in primary care.4 while commonly used approaches to identify patients for pharmacist care include physician referrals , drug- or disease - specific programs ( eg , chronic disease management ) , and patients recently discharged from hospital , an alternative strategy is to focus on patients at increased risk of medication - related problems ( mrps).2,57 mrps are events or circumstances involving drug therapy that actually , or potentially , interfere with an optimum outcome for the patient.8,9 several studies have identified factors associated with an increased risk of mrps,1014 and short , self - administered surveys have been developed to help identify patients at risk of mrps.12,15 our objective was to determine the feasibility of using a locally adapted , self - administered , paper - based , patient survey , to identify patients at risk of mrps , in a convenience sample of patients at a single academic family medicine clinic .", "we conducted a cross - sectional pilot study based on a convenience sample of 100 completed surveys at the university of alberta hospital family medicine clinic in edmonton . at the time of the survey , seven family physicians , two family medicine resident doctors , two full time clinic nurses ( one dedicated to chronic disease management ) , and two academic clinical pharmacists ( each working ~0.2 full - time equivalent ) provided service to approximately 7,000 patients . all patients registered to be seen by one of the clinic physicians were eligible to participate in this survey , unless they were less than 18 years old , could not read english , or were not responsible for self - management of their medications . after registration at the reception desk , a medical office assistant asked eligible patients if they would be willing to participate . those who expressed interest were given an information letter and a copy of the survey as they were placed in an examination room , and asked to complete the survey while waiting to be seen by their physician . the university of alberta health research ethics board ( reb ) approved the study protocol . we used the previously validated , ten - item mrp questionnaire , developed by barenholtz levy , and the modified five - item questionnaire , developed by langford et al , to create an adapted ten - item questionnaire ( figure 1).12,15 to adapt the questionnaire for the primary care ambulatory population , several changes were made . the two most significant changes made were : 1 ) the focus of question 3 ( present in both surveys ) was changed from high risk medications to a chronic diseases focus , and 2 ) question 9 , does someone else bring any of your medications to your home for you ? was replaced ( as all patients attending the family medicine clinic were ambulatory ) with do you sometimes worry about the long - term effects of your medications ? to determine feasibility , we recorded patient - reported time to complete the questionnaire , and we solicited opinions from the medical office assistants and physicians about the impact of the questionnaire on clinic flow . to estimate the number of patients at high risk for an mrp , we calculated a summary risk score . responses , after collapsing question 3 into a single yes or no response . as done by others in the literature , we defined patients who answered yes to three or more questions as being at high risk for a mrp.11,15,16 to allow further comparisons , we did a sensitivity analysis , in which we examined the number of patients who were at risk , while restricting analysis to the five questions developed by langford et al.15", "we conducted a cross - sectional pilot study based on a convenience sample of 100 completed surveys at the university of alberta hospital family medicine clinic in edmonton . at the time of the survey , seven family physicians , two family medicine resident doctors , two full time clinic nurses ( one dedicated to chronic disease management ) , and two academic clinical pharmacists ( each working ~0.2 full - time equivalent ) provided service to approximately 7,000 patients . all patients registered to be seen by one of the clinic physicians were eligible to participate in this survey , unless they were less than 18 years old , could not read english , or were not responsible for self - management of their medications . after registration at the reception desk , a medical office assistant asked eligible patients if they would be willing to participate . those who expressed interest were given an information letter and a copy of the survey as they were placed in an examination room , and asked to complete the survey while waiting to be seen by their physician . the university of alberta health research ethics board ( reb ) approved the study protocol .", "we used the previously validated , ten - item mrp questionnaire , developed by barenholtz levy , and the modified five - item questionnaire , developed by langford et al , to create an adapted ten - item questionnaire ( figure 1).12,15 to adapt the questionnaire for the primary care ambulatory population , several changes were made . the two most significant changes made were : 1 ) the focus of question 3 ( present in both surveys ) was changed from high risk medications to a chronic diseases focus , and 2 ) question 9 , does someone else bring any of your medications to your home for you ? was replaced ( as all patients attending the family medicine clinic were ambulatory ) with do you sometimes worry about the long - term effects of your medications ? ", "to determine feasibility , we recorded patient - reported time to complete the questionnaire , and we solicited opinions from the medical office assistants and physicians about the impact of the questionnaire on clinic flow . to estimate the number of patients at high risk for an mrp , we calculated a summary risk score . responses , after collapsing question 3 into a single yes or no response . as done by others in the literature , we defined patients who answered yes to three or more questions as being at high risk for a mrp.11,15,16 to allow further comparisons , we did a sensitivity analysis , in which we examined the number of patients who were at risk , while restricting analysis to the five questions developed by langford et al.15", "demographic characteristics , summary risk scores , and time to complete the questionnaire are presented in table 1 . from patient reports in answer to the questionnaire , the median time to complete the questionnaire was 2 minutes ( interquartile range : 1.53 minutes ) . there were no comments from the medical office assistants , or physicians approached during regular staff meetings , to suggest that the questionnaire was intrusive on the clinic s workflow . the questionnaire was not distributed by medical office assistants to consecutive patients , because research was not considered to be a priority in their responsibilities , and clinical tasks sometimes intervened . forty - three respondents had three or more risk factors identified by the full ten - point questionnaire , and 26 respondents met this criterion in the sensitivity analysis . in view of the limited data , and bearing in mind that this was a pilot study , no further mining of the data was undertaken .", "the findings of this pilot study suggest that distribution of a mrp questionnaire at the point of care is feasible in a busy family medicine clinic , and that there was a strong signal for potential mrps in the sample studied . however , there may have been a selection bias introduced by the office assistants , in terms of who they approached to participate , especially when they were busy . we have identified issues relating to the efficiency of this approach ( consecutive patients were not all approached ) , and we suggest that asynchronous methods to systematically screen larger numbers of patients , such as the use of electronic medical record data , should also be explored to supplement the direct approach . this anonymous pilot study , as approved by the reb , did not permit the pharmacists to act upon the mrps identified , whereas a chart survey would allow this . this study adds to existing literature that demonstrates in practice the clinical utility of a mrp screening tool , and demonstrates its potential to generate pharmacist referrals . while our overall estimate of patients at high risk for an mrp was double that obtained by langford et al15 ( 43% versus 18% , respectively ) , our sensitivity analysis , using only langford s questions , demonstrated more similar proportions ( 26% versus 18% ) . the difference is likely related to the change in focus of one question , from high risk medications to the presence of common chronic diseases . in view of the differences between the two studies , unfortunately , comparisons with the sample studied by barenholtz levy are not possible , as they did not report summary risk scores , nor use a cut - off to define patients as being at high risk.12 perhaps one of our study s biggest strengths was its use of medical office assistant staff to screen patients , rather than using research personnel or clinic pharmacists . from a practical standpoint , part - time clinic pharmacists are not available to screen all patients attending clinics . despite this strength , since our study was an uncontrolled , single site , cross sectional survey based on a convenience sample , the time taken to achieve 100 completed surveys , as well as our estimates of the proportions of patients at high risk of mrps , may be unreliable . framing the survey as research to the office assistants ( rather than quality improvement ) may have contributed to the prolonged recruitment period ; this was also contributed to by the deliberate arms length policy of the investigators , to test the feasibility of the intervention in the practice context . volunteer bias or preselection by medical office assistants could have led to overestimation of those at high risk . finally , as demonstrated by our sensitivity analysis , the reliability of this estimate is influenced by the number of questions included in the survey , and the arbitrary nature of the cut - off points to define high risk . future study is required to compare different methods of patient identification for primary care pharmacists , determine what threshold accurately defines high risk of an mrp , and , ultimately , the effect of pharmacist intervention on risk of mrps . in conclusion , our results suggest that screening patients at the point of care , using a self - administered mrp questionnaire , was feasible , and that a large proportion of those who complete the questionnaire will have multiple risk factors for mrps . this is similar to the opportunity found in our dietician study.4 however , other strategies , including electronic medical record screening or focusing on patients attending the chronic disease management nurse , may represent complementary methods to increase screening efficiency , and allow for more systematic screening of clinic patients for risk of mrps . our findings support the integration of a pharmacist into family medicine practices , as a member of the patient - centered medical home . future work will include further content and construct validation of the revised tool and its predictive ability for measures of health care utilization . additionally , we plan to compare the predictive accuracy and feasibility of an automated electronic medical record medication list screening approach for identifying patients at high risk of mrps ." ]

[ "obesity has become a heavy public health problem in the united states , with a prevalence among adults increasing to 32% from 13% between the 1960s and 2004 . currently , 66% of adults and 16% of children and adolescents are overweight or obese . although obesity has long been recognized as an independent risk factor for cardiovascular diseases and diabetes mellitus , newer research points to obesity as an important risk factor for chronic kidney diseases ( ckds ) [ 24 ] . in 1974 , weisinger et al . subsequent studies confirmed that obesity could induce renal injury , namely , obesity - related glomerulopathy ( org ) [ 68 ] . a large - scale clinicopathologic study including 6818 renal biopsies from 1986 to 2000 revealed a progressive increase in biopsy incidence of org from 0.2% in 19861990 to 2.0% in 19962000 . the tenfold increase in incidence of org over 15 years suggests a newly emerging epidemic . the clinical characteristics of subjects with org typically manifest with nephrotic or subnephrotic proteinuria , accompanied by renal insufficiency [ 810 ] . histologically , org presents as focal segmental glomerulosclerosis ( fsgs ) and glomerular hypertrophy or glomerular hypertrophy alone and relatively decreased podocyte density and number and mild foot process fusion [ 8 , 11 , 12 ] . clinically , it is distinguished from idiopathic fsgs ( i - fsgs ) by its lower incidence of nephrotic syndrome , more benign course , and slower progression of proteinuria and renal failure [ 8 , 11 ] . potential mechanisms by which obesity affects renal physiology include altered renal hemodynamics , insulin resistance , hyperlipidemia , activation of renin - angiotensin - aldosterone system ( raas ) , inflammation , and oxidative stress . increases in both glomerular filtration rate ( gfr ) and renal plasma flow ( rpf ) were observed in obese subjects and animals [ 13 , 14 ] . this likely occurs because of afferent arteriolar dilation as a result of proximal salt reabsorption , coupled with efferent renal arteriolar vasoconstriction as a result of elevated angiotensin ii ( angii ) . these effects may contribute to hyperfiltration , glomerulomegaly , and later focal glomerulosclerosis [ 8 , 9 ] . insulin resistance can raise the transcapillary pressure gradient and cause hydrostatic pressure and hyperfiltration by reducing norepinephrine - induced efferent arteriolar constriction , leading to glomerular hypertrophy and sclerosis . hyperinsulinemia also has been shown to stimulate the synthesis of growth factors such as insulin - like growth factor- ( igf- ) 1 and igf-2 and transforming growth factor-1 ( tgf-1 ) , which accelerate production of extracellular matrix and promote glomerular hypertrophy and sclerosis [ 17 , 18 ] . hyperlipidemia may promote glomerulosclerosis through mechanisms that involve engagement of low - density lipoprotein receptors on mesangial cells , direct podocyte toxicity , oxidative cellular injury , macrophage chemotaxis , and increase renal expression of sterol regulatory element - binding proteins ( srebp-1 and srebp-2 ) , resulting in the renal accumulation of cholesterol and triglycerides and together with significant renal increase of fibrogenic cytokines [ 19 , 20 ] . obese subjects usually have increases in plasma renin activity , angiotensinogen , angiotensin - converting enzyme activity , and circulating angii , which trigger or promote renal damage by renal hemodynamic changes and nonhemodynamic pathways such as hyperinsulinemia , oxidative stress , and inflammation [ 2123 ] . inflammatory abnormalities and oxidative stress are characteristic findings of obesity and play important roles in the renal damage associated with obesity , which will be discussed in detail in the following .", "recent studies have demonstrated that obesity causes chronic low - grade systemic inflammation and thus contributes to the development of systemic metabolic dysfunction that is associated with obesity - related disorders and renal disease [ 2427 ] . levels of some inflammatory markers and cytokines such as c - reactive protein ( crp ) , tumor necrosis factor- ( tnf- ) , interleukin-6 ( il-6 ) , and macrophage migration inhibitory factor ( mif ) are elevated , whereas concentrations of adiponectin , a protein hormone that exerts anti - inflammatory activities , are reduced in obesity [ 2834 ] . leptin is a 16-kda - peptide hormone encoded by obese ( ob ) gene that is mainly produced by adipose tissue . leptin serves as a regulator of energy balance by binding to the full - length leptin receptors obese receptor b ( ob - rb ) in the hypothalamus , leading to reduction in food intake and elevation in temperature and energy expenditure . leptin receptors can be classified as secreted - forms ( ob - re ) , short - forms ( ob - ra , c , d , and f ) mainly expressed in peripheral tissue , and long - forms ( ob - rb ) predominantly expressed in hypothalamus . the kidney expresses abundant concentrations of the truncated isoform of the leptin receptor ob - ra , but only a small amount of the full - length receptor ob - rb . leptin production is associated with increased size of adipocytes and is positively correlated with the body mass index ( bmi ) . increased circulating leptin , a marker of leptin resistance , is common in obesity . obesity - induced leptin resistance injures numerous peripheral tissues including kidney , liver , myocardium , and vasculature [ 36 , 38 ] . leptin results in the development of renal disease by binding to its specific receptors in renal endothelial cells and mesangial cells . in glomerular endothelial cells , leptin stimulates cellular proliferation , tgf-1 synthesis , and type iv collagen production [ 36 , 39 ] . in mesangial cells , leptin upregulates synthesis of the tgf- type ii receptor , but not tgf-1 , and stimulates glucose transport and type i collagen production through signal transduction pathways involving phosphatidylinositol-3-kinase . however , both those cell types increase their expression of extracellular matrix in response to leptin . transgenic mice with leptin overexpression demonstrated an increase in collagen type iv and fibronectin mrna in the kidney . leptin is involved in the development of glomerulosclerosis through a paracrine tgf- pathway ( between glomerular endothelial and mesangial cells ) that promotes the deposition of extracellular matrix , proteinuria , and , eventually , glomerulosclerosis . infusion of leptin into normal rats for 3 weeks fosters the development of focal glomerulosclerosis and proteinuria . leptin also has proinflammatory actions through its interaction with mediators of innate and adaptive immunity and crp . leptin regulates components of innate and adaptive immunity , including t lymphocytes and monocytes / macrophages [ 42 , 43 ] . central leptin administration in ob / ob mice accelerates renal macrophage infiltration through the melanocortin system . leptin stimulates central t - cell production and a peripheral shift in favor of t helper ( th ) 1 adaptive immune responses ( proinflammatory ) as opposed to th2 responses ( anti - inflammatory ) . leptin has been shown to modulate adaptive immunity by enhancing t - cell survival and stimulating production of proinflammatory cytokines such as ifn- and il-2 . leptin also has structural and functional resemblance to proinflammatory cytokines , such as il-6 , and may modulate crp , a leptin - interacting protein . therefore , these direct and indirect effects of leptin on the kidney , including stimulating cellular proliferation and hypertrophy , increasing extracellular matrix expression , and exhibiting proinflammatory activities , may partially explain obesity - related kidney disease . adiponectin is a 30 kda adipocyte - derived protein hormone encoded by the adipose most abundant gene transcript 1 ( apm1 ) , which plays a role in the suppression of inflammation - associated metabolic disorders . adiponectin is highly abundant in human serum , but its level is decreased in most obese animal and human subjects , particularly in those with visceral obesity [ 4951 ] . recent clinical studies show a negative association of adiponectin in obese patients , [ 52 , 53 ] suggesting that adiponectin may play a key role in the development of obesity - related albuminuria and alteration of renal function . studies with the adiponectin knockout mouse provide evidence that adiponectin can regulate podocyte function and thus contribute to the initial development of albuminuria [ 37 , 53 ] . sharma et al . showed that knockout of adiponectin in mice increased albuminuria and caused fusion of podocyte foot processes . in cell culture studies with podocytes , ( zo-1 ) to the plasma membrane , and reduced the renal predominant nadph oxidase nox 4 , largely via a 5-amp - activated - protein kinase- ( ampk- ) dependent pathway . treatment of the adiponectin knockout mice with exogenous adiponectin was able to decrease albuminuria and improve podocyte morphology . chronic hyperadiponectinemia significantly alleviated the progression of proteinuria in early - stage diabetic nephropathy by several mechanisms . it led to an increase in nephrin expression , improvement of the endothelial dysfunction due to decreases in endothelin 1 ( et-1 ) and plasminogen activator inhibitor 1 ( pai-1 ) , and an increase in endothelial nitric oxide synthase ( enos ) expression in the renal cortex . recent studies suggest that adiponectin exerts anti - inflammatory effects by suppressing tnf--induced activation of nuclear factor-b ( nf-b ) in human aortic endothelial cells and aortic smooth muscle cells through inhibition of ib phosphorylation [ 55 , 56 ] and inhibition of vascular cell adhesion molecule 1 ( vcam-1 ) and intercellular adhesion molecule 1 ( icam-1 ) expression , thereby reducing monocyte adhesion and macrophage - induced cytokine production and crp expression in human adipose tissue . human adipose tissue expressed crp , which was negatively correlated with adiponectin expression in adipose tissue . low levels of adiponectin are associated with higher levels of highly - sensitive c - reactive protein ( hs - crp ) and il-6 , two inflammatory mediators that are involved in the initiation and progression of atherosclerosis and renal disease . therefore , hypoadiponectinemia contributes to development of a low - grade systemic chronic inflammation state , suggesting that hypoadiponectinemia may play a causative role in the systemic and vascular inflammation commonly found in obesity and obesity - related disorders , including renal injury , through its proinflammatory effects . resistin , also known as adipocyte - specific secretory factor ( adsf ) or as found in inflammatory zone ( fizz ) , is a cysteine - rich 12.5-kda polypeptide that belongs to a small family called resistin - like molecules ( relms ) [ 60 , 61 ] . in rodents , resistin is secreted from white adipocytes [ 62 , 63 ] . in human , it is produced largely by macrophages and expressed in adipose tissue predominantly by nonadipocyte resident inflammatory cells [ 6466 ] . current evidence suggests that resistin has been variably associated with obesity , insulin resistance , inflammation , and renal dysfunction . resistin levels are elevated in both genetic and diet - induced animal models of obesity [ 62 , 67 ] . studies of obese subjects have frequently noted higher serum levels of resistin as well as direct correlations between resistin level and adiposity as measured by bmi [ 68 , 69 ] . there has been a link between circulating resistin and low - grade inflammation that accompany obesity . resistin is associated with elevated crp and white blood cells , suggesting that the role of resistin may be a component of obesity - related inflammation . it has recently been found that resistin involves in the regulation of proinflammatory cytokine expression . resistin strongly upregulates il-6 and tnf- in human peripheral blood mononuclear cells ( pbmcs ) via nf-b pathway . human resistin enhanced secretion of proinflammatory cytokines , tnf- and il-12 in macrophages by nf-b - dependent pathway . studies also show that increased levels of resistin in patients with ckd are associated with declined renal function and inflammation [ 73 , 74 ] . this suggests that resistin may play an important role in obesity and obesity - associated disease by triggering the release of other proinflammatory cytokines . a growing body of evidence indicates that obesity - related glomerulopathy is associated with upregulation of inflammatory mediators . obesity leads to adipose tissue macrophage infiltration in white adipose tissue and increased levels in proinflammatory cytokines . several inflammatory mediators released from adipocytes and macrophages , such as tnf- , il-6 , il-1 , crp , monocyte chemoattractant protein 1 ( mcp-1 ) , pai-1 , and mif , contribute to a low level of chronic inflammatory state in obesity and may be responsible for renal injury in obesity - associated glomerulopathy . an emerging pattern of gene expression was observed in adipose tissue in mice fed high fat - fed diets , indicating a shift toward global upregulation of inflammatory genes , including tnf- , il-6 , and mcp-1 . tnf- , a proinflammatory cytokine , is predominantly produced by macrophages infiltrating adipose tissue [ 77 , 78 ] and can also be produced by the kidney . this cytokine is involved in the genesis of inflammation and contributes to obesity - associated insulin resistance [ 8082 ] . within the kidney , angii , advanced glycation end - products ( ages ) , and oxidized low - density lipoprotein ( ldl ) a recent study demonstrates that tnf- reduces the expression of klotho , a protein expressed by renal cells , through an nf-b - dependent mechanism , which contribute to renal injury . tnf- enhances the expression of pai-1 in human adipose tissue and plasma pai-1 levels in obesity subjects and is responsible for reduced fibrinolysis and also a component of extracellular matrix , leading to renal fibrosis and terminal renal failure [ 8890 ] . tnf- also has been shown to induce the expression of mcp-1 via p38 mitogen - activated protein kinase ( mapk ) signaling pathway in renal mesangial cells . mcp-1 , a key regulator in recruiting monocytes to the glomeruli , may also contribute to renal damage at a later stage of kidney disease in obesity . il-6 is another important proinflammatory mediator systemically secreted from adipose tissue and locally produced in the kidney . studies have demonstrated the positive relationship between bmi and plasma il-6 concentrations [ 9294 ] . studies also suggest that il-6 plays a key role in the development of renal disease . endogenous il-6 enhances the degree of renal injury , dysfunction , and inflammation caused by ischemia / reperfusion by promoting the expression of adhesion molecules and subsequent oxidative stress . transgenic knockout of il-6 ameliorates renal injury as measured by serum creatinine and histology . blocking the il-6 receptor prevents progression of proteinuria and renal lipid deposit as well as the mesangial cell proliferation associated with severe hyperlipoproteinemia . il-6 also stimulates the synthesis of crp , which is well known as both a marker and important risk factor of atherosclerosis in the general population and ckd patients . mif was initially described as an immunomodulatory factor isolated from the supernatants of t lymphocytes and was found to inhibit the random migration of macrophages . subsequent studies have indicated that mif acts as a proinflammatory cytokine and pituitary - derived hormone that potentiates endotoxemia . immunologically induced crescentic antiglomerular basement membrane glomerulonephritis , treatment with anti - mif antibody reduced proteinuria , prevented the loss of renal function , attenuated histological damage including glomerular crescent formation , inhibited renal leukocytic infiltration and activation , and reduced il-1 expression by both intrinsic kidney cells and macrophages . thus , mif is a key mediator of the inflammatory and immune response and plays a pathological role in immune - mediated renal injury .", "oxidative stress is caused by an imbalance between increased production of reactive oxygen species ( ros ) and/or reduced antioxidant activity , leading to oxidative damage to cells or tissue including lipids , proteins and dna . it is known that oxidative stress is involved in pathological processes of various diseases , such as cancer , diabetes mellitus , hypertension , and cardiovascular disease . studies have suggested that obesity is associated with increased oxidative stress [ 103 , 104 ] . analysis of oxidative markers in obesity subjects indicates that oxidative damage is associated with increased bmi and percentage of body fat [ 105 , 106 ] . conversely , parameters of antioxidant capacity are inversely related to the amount of body fat and central obesity [ 107 , 108 ] . the possible mechanisms of obesity - related oxidative stress include increased oxygen consumption and subsequent production of free radicals derived from the increase in mitochondrial respiration , diminished antioxidant capacity , fatty acid oxidation , lipid oxidizability , and cell injury causing increased rates of free radical formation [ 104 , 109 , 110 ] . it is also reported that the increase in obesity - associated oxidative stress is due to the presence of excessive adipose tissue accumulation . accumulated adipose tissue generates an immune response leading to the secretion of proinflammatory cytokines , including tnf- , il-1 , and il-6 , which lead to increased generation of ros . excessive fat accumulation also stimulates nicotinamide adenine dinucleotide phosphate ( nadph ) oxidase activity , which contributes to ros production . ros , in return , augmented the expressions of nadph oxidase ( nox ) subunits , including nox4 and pu.1 in adipocytes , establishing a vicious cycle that augments oxidative stress in white adipose tissue and blood . oxidative stress in adipocyte seems to be responsible for the low - grade proinflammatory state commonly observed in obesity [ 113 , 114 ] . oxidative stress is increasingly viewed as a major upstream component in cell - signaling cascades involved in inflammatory responses , stimulating the expression of proinflammatory cytokines . ros activate redox - sensitive transcription factors , particularly nf-b , inducing the release of proinflammatory cytokines and the expression of adhesion molecules and growth factors , including tnf- , il-6 , il-1 , tgf-1 , connective tissue growth factor , igf-1 , platelet - derived growth factor , and vcam-1 [ 115 , 116 ] . h2o2 stimulates il-4 and il-6 gene expression and cytokine secretion by an apurinic / apyrimidinic - endonuclease / redox - factor-1- ( ape / ref-1- ) dependent pathway . ros increased the expression levels of pai-1 , il-6 , and mcp-1 through nadph oxidase pathway . oxidized high - density lipoprotein ( hdl ) enhances proinflammatory properties such as tnf- and mcp-1 in renal mesangial cells partly via cd36 and ldl receptor-1 and via mapk and nf-b pathways . oxidized ldl can stimulate tnf- synthesis from renal cells and initiate local effects of renal damage . increased ros production and mcp-1 secretion from accumulated fat may cause infiltration of macrophages and inflammation in adipose tissue of obesity . moreover , enhanced macrophage migration induces the release of proinflammatory cytokines , which further stimulates the generation of ros [ 119 , 120 ] . therefore , oxidative stress - induced cytokine production is likely to further increase oxidative stress levels , setting a vicious cycle that may promote the progression of kidney damage in obesity . oxidative stress has been commonly identified in obesity - related renal diseases and may be the mechanism underlying the initiation or progression of renal injury in obesity [ 122 , 123 ] . previous studies suggest that oxidative stress triggers , at an early age , the onset of kidney lesions and functional impairment in zucker obese ( zo ) fa / fa rats , a good model of obesity - related renal disease , in absence of hyperglycaemia , hypertension , and inflammation . ros are highly reactive molecules that oxidize lipids and proteins , cause cellular injury , and promote glomerular and renal tubule injury and associated proteinuria . ros are produced by various cells , such as vascular cells , inflammatory cells , and renal cells , and have distinct function on different types of cells , such as endothelial dysfunction , inflammatory gene expression , and renal tubule ion transport . a major source for vascular and renal ros is a nox family of nonphagocytic nad(p)h oxidases , including the prototypic nox2 homolog - based nad(p)h oxidase , as well as other nad(p)h oxidases , such as nox1 and nox4 . numerous reports indicate that within the kidney , nad(p)h oxidase , an enzyme that produces superoxide ( o2 ) by transferring electrons from nadh / nadph to molecular oxygen and thereby forming o2 , h , and nad / nadp , is capable of modulating renal epithelial ion transport [ 130 , 131 ] . nap(d)h oxidase - derived ros can alter renal pressure natriuresis and blood pressure regulation through its effects on renal hemodynamics and renal tubular sodium transport . recent data suggest that nadph oxidase - mediated oxidative injury to the proximal tubule , like that seen in the glomerulus , contributes to proteinuria in insulin - resistant states . oxidative stress also plays an important role in the pathogenesis of renal damage through its effects on vascular biology . ros are generated by all types of vascular cells , including endothelial , smooth muscle , and adventitial cells . ros influence vascular cell growth , migration , proliferation , and activation [ 132 , 133 ] . physiologically , ros can mediate cellular function , receptor signals , and immune responses on vascular cells . in pathophysiological condition , ros contribute to progressive vascular dysfunction and remodeling through oxidative damage caused by decreased nitric oxide ( no ) bioavailability , impaired endothelium - dependent vasodilatation and endothelial cell growth , apoptosis or anoikis , endothelial cell migration , and activation of adhesion molecules and inflammatory reactions [ 134 , 135 ] .", "obesity accelerates the progression of renal injury , associated with augmented inflammation in adipose and kidney tissues . studies in il-6 transgenic mice suggested that high concentrations of il-6 contribute to development of renal injury . treatment with anti - il-6 receptor antibody mr16 - 1 prevented progression of proteinuria , renal lipid deposit , and the mesangial cell proliferation in hypercholesterolemia - induced renal injury . inhibition of tnf- by etanercept , a tnf- antagonist , also decreased blood pressure and protected the kidney through reduction of renal nf-b , oxidative stress , and inflammation . tnf- blockade increases renal cyp2c23 expression and slows the progression of renal damage in salt - sensitive hypertension . tnf- inhibition also reduces renal injury in deoxycorticosterone - acetate- ( doca- ) salt hypertensive rats via suppression of renal cortical nf-b activity . treatment of adiponectin - knockout mice with adenovirus - mediated adiponectin results in amelioration of albuminuria , glomerular hypertrophy , and tubulointerstitial fibrosis and reduces the elevated levels of vcam-1 , mcp-1 , tnf- , tgf-1 , collagen type i / iii , and nadph oxidase components . excessive fat accumulation contributes to macrophage infiltration in adipose tissue and increased production of proinflammatory cytokines , such as tnf- , il-8 , and il-6 [ 142145 ] . consequently , it is possible that weight loss may be a potential method to reduce inflammation . evidence indicates that weight loss induced by nutritional intervention or gastric surgery markedly improves the systemic and adipose tissue inflammatory states linked to obesity [ 143 , 146 , 147 ] . studies by gene profiling analysis have shown that caloric restriction - induced weight reduction leads to the regulation of a wide variety of inflammation - related molecules in human adipose tissue . weight loss globally improves the inflammatory profile of obese subjects through a decrease of proinflammatory factors and an increase of anti - inflammatory molecules in white adipose tissue . roux - en - y - gastric - bypass- ( rygb- ) induced weight loss has been shown to reduce mcp-1 , il-18 , il-6 , and tnf- concentrations [ 149 , 150 ] . a longer - term weight reduction induced by rygb in corpulence also prevails in regulating circulating cytokine concentrations . weight loss also ameliorates the low - grade inflammation state that leads to glomerular dysfunction in obesity . in morbidly obese individuals with glomerular hyperfiltration , weight loss by surgical interventions normalizes glomerular filtration rate ( gfr ) and reduces blood pressure and microalbuminuria . weight loss improves renal function as shown by reduced levels of serum creatinine and improved creatinine clearance . since ros play a key role in the pathogenesis of renal injury such as glomerulosclerosis and tubulointerstitial fibrosis , approaches to reduce oxidative stress by antioxidants supplementation , nutritional and surgical interventions may have renoprotective effects . garcinia protects against obesity - induced nephropathy by attenuating oxidative stress through reduced lipid peroxidation and levels of oxidized ldl . the obese zucker rat is a good model for studying obesity - related kidney disease because it develops proteinuria , glomerular hypertrophy , and focal segmental glomerulosclerosis [ 155157 ] . using these rats , it has been demonstrated that nephropathy is associated with oxidative stress , and supplementation with an antioxidant ebselen improved kidney damage by ameliorating proteinuria and renal focal and segmental sclerosis . chronic ebselen therapy also improved vasculopathy with lipid deposits , tubulointerstitial scarring , and inflammation . for example , administration of grape seed proanthocyanidin extract ( gspe ) , an efficient phytochemical antioxidant , can protect against the nephrotoxicity effects induced by cisplatin and gentamicin [ 159 , 160 ] and reverse experimental myoglobinuric acute renal failure . quercetin , a flavonoid that exhibits antioxidant properties in many diseases , could also protect the rat kidney against lead - induced injury and improve renal function . thus , antioxidants may be a potential therapeutic to prevent the renal damage in org . nutritional and surgical interventions are additional approaches to reduce oxidative stress and prevent kidney injury in obesity . caloric restriction and protein restriction reduce free radicals and ros formation and inhibit accumulation of oxidative biomarkers in animal models . in genetically obese animals , diet restriction can prevent or greatly delay the onset of specific degenerative lesions , in particular glomerulonephritis associated with obesity . since adipose tissue mass in obesity contributes to oxidative stress , bariatric surgery - induced weight loss also results in decreasing systemic oxidative stress in adiposity . weight loss induced by diet restriction or bariatric surgery not only improves inflammation state but also reduces oxidative stress state in obesity , which may protect renal function in obesity - related glomerulopathy .", "obesity causes chronic low - grade inflammation and systemic and local oxidative stress , which may play a pivotal role in the initiation or progression of obesity - associated glomerulopathy . elevated inflammation in obesity is the result of the production of adipokines and increased inflammatory cytokines and decreased anti - inflammatory factors . oxidative stress is triggered by an imbalance between increased production of ros and/or reduced antioxidant activity . both inflammation and oxidative stress induce damage to renal tubule and glomerulus and result in endothelial dysfunction in the kidney . therefore , anti - inflammation and antioxidant interventions may be the potential therapies to prevent and treat obesity - related renal diseases ." ]

[ "electropolymerization was performed using a homemade electrochemical cell coupled with a high performance potentiostat galvanostat ( pgstat128n series ; metrohm - autolab b.v . , all chemicals and reagents were of high performance liquid chromatography ( hplc ) or analytical grade . pyrrole ( 99 % ) , thiophene ( 99 % ) , and tetrabutylammonium perchlorate ( 98 % ) were purchased from sigma - aldrich ( schnelldorf , germany ) . medical steel wires ( ni cr , = 750 m ) were purchased from b. braun surgical s.a . polypyrrole ( ppy ) and polythiophene ( pth ) coatings were synthesized by electropolymerization with a linear sweep voltammetry technique . the three - electrode cell was filled with electrolyte solution consisting of 100 mm pyrrole / thiophene ( fig . 4 ) and 250 mm tetrabutylammonium perchlorate in acetonitrile.fig . 4chemical structures of ppy ( a ) and pth ( b ) spme fiber coatings chemical structures of ppy ( a ) and pth ( b ) spme fiber coatings polymerization was performed using medical steel wires as working electrodes . an ag / ag electrode was applied as a reference electrode , with platinum net bent into a cylinder as a counterelectrode . for ppy , a potential range from 0.2 to + 2.5 v and seven scans second , an additional 14 scans in the higher potential range from 0.2 to 2.7 v were used ( fig . 5electropolymerization of pyrrole ( a ) and thiophene ( b ) electropolymerization of pyrrole ( a ) and thiophene ( b ) the morphologies of the polypyrrole and polythiophene fibers were investigated using a scanning electron microscope ( leo 1430vp ; carl zeiss smt , oberkochen , germany ) coupled with a backscattered electron ( bse ) detector . small angle x - ray scattering ( saxs ) data were collected using a nanostar system ( bruker axs gmbh , karlsruhe , germany ) with pinhole collimation and a two - dimensional hi star detector with resolution of 1,024 1,024 pixels , mounted on an x - ray tube with a copper anode and equipped with crossed gbel focusing mirrors . samples were mounted between two mica windows in sample holders with thickness of 1 or 2 mm . the sample - to - detector distance was 650 mm , and the exposure time for a single frame was 10,000 s. each sample was measured three times , and the obtained data were averaged . the saxs data were recorded within the scattering vector range of 0.15 nm < s < 3.5 nm ( where s = 4sin/ , 2 is the scattering angle , and is the x - ray wavelength ) . the saxs data were corrected for the detector response and normalized to the intensity of the incident beam , and the background scattering ( empty holder ) was subtracted using the saxs_nt v4.1 program package ( bruker axs gmbh , karlsruhe , germany ) .", "electropolymerization was performed using a homemade electrochemical cell coupled with a high performance potentiostat galvanostat ( pgstat128n series ; metrohm - autolab b.v . , all chemicals and reagents were of high performance liquid chromatography ( hplc ) or analytical grade . pyrrole ( 99 % ) , thiophene ( 99 % ) , and tetrabutylammonium perchlorate ( 98 % ) were purchased from sigma - aldrich ( schnelldorf , germany ) . medical steel wires ( ni cr , = 750 m ) were purchased from b. braun surgical s.a . polypyrrole ( ppy ) and polythiophene ( pth ) coatings were synthesized by electropolymerization with a linear sweep voltammetry technique . the three - electrode cell was filled with electrolyte solution consisting of 100 mm pyrrole / thiophene ( fig . 4 ) and 250 mm tetrabutylammonium perchlorate in acetonitrile.fig . 4chemical structures of ppy ( a ) and pth ( b ) spme fiber coatings chemical structures of ppy ( a ) and pth ( b ) spme fiber coatings polymerization was performed using medical steel wires as working electrodes . an ag / ag electrode was applied as a reference electrode , with platinum net bent into a cylinder as a counterelectrode . for ppy , a potential range from 0.2 to + 2.5 v and seven scans second , an additional 14 scans in the higher potential range from 0.2 to 2.7 v were used ( fig . 5electropolymerization of pyrrole ( a ) and thiophene ( b ) electropolymerization of pyrrole ( a ) and thiophene ( b )", "the morphologies of the polypyrrole and polythiophene fibers were investigated using a scanning electron microscope ( leo 1430vp ; carl zeiss smt , oberkochen , germany ) coupled with a backscattered electron ( bse ) detector .", "small angle x - ray scattering ( saxs ) data were collected using a nanostar system ( bruker axs gmbh , karlsruhe , germany ) with pinhole collimation and a two - dimensional hi star detector with resolution of 1,024 1,024 pixels , mounted on an x - ray tube with a copper anode and equipped with crossed gbel focusing mirrors . samples were mounted between two mica windows in sample holders with thickness of 1 or 2 mm . the sample - to - detector distance was 650 mm , and the exposure time for a single frame was 10,000 s. each sample was measured three times , and the obtained data were averaged . the saxs data were recorded within the scattering vector range of 0.15 nm < s < 3.5 nm ( where s = 4sin/ , 2 is the scattering angle , and is the x - ray wavelength ) . the saxs data were corrected for the detector response and normalized to the intensity of the incident beam , and the background scattering ( empty holder ) was subtracted using the saxs_nt v4.1 program package ( bruker axs gmbh , karlsruhe , germany ) ." ]

[ "the maxillary anterior teeth should be positioned as close as possible to the positions originally occupied by natural teeth to achieve a natural appearance in making complete dentures.1 it is necessary to refer to anatomic landmarks to achieve this goal.2 the incisive papilla is a small , pear shaped eminence composed of a pad of fibrous connective tissue overlying bony exit of nasopalatine blood vessels - nerves and one of the significant anatomical landmark in locating maxillary anterior central incisors position in complete denture fabrication procedure . harper3 stated that the incisive papilla was stable that was obtained by caliper measurements on pre - extraction and post - resorption models of the same cases over seven years . although the shape and the localization of the papilla shows a wide range of variation,58 the centre of the papilla is commonly used as a reference point in denture construction and studies.24,915 when artificial teeth is set in proper positions , which were determined from the incisive papilla , the foundation is correctly laid for natural speech , pleasing appearances and normal function.3 the horizontal relationship between incisive papilla and maxillary central incisors has been already investigated by several authors . harper3 suggested that the incisal edges of the maxillary central incisors should be 5 to 8 mm at the horizontal direction in front of the centre of the papilla . ortman and tsao6 stated that the most anterior part of the maxillary central incisors and the posterior of the incisive papilla was 12.45 mm . many studies showed that incisal edges of the maxillary central incisors were positioned 8 to 10 mm in front of the centre of the incisive papilla.2,4,68,1020 another anatomical landmark that have been suggested in complete denture construction is pterygomaxillary notch - incisive papilla plane.21 fu et al22 found that the pterygomaxillary notch - incisive papilla plane tends to be more parallel to the occlusal plane that uses mesio - labial incisal edge of maxillary right central incisor as anterior reference point and mesio - buccal cusp tips of maxillary first or second molars as posterior reference point . although , several studies investigated the horizontal relationship between incisive papilla and the maxillary central incisors,24,10,11,16,17,20,21,2325 the authors did not find specific information in the literature relative to vertical distance between the incisive papilla and the maxillary central incisors . thus , the aim of the present study was to determine the vertical distance between the maxillary central incisors and centre of the incisive papilla .", "dental student volunteers from suleyman demirel university faculty of dentistry were solicited by a written announcement to participate in the study . the inclusion criteria were : no restoration and tooth loss on the upper jaw , well - arranged maxillary anterior teeth , no orthodontic treatment , angle class i maxillomandibular relationship , no pathology that affects the dentition or the surface texture and shape of teeth , no incisal wear on the dentition and no gummy smile in the subjects . the volunteers were examined by one of the investigators of the study and one hundred volunteers ( 58 women , 42 men ) were selected from the students that have the inclusion criteria with a draw . the ages of the subjects were ranged from 19 to 22 years , with a mean age of 20.371.1 years . upper jaw impressions were taken on the subjects by using stock impression trays ( teknik dental rostfrei , istanbul , turkey ) and irreversible hydrocolloid impression material ( tulip , cavex holland , haarlem , holland ) . in order to correctly register the incisive papilla and reduce soft tissue distortion the stone casts were obtained using ada type iii dental stone ( gilidur , fachbereich dental , ludwigshafen , germany ) . each stone cast was trimmed following the same procedures to produce a flat base that was parallel to the occlusal plane ( anterior reference point : mesio - labial incisal edge of upper right central incisors ; posterior reference points : mesio - buccal cusp tips of upper first molars ) . two mm standard transparent resin plate and a spirit level ( veto , istanbul , turkey ) were used to achieve the parallelism of the described occlusal plane to the base of the stone casts . the centre of the incisive papilla was found measuring the planar distance between the most anterior and posterior borders of the incisive papilla that was on the median line of the palate using a digital caliper which had a precision level at 0.01 mm ( mitutoyo corporation , tokyo , japan ) ( figure 1 ) and halving this distance . the midpoints of the incisive papilla were marked on each stone cast ( figure 2 ) . a digital caliper that was fixed perpendicular to the horizontal bar of a surveyor ( rotaks dent a.s . , istanbul , turkey ) was used as a measurement instrument ( figure 3 ) . stone casts were mounted to the surveyor by the help of a spirit level to obtain a certain level in the horizontal plane . the midpoint of the flat edge panel of the depth caliper was marked with graphite . the flat edge plane of the depth caliper had contacted to the mesial incisal edges of the maxillary central incisors and the midpoint of the flat edge plane of the depth caliper was matched with the midpoint of the maxillary central incisors ( figure 4 ) . the pin of the depth caliper was touched to the midpoint mark of the incisive papilla . the mounted casts were moved by sliding and the first measurement accepted as a starting point . the measurements were done by two investigators using the same instrument at two separate times . the average values of two investigators measurements were calculated and analyzed with spss 13.0 for windows ( spss inc . ,", "the kappa scores for the assessment of intra and inter observer agreement were higher than 0.75 which implies substantial agreement between the observers ( tables 1 and 2 ) . the mean vertical distance between maxillary central incisors and midpoint of the incisive papilla on the stone casts was 6.700.81 mm .", "a common average vertical position of the maxillary anterior teeth to the constant landmarks has been found by measuring lots of casts of natural healthy teeth . it is important to orient the stone casts in a standardized manner during measurements so that the results achieved can be applied when artificial teeth are being set in complete denture bases . the occlusal plane is a common reference for mounting stone casts and in general , occlusal plane done parallel to the bases of stone cast in standardize manner . fu et al22 found the pterygomaxillary notch - incisive - papilla plane ( hip ) tends to be parallel to the natural occlusal plane that defined as anterior reference point was mesio - labial incisal edge of upper right central incisor and posterior reference points were mesio - buccal cusp tips of upper first or second molars . in the present study , a standard transparent resin sheet was positioned in contact with the maxillary right central incisors and the mesio - buccal cusp tips of first molars to orient the stone cast to the standardized occlusal plane . this occlusal plane orientation was used , for it is more comparable to the pterygomaxillary notch - incisive - papilla occlusal plane.22 harper3 stated that the incisive papilla is a dependable basis for reproducing the horizontal and vertical position of the maxillary central incisors . watt9 stated that the anterior portion of the maxilla undergoes extensive resorptive changes following tooth removal and the anatomic location of the incisive papilla can be used only as a guide in the setting of denture teeth . grave16 suggested that using the border of the incisive papilla as the part likely to be least affected by the changes in anatomy of the region . in the previous studies , the midpoint of the incisive papilla has been used as a reference point for tooth arrangement . most of the previous studies used the midpoint of the incisive papilla as a reference point.2,4,1015,25,26 the midpoint of the incisive papilla has also been used as a reference point for tooth arrangement and occlusion rims in contemporary dental practice . pterygomaxillary notch - incisive - papilla occlusal plane also uses the centre of the incisive papilla as anterior reference point.22 for these reasons ; the centre of the incisive papilla was used as a reference point for measurements in the present study . ortman and tsao6 stated that the most anterior part of the maxillary central incisors and the posterior of the incisive papilla was 12.45 mm . the measurement methods of all these studies were in 2-dimensional , however park et al28 found similar results ( 11.961.37 ) in their 3-dimensional measurement on a virtual model between the maxillary anterior teeth and incisive papilla . the results of these studies that used 2 and 3 dimensional measuring methods show that a sensitive measuring method is reliable and can be repeatable . chalsuthipan and boonsiri29 investigated the relationship between the incisive papilla , maxillary central incisors and canines in thai population . the authors found that the vertical distance from the most distal point of the incisive papilla to incisal edge of the central incisors was ranged from 6.94 to 7.23 mm with the mean of 7.08 mm.29 the present study supports the findings of chalsuthipan and boonsiri.29 the mean value difference between chalsuthipan and boonsiri s study29 and the present study was 0.38 mm . the mean value variation may be caused by the reference point differences on the incisive papilla because the authors used the most posterior border of the incisive papilla . the most posterior part of incisive papilla is the most far from the occlusal plane and maxillary central incisors . on the anterior region of the maxilla , the vertical relationship between incisive papilla and central incisors can be used in determination of occlusal plane with the reference of other anatomical landmarks together ; however the clinical applicability of this relationship in complete denture construction needs further investigation .", "this study investigated the vertical distance between maxillary central incisors and centre of incisive papilla . the dentist and laboratory technician can be use the results of this study as a guideline in fabrication of maxillary occlusal rims and in determination of the position of the occlusal plane with the reference of other anatomical landmarks together . however , the wax rim should be modified intraorally to incorporate individual characteristics , and the anterior tooth should be arranged on modified wax rim . also , further investigations are needed in different age , gender , race , dental and skeletal morphology groups ." ]

[ "in recent years , nanoparticles \n have been used increasingly for \n biomedical applications , including drug or gene delivery , imaging , \n sensing , or photothermal therapy . in particular , gold nanoparticles ( nps ) have been suggested as highly \n useful sensitizing agents in phototherapy due to their unique size \n and shape - dependent optical properties , high absorption coefficients , \n ease of synthesis , biocompatibility , and their ability to hold a variety \n of functional ligands . it is well - known that citrate - stabilized gold nps \n are endocytosed by cells and remain in intracellular vesicles . moreover , targeting of specific sites inside \n the cell by functionalization of the surface with cell penetrating \n peptides or peptides containing nuclear localization sequences has \n been reported . alternatively , to selectively \n target specific types of cells , gold nps can be modified with suitable \n antibodies . this approach has been studied for potential use in photothermal \n cancer therapy , where cancer cells overexpressing human epidermal \n growth factor receptor 2 ( her2 ) or epithelial growth factor receptor \n ( egfr ) were incubated with gold nps conjugated to anti - her2 or anti - egfr antibodies , respectively . \n the nps were then irradiated with light within their plasmon resonance \n absorbance band to heat the cells to temperatures leading to cell \n death . most interestingly , it has also been demonstrated \n that endocytosis \n of gold nps by cancer cells and subsequent irradiation of such intracellular \n nps can lead to cell death even at irradiation levels that are not \n high enough to cause significant heating . this nonthermal \n route to laser - induced cell death has been ascribed to an as yet not \n fully characterized photochemical reaction , although irradiation of \n endocytosed nps was reported to be accompanied by increased levels \n of reactive oxygen species . the use of such a photochemical \n mechanism could be of great advantage in situations where different \n types of cells coexist in close vicinity , since it would allow for \n more selective targeting of particular cells , whereas due to \n the fast diffusion of heat over the relevant length scales photothermally \n induced cell death will affect all cells within the irradiated volume \n more or less indiscriminately , as long as some of them contain nps . irradiation of gold nps with continuous wave ( cw ) lamp or laser \n light has been shown to lead to photogeneration of singlet oxygen \n ( o2 ) in vitro , suggesting that this highly reactive species , which is widely used \n in photodynamic therapy , may be involved in \n the photochemical pathway of cell killing by gold nps . in \n vitroo2 photogeneration by irradiation \n of spherical gold nps with short laser pulses or cw \n laser light at comparable powers and intensities has also been reported . in this study , it was suggested that the mechanism \n of singlet oxygen photogeneration may involve hot \n electrons , i.e. , the highly excited conduction band electrons which \n upon absorption of a short laser pulse by a np can reach quasi - equilibrated \n energy distributions corresponding to temperatures of several thousand \n degrees . this seems somewhat surprising , since cw \n light at the intensities used does not yield hot here we present new experimental results on the photogeneration \n of singlet oxygen by irradiation of gold nanoparticles with continuous or pulsed laser light , as well as theoretical work pertaining \n to the underlying mechanism(s ) , which so far had not been addressed . \n we show that electron temperatures in excess of 2000 c are easily \n achieved in pulsed laser irradiation experiments , whereas cw light \n under similar conditions yields electronic temperatures of at most \n 10 c above room temperature . thus , the photogeneration of o2 by gold nps proceeds by different mechanisms \n under different irradiation conditions ; the implications for the further \n development of medical applications of the effect are discussed in \n detail . furthermore , we also found that even a moderately thick , but \n dense , ligand layer significantly reduces the efficiency of o2 photogeneration at the np surface , which also has important \n consequences for practical applications .", "citrate - stabilized spherical \n gold nps with 15 and 46 nm diameter were prepared according to the \n turkevich frens and a seeded growth method , respectively . gold nanorods ( nrods ) were synthesized \n using the seeded - growth method reported by dickerson et al . with slight modifications . more details of the \n np preparation are provided in the supporting information . nps were characterized using uv vis spectroscopy \n ( genesys 10 uv ) , differential centrifugal sedimentation ( cps instruments \n dc24000 ) , and transmission electron microscopy ( tem , fei tecnai spirit \n microscope at 120 kv ) ( see figures s1 and s2 in the supporting information ) . the uv vis absorbance spectrum \n of nrods showed a transverse plasmon resonance band at 522 nm , and \n a longitudinal plasmon resonance band at 798 nm and tem revealed nrods \n to have a length of 40 nm and a diameter of 12 nm . functionalization \n with thiolated peg ligands or peptides was achieved by overnight incubation \n with excess ligand , followed by repeated centrifugation for excess \n ligand removal . the capping ligands used here were peg - oh ( hs-(ch2)11-(eg)4-oh ) , mpeg5000 ( hs-(ch2)2-(eg)n - o - ch3 , average mw 5000 g mol ) and peptide c - tat ( primary \n sequence calnnagrkkrrqrrr ) ; see figure s3 for the structures of the peg ligands . o2 was \n detected via the bleaching of 1,3-diphenylisobenzofuran ( dpbf ) , \n which is widely used for this purpose . all experiments involving dpbf \n were carried out in the dark . because dpbf is not soluble in neat \n water , all experiments were conducted \n in 50/50 ( v / v ) mixtures of water and ethanol . a fresh solution of \n dpbf ( 3.1 mg , 0.115 mm , a412 nm = 2 ) in etoh ( 100 ml ) was kept stirring in the dark . a 10 mm quartz \n cuvette and a 3 mm stirrer bar were left in aqua regia ( 1:3 hno3:hcl ) for 15 min and thoroughly rinsed multiple times with \n milli - q water ( mq h2o ) and etoh . in the clean cuvette with \n stirrer bar , either mq h2o ( 600 l ) or np solution \n ( 600 l ) was mixed with the ethanolic dye solution ( 600 l ) . \n where appropriate , the np concentration was adjusted prior to mixing \n to yield an absorbance of 0.4 at 532 nm in the final solution . the \n cuvette was sealed with an airtight lid and parafilm , and the uv vis \n absorbance spectrum was recorded ( genesys 10 uv ) . the sample was then \n placed on a stirring plate ( in the dark ) , and its absorbance spectrum \n measured every 10 min for 30 min to ensure the solution was stable . for the irradiation experiments , the cuvette containing 1200 l \n of sample solution was placed on a stirring plate in front of the \n laser . for experiments with nps , the cuvette was fitted with heat \n fins using non - silicone heat transfer paste and cooled using a fan . \n no significant increase of the cuvette temperature beyond a slight \n warming was observed . for most experiments , the sample was irradiated \n at 1000 mw ( unless stated otherwise ) using a 532 nm continuous - wave \n diode pumped solid state laser ( laser quantum opus 532 ) with a 1/e beam diameter of 1.85 mm ; for some experiments \n the beam was expanded to 8 mm diameter using a lens , as stated explicitly . \n the sample was irradiated for 10 min and then removed from the laser \n setup to record the absorbance spectrum ; this was repeated until the \n sample had been irradiated for 60 min in total . irradiation of gold \n nrods in the longitudinal plasmon resonance band at 800 nm was performed \n in the same setup , but using a titanium : sapphire laser ( coherent mira \n 900 ) , aligned on the auxiliary cavity , which prevents mode - locking \n and thus provides continuous - wave laser operation at 800 nm with 1000 \n mw power ; the beam was expanded to 3.4 mm using two lenses . pulsed \n laser irradiation was performed in the same setup , but using the second \n harmonic of a q - switched nd : yag laser ( quantel brilliant ) ( 532 nm , \n 5 ns pulse length , 10 hz repetition rate , 3.5 mm beam size , 15 mj \n pulse energy ) . for experiments that included nps , the absorbance \n of dpbf at 412 \n nm was calculated by subtracting the np absorbance at 412 nm ( obtained \n from neat np samples ) from the measured sample absorbance . the concentration \n of dpbf in the samples always yielded an initial absorbance very close \n to 1 at 412 nm ; for comparative data analysis , the irradiation - time - dependent \n dpbf absorbance at 412 nm was therefore normalized to 1 at zero irradiation \n time .", "1,3-diphenylisobenzofuran \n ( dpbf ) \n readily undergoes a 1,4-cycloaddition on reaction with o2 to form endoperoxides which irreversibly yield 1,2-dibenzoylbenzene . \n dpbf strongly absorbs light at 412 nm ( figure 1a ) , but due to the loss of the -system \n of isobenzofuran , the product does not absorb light at this wavelength ; \n it is this loss of absorbance that is used to detect the presence \n of singlet oxygen . here , dpbf was chosen \n as singlet oxygen sensor as it has no absorbance at 532 or 800 nm , \n the wavelengths used for laser irradiation of gold nanoparticles in \n their plasmon resonance bands , and no photobleaching of dpbf was expected \n to occur upon irradiation in the absence of nps . contrary to this \n expectation , some bleaching was observed under our cw irradiation \n conditions , with the dpbf absorbance decreasing by ca . 10% upon irradiation \n at 1 w ( 37 w cm ) for 60 min ( figure 1a ) . as shown in figure 2a , and described in more detail \n in the supporting information , this photobleaching \n of dpbf has two phases : ( i ) a rapid phase , extending over the first \n 1020 min of irradiation under the conditions used here , and \n ( ii ) a slower phase , which on the time scale investigated appears \n linear with time . we found that phase i depends on laser power , beam \n size , and the presence of oxygen , whereas the slope of the time dependent \n bleaching after 20 min , phase ii , is essentially independent of the \n presence of oxygen and of the beam power / intensity in the range used \n here ( see figure s4 for details ) . it should \n be noted that similar effects have been reported previously , with \n cw irradiation at 514 nm for 60 min at significantly lower powers \n ( 40 mw ) than employed here , leading to a ca . 5% decrease of the absorbance \n of dpbf in benzene , although no explanation was suggested in that \n report . the oxygen dependence of phase \n i is in agreement with direct photogeneration of singlet oxygen by \n visible light , which has been suggested to be the reason for dpbf \n photobleaching upon irradiation with light at wavelengths above 470 \n nm . however , the slower phase ii is not \n affected by removing oxygen from the solution ( see figure s4b and figure 3 ) and hence can not be ascribed to singlet oxygen formation . \n at the present moment , the mechanism of both phases of dpbf photobleaching \n remains unclear , although both effects are highly reproducible and \n bleaching does not occur when the sample is not exposed to light ( see figure s4a ) . photobleaching of the dpbf absorbance \n upon cw irradiation at 532 \n nm , 1 w ( 37 w cm ) , in a 50/50 ( v / v ) mixture of \n water and ethanol : ( a ) in the absence of nps and ( b ) in the presence \n of 15 nm citrate - stabilized spherical gold nps . shown are absorbance \n spectra taken at intervals of 10 min from before the irradiation up \n to a maximum irradiation time of 60 min ; the arrows indicate the direction \n of change . ( a ) time dependence of the photobleaching of \n the dpbf absorbance \n at 412 nm , a412(dpbf ) , upon cw irradiation \n at 532 nm , 1 w ( 37 w cm ) , in the absence and presence \n of citrate - stabilized spherical nps with 15 and 46 nm diameter ; shown \n here are the results from several individual experiments ( dashed lines ) \n and the average ( solid lines ) , after subtraction of the np absorbance \n and normalization to 1 at time zero ; see the experimental \n section for details of data treatment and analysis . ( b ) effect \n of the nps alone , calculated by subtracting the photobleaching effect \n of dpbf in the absence of nps from the results obtained in the presence \n of nps ; the solid lines in ( b ) are linear fits of the data in the \n range 2060 min . gradient of the time - dependent dpbf absorbance photobleaching in \n the irradiation time window 2060 min for different samples \n under cw irradiation at 532 nm , 1 w ( 37 w cm ) . \n for experiments in the presence of nps , the np concentration was adjusted \n to yield an absorbance of 0.4 at 532 nm . experiments for dpbf in the \n absence of nps and in the presence of 15 nm spherical nps were also \n undertaken after bubbling the sample with nitrogen for 10 min , as \n indicated ( + n2 ) . the error bars correspond to the standard deviation \n of several repeat experiments , and indicates \n statistically significant differences with respect to the experiment \n on dpbf only , as determined by the anova f - test at p < 0.001 ; it should be noted that the results for 15 or 46 nm \n spherical nps without nitrogen bubbling or a peg capping layer were \n found to be different to all other results at this statistical significance \n level . no repeat experiment was undertaken for irradiation of nanorods \n at 532 nm , but the same result ( no additional bleaching in the presence \n of nanorods ) was obtained for irradiation at 800 nm ( figure s5 ) . in the presence \n of citrate - stabilized spherical gold nps , the photobleaching \n of dpbf upon irradiation at 532 nm , i.e. , within the nanoparticle \n plasmon resonance band , is significantly increased ( figure 1b ) . the time dependence of \n the dpbf photobleaching in the absence of nps and in the presence \n of nps with 15 and 46 nm diameter is shown in figure 2a . it can be clearly seen that the \n presence of nps leads to a significant increase of the dpbf photobleaching \n effect and that larger nps yield a larger effect although the np absorbance \n was adjusted to be the same for all samples , so that the same amount \n of light was absorbed . the additional photobleaching of dpbf caused \n by the presence of nps can be clearly ascribed to the generation of \n reactive oxygen species ( ros ) , since purging of the samples with nitrogen \n removes this effect ( see below ) . moreover , previous experiments had \n shown that light irradiation of citrate - stabilized spherical gold \n nps leads to the characteristic luminescence of o2 at 1280 nm and does not result \n in the generation of superoxide , o2 , \n or hydroxyl , oh , radicals , which leads \n us to conclude that the predominant ros species produced here is singlet \n oxygen , o2 . the fact that significant \n dpbf photobleaching occurs even in the absence of nps requires careful \n consideration for the analysis of these data . like dpbf photobleaching \n in the absence of nps , the effect in the presence of nps shows two \n phases . however , subtraction of the effect observed when only the \n dye is present yields an essentially linear time dependence for the \n additional effect ascribed to the nps on the time scale of the experiment \n ( figure 2b ) , indicating \n that the np - induced photogeneration of o2 is \n essentially constant over our experimental time interval . moreover , \n the second phase of direct dpbf photobleaching is largely independent \n of the laser power , at least down to 0.1 w , i.e. , significantly lower \n powers than those used for most of the experiments reported here , \n as shown in detail in the supporting information ( figure s4a ) , and therefore should be independent of the presence \n of nps whose absorbance leads to a decrease of the laser power along \n the beam path , allowing a direct comparison of the results obtained \n in the time frame of 2060 min . moreover , this slower phase \n of direct dpbf photobleaching is also independent of the presence \n of oxygen . for these reasons , only data starting at 20 min irradiation \n will be used for quantitative comparisons . figure 3 summarizes \n the main results obtained here after np photoexcitation at 532 nm . \n it is very obvious that citrate - stabilized spherical gold nps induce \n significantly faster dpbf photobleaching than is observed in their \n absence ( black ) ; this is found for 15 nm gold nanoparticles ( red ) \n but is even more pronounced for larger nps with 46 nm diameter ( blue ) . \n increasing the size of the spherical nps from 15 to 46 nm increases \n the np - induced dpbf photobleaching ( i.e. , the additional effect , after \n subtracting the direct dpbf photobleaching effect ) by ca . it is interesting to note \n that unlike the direct dpbf photobleaching , which is not affected \n by nitrogen purging , the np - induced additional bleaching is significantly \n reduced by nitrogen purging , almost down to the level of the direct \n dpbf photobleaching effect . this strongly supports the conclusion \n that the np - induced effect is caused by the formation of o2 which then leads to dpbf photobleaching . furthermore , \n capping the 15 nm gold nps with peg - oh , a moderately large ligand \n ( figure s3 ) , essentially removes the np - induced \n bleaching and reduces the observed effect to the level of the direct \n dpbf photobleaching . gold nanorods ( nrods ) with a length of \n 40 nm and a diameter of \n 12 nm , which have two plasmon resonance bands , namely the transverse \n band at 522 nm and the longitudinal one at 798 nm ( figure s1a ) , stabilized with a capping layer consisting of \n a mixture of a peg polymer ( mpeg5000 ) and a peptide ( c - tat ) ( see the experimental section ) , were also investigated . irradiation \n at 532 nm , i.e. , in the transverse plasmon resonance band , showed \n no additional dpbf bleaching above the direct dpbf effect ( figure 3 ) . irradiation with \n 1 w ( 11 w cm ) cw laser power at 800 nm , i.e. , \n in the longitudinal plasmon resonance band ( figure s5 ) , showed reduced direct dpbf photobleaching compared to \n irradiation at 532 nm , but again no additional photobleaching was \n observed in the presence of nrods . these results show that the nrods \n used here , which have a ligand layer consisting of a mixture of a \n peg polymer and a peptide , do not induce the formation of o2 upon laser irradiation , independent of the plasmon \n resonance which is photoexcited . irradiation at 532 nm in the \n presence and absence of citrate - stabilized \n spherical gold nps with 15 nm diameter was also investigated using \n laser pulses with a pulse duration of 5 ns ( figure 4 ) . in this case , the uv vis spectra \n show a slight broadening of the np plasmon resonance band at 520 nm \n during the first 5 min of irradiation , suggesting that some aggregation \n occurs ; after the initial 5 min , however , the nps remain stable . analysis \n of the spectra shows that photobleaching of dpbf in the absence of \n gold nps is similar to the results obtained using cw laser irradiation \n at comparable average powers ( figure s4a ) . in the presence of gold nps with 15 nm diameter , on the other \n hand , the np - induced bleaching effect ( i.e. , the effect remaining \n after subtracting the direct dpbf photobleaching effect ) which is \n induced by pulsed laser irradiation is larger than that caused by \n cw irradiation by almost 1 order of magnitude , in spite of the significantly \n lower laser power employed during the pulsed irradiation experiments \n ( 0.15 w vs 1 w ) , which leads to a correspondingly lower number of \n absorbed photons ; compare figure 4b ( pulsed irradiation ) with figure 3 ( cw irradiation ) . photobleaching of the \n dpbf absorbance upon laser irradiation with \n 5 ns laser pulses at 532 nm , 0.15 w , 10 hz repetition rate ( corresponding \n to a power density of 1.5 w cm and a pulse energy \n density of 0.15 j cm ) . ( a ) absorbance spectra \n in the presence of citrate - stabilized spherical gold nps with 15 nm \n diameter , taken at intervals of 5 min from before the irradiation \n ( gray ) up to a maximum irradiation time of 30 min ; the arrow indicates \n the direction of change . ( b ) gradient of the time - dependent dpbf photobleaching \n ( measured at 412 nm ) in the irradiation time window of 2030 \n min in the absence and presence of nps . it is straightforward to estimate the quantum yield of np - induced \n dpbf photobleaching from the absorbed laser power and the observed \n rate of absorbance bleach . for the citrate - stabilized spherical nps \n with 15 and 46 nm diameter under cw irradiation at 532 nm , this yields \n values of 5 10 and 8 10 , respectively . thus , less than one of each 1 million photons absorbed \n by a np leads to the photobleaching of a dpbf molecule under cw irradiation . \n for irradiation of 15 nm nps with 5 ns laser pulses , on the other \n hand , the quantum yield of np - induced dpbf photobleaching is 3.5 \n 10 , i.e. , almost 2 orders of magnitude larger \n than for cw irradiation , but still very small . diffusion of \n oxygen over the lifetime of o2 ( approximately 6 s in 50/50 water / ethanol ) covers a distance larger than the average distance \n between dpbf molecules at the concentrations used here , and hence \n it can be concluded that a significant fraction of the photogenerated o2 which escapes from the np surface should be \n detected . 10 m , which is close to the \n concentration used here , is sufficient to detect 50% of photogenerated o2 . thus , the observed \n low quantum yield of dpbf photobleaching indicates a very low quantum \n yield of o2 photogeneration by gold nps ( i.e. , \n number of o2 generated for each photon absorbed ) , \n having values of the order of ca . 10 for cw irradiation \n and ca . 10 for irradiation with nanosecond laser \n pulses . our results show that irradiation of citrate - stabilized \n spherical gold nps at 532 nm , i.e. , in their surface plasmon band , \n with pulsed or cw laser light leads to the production of o2 , detected here by monitoring the bleaching of dpbf \n absorbance . however , the rate of o2 production \n is much larger when using short laser pulses than when using cw light \n of comparable intensity . whereas ca . 24% of the dye is bleached after \n only 10 min of irradiation of 15 nm nps with 5 ns laser pulses at \n an average power of 150 mw ( figure 4 ) , only 12% of the dye is bleached over this \n time by irradiation of the same nps with cw light at significantly \n higher power ( 1 w ) ( figure 2 ) . this difference allows one to draw important conclusions \n on the mechanism of o2 production by irradiation \n of gold nanoparticles . irradiation \n of spherical gold nps at 532 nm leads to the excitation of their plasmon \n resonance , which can be described as a coherent oscillatory motion \n of the conduction band electrons ; this oscillation dephases and decays \n on the sub-100 fs time scale , with only a very small radiative contribution , \n so that most of the excitation energy is retained as electronic excitation \n in the form of electron hole pairs . since the photon energy \n is close to the minimum energy required for direct excitation of d - band \n electrons into the conduction band of gold , a minor contribution of \n this excitation mechanism can not be ruled out ; however , d - band holes \n are filled by conduction band electrons on the 10 fs time scale , yielding \n essentially the same outcome as excitation of the plasmon resonance \n band . the excited electrons initially \n have a nonthermal energy distribution , and often are referred to as \n primary hot electrons , although the concept of temperature \n does not strictly apply to such a distribution . they rapidly ( within \n less than 500 fs ) equilibrate by electron electron scattering \n to yield a fermi distribution corresponding to an elevated temperature \n and can then be referred to as hot electrons . thiol bond dissociation \n at the surface of gold nps and have been \n suggested to be responsible for the creation of o2 by irradiation of gold nps with nanosecond laser pulses . they lose their energy on the time scale of \n a few picoseconds by interaction with the lattice ( electron phonon \n scattering ) with coupling times that are essentially size - independent \n for nps above 10 nm in diameter , although they strongly depend on \n the amount of energy deposited due to the temperature - dependent electronic \n heat capacity . because the lattice heat capacity \n is much larger than the electronic heat capacity , this leaves the \n electrons and the lattice in equilibrium at a temperature which is \n significantly lower than the initial electronic temperature ; finally , \n cooling occurs by heat transfer to the solvent and heat diffusion \n on the time scale of 10 to a few 100 ps , strongly depending on np \n size . since the dissipation of \n the absorbed energy proceeds on the picosecond time scale , excitation \n with a nanosecond laser pulse yields a highly nonequilibrium situation \n during the duration of the laser pulse where energy is continuously \n deposited into the electronic system and at the same time flows through \n the lattice into the surrounding solvent . it is therefore not straightforward \n to predict the temperature of the hot electrons achieved in such experiments . \n we used the two - temperature model for the electron \n and phonon heat baths coupled to finite - element heat transfer \n and diffusion simulations in the surrounding solvent to estimate this \n temperature ; details of these simulations are given in the supporting information . as shown in figure 5 , under our conditions \n the electrons are expected to reach a temperature of 2100 c , \n whereas the lattice reaches temperatures of the order of 1400 c \n and the solvent in the immediate vicinity of the np a temperature \n of about 900 c . time - dependent temperatures of the conduction band electrons \n ( red ) , \n lattice ( black ) , and first solvent layer ( blue ) , calculated for our \n experiments using nanosecond - laser pulse excitation ( 15 nm spherical \n nps in 50/50 etoh / water , 5 ns laser pulses with 0.15 j cm intensity , solid lines ) and for the experiments described in ref ( 24 ) ( 40 nm spherical nps in \n 80/20 etoh / water , 7 ns laser pulses with 0.03 j cm intensity , dashed lines ) using the two - temperature model \n for the electron and phonon heat baths , coupled to finite - element \n heat transfer and diffusion simulations in the surrounding solvent \n ( see supporting information for details ) ; \n time zero corresponds to the center of the laser pulse . a lattice temperature of 1400 c , which is \n above the melting \n temperature for solid gold , albeit present for only a few nanoseconds , \n might be sufficient to cause temporary melting of the nanoparticle , although the pulse intensities used here are \n still below the reported threshold for size reduction of gold nps \n of 15 nm diameter by nanosecond laser pulses . nevertheless , a minor effect on the shape and/or size of the nps , \n especially at the upper end of the size distribution , can not be ruled \n out and may be related to the minor np spectral changes observed upon \n irradiation which suggest some aggregation to occur ( see above ) . because \n of the slower heat dissipation around larger nps , these are heated \n to higher temperatures and hence are more likely to fragment , in agreement \n with experimental results on nanosecond - laser pulse induced fragmentation , \n which also show that fragmentation is finished after 5 min under conditions \n similar to the ones used here . since \n our nps were prepared without excess citrate , the resulting increase \n in the ratio of surface area to volume may indeed cause some aggregation . \n it also can not be ruled out that the solvent near the np surface temporarily \n forms bubbles , although the pulse intensities \n used here are still below the reported threshold for bubble formation \n by nanosecond pulses for 15 nm nps . such \n bubbles could lead to better thermal insulation , thus potentially \n increasing the maximum temperatures , but on the other hand , they might \n prevent oxygen from reaching the np surface , thus reducing the chance \n of o2 formation . the formation of o2 from the triplet ground state by interaction with a \n photoexcited sensitizer requires a change of the electron spin and \n hence can not result from dipole dipole ( frster ) interaction , \n but only from dexter - type electron exchange coupling ; the latter can \n be described as simultaneous transfers of an electron from one of \n the 2 * molecular orbitals on oxygen to a photogenerated hole \n on the sensitizer and of an electron with opposite spin from a high - energy \n excited sensitizer level to the same or the other 2 * orbital , \n resulting in the formation of the or singlet oxygen state , respectively . this mechanism requires significant overlap of the relevant electronic \n wave functions and hence only occurs at short distances of at most \n 10 . since the photoexcited hot electrons have such a short \n lifetime , this reaction can only occur if an oxygen molecule happens \n to be in the vicinity of the np or is temporarily adsorbed to its \n surface at the moment of excitation . a similar reaction occurs on \n photoexcited si nanocrystals , although in this case \n the reaction is more efficient than for au nps due to the much longer \n lifetime of the photoexcited excitons in si . thus , the short lifetime \n of hot electron excitation in gold nanoparticles easily explains the \n low quantum yield of o2 photogeneration which \n is observed here . the excitation of electrons to temperatures \n exceeding 2000 c means that a significant number have sufficient \n energy to excite an oxygen molecule to the state , \n which has an energy of 0.98 ev above the ground \n state ( see figure 6 ) . the number of hot electrons available \n at the higher energy and the number of holes available at the \n lower energy involved in this two - electron exchange reaction can \n be estimated from the density of states of gold and the fermi distribution , \n as described in more detail in the supporting information . for example , for a spherical np with 15 nm diameter \n at an electron temperature of 2100 c , there are 260 electrons \n within an energy interval of 0.1 ev around the \n state . ( it should be noted that the relevant energy interval is the \n width of this state for an oxygen in the vicinity or temporarily adsorbed \n onto a np , which is not known , so only relative numbers will be used \n here . ) thus , the electron temperatures achieved in our experiment \n are sufficient for a significant population of hot electrons and holes \n at the relevant levels . schematic diagram showing the population probability f(e ) for a np electron state at energy e near the fermi level , ef , \n under different \n conditions : ( a ) in equilibrium at room temperature , ( b ) at an electron \n temperature of te = 2100 c after \n electron electron equilibration ( hot electrons ) , \n and ( c ) immediately after the absorption of photons by single electrons \n ( primary hot electrons , with population changes highly \n exaggerated to make them visible ) . also shown are the energies of \n the ground - state triplet ( ) and lowest - excited \n singlet state ( ) of oxygen as well as the next \n singlet state ( ) under the assumption that ef is equidistant from the \n and energies . excitation of an oxygen molecule \n to o2 requires the simultaneous transfer of \n an electron from the oxygen to a hole at the energy of the state and of a hot electron with the opposite spin and an \n energy at the ( or ) level to \n the oxygen molecule . the large number of equilibrated hot electrons available \n during \n a nanosecond - laser pulse also rules out the primary hot electrons \n as the main source of o2 photogeneration . the \n number of primary hot electrons available during the pulse duration \n can be estimated from the number of photons which are absorbed per \n nanosecond , multiplied by their lifetime , which is less than 500 fs . however , these primary hot electrons populate \n np states at energies from the fermi level ef to ef + 2.34 ev ( the energy of \n a photon at 532 nm ) ( see figure 6c ) . for 15 nm nps and excitation with 5 ns laser pulses \n with 0.15 j cm intensity , this predicts that not \n more than three primary hot electrons are available within an energy \n interval of 0.1 ev around the level at any time \n during the laser pulse , assuming that all levels are equally populated \n and taking into account that the density of states for gold has an \n essentially constant value in the relevant energy range around ef . this is significantly \n less than the number of hot ( equilibrated ) electrons available at \n the maximum electron temperature , which was estimated to be on the \n order of 260 for the same energy interval ( see above ) . thus , we can \n conclude that it is indeed the hot electrons which are responsible \n for the observed o2 photogeneration when using \n short laser pulses for excitation . similar results to those \n obtained here using pulsed irradiation \n have recently been reported for spherical gold nps with 40 nm diameter . although a significantly lower laser pulse energy \n density ( 0.03 j cm , compared to 0.15 j cm here ) was used under otherwise similar experimental \n conditions , slightly faster o2 photogeneration \n was observed in this study , with the dpbf absorbance decreasing by \n ca . 24% decrease \n observed here in the first 10 min ( figure 4a ) . in this context , it is interesting to \n note that in spite of the lower laser pulse energy density the nps \n are heated to almost the same electron temperature as in our experiments \n ( see figure 5 ) . this \n is largely due to ( i ) heat dissipation from larger nps being slower and ( ii ) the use of 80/20 etoh / water as solvent \n in ref ( 24 ) , which \n has slower heat transport than a 50/50 etoh / water mixture . an explicit \n calculation of the number of hot electrons available at the energy \n of the oxygen state for the two experiments is \n given in the supporting information ; together \n with a detailed consideration of all other experimental differences , \n these numbers yield very good agreement between the expected and the \n observed relative o2 photogeneration rates \n ( see the supporting information for details ) . \n this provides further support for the conclusion that o2 photogeneration is mediated by the equilibrated hot \n electrons of gold nanoparticles under nanosecond - pulsed laser irradiation . compared to the \n effect of pulsed irradiation , the rate of o2 photogeneration is much smaller when using cw light of comparable \n intensity . whereas ca . 24% of the dye is bleached after only 10 min \n of irradiation of spherical nps with 15 nm diameter with 5 ns laser \n pulses at an average power of 150 mw ( figure 4 ) , only 12% of the dye is bleached \n over this time by irradiation of the same nps with cw light at significantly \n higher power ( 1 w ) ( figure 2 ) . in a previous publication , significantly higher rates of dpbf photobleaching had been reported \n under conditions which appear to be similar to the ones used here . \n in this context , we note that we also observed such significantly \n higher rates of bleaching , but only when the sample preparation protocol \n described in the experimental section was \n not followed accurately ; for example , the use of dye solution that \n had not been freshly prepared or of a cuvette that had not been cleaned \n thoroughly and rinsed multiple times with mq water or the use of a \n cell that was not sealed during irradiation , leading to some loss \n of ethanol from the solution , all resulted in larger and highly irreproducible \n bleaching of dpbf under cw irradiation , up to levels comparable to \n those reported in ref ( 24 ) , even in the absence of nps . in the following , we will show \n that photogeneration of o2 under cw irradiation , \n unlike pulsed irradiation , is mediated by the initially created primary \n hot electrons ; i.e. , it occurs during the short time during \n which the excited electrons have not yet relaxed to a thermal distribution \n ( see figure 6c ) . under \n the cw irradiation conditions used here ( 1 w , 1.85 mm beam diameter ) , \n a spherical np with 15 nm diameter absorbs photons at an average rate \n of 1.3 10 s , as estimated from \n the absorption cross section and the \n beam intensity . this means that after absorption of a photon there \n is enough time for full relaxation and transfer of the photon energy \n into the solvent , which occurs in less than 100 ps , before absorption of the next photon . absorption of one \n photon by a 15 nm np yields hot electrons at a temperature \n of 10 k above the surrounding after electron electron equilibration ; \n these hot electrons lose their energy by electron phonon \n scattering within a few picoseconds to yield a np whose temperature \n is only 80 mk above the surrounding . neither of these effects is expected \n to yield any significant photochemical effects ; estimates analogous \n to those described above predict that for a spherical 15 nm np with \n an electron temperature of 35 c there are ca . 2.6 10 electrons \n within an energy interval of 0.1 ev around the oxygen state and the same number of holes around the state energy . this means that there are on the order of 10 times less hot electrons and 10 times less holes \n available for the photoreaction than under our pulsed laser irradiation \n conditions , which rules out any significant reaction ; this is also \n confirmed by the fact that these population numbers are less than \n a factor 2 larger than those for room temperature , where no o2 is generated in the absence of light . this leaves \n only the primary hot electrons , i.e. , those electrons \n that are excited upon absorption of a photon but have not yet equilibrated \n by electron electron scattering , as potential cause for photogeneration \n of o2 . absorption of a single photon can potentially \n excite electrons to energies of up to 2.34 ev above the fermi level . \n if one assumes excitation of only one electron by each photon and \n equal excitation probability for all available electrons , as shown \n in figure 6c , on average \n there will be 0.043 electrons within an energy interval of 0.1 ev \n around the oxygen state , which is approximately \n 5000 times less than during a single laser pulse in the pulsed experiments , \n see above . taking into account that the same factor also applies to \n the holes required for the dexter mechanism , but correcting for the \n lifetime of the excitation ( 500 fs for primary hot electrons created \n during cw irradiation , ca . 3 ns for the hot electron distribution induced \n by a single laser pulse ; see figure s6 ) \n and the repetition rates ( 1.3 10 s for single photon absorption during cw irradiation , 10 s for the pulsed laser irradiation ) , one would predict a rate of o2 photogeneration under our cw irradiation conditions \n which is smaller than that expected for our pulsed irradiation conditions \n by a factor on the order of 10 . this is significantly \n closer to the experimental results ( ratio of o2 photogeneration rates under pulsed vs cw irradiation of 1020 ) \n than any estimate based on the equilibrated hot electrons \n after the absorption of a single photon . the main discrepancy between \n the predicted and observed cw results arises from the assumption of \n direct excitation of single electrons in the above estimate , which \n is not valid for irradiation at 532 nm , i.e. , in the gold np plasmon \n resonance band , since this leads to the coherent excitation of many \n electrons which rapidly dephases without the electrons exchanging \n energy . thus , absorption of a single photon yields more than one primary \n hot electron , with the photon s energy distributed over all \n of them . consequently , the energy distribution even of the primary \n hot electrons will not extend up to 2.34 ev above ef but will be shifted toward the states nearer the fermi \n level , thus increasing the population of states around the oxygen energy and hence the yield of o2 . a more quantitative estimate of this effect is beyond the scope \n of this paper . an alternative possibility for the mechanism \n of o2 photogeneration by gold nps could be \n envisaged , which is \n based on increased direct photoexcitation of oxygen due to the well - known \n local electric field enhancement in the vicinity of metal nps by the \n plasmon electrons . however , significant field enhancement extends \n to distances comparable to the dimensions of the nanoparticle , so that this mechanism is in disagreement with \n our observation that a peg - oh capping layer , which has a thickness \n of only 2 nm , completely inhibits o2 photogeneration ( figure 3 ) . dexter - type electron exchange coupling , \n on the other hand , is known to be of significance only over distances \n of less than 1 nm , as discussed above , and thus is further supported \n by this observation . in conclusion , cw irradiation is less efficient \n than pulsed laser \n irradiation in photogenerating o2 since the \n ( equilibrated ) hot electrons , which are the main mediator of the photochemistry \n in the case of pulsed irradiation , do not have sufficient energy / temperature \n to drive o2 photogeneration . nevertheless , \n our results confirm that cw irradiation of spherical gold nps produces \n detectable amounts of o2 . the absence of significant amounts of ( equilibrated ) hot electrons \n means that o2 photogeneration proceeds via \n a different mechanism under cw irradiation compared to pulsed laser \n generation ; the above estimates indicate that it is the primary hot \n electrons , i.e. , the directly photoexcited electrons , which are responsible \n for the photochemistry here and that the photochemical reaction must \n occur before these equilibrate by electron electron thermalization . photodynamic \n therapy holds great promises for medical applications , such as the \n treatment of cancer , because of the ability to selectively affect \n diseased tissue only . however , wider use of photodynamic \n cancer therapy is currently prevented by several limitations imposed \n by the available photosensitizers . these limitations include toxicity , \n poor stability and photostability , poor selectivity for cancer tissue , \n and the need of using visible light with poor tissue penetration . \n all of these limitations , in principle , can be overcome by the use \n of gold nanoparticles , which are nontoxic , have excellent stability \n even under irradiation , can target cancer tissue either passively \n by the enhanced penetration and retention ( epr ) effect or by active targeting , have extinction coefficients \n that are larger than those of dye molecules by several orders of magnitude , \n and can be tuned to absorb in the near - infrared spectral region for \n maximum tissue penetration . gold nanoparticles have been reported \n to have three potential modes of operation for inducing cell death \n by irradiation , namely ( i ) hyperthermia , which is based on the rapid \n conversion of the absorbed light energy into heat , ( ii ) np - assisted \n photodynamic therapy , in which the efficiency of a standard sensitizer \n is amplified by the np plasmon field enhancement effect , or ( iii ) \n a direct photochemical mechanism without involvement of a photosensitizer . the feasibility of photothermal therapy has been clearly established \n by careful experiments in vitro(1113,15,54 ) and has been shown \n to work in vivo . however , it should be noted that most studies reporting successful \n photothermal therapy made no attempt to either confirm significant \n heating or rule out photochemical effects , which means that some of \n these reported results could in fact arise from photochemical rather \n than photothermal effects or from a synergistic combination of photochemical \n and photothermal effects . as has been pointed out recently , another problem with many reports on photoinduced \n hyperthermia using nps is the relatively high light intensity required \n to reach sufficient temperatures , which often were well above the \n generally accepted skin tolerance threshold . the presence of \n gold nps can also lead to increased photogeneration \n of o2 by traditional photodynamic sensitizers , which can be ascribed to the local electric field enhancement in \n the vicinity of metal nps by the plasmon electrons , similar to the \n well - studied sers ( surface - enhanced raman spectroscopy ) effect . this \n approach might help to alleviate some of the drawbacks of traditional \n photodynamic therapy but does not directly overcome them , since it \n still requires the presence of a sensitizer . in fact , the requirement \n for two active components , gold np and sensitizer , which must be colocalized , \n introduces an additional complication , and care must be taken to avoid \n a reduction of the photosensitizer effect due to quenching of its \n excited state by the metal np . in an alternative approach , cell death has been demonstrated to \n occur following irradiation of intracellular ( endocytosed ) gold nps \n even at irradiation levels that are not high enough to cause significant \n heating . this photochemical effect has been related to the \n observation that irradiation of nps in vitro results \n in the formation of singlet oxygen , which is the active species in traditional \n photodynamic therapy . because of the short lifetime of o2 ( 3.4 s in water ) , its action is highly localized an oxygen molecule only \n diffuses over the length scale of 100 \n nm in this time . for this reason , only intracellular nps are expected \n to trigger cell death by the photochemical route , although they may \n initially be located inside endosomes which are known to be broken \n up by nps under cw irradiation . on the \n other hand , the localization of the photochemical effects within individual \n cells also means that in situations where different types of cells \n coexist in close vicinity , selective targeting of particular cells \n and minimization of collateral damage should be achievable . this is \n different than photothermally induced cell death , which affects all \n cells within the irradiated volume more or less indiscriminately , \n as long as some of them contain nps , because of the fast diffusion \n of heat over the relevant length scales . the results of the \n experiments described here provide more insight \n into the direct photochemical mechanism and allow some important conclusions \n to be made for the further development of practical applications . \n they show that detectable amounts of o2 are \n generated by irradiation of nps with short laser pulses or cw light \n even in the absence of a photodynamic sensitizer , albeit with low \n quantum yield . short laser pulses are significantly more efficient \n at this process , since they can heat a significant fraction of the \n np conduction band electrons to high enough temperatures to excite \n oxygen to the singlet state . however , this requires pulse energy densities \n that are well above generally accepted safe levels for the irradiation \n of skin with pulsed laser light . since the effect requires the absorption \n of many photons by a np during one laser pulse , it is highly nonlinear \n with respect to irradiation intensity , and thus it will not be possible \n to compensate lower irradiation levels by longer irradiation times . \n furthermore , pulsed irradiation at the required intensities also can \n cause other effects , such as np fragmentation or bubble formation , and it is not clear what consequences \n these effects may have when occurring in tissue . although the use \n of femtosecond laser pulses , as compared to the nanosecond pulses \n used here , might alleviate some of these problems to some extent , \n they would require even more sophisticated equipment which may not \n be suitable for a clinical environment . the use of cw light , \n on the other hand , is straightforward and \n does not even require a laser but can be achieved with simple lamps . \n in spite of the lower quantum yield of o2 photogeneration \n by nps under cw irradiation which is reported here , cell death induced \n by cw light in the presence of nps has been reported in vitro and in vivo . although the experiments \n described here , which did not involve any biological material , were \n undertaken at light intensities that are above safe irradiation levels , \n the mechanism of o2 photogeneration by nps \n under cw illumination is shown to be based on the absorption of single \n photons . this means that lower light intensities can be compensated \n for by longer irradiation times , allowing one to reduce the intensity \n to safe levels for biological or medical applications without affecting \n the amount of o2 generated . o2 photogeneration and cancer cell destruction in vitro and in vivo have indeed been observed upon irradiation \n on the minute time scale at intensities below safe levels and using \n nonlaser light sources . the results presented here indicate another design criterion \n which \n needs to be fulfilled for successful implementation of the photochemical \n route of inducing cell death by irradiation of nps , namely that the \n nps must not possess a dense capping layer . even the thin capping \n layer formed by peg - oh , with a thickness of only 2 nm , is sufficient to completely inhibit o2 photogeneration ( figure 3 ) . this is in full agreement with the suggestion that \n energy exchange between the np and the oxygen molecule occurs via \n the dexter ( two electron exchange ) mechanism , which is limited to \n distances of less than 1 nm . similar effects have been reported for o2 photogeneration by si nanocrystals , which is \n significantly affected by a thin oxide layer . it is important to point out that the assay used here for o2 detection ( bleaching of dpbf ) only reports on singlet \n oxygen found outside the np capping layer ; thus , it can not be ruled \n out that even on nps with a peg - oh capping layer some o2 is photogenerated at the np surface but reacts with \n the np ligands and hence is quenched . on the other hand , such singlet \n oxygen would not be of any direct use for practical applications , \n such as the induction of cell death , so that the assay results in \n fact report the effects relevant for such applications . uncapped ( citrate ) \n nps are rapidly covered by a protein corona after they have been taken \n up into live cells , but it appears that \n this corona is permeable enough for oxygen to not completely prevent \n singlet oxygen formation , as evidenced by fact that cell death by \n the photochemical route has been observed with such nps ; it may be that because of their size proteins \n are not able to form a capping layer ( corona ) of similar density as \n the smaller ligands used here . similarly , a lipid bilayer appears \n to allow oxygen access to the surface of gold nanorods , whereas a dense pentapeptide ( calnn ) capping \n layer on spherical gold nanoparticles is sufficient to suppress the \n photochemical mechanism of cell death . it also seems likely that the thick peg capping layer present \n on \n the nanorods used here is the main reason for the absence of o2 photogeneration by nanorods upon irradiation \n in either the transversal or the longitudinal plasmon resonance band \n that was observed here . because of their longitudinal plasmon resonance \n band , which is in the near - ir spectral region with high tissue penetration , \n nanorods are more suitable for practical photodynamic applications \n in tissue . however , standard synthesis protocols yield nanorods within \n a bilayer of cytotoxic ctab , so that ligand exchange is required before \n any biological or medical application , and care will need to be taken \n to choose suitable ligands to allow access of oxygen to the nanorod \n surface . potential examples for these are poly(vinylpyrrolidone ) , \n lipid bilayers , or mesoporous silica , all of which have been used \n successfully in experiments showing photochemically induced cell death \n using gold nanorods .", "dpbf has been used successfully to detect \n singlet oxygen that is \n formed when spherical nps are irradiated at 532 nm , either with cw \n or pulsed laser irradiation . singlet oxygen generation by pulsed laser \n irradiation has been shown to act via the equilibrated hot electrons \n that can reach temperatures of several thousand degrees during the \n laser pulse ; cw irradiation , on the other hand , can act only via the \n directly excited primary hot electrons , which rapidly \n lose their energy by electron electron equilibration , and hence \n is significantly less efficient for the formation of singlet oxygen . \n nevertheless , even cw irradiation can produce enough singlet oxygen \n for photodynamic therapy applications and will allow practical applications \n of the effect at safe irradiation levels . photodynamic therapy using \n gold nanoparticles will also require careful design of the nanoparticles \n with respect to size , shape , and capping layer and will require internalization \n of the nps , not just attachment to the cell surface , which is sufficient \n for photothermal therapy ." ]

[ "supernumerary teeth are a relatively frequent disorder of odontogenesis characterized by an excess number of teeth , with mesiodens being the most frequent.1 the term of mesiodens is used to refer to an unerupted supernumerary tooth in the midline of maxilla , between the central incisors.2,3 the incidence of mesiodens among caucasians is 0.153% for permanent teeth,4,5 and is found two times more frequently in males , compared to females.46 a mesiodens can manifest bilaterally,46 or in an inverted ( upward ) position.4,5 the frequency of inverted mesiodens constitutes to approximately 967% of all reported cases.711 to date , a total of 278 single inverted mesiodentes have been reported ( table 1 ) . the mesiodens may present a rudimentary morphology with a cone - shaped crown , generally smaller in size than the adjacent normal teeth . it may also be found to mimic a natural tooth shape . the root is often totally formed , and may be curved or globular.12 mesiodens is frequently associated with several craniofacial disturbances , including cleft lip and palate13 and cleidocranial dysostosis;14 and to a lesser extent with gardner s syndrome or chondorectodermal dysplasia.15 only 25% of all mesiodentes spontaneously erupt into the oral cavity.6 in general , they remain impacted and asymptomatic ; and are commonly discovered during routine radiographic examination.16 the most common complications of mesiodentes are the delay or prevention of eruption ( 2652% ) and displacement / rotation ( 2860% ) of maxillary permanent incisors . relatively less common complications include crowding , diastema , dilaceration of permanent teeth , cyst formation and eruption into the nasal cavity.6 this case reports presents radiographic features of a unique occurrence of two impacted , inverted mesiodentes in a child patient ; and the 24-month radiographic and clinical follow - up after surgical removal .", "an 8-year - old boy was referred to the department of pediatric dentistry with a chief complaint of crowding in the maxillary anterior region . being born to non - consanguineous parents , the patient s health status was excellent . panoramic radiograph revealed the presence of two supernumerary teeth , located between the roots of the permanent central incisors ( figure 2 ) . occlusal and periapical radiographs of the region showed that these two teeth were inverted mesiodentes ( figure 3 ) . apparently , the roots of the permanent central incisors had migrated distally , owing to the presence of the supernumerary teeth . the lateral cephalogram showed that the mesiodentes were located buccally to the apices of the central incisors ( figure 4 ) . the treatment plan consisted of surgical removal the inverted mesiodentes . the teeth were removed under general anaesthesia without any complication ( figure 5 ) . following removal of sutures one week later , the patient was scheduled for bimonthly recalls . at the second - month recall , the extraction cavities were completely filled with bone ( figure 6 ) and the permanent central incisors responded favorably to thermal and electrical pulp tests . removal of the inverted mesiodentes had slightly aided to self - correction of the axial rotations of the central incisors .", "although mesiodentes are often located palatally,2 the present case demonstrates the possibility of their occurrence in a more vestibular location . in addition to routine ortopantomography or periapical / occlusal radiographs which can rapidly solve the initial diagnostic phase of mesiodentes , further techniques may be required to better define their position in relation to the adjacent teeth and anatomic structures . in the present case , the exact route ( i.e. , buccal / palatal ) for surgical removal of the inverted mesiodentes undoubtedly , greater detail concerning their location could be obtained through computerized axial tomography ( cat),17 which also can help evaluate their relationship with the adjacent tooth structures . in this regard , periapical and occlusal radiographs were used in conjunction with the lateral cephalogram , since cat was not available in our centre . extraction is not the only treatment choice for impacted mesiodentes . if the mesiodens remains in place without symptoms and does not adversely affect the adjacent teeth , it may be left in place and observed periodically.16 in the present case , surgical removal of the mesiodentes was judged necessary , since these teeth had caused axial rotation of the permanent central incisors . there are no precise indications concerning the ideal time for surgical removal of impacted mesiodens . according to atwan,18 a mesiodens can be best removed when the permanent central incisors start to erupt , but this may not be always possible . contrary , primosh19 discourages early extraction of mesiodentes due to the risk of iatrogenic damage to the developing adjacent permanent teeth . in the present case , the timing for surgical removal of the inverted mesiodentes was judged to be suitable , since both maxillary central incisors had totally erupted , showing complete root formation and advanced apical closure . the case presented herein was considered to be extremely rare , since inversion of two mesiodentes in a single patient has only been reported once20 ( table 1 ) . the clinical significance of the inversion is questionable , since both mesiodentes remained intact ; i.e. , did not erupt ectopically . however , the occurrence of two mesiodentes was definitely regarded as a contributory factor to the crowding in the anterior maxillary region , as evidenced by the deviation in the vertical axis of both central incisor teeth . fortunately , the post - surgical phase was uneventful , and did not affect commencement of fixed orthodontic therapy after a relatively short follow - up period ." ]

[ "the whole school , whole community , whole child ( wscc ) model ( figure 1 ) , developed by ascd ( formerly the association for supervision and curriculum development ) and the us centers for disease control ( cdc ) , provides a framework and a call for the health and education sectors to work toward greater alignment and coordination of policy , process , and practice . the wscc model , released in 2014 , was developed to ensure that school - community and education - health sector alignments are front and center . the model expands the traditional 8-component coordinated school health ( csh ) approach and deliberately places it inside the larger community in order to emphasize this connection and role . this includes not only an increase in the number of school health components from 8 to 10 but also the alignment of an education focus with a health focus . the new model calls for a greater collaboration across the community , school , and health and education sectors to meet the needs and support the full potential of each child.8 whole school , whole community , whole child ( wscc ) model . the benefits of collaboration , alignment , and integration between health and education can best be viewed in 3 key areas : leveraging resources , utilizing resources efficiently , and improving both health and education outcomes . a 2010 report from the coalition of community schools cited 5 key ways that schools can efficiently leverage resources.9 this includes collaborative efforts to strengthen the core instructional mission of schools , leveraging community - wide financial resources , developing collaborative leadership structures and ownership , enhancing public / private partnerships , and providing coordination between and across systems . in terms of more efficient utilization , increased integration of health and education can improve usage of facilities and resources . leveraging resources from across the community , as illustrated in the wscc model , can aid both the establishment of school - community connections and health and educational outcomes . examples include shared use agreements that can lead to improved community access to facilities for physical activity10,11 and school - based health centers that have been shown to reduce a community 's inappropriate emergency room use , increase use of primary care , and result in fewer hospitalizations among regular users.1214 finally , strengthening the integration and collaboration between health and education benefits both sectors because research suggests that educational attainment is important to achieving better health outcomes,15,16 and health is key to better educational outcomes.2,3 operationalizing wscc requires moving from theoretical model to practical implementation through the development of state and local school policies and practices . although the model is new , a number of states and local school districts have made significant strides over the last few years in their efforts to better align health and education by building a collaborative , integrated approach . arkansas , kentucky , colorado , maine regional school unit # 22 , and denver public schools ( dps ) all provide valuable examples of states and local districts that embraced wscc 's core vision of aligning health and education through the adoption of integrated policies and initiatives . their trailblazing efforts demonstrate both the feasibility of implementing the wscc model and the visionary leadership required to do so .", "we conducted a search of the health and education department 's websites in arkansas , colorado , and kentucky , and the school districts ' websites in maine regional school unit # 22 , and dps for materials demonstrating adoption and implementation of csh and its respective components or the wscc model . we also conducted a search of the respective state legislative databases , state health and education department websites , and local school district websites for adopted policy relevant to csh and the wscc model . we searched pubmed , web of science , and academic search premier and for relevant peer - reviewed articles around the implementation of aspects of csh or wscc in the 4 states and 2 local school districts . in addition , we searched online for news articles and gray literature highlighting the implementation of csh or the wscc model , or aspects of them , in each state and local school district . secondarily , we conducted informal reinforcing interviews via phone with 1 person in a key school health leadership role related to the promotion and implementation of the wscc model in each district and state : arkansas , kentucky , and colorado , maine regional school unit # 22 and dps .", "in arkansas , the state health and education departments have a long history of working collaboratively and with partners to improve the health of students . the 2 agencies have jointly operated the coordinated school health program ( cshp ) for over 2 decades , working together to expand csh in school districts across the state . they have been supported by a series of state laws that have created a strong and long - lasting foundation for their actions . in 2003 , the arkansas legislature adopted act 1220 , landmark legislation addressing multiple areas of a healthy school environment.17 the legislation included requirements for body mass index ( bmi ) screening in schools ; the establishment of a state - level child health advisory committee ( chac ) to develop rules and regulations for school nutrition , physical education ( pe ) , and physical activity , and to make recommendations to the arkansas state board of education and board of health ; and the establishment of a wellness committee in each local school district . act 1220 also created positions for regional community health promotion specialists ( chps ) specifically to assist schools and led to new standards for competitive foods and physical activity in schools , created by the arkansas chac . act 201 followed in 2007 to make modifications to the bmi screening and grade level requirements , provide a parental opt - out clause , and require the department of health to assign regional community health nurse specialists ( chns ) to work with schools to ensure appropriate protocols and follow up . act 317 ( 2007 ) later strengthened the chac recommended physical activity requirements and required a half credit of pe for high school graduation.18 in 2009 , act 180 ( tobacco excise tax ) made possible a state - level joint use agreement grant program collaboratively run by the arkansas department of education ( ade ) , the arkansas department of health ( adh ) , and the arkansas center for health improvement.19 the program assists schools in the adoption and implementation of shared use policies and the formation of collaborative community partnerships to increase opportunities for physical activity . act 180 also provided funding for an annual school - based health center grant , a collaboration among ade , adh , the arkansas department of human services , and medicaid in the schools . this funding led to the establishment of 22 school - based health centers across the state , providing on - site clinical services for students and connecting them with community partners in an effort to meet their needs better . , post - implementation data indicate that act 1220 and the related policies have resulted in substantial positive changes in school health environments , policies , and practices , with widespread parental support and no increase in negative consequences such as weight - based teasing.2022 among the changes are improved nutrition environments in school districts across the state , including reduced access to less healthy competitive foods and beverages and increased access to healthier options , and stronger policies to promote physical activity and recess.20,21,2327 the chps , employed by the state health department but housed in regional educational cooperatives , have worked together with chns to support local school districts , including assisting with the creation of wellness committees and policies , administering the school health index , and developing strategies to improve nutrition , physical activity , and health environment policies and programs . the state health and education departments have worked collaboratively , with initiatives guided by a school health services team comprised of staff from various areas in each agency , including school health services , human immunodeficiency virus infection / acquired immune deficiency syndrome , mental health , arkansas medicaid in the schools , the state school nurse consultant , act 1220 , and csh . team members provide ongoing support , training , and professional development to school districts throughout the state and to the chps and chns on topics such as wellness policies and administration of the school health index . kentucky provides another valuable example of a state that has worked to integrate health and education through the alignment of policy at the state level . kentucky 's state health and education departments have enjoyed a strong , collaborative relationship in recent years . together , staff from each agency have worked together to increase awareness of csh among education leaders across the state , including superintendents , principals , and school board members . they have collaborated with state associations including the kentucky association of school councils , kentucky school nutrition association , kentucky family resource youth service centers , and kentucky association of health , physical education , recreation , and dance to promote understanding , adoption , and implementation of csh at the local level . the state agencies have focused on helping education leaders make the connection between the csh components and educational outcomes of academic achievement , discipline , and attendance . this foundation , along with strong relationships with state - level organizations including the kentucky school boards association and the kentucky superintendents association , has facilitated the adoption of education policies that support healthier schools and students . an example of this can be seen in kentucky 's program review process . in 2009 , the kentucky legislature adopted senate bill 1 , which established program review as a part of a new assessment and accountability model.28 in addition to ensuring district and school accountability for student achievement , the annual program reviews are designed to both audit and provide feedback and recommendations focused on improving the quality of educational experiences available to students . the program review includes a practical living / career studies component , encompassing health education and pe . the state health and education departments worked collaboratively to draft the program review rubric for the practical living / career studies component , setting a high baseline to support and strengthen the integration of health into school - level policies and practices . schools receive one of 4 designations during the program review process : no implementation , needs improvement , proficient , and distinguished . to achieve the proficient level for health education in the practical living / career studies program review , a school must have a csh committee that is used as a support and resource for collaboration and integration of health education instruction in the school environment ( table 1 ) . similarly , to achieve proficient in the practical living / administrative leadership / support and monitoring component , a school must convene its csh committee at least twice a year , implement the district - level wellness policy via a school - level wellness policy reviewed annually , and include goals for school wellness in the comprehensive school improvement plan ( csip ) . for pe , the csh committee of a proficient school must utilize a comprehensive school physical activity program ( cspap ) to increase quality pe and physical activity opportunities , and integrate the pe curriculum , providing opportunities for cross - disciplinary connections ( table 1 ) . whereas schools may go beyond these standards to achieve the distinguished level , setting high requirements for the proficient level has greatly incentivized the establishment of coordinated school health committees in schools across the state , creation of school - level wellness policies , integration of wellness goals into the school improvement plans , and expansion of cspap . using a common sense approach , kentucky has intentionally and thoughtfully woven health into education measures , and in doing so , has taken a major step toward ensuring better integration of health and education at the local level . examples from kentucky practical living / career studies ( plcs ) program review rubric * adapted from the kentucky department of education . in colorado , strong collaboration and leadership between health and education partners , both public and private , have been instrumental in advancing the integration of health and education across the state . cooperative relationships have been created by aligning the priorities of local foundations with state - level initiatives rather than passing policy . in addition to a solid working partnership between the state health and education agencies , the state has benefitted from the strategic support of local foundations and organizations including : the colorado education initiative , rmc health , the colorado health foundation , and kaiser permanente . in 2010 , these partners came together to develop and disseminate the healthy school champion score card , a voluntary online school - level assessment tool and associated statewide recognition program that provides financial incentives for creating healthy schools ( tables 2 and 3 ) . whereas the design is based on the school health index , it also takes into consideration colorado legislation and policies . the use of the score card grew significantly from its initiation in 2010 , when 100 schools participated . in the 20132014 school year , 216 schools , covering over 100,000 students , participated.29,30 colorado healthy school champions score card sections * colorado coalition for healthy schools . score card overview . colorado is working to build on the success of the score card by developing the next phase of a district- and school - level assessment and implementation tool for health policies and practices , called healthy schools smart source . this effort is funded by kaiser permanente , in partnership with the colorado department of education , the colorado department of public health and the environment , the colorado education initiative , and the colorado coalition for healthy schools . the goal of smart source is to create a single assessment tool that will incorporate questions from the score card and other statewide school health assessments , streamlining data collection and submission for schools.31 to create the assessment , partners conducted an extensive literature review and convened over 200 stakeholders in order to determine the correct data points . a pilot of smart source was launched in the 20142015 school year with 90 schools . by the 20162017 school year , smart source will collect and synthesize data about school and district health policies and practices into a statewide system . smart source will provide schools with school - level reports that offer objective , meaningful comparisons to the aggregated results from all respondents statewide . these reports are designed to provide useful , actionable data as well as recommendations for implementing best practices related to health and wellness ( table 4 ) . reports will provide school- and district - level leaders with a more robust picture of the effect their school health efforts have on student performance and classroom behavior , informing the school improvement planning process.31,32 overview of colorado healthy schools smart source * colorado education initiative . in addition to the smart source initiative , collaborating partners have also launched the colorado healthy schools collective impact ( hsci ) , a purposeful and strategic effort to define healthy schools , prioritize school health - related work in the state , minimize duplication of initiatives , and better leverage resources and funding for healthy schools . healthy schools collective impact members include 20 foundations and nearly 100 community organizations , state agencies , school districts and schools , and nonprofits that are all interested in strengthening the school health environment . the 4 focus areas , each represented by a work group that meets monthly , are pe and physical activity , nutrition , behavioral health ( social , emotional , and mental ) , and student health services . healthy schools collective impact 's objectives are to ( 1 ) coordinate efforts and ensure resources are allocated based on identified needs ; ( 2 ) engage those impacted by the work , including districts and schools , parents , students , funders , and organizations that champion healthy schools ; ( 3 ) gather consistent data by coordinating surveys and systems to collect data from schools ; ( 4 ) use consistent communication by creating a shared definition of a healthy school , possibly producing a one - stop shop for data and resources related to priority school health strategies ; ( 5 ) support core capacity for schools and districts ; and ( 6 ) develop shared policy priorities.33 at the local level , many school districts nationally have embraced the importance of addressing the whole child through better alignment and integration of health and education . two districts that provide strong examples of application and implementation of coordinated school health and/or the wscc model include maine regional school unit ( rsu ) # 22 and denver public schools in colorado . both have benefited from the leadership of superintendents and local school boards that have embraced the importance of educating the whole child . maine regional school unit # 22 , located in the hampden area , began to intentionally focus on the health and wellness of students and staff in 1991 . the vision to strengthen school health and wellness has been driven by rick lyons , district superintendent since 1992 . the district began by forming a wellness team long before the concept had gained widespread popularity . the wellness team , comprised administrators , faculty , support staff , and community members , has been a key factor behind the district 's school health achievements since its establishment . in 2001 , the district also founded a school health advisory council ( shac ) comprised of administrators , healthcare professionals , and community members . the shac oversees the cshp , which is headed by a school health coordinator employed by the district . given rsu # 22 's small size ( approximately 2,200 students and 375 employees ) , the financial commitment to support a full - time school health coordinator speaks to the high priority that school health has in this district . over the last 20 years , rsu # 22 has taken many proactive steps toward strengthening school health in each of its 6 schools . it increased the number of positions and staffing time for health education teachers , nurses , and guidance counselors and forged partnerships with behavioral health providers . the district promotes active transport to school with a walking school bus , works with the hampden town council to repair bike lanes and install street lights , and partners with the hampden recreation department to provide before- and after - school programming . regional school unit # 22 implemented a comprehensive k12 health / pe curriculum focusing on lifelong activities ( such as weight training , aerobics , bicycling , and running ) along with activities such as skiing , tennis , snowshoeing , and swimming and uses recess rocks in k2 classrooms . in 2002 , the wellness coordinator and nutrition director worked with students and suppliers to change the content of all food offered in vending machines to healthy choices . regional school unit # 22 's schools offer salad bars at lunch , and the food service director strives to use locally sourced foods as much as possible . in 2010 , parents , students , the district 's food service director , wellness coordinator , and community partners collaborated to plant apple orchards and a vegetable garden . leadership from school board members and the superintendent has been key in rsu # 22 's transformation into a district where health and wellness is a top priority and a value embraced by the school employees and the community . the shac , wellness team , and school health coordinator have also been integral to the process . they have worked together to facilitate the integration of health and education in numerous areas , including the district 's strategic plan and the interview process for prospective employees , which includes questions about their thoughts on the district 's wellness priorities . the district has focused on improving student and staff health and dedicated significant financial resources to both . the local school board allocates approximately $ 40,000 per year for employee wellness incentives for those who did not use more than 3 sick days the previous year , and 60% of staff members have qualified for this reward . regional school unit # 22 promotes periodic wellness challenges for staff and a 7-week total health track through adult education . in 2012 , it opened a new high school that includes a fitness center , track , and tennis courts open to students , staff , and the community . as a result of these efforts , the wellness council of america awarded the district their gold level workplace wellness award . superintendent leadership also has been critical to the success of dps ' strategic inclusion of student health in the district 's overall priorities and plan . since being appointed to the position by the board of education in 2009 , superintendent tom boasberg has managed the district 's 183 schools , which serve a diverse population of over 90,000 children . seventy - two percent of students qualify for free or reduced - price lunch , and 41% speak a language other than english at home.34 the district has also committed significant resources to student health , and made it a part of the district 's strategic planning process . in 2010 , boasberg oversaw the denver school health advisory council 's creation of the dps health agenda 2015 , which outlines the districts ' health priorities.35 the district has since invested over $ 22 million toward its health goals , including the addition of 6 full - time district - funded employees . some of the significant accomplishments include dramatically increasing the number of schools that serve breakfast after the bell ; adding pe teachers to elementary schools , which has led to an additional 22 minutes per week of pe classes ; and increasing the number of school counselors , which has corresponded with a decrease in expulsion and suspension rates . denver public school has also added a school - based health center every year since 2010 , with a fifth scheduled to open in 2015 . in august 2014 , dps released the current version of its 5-year strategic plan , denver plan 2020 . this version , which was developed with the input of nearly 3,000 educators , parents , students , community partners , and city leaders , includes support for the whole child as one of 5 goals . using the wscc model as a backbone , an advisory committee is assessing dps ' activities , and will present recommendations for creating a metric to determine future progress in early 2015.36 denver public school is also working on the development of the next generation of their health agenda , dps health agenda 2020 . the district has solicited input from students , parents , and community members through a widely disseminated survey about health issues that most impact students . survey topics include asthma , vision , oral health , nutrition , physical activity , social / emotional health , substance abuse , teen pregnancy , and school culture . denver public school plans to use the results of this survey to improve student health and wellness going forward .", "in arkansas , the state health and education departments have a long history of working collaboratively and with partners to improve the health of students . the 2 agencies have jointly operated the coordinated school health program ( cshp ) for over 2 decades , working together to expand csh in school districts across the state . they have been supported by a series of state laws that have created a strong and long - lasting foundation for their actions . in 2003 , the arkansas legislature adopted act 1220 , landmark legislation addressing multiple areas of a healthy school environment.17 the legislation included requirements for body mass index ( bmi ) screening in schools ; the establishment of a state - level child health advisory committee ( chac ) to develop rules and regulations for school nutrition , physical education ( pe ) , and physical activity , and to make recommendations to the arkansas state board of education and board of health ; and the establishment of a wellness committee in each local school district . act 1220 also created positions for regional community health promotion specialists ( chps ) specifically to assist schools and led to new standards for competitive foods and physical activity in schools , created by the arkansas chac . act 201 followed in 2007 to make modifications to the bmi screening and grade level requirements , provide a parental opt - out clause , and require the department of health to assign regional community health nurse specialists ( chns ) to work with schools to ensure appropriate protocols and follow up . act 317 ( 2007 ) later strengthened the chac recommended physical activity requirements and required a half credit of pe for high school graduation.18 in 2009 , act 180 ( tobacco excise tax ) made possible a state - level joint use agreement grant program collaboratively run by the arkansas department of education ( ade ) , the arkansas department of health ( adh ) , and the arkansas center for health improvement.19 the program assists schools in the adoption and implementation of shared use policies and the formation of collaborative community partnerships to increase opportunities for physical activity . act 180 also provided funding for an annual school - based health center grant , a collaboration among ade , adh , the arkansas department of human services , and medicaid in the schools . this funding led to the establishment of 22 school - based health centers across the state , providing on - site clinical services for students and connecting them with community partners in an effort to meet their needs better . , post - implementation data indicate that act 1220 and the related policies have resulted in substantial positive changes in school health environments , policies , and practices , with widespread parental support and no increase in negative consequences such as weight - based teasing.2022 among the changes are improved nutrition environments in school districts across the state , including reduced access to less healthy competitive foods and beverages and increased access to healthier options , and stronger policies to promote physical activity and recess.20,21,2327 the chps , employed by the state health department but housed in regional educational cooperatives , have worked together with chns to support local school districts , including assisting with the creation of wellness committees and policies , administering the school health index , and developing strategies to improve nutrition , physical activity , and health environment policies and programs . the state health and education departments have worked collaboratively , with initiatives guided by a school health services team comprised of staff from various areas in each agency , including school health services , human immunodeficiency virus infection / acquired immune deficiency syndrome , mental health , arkansas medicaid in the schools , the state school nurse consultant , act 1220 , and csh . team members provide ongoing support , training , and professional development to school districts throughout the state and to the chps and chns on topics such as wellness policies and administration of the school health index . kentucky provides another valuable example of a state that has worked to integrate health and education through the alignment of policy at the state level . kentucky 's state health and education departments have enjoyed a strong , collaborative relationship in recent years . together , staff from each agency have worked together to increase awareness of csh among education leaders across the state , including superintendents , principals , and school board members . they have collaborated with state associations including the kentucky association of school councils , kentucky school nutrition association , kentucky family resource youth service centers , and kentucky association of health , physical education , recreation , and dance to promote understanding , adoption , and implementation of csh at the local level . the state agencies have focused on helping education leaders make the connection between the csh components and educational outcomes of academic achievement , discipline , and attendance . this foundation , along with strong relationships with state - level organizations including the kentucky school boards association and the kentucky superintendents association , has facilitated the adoption of education policies that support healthier schools and students . an example of this can be seen in kentucky 's program review process . in 2009 , the kentucky legislature adopted senate bill 1 , which established program review as a part of a new assessment and accountability model.28 in addition to ensuring district and school accountability for student achievement , the annual program reviews are designed to both audit and provide feedback and recommendations focused on improving the quality of educational experiences available to students . the program review includes a practical living / career studies component , encompassing health education and pe . the state health and education departments worked collaboratively to draft the program review rubric for the practical living / career studies component , setting a high baseline to support and strengthen the integration of health into school - level policies and practices . schools receive one of 4 designations during the program review process : no implementation , needs improvement , proficient , and distinguished . to achieve the proficient level for health education in the practical living / career studies program review , a school must have a csh committee that is used as a support and resource for collaboration and integration of health education instruction in the school environment ( table 1 ) . similarly , to achieve proficient in the practical living / administrative leadership / support and monitoring component , a school must convene its csh committee at least twice a year , implement the district - level wellness policy via a school - level wellness policy reviewed annually , and include goals for school wellness in the comprehensive school improvement plan ( csip ) . for pe , the csh committee of a proficient school must utilize a comprehensive school physical activity program ( cspap ) to increase quality pe and physical activity opportunities , and integrate the pe curriculum , providing opportunities for cross - disciplinary connections ( table 1 ) . whereas schools may go beyond these standards to achieve the distinguished level , setting high requirements for the proficient level has greatly incentivized the establishment of coordinated school health committees in schools across the state , creation of school - level wellness policies , integration of wellness goals into the school improvement plans , and expansion of cspap . using a common sense approach , kentucky has intentionally and thoughtfully woven health into education measures , and in doing so , has taken a major step toward ensuring better integration of health and education at the local level . examples from kentucky practical living / career studies ( plcs ) program review rubric * adapted from the kentucky department of education . in colorado , strong collaboration and leadership between health and education partners , both public and private , have been instrumental in advancing the integration of health and education across the state . cooperative relationships have been created by aligning the priorities of local foundations with state - level initiatives rather than passing policy . in addition to a solid working partnership between the state health and education agencies , the state has benefitted from the strategic support of local foundations and organizations including : the colorado education initiative , rmc health , the colorado health foundation , and kaiser permanente . in 2010 , these partners came together to develop and disseminate the healthy school champion score card , a voluntary online school - level assessment tool and associated statewide recognition program that provides financial incentives for creating healthy schools ( tables 2 and 3 ) . whereas the design is based on the school health index , it also takes into consideration colorado legislation and policies . the use of the score card grew significantly from its initiation in 2010 , when 100 schools participated . in the 20132014 school year , 216 schools , covering over 100,000 students , participated.29,30 colorado healthy school champions score card sections * colorado coalition for healthy schools . score card overview . colorado is working to build on the success of the score card by developing the next phase of a district- and school - level assessment and implementation tool for health policies and practices , called healthy schools smart source . this effort is funded by kaiser permanente , in partnership with the colorado department of education , the colorado department of public health and the environment , the colorado education initiative , and the colorado coalition for healthy schools . the goal of smart source is to create a single assessment tool that will incorporate questions from the score card and other statewide school health assessments , streamlining data collection and submission for schools.31 to create the assessment , partners conducted an extensive literature review and convened over 200 stakeholders in order to determine the correct data points . a pilot of smart source was launched in the 20142015 school year with 90 schools . by the 20162017 school year , smart source will collect and synthesize data about school and district health policies and practices into a statewide system . smart source will provide schools with school - level reports that offer objective , meaningful comparisons to the aggregated results from all respondents statewide . these reports are designed to provide useful , actionable data as well as recommendations for implementing best practices related to health and wellness ( table 4 ) . reports will provide school- and district - level leaders with a more robust picture of the effect their school health efforts have on student performance and classroom behavior , informing the school improvement planning process.31,32 overview of colorado healthy schools smart source * colorado education initiative . in addition to the smart source initiative , collaborating partners have also launched the colorado healthy schools collective impact ( hsci ) , a purposeful and strategic effort to define healthy schools , prioritize school health - related work in the state , minimize duplication of initiatives , and better leverage resources and funding for healthy schools . healthy schools collective impact members include 20 foundations and nearly 100 community organizations , state agencies , school districts and schools , and nonprofits that are all interested in strengthening the school health environment . the 4 focus areas , each represented by a work group that meets monthly , are pe and physical activity , nutrition , behavioral health ( social , emotional , and mental ) , and student health services . healthy schools collective impact 's objectives are to ( 1 ) coordinate efforts and ensure resources are allocated based on identified needs ; ( 2 ) engage those impacted by the work , including districts and schools , parents , students , funders , and organizations that champion healthy schools ; ( 3 ) gather consistent data by coordinating surveys and systems to collect data from schools ; ( 4 ) use consistent communication by creating a shared definition of a healthy school , possibly producing a one - stop shop for data and resources related to priority school health strategies ; ( 5 ) support core capacity for schools and districts ; and ( 6 ) develop shared policy priorities.33", "in arkansas , the state health and education departments have a long history of working collaboratively and with partners to improve the health of students . the 2 agencies have jointly operated the coordinated school health program ( cshp ) for over 2 decades , working together to expand csh in school districts across the state . they have been supported by a series of state laws that have created a strong and long - lasting foundation for their actions . in 2003 , the arkansas legislature adopted act 1220 , landmark legislation addressing multiple areas of a healthy school environment.17 the legislation included requirements for body mass index ( bmi ) screening in schools ; the establishment of a state - level child health advisory committee ( chac ) to develop rules and regulations for school nutrition , physical education ( pe ) , and physical activity , and to make recommendations to the arkansas state board of education and board of health ; and the establishment of a wellness committee in each local school district . act 1220 also created positions for regional community health promotion specialists ( chps ) specifically to assist schools and led to new standards for competitive foods and physical activity in schools , created by the arkansas chac . act 201 followed in 2007 to make modifications to the bmi screening and grade level requirements , provide a parental opt - out clause , and require the department of health to assign regional community health nurse specialists ( chns ) to work with schools to ensure appropriate protocols and follow up . act 317 ( 2007 ) later strengthened the chac recommended physical activity requirements and required a half credit of pe for high school graduation.18 in 2009 , act 180 ( tobacco excise tax ) made possible a state - level joint use agreement grant program collaboratively run by the arkansas department of education ( ade ) , the arkansas department of health ( adh ) , and the arkansas center for health improvement.19 the program assists schools in the adoption and implementation of shared use policies and the formation of collaborative community partnerships to increase opportunities for physical activity . act 180 also provided funding for an annual school - based health center grant , a collaboration among ade , adh , the arkansas department of human services , and medicaid in the schools . this funding led to the establishment of 22 school - based health centers across the state , providing on - site clinical services for students and connecting them with community partners in an effort to meet their needs better . , post - implementation data indicate that act 1220 and the related policies have resulted in substantial positive changes in school health environments , policies , and practices , with widespread parental support and no increase in negative consequences such as weight - based teasing.2022 among the changes are improved nutrition environments in school districts across the state , including reduced access to less healthy competitive foods and beverages and increased access to healthier options , and stronger policies to promote physical activity and recess.20,21,2327 the chps , employed by the state health department but housed in regional educational cooperatives , have worked together with chns to support local school districts , including assisting with the creation of wellness committees and policies , administering the school health index , and developing strategies to improve nutrition , physical activity , and health environment policies and programs . the state health and education departments have worked collaboratively , with initiatives guided by a school health services team comprised of staff from various areas in each agency , including school health services , human immunodeficiency virus infection / acquired immune deficiency syndrome , mental health , arkansas medicaid in the schools , the state school nurse consultant , act 1220 , and csh . team members provide ongoing support , training , and professional development to school districts throughout the state and to the chps and chns on topics such as wellness policies and administration of the school health index .", "kentucky provides another valuable example of a state that has worked to integrate health and education through the alignment of policy at the state level . kentucky 's state health and education departments have enjoyed a strong , collaborative relationship in recent years . together , staff from each agency have worked together to increase awareness of csh among education leaders across the state , including superintendents , principals , and school board members . they have collaborated with state associations including the kentucky association of school councils , kentucky school nutrition association , kentucky family resource youth service centers , and kentucky association of health , physical education , recreation , and dance to promote understanding , adoption , and implementation of csh at the local level . the state agencies have focused on helping education leaders make the connection between the csh components and educational outcomes of academic achievement , discipline , and attendance . this foundation , along with strong relationships with state - level organizations including the kentucky school boards association and the kentucky superintendents association , has facilitated the adoption of education policies that support healthier schools and students . an example of this can be seen in kentucky 's program review process . in 2009 , the kentucky legislature adopted senate bill 1 , which established program review as a part of a new assessment and accountability model.28 in addition to ensuring district and school accountability for student achievement , the annual program reviews are designed to both audit and provide feedback and recommendations focused on improving the quality of educational experiences available to students . the program review includes a practical living / career studies component , encompassing health education and pe . the state health and education departments worked collaboratively to draft the program review rubric for the practical living / career studies component , setting a high baseline to support and strengthen the integration of health into school - level policies and practices . schools receive one of 4 designations during the program review process : no implementation , needs improvement , proficient , and distinguished . to achieve the proficient level for health education in the practical living / career studies program review , a school must have a csh committee that is used as a support and resource for collaboration and integration of health education instruction in the school environment ( table 1 ) . similarly , to achieve proficient in the practical living / administrative leadership / support and monitoring component , a school must convene its csh committee at least twice a year , implement the district - level wellness policy via a school - level wellness policy reviewed annually , and include goals for school wellness in the comprehensive school improvement plan ( csip ) . for pe , the csh committee of a proficient school must utilize a comprehensive school physical activity program ( cspap ) to increase quality pe and physical activity opportunities , and integrate the pe curriculum , providing opportunities for cross - disciplinary connections ( table 1 ) . whereas schools may go beyond these standards to achieve the distinguished level , setting high requirements for the proficient level has greatly incentivized the establishment of coordinated school health committees in schools across the state , creation of school - level wellness policies , integration of wellness goals into the school improvement plans , and expansion of cspap . using a common sense approach , kentucky has intentionally and thoughtfully woven health into education measures , and in doing so , has taken a major step toward ensuring better integration of health and education at the local level . examples from kentucky practical living / career studies ( plcs ) program review rubric * adapted from the kentucky department of education .", "in colorado , strong collaboration and leadership between health and education partners , both public and private , have been instrumental in advancing the integration of health and education across the state . cooperative relationships have been created by aligning the priorities of local foundations with state - level initiatives rather than passing policy . in addition to a solid working partnership between the state health and education agencies , the state has benefitted from the strategic support of local foundations and organizations including : the colorado education initiative , rmc health , the colorado health foundation , and kaiser permanente . in 2010 , these partners came together to develop and disseminate the healthy school champion score card , a voluntary online school - level assessment tool and associated statewide recognition program that provides financial incentives for creating healthy schools ( tables 2 and 3 ) . whereas the design is based on the school health index , it also takes into consideration colorado legislation and policies . the use of the score card grew significantly from its initiation in 2010 , when 100 schools participated . in the 20132014 school year , 216 schools , covering over 100,000 students , participated.29,30 colorado healthy school champions score card sections * colorado coalition for healthy schools . colorado is working to build on the success of the score card by developing the next phase of a district- and school - level assessment and implementation tool for health policies and practices , called healthy schools smart source . this effort is funded by kaiser permanente , in partnership with the colorado department of education , the colorado department of public health and the environment , the colorado education initiative , and the colorado coalition for healthy schools . the goal of smart source is to create a single assessment tool that will incorporate questions from the score card and other statewide school health assessments , streamlining data collection and submission for schools.31 to create the assessment , partners conducted an extensive literature review and convened over 200 stakeholders in order to determine the correct data points . a pilot of smart source was launched in the 20142015 school year with 90 schools . by the 20162017 school year , smart source will collect and synthesize data about school and district health policies and practices into a statewide system . smart source will provide schools with school - level reports that offer objective , meaningful comparisons to the aggregated results from all respondents statewide . these reports are designed to provide useful , actionable data as well as recommendations for implementing best practices related to health and wellness ( table 4 ) . reports will provide school- and district - level leaders with a more robust picture of the effect their school health efforts have on student performance and classroom behavior , informing the school improvement planning process.31,32 overview of colorado healthy schools smart source * colorado education initiative . in addition to the smart source initiative , collaborating partners have also launched the colorado healthy schools collective impact ( hsci ) , a purposeful and strategic effort to define healthy schools , prioritize school health - related work in the state , minimize duplication of initiatives , and better leverage resources and funding for healthy schools . healthy schools collective impact members include 20 foundations and nearly 100 community organizations , state agencies , school districts and schools , and nonprofits that are all interested in strengthening the school health environment . the 4 focus areas , each represented by a work group that meets monthly , are pe and physical activity , nutrition , behavioral health ( social , emotional , and mental ) , and student health services . healthy schools collective impact 's objectives are to ( 1 ) coordinate efforts and ensure resources are allocated based on identified needs ; ( 2 ) engage those impacted by the work , including districts and schools , parents , students , funders , and organizations that champion healthy schools ; ( 3 ) gather consistent data by coordinating surveys and systems to collect data from schools ; ( 4 ) use consistent communication by creating a shared definition of a healthy school , possibly producing a one - stop shop for data and resources related to priority school health strategies ; ( 5 ) support core capacity for schools and districts ; and ( 6 ) develop shared policy priorities.33", "at the local level , many school districts nationally have embraced the importance of addressing the whole child through better alignment and integration of health and education . two districts that provide strong examples of application and implementation of coordinated school health and/or the wscc model include maine regional school unit ( rsu ) # 22 and denver public schools in colorado . both have benefited from the leadership of superintendents and local school boards that have embraced the importance of educating the whole child . maine regional school unit # 22 , located in the hampden area , began to intentionally focus on the health and wellness of students and staff in 1991 . the vision to strengthen school health and wellness has been driven by rick lyons , district superintendent since 1992 . the district began by forming a wellness team long before the concept had gained widespread popularity . the wellness team , comprised administrators , faculty , support staff , and community members , has been a key factor behind the district 's school health achievements since its establishment . in 2001 , the district also founded a school health advisory council ( shac ) comprised of administrators , healthcare professionals , and community members . the shac oversees the cshp , which is headed by a school health coordinator employed by the district . given rsu # 22 's small size ( approximately 2,200 students and 375 employees ) , the financial commitment to support a full - time school health coordinator speaks to the high priority that school health has in this district . over the last 20 years , rsu # 22 has taken many proactive steps toward strengthening school health in each of its 6 schools . it increased the number of positions and staffing time for health education teachers , nurses , and guidance counselors and forged partnerships with behavioral health providers . the district promotes active transport to school with a walking school bus , works with the hampden town council to repair bike lanes and install street lights , and partners with the hampden recreation department to provide before- and after - school programming . regional school unit # 22 implemented a comprehensive k12 health / pe curriculum focusing on lifelong activities ( such as weight training , aerobics , bicycling , and running ) along with activities such as skiing , tennis , snowshoeing , and swimming and uses recess rocks in k2 classrooms . in 2002 , the wellness coordinator and nutrition director worked with students and suppliers to change the content of all food offered in vending machines to healthy choices . regional school unit # 22 's schools offer salad bars at lunch , and the food service director strives to use locally sourced foods as much as possible . in 2010 , parents , students , the district 's food service director , wellness coordinator , and community partners collaborated to plant apple orchards and a vegetable garden . leadership from school board members and the superintendent has been key in rsu # 22 's transformation into a district where health and wellness is a top priority and a value embraced by the school employees and the community . the shac , wellness team , and school health coordinator have also been integral to the process . they have worked together to facilitate the integration of health and education in numerous areas , including the district 's strategic plan and the interview process for prospective employees , which includes questions about their thoughts on the district 's wellness priorities . the district has focused on improving student and staff health and dedicated significant financial resources to both . the local school board allocates approximately $ 40,000 per year for employee wellness incentives for those who did not use more than 3 sick days the previous year , and 60% of staff members have qualified for this reward . regional school unit # 22 promotes periodic wellness challenges for staff and a 7-week total health track through adult education . in 2012 , it opened a new high school that includes a fitness center , track , and tennis courts open to students , staff , and the community . as a result of these efforts , the wellness council of america awarded the district their gold level workplace wellness award . superintendent leadership also has been critical to the success of dps ' strategic inclusion of student health in the district 's overall priorities and plan . since being appointed to the position by the board of education in 2009 , superintendent tom boasberg has managed the district 's 183 schools , which serve a diverse population of over 90,000 children . seventy - two percent of students qualify for free or reduced - price lunch , and 41% speak a language other than english at home.34 the district has also committed significant resources to student health , and made it a part of the district 's strategic planning process . in 2010 , boasberg oversaw the denver school health advisory council 's creation of the dps health agenda 2015 , which outlines the districts ' health priorities.35 the district has since invested over $ 22 million toward its health goals , including the addition of 6 full - time district - funded employees . some of the significant accomplishments include dramatically increasing the number of schools that serve breakfast after the bell ; adding pe teachers to elementary schools , which has led to an additional 22 minutes per week of pe classes ; and increasing the number of school counselors , which has corresponded with a decrease in expulsion and suspension rates . denver public school has also added a school - based health center every year since 2010 , with a fifth scheduled to open in 2015 . in august 2014 , dps released the current version of its 5-year strategic plan , denver plan 2020 . this version , which was developed with the input of nearly 3,000 educators , parents , students , community partners , and city leaders , includes support for the whole child as one of 5 goals . using the wscc model as a backbone , an advisory committee is assessing dps ' activities , and will present recommendations for creating a metric to determine future progress in early 2015.36 denver public school is also working on the development of the next generation of their health agenda , dps health agenda 2020 . the district has solicited input from students , parents , and community members through a widely disseminated survey about health issues that most impact students . survey topics include asthma , vision , oral health , nutrition , physical activity , social / emotional health , substance abuse , teen pregnancy , and school culture . denver public school plans to use the results of this survey to improve student health and wellness going forward .", "maine regional school unit # 22 , located in the hampden area , began to intentionally focus on the health and wellness of students and staff in 1991 . the vision to strengthen school health and wellness has been driven by rick lyons , district superintendent since 1992 . the district began by forming a wellness team long before the concept had gained widespread popularity . the wellness team , comprised administrators , faculty , support staff , and community members , has been a key factor behind the district 's school health achievements since its establishment . in 2001 , the district also founded a school health advisory council ( shac ) comprised of administrators , healthcare professionals , and community members . the shac oversees the cshp , which is headed by a school health coordinator employed by the district . given rsu # 22 's small size ( approximately 2,200 students and 375 employees ) , the financial commitment to support a full - time school health coordinator speaks to the high priority that school health has in this district . over the last 20 years , rsu # 22 has taken many proactive steps toward strengthening school health in each of its 6 schools . it increased the number of positions and staffing time for health education teachers , nurses , and guidance counselors and forged partnerships with behavioral health providers . the district promotes active transport to school with a walking school bus , works with the hampden town council to repair bike lanes and install street lights , and partners with the hampden recreation department to provide before- and after - school programming . regional school unit # 22 implemented a comprehensive k12 health / pe curriculum focusing on lifelong activities ( such as weight training , aerobics , bicycling , and running ) along with activities such as skiing , tennis , snowshoeing , and swimming and uses recess rocks in k2 classrooms . in 2002 , the wellness coordinator and nutrition director worked with students and suppliers to change the content of all food offered in vending machines to healthy choices . regional school unit # 22 's schools offer salad bars at lunch , and the food service director strives to use locally sourced foods as much as possible . in 2010 , parents , students , the district 's food service director , wellness coordinator , and community partners collaborated to plant apple orchards and a vegetable garden . leadership from school board members and the superintendent has been key in rsu # 22 's transformation into a district where health and wellness is a top priority and a value embraced by the school employees and the community . the shac , wellness team , and school health coordinator have also been integral to the process . they have worked together to facilitate the integration of health and education in numerous areas , including the district 's strategic plan and the interview process for prospective employees , which includes questions about their thoughts on the district 's wellness priorities . the district has focused on improving student and staff health and dedicated significant financial resources to both . the local school board allocates approximately $ 40,000 per year for employee wellness incentives for those who did not use more than 3 sick days the previous year , and 60% of staff members have qualified for this reward . regional school unit # 22 promotes periodic wellness challenges for staff and a 7-week total health , it opened a new high school that includes a fitness center , track , and tennis courts open to students , staff , and the community . as a result of these efforts , the wellness council of america awarded the district their gold level workplace wellness award .", "superintendent leadership also has been critical to the success of dps ' strategic inclusion of student health in the district 's overall priorities and plan . since being appointed to the position by the board of education in 2009 , superintendent tom boasberg has managed the district 's 183 schools , which serve a diverse population of over 90,000 children . seventy - two percent of students qualify for free or reduced - price lunch , and 41% speak a language other than english at home.34 the district has also committed significant resources to student health , and made it a part of the district 's strategic planning process . in 2010 , boasberg oversaw the denver school health advisory council 's creation of the dps health agenda 2015 , which outlines the districts ' health priorities.35 the district has since invested over $ 22 million toward its health goals , including the addition of 6 full - time district - funded employees . some of the significant accomplishments include dramatically increasing the number of schools that serve breakfast after the bell ; adding pe teachers to elementary schools , which has led to an additional 22 minutes per week of pe classes ; and increasing the number of school counselors , which has corresponded with a decrease in expulsion and suspension rates . denver public school has also added a school - based health center every year since 2010 , with a fifth scheduled to open in 2015 . in august 2014 , dps released the current version of its 5-year strategic plan , denver plan 2020 . this version , which was developed with the input of nearly 3,000 educators , parents , students , community partners , and city leaders , includes support for the whole child as one of 5 goals . using the wscc model as a backbone , an advisory committee is assessing dps ' activities , and will present recommendations for creating a metric to determine future progress in early 2015.36 denver public school is also working on the development of the next generation of their health agenda , dps health agenda 2020 . the district has solicited input from students , parents , and community members through a widely disseminated survey about health issues that most impact students . survey topics include asthma , vision , oral health , nutrition , physical activity , social / emotional health , substance abuse , teen pregnancy , and school culture . denver public school plans to use the results of this survey to improve student health and wellness going forward .", "the wscc model provides a framework for the health and education sectors at the state and local levels to work toward stronger alignment , integration , and collaboration . as lloyd kolbe stated , the wscc model provides a new opportunity , impetus , and sense of purpose for both sectors : if we prudently could unite the expertise , perspectives , resources , communication , visibility , and political support of national education , health , and related organizations to help schools and colleges implement a wscc approach , we synergistically could increase the impact of each sector , each organization involved ; we could build a lasting national base for new means to improve both education and health ; we could accomplish that which we otherwise cannot.37 whereas the wscc model is new , having been launched in march 2014 , many states and local school districts are already in the process of adapting and adjusting practices and processes to suit the expanded version of csh . the 3 states and 2 school districts highlighted all provide strong examples of implementation of various aspects of the wscc model . they demonstrate that it is possible to prioritize student health within the education system , for the purpose of better addressing the needs of the whole child . they also demonstrate the importance of strategic leadership at the state and local level , including support from policymakers , superintendents , local school boards , and community organizations that recognize and seize opportunities to integrate and better align the health and education sectors . moving forward , state and local health and education agencies should focus greater attention toward ensuring the adoption and implementation of policies that serve both sectors and students , and promote the formation of collaborative partnerships . policymakers need to focus on adoption and implementation of policies and practices that build on the connection between the school health components and educational outcomes , both academic and behavioral . when those in decision - making roles understand and embrace the wscc model , they are able to create programs and policies that lead to collaboration and more effective use of resources , creating healthier school environments that address the needs of the whole child . information contained in this article was not deemed to involve human subjects , and therefore , was considered exempt from institutional review board examination .", "information contained in this article was not deemed to involve human subjects , and therefore , was considered exempt from institutional review board examination ." ]

[ "juvenile ossifying fibroma ( jof ) is an actively growing aggressive lesion containing cells rich in the fibrous stroma containing cellular osteoid , trabeculae of woven bone without osteoblastic lining , with or without clusters of giant cells . el - mofty first identified the two histopathological variants of jof : trabecular and psammomatoid . rare cases of mandibular jof have been reported . clinical examination reveals it as asymptomatic slow growing swelling causing facial asymmetry , extending to a substantial size and behaving as an aggressive lesion . it has higher male predominance with the mean age of occurrence ranging from 2 to 15 years . the radiographic features show unilocular or multilocular radiolucency with well - delineated borders and few opacification in the center . the suggestive features of the aggressive variant are the presence of the perforation , cortical thinning , tooth displacement , and root resorption . the advanced imaging studies show more invasive and destructive features apart from conventional radiographic features . histopathologically both the entities are nonencapsulated , well demarcated from the surrounding bone consisting of neoplastic cellular stroma formed by spindle shape fibroblast . in contrast , the psammomatoid pattern forms concentrically laminated ossicles that vary in shape and having peripheral eosinophilic osteoid rims with basophilic centers typically . the overall clinical features , histopathological features , and advanced imaging were indispensable to provide the diagnosis as jof . a nonaggressive form of this lesion is managed by curettage and local excision whereas aggressive form is managed by complete surgical excision , en - bloc resection or hemimaxillectomy to prevent recurrences . reported here is a case of a trabecular variant of jof in a 17-year - old female patient .", "a 17-year - old female represented with a chief complaint of pain and slowly progressive swelling of the left side of face along with reduced mouth opening for the last 2 months . initially , the swelling was smaller in size and increased progressively to attain final size causing facial asymmetry with reduced mouth opening . extraoral examination revealed an ill - defined , diffuse , and bony hard swelling of approximately measuring about 6 cm 4 cm on the left side of the face , extending superior - inferiorly from left zygomatic arch till angle of the mandible and anteroposteriorly from nasolabial fold till tragus of the left ear with normal overlying skin [ figure 1a and b ] intraorally , swelling was solitary , slightly tender with the expansion of buccal and lingual cortical plates with no pathological changes to the overlying mucosa . there was reduce mouth opening measuring about 15 mm in the greatest dimension [ figure 2a ] . orthopantomogram revealed a large well - bordered , well - defined unilocular , circular , mixed lesion ( calcification specks ) approximately , 2 cm in size involving the ramus , coronoid , and angle of the mandible [ figure 3 ] posteroanterior view of mandible showed a well - defined , mixed lesion involving the coronoid process , ramus and the angle of the left side of the mandible [ figure 4 ] . axial and sagittal view of computed tomography confirmed the routine radiographic features and further showed a heterogeneously enhancing mixed lytic - sclerotic lesion with a sclerotic rim extending into adjacent soft tissue measuring 5.12 cm 3.83 cm with exterior and superior extension into the left infratemporal fossa and medial extension up to pterygoid muscle [ figure 5a and b ] . biopsy was taken from the involved site , and the h and e section showed the presence of trabeculae of fibrillar osteoid and woven bone . in few areas , multinucleated giant cells were also noted . based on clinical , radiographic , advanced imaging studies and histopathological report provided the diagnosis of the trabecular variant of jof affecting left mandible with differential diagnosis of cement - ossifying fibroma , osteoblastoma , and osteogenic sarcoma . the lesion was excised by segmental resection using transmandibular approach and reconstruction was done [ figure 7a and b ] . postoperative healing was uneventful ; mouth opening was increased to the value of 30 mm [ figure 2b ] . postoperatively , the esthetics , oral function of the patient such as speaking , and chewing were intact and normal [ figure 8a and b ] . the patient is still under regular follow - up as the lesion has high recurrence rate . ( a and b ) preoperative extraoral view of the patient showing swelling on the left side of face ( a ) preoperative mouth opening , ( b ) postoperative mouth opening preoperative panoramic view of the patient preoperative posteroanterior view of the patient ( a ) axial view of computed tomography , ( b ) sagittal view of computed tomography histopathological section ( a ) postoperative panoremic view of the patient , ( b ) postoperative posteroanterior view of the patient ( a and b ) postoperative extraoral view of the patient with intact facial symmetry", "jof mostly strikes children with slightly male predominance with a mean age of 11 years . clinically , it is asymptomatic , however rarely it is aggressive and symptomatic . in the present case , both maxilla and mandible are affected with more prevalence for maxilla . in the present case , the duration of swelling noted was 2 months . the characteristic radiographic features reveal a central opacification with well - defined unilocular to multilocular pattern giving it a ground glass appearance . the 50% of cases of jof are recorded in multiple sinuses with few occurring in the single sinus . the duration , clinical and radiographic features and extension of the lesion into the adjacent soft tissue favors the aggressive type of jof as previously reported . the features like resorption and perforation of cortices , tooth displacement and aggressive growth of the lesion favor the diagnosis as an aggressive variety of jof . in this case , except for tooth resorption all other features like perforation of the cortices and extension into the surrounding tissue was noted . because of the high recurrence rate , there is no follow - up protocol mentioned in the literature and immediate reconstruction is not advised . resection is considered where there is recurrence , invasion into surrounding tissues , or when preserving the inferior border is not feasible .", "jof is an uncommon clinical entity , because of its aggressiveness and high recurrence rate it is necessary to arrive at an early diagnosis , applying the appropriate clinical , diagnostic approaches , and appropriate treatment for effective results and long - standing follow - up . the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed there are no conflicts of interest .", "the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s ) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed", "", "" ]

[ "mr urography ( mru ) can be performed with two different imaging strategies : unenhanced mr urography ( t2w - mru ) , based on heavily t2-weighted fse sequences and contrast - enhanced excretory mr urography ( ce - mru ) , which is performed with a 3d ge t1-weighted sequences following intravenous gadolinium administration . a heavily t2-weighted sequence , utilizing the single - shot fast spin - echo ( ssfse ) sequence , provides static hydrographic images of fluid inside the urinary tract . it is known to be mainly useful for only evaluating dilated systems , , because of its inability to visualize entirely non - dilated ureters . so that , in routine practice , excretory ce - mru is the technique most commonly used to evaluate the non - dilated collecting system . low - dose ( 520 mg ) intravenous furosemide administration has been recommended for excretory ce - mru to improve the visualization of the non - dilated upper urinary tract , . to the best of our knowledge , no recent paper has explored the potential of the repetition of this simple breath hold t2w - mru sequence . our hypothesis was that by using low - dose of furosemide and additional multiple rotations around the ureter may improve the detection of the whole non - dilated ureter as well as mru with intravenous administration of contrast medium and provides a pertinent information . the purpose of this prospective study was to compare those two types of mru in terms of ureteral visualization in patients with non - dilated ureters , using diuretic administration by means of a direct confrontation of each technique in the same patient . to the best of our knowledge , there is no paper in the literature concerning this evaluation .", "this prospective , single - institution study received approval by our local ethical committee and no written informed consent was necessary . however all patients were orally informed just before the examination of this additional exploration of their urinary tract regarding to the use of a supplementary drug . during a period of 12 months from april 2012 through april 2013 , among a cohort of 155 patients referred to our mr department for a pelvic with retroperitoneal mr exploration , we have selected 126 patients ( extensive endometriosis : 38 patients , various pelvic carcinoma staging : 54 patients , suspicion for retroperitoneal fibrosis : 15 patients , lymph nodes or retroperitoneal masses recurrency : 19 patients ) with bilateral non - dilated ureter who underwent both a 2d t2w - mru immediately followed by an excretory 3d ce - mru . we have excluded from the study the patients with urinary tract dilatation ( n = 6 ) or urinary symptoms ( n = 2 ) , moderate renal deficiency or known allergic reaction to medication ( n = 6 ) and those who had undergone a previous pelvic surgery ( n = 15 ) ( fig . mr examinations were performed on a 3.0 t mr unit ( achieva , philips medical systems , best , the netherlands ) by using the phased array dedicated coil for pelvic and lower abdomen exploration . the field of view was large enough to cover entirely the retroperitoneum with the exploration of the whole ureter from the uretero pelvic junction to the bladder neck . each patient was explored in addition to our routine protocol for the clinical context with two breath - hold mru sequences in coronal orientation . patients were asked to void their bladder before mr examination and received an intravenous antispasmodic medication as glucagon at the dose of 1 mg ( glucagen lab novo nordisk pharmaceutics sa ) at the beginning of the exploration . if no urinary dilatation was observed on the first non - injected routine sequences , the patient received an intravenous injection of furosemide at the dose of 20 mg ( lasilix lab sanofi - aventis ) ( fig . patients were orally informed of potential side effects such as increased urination at the end of the exploration . five minutes later a 2d coronal single shot turbo fast se t2-weighted static fluid mru ( t2w - mru ) was performed localized on the right and the left urinary tract with an acquisition time of 5 s. each sequence was repeated nine times with coronal rotations and cine loop registration on each urinary tract . at the end of this first mru sequence , an intravenous injection of 0.2 ml / kg body weight gadolinium - based contrast medium , ( dotarem , guerbet , roissy cdg , france ) followed by a 15 ml saline five minutes later , coronal excretory phase mr urogram was obtained by means of a 3d breath - hold t1-weighted ge sequence with an acquisition time from 15 to 18 s ( table 1 ) . maximum - intensity - projection ( mip ) images were processed from the original source images of this excretory mru . the two types of mru , including original source images and mip reformations available from the workstation , were retrospectively reviewed . each sequence was evaluated separately and independently by two radiologists ( phh , ga ) with five and three years of experience in abdominal mr imaging , respectively . disagreements were checked by consensus with a third radiologist ( cr ) with twenty years experienced in that field to find a consensus . the readings were done in several sessions and in each session ; it was given to each reviewer a random combination of isolated mru with native images and mip . readers were neither informed of the clinical context nor the radiological report done by the radiologist who was in charge of the examination . readers evaluated the whole technical image quality with the precise criterion of the presence of disturbing artifacts such as ghosting or pulsation artifacts using a four points subjective scale ( 1 poor , 2 fair , 3 good , 4 excellent ) . the distension of the ureter on ce - mru was compared with that of obtained on t2w - mru and quoted as higher or equal . based on visual assessment , readers were asked to score four segments of each ureter . the lumbar ureter was divided in two segments : a proximal part ( from the pelvic junction to the level of the lower pole of the kidney ) ( pl ) and a distal part ( from the lower pole of the kidney and iliac crest ) ( dl ) . the pelvic ureter was also checked with two segments : an upper portion from iliac vessels to mid pelvic cavity ( pp ) and an inferior portion from mid pelvic cavity to the bladder ( dp ) . , readers were asked to evaluate independently one rotation ( chosen as strictly anteroposterior view ) , 6 rotations and finally the entire sequence ( 9 rotations ) . for ce - mru , they were asked to quote the number of entire ureter identified on mip images . the kappa statistic was used to measure the degree of interobserver agreement for qualitative assessment . strength of agreement was classified as poor ( value less than or equal to 0.20 ) , fair ( value between 0.21 and 0.40 ) , moderate ( value between 0.41 and 0.60 ) , substantial ( value between 0.61 and 0.80 ) , or excellent ( value between 0.81 and 1.0 ) . we used the wilcoxon test to compare the scoring of the segmental analysis of our two mru techniques . a p value less than 0.05 was considered to be statistically significant .", "this prospective , single - institution study received approval by our local ethical committee and no written informed consent was necessary . however all patients were orally informed just before the examination of this additional exploration of their urinary tract regarding to the use of a supplementary drug . during a period of 12 months from april 2012 through april 2013 , among a cohort of 155 patients referred to our mr department for a pelvic with retroperitoneal mr exploration , we have selected 126 patients ( extensive endometriosis : 38 patients , various pelvic carcinoma staging : 54 patients , suspicion for retroperitoneal fibrosis : 15 patients , lymph nodes or retroperitoneal masses recurrency : 19 patients ) with bilateral non - dilated ureter who underwent both a 2d t2w - mru immediately followed by an excretory 3d ce - mru . we have excluded from the study the patients with urinary tract dilatation ( n = 6 ) or urinary symptoms ( n = 2 ) , moderate renal deficiency or known allergic reaction to medication ( n = 6 ) and those who had undergone a previous pelvic surgery ( n = 15 ) ( fig .", "mr examinations were performed on a 3.0 t mr unit ( achieva , philips medical systems , best , the netherlands ) by using the phased array dedicated coil for pelvic and lower abdomen exploration . the field of view was large enough to cover entirely the retroperitoneum with the exploration of the whole ureter from the uretero pelvic junction to the bladder neck . each patient was explored in addition to our routine protocol for the clinical context with two breath - hold mru sequences in coronal orientation . patients were asked to void their bladder before mr examination and received an intravenous antispasmodic medication as glucagon at the dose of 1 mg ( glucagen lab novo nordisk pharmaceutics sa ) at the beginning of the exploration . if no urinary dilatation was observed on the first non - injected routine sequences , the patient received an intravenous injection of furosemide at the dose of 20 mg ( lasilix lab sanofi - aventis ) ( fig . patients were orally informed of potential side effects such as increased urination at the end of the exploration . five minutes later a 2d coronal single shot turbo fast se t2-weighted static fluid mru ( t2w - mru ) was performed localized on the right and the left urinary tract with an acquisition time of 5 s. each sequence was repeated nine times with coronal rotations and cine loop registration on each urinary tract . at the end of this first mru sequence , an intravenous injection of 0.2 ml / kg body weight gadolinium - based contrast medium , ( dotarem , guerbet , roissy cdg , france ) followed by a 15 ml saline flush was performed . five minutes later , coronal excretory phase mr urogram was obtained by means of a 3d breath - hold t1-weighted ge sequence with an acquisition time from 15 to 18 s ( table 1 ) . maximum - intensity - projection ( mip ) images were processed from the original source images of this excretory mru .", "the two types of mru , including original source images and mip reformations available from the workstation , were retrospectively reviewed . each sequence was evaluated separately and independently by two radiologists ( phh , ga ) with five and three years of experience in abdominal mr imaging , respectively . disagreements were checked by consensus with a third radiologist ( cr ) with twenty years experienced in that field to find a consensus . the readings were done in several sessions and in each session ; it was given to each reviewer a random combination of isolated mru with native images and mip . readers were neither informed of the clinical context nor the radiological report done by the radiologist who was in charge of the examination .", "readers evaluated the whole technical image quality with the precise criterion of the presence of disturbing artifacts such as ghosting or pulsation artifacts using a four points subjective scale ( 1 poor , 2 fair , 3 good , 4 excellent ) . the distension of the ureter on ce - mru was compared with that of obtained on t2w - mru and quoted as higher or equal . based on visual assessment , readers were asked to score four segments of each ureter . the lumbar ureter was divided in two segments : a proximal part ( from the pelvic junction to the level of the lower pole of the kidney ) ( pl ) and a distal part ( from the lower pole of the kidney and iliac crest ) ( dl ) . the pelvic ureter was also checked with two segments : an upper portion from iliac vessels to mid pelvic cavity ( pp ) and an inferior portion from mid pelvic cavity to the bladder ( dp ) . , readers were asked to evaluate independently one rotation ( chosen as strictly anteroposterior view ) , 6 rotations and finally the entire sequence ( 9 rotations ) . for ce - mru , they were asked to quote the number of entire ureter identified on mip images .", "the kappa statistic was used to measure the degree of interobserver agreement for qualitative assessment . strength of agreement was classified as poor ( value less than or equal to 0.20 ) , fair ( value between 0.21 and 0.40 ) , moderate ( value between 0.41 and 0.60 ) , substantial ( value between 0.61 and 0.80 ) , or excellent ( value between 0.81 and 1.0 ) . we used the wilcoxon test to compare the scoring of the segmental analysis of our two mru techniques .", "no patient suffered from any serious side effects of furosemide , especially no sign of allergic reaction was recorded . a total of 252 ureters were evaluated and 1008 segments were checked . for the assessment of image quality , readers judged both sequences good or excellent for all segments ( score 3 : 35/252 ; 51/252 ; score 4 : 217/252 ; 201/252 for t2w - mru and ce - mru , respectively ) . comparison of the two sets of images revealed no significant difference with respect to overall image quality ( 3.8 0.5 vs 3.6 0.7 , p value : 0.13 ) . statistically , the interobserver agreement was excellent with a correlation coefficient as high as 0.89 for t2w - mru and 0.92 for ce - mru , respectively . one rotation of t2w - mru was found clearly inaccurate to assess the non - dilated ureter . increasing the number of rotations , significantly improved the visualization of all segments . with 9 rotations and regard to identification , there were no statistically significant differences in the visualization of the ureteral segments between the two types of mru . the lumbar distal portion of the ureter was less pertinent for identification on t2w - mru with a sensitivity of 71% vs 78% for ce - mru ( table 2 ) . both segments up and down the pelvic ring have less than excellent identification in both mru techniques by comparison with two others . a bilateral entirely opacified ureteral column until the vesicoureteral junction was found in 236/252 cases with ce - mru , simulating a mild obstruction . on the contrary , on one rotation of t2w - mru , it was constantly found at least one segment , indicating that the others were collapsed . the ureter was visualized as a linear thicker structure than on t2w - mru ( fig . deviation of the pelvic ureter due to the genital tract was found in 118 ureteral segments equally recognized on both sequences ( fig . deviations due to ovarian cysts and follicules were easier to diagnose on t2w - mru than on standard t2 sequence .", "mru permits an evaluation of the urinary tract without the exposure to ionizing radiation and iodinated contrast medium . t2w - mru was the first sequence to be proposed to study excretory urinary tract . its initial relatively low resolution has been improved by the development of heavily t2-weighted turbo spin - echo sequences such as rapid acquisition with relaxation enhancement ( rare ) utilizing multiple thin - slices with mip or a thick - slice imaging technique and half - fourier acquisition single - shot turbo spin - echo ( haste ) sequences . high - resolution thick - slice images can be achieved during a single breath - hold . however , in the literature , t2w - mru is known to have several limitations . these include the superimposition of hyperintense extraurinary fluid collections on thick slice mru , no functional information and undetectable small stones . the inability of t2-w mru to fully visualize complete non - dilated ureters is cited as a major negative point in many papers , , , . finally , t2w - mru has been shown to be a rapid and non - invasive imaging technique which is useful and reliable to reveal hydronephrosis and perirenal high - intensity signals in acute urinary obstruction , , , . the concept of ce - mru has been developed later . in his initial experimental study , nolte - ernsting et al . presented the advantages of this imaging technique in retrieving high - spatial - resolution images in non - obstructed urinary tracts . the use of a paramagnetic contrast agent permits the evaluation of renal excretory function and better visualization of the non - dilated urinary tract . several papers , , , have advocated the need for a diuretic to optimize the endoluminal concentration of gadolinium and to produce accelerated distention . these authors stated that furosemide initially induces an increase in urine volume resulting in mild distention of the urinary tract , and then there is a dilution effect on the excreted gadolinium . the limitation of ce - mru is for cases with delayed excretion due to severe obstruction , the long duration of examination for revealing the underlying abnormality increasing patient and personnel inconvenience , . as several previous papers have shown the superiority of enhanced over unenhanced imaging , ce - mru is becoming in clinical routine practice the technique most commonly used to evaluate the non - dilated collecting system . to our knowledge , in the previous recent literature in adult , there are no clinical studies comparing those two mru techniques in the same patient . additionally , our evaluation has been focused on the non - dilated ureter , which is considered as being the most difficult task to obtain a visualization of all segments . as the use of low - dose ( 520 mg ) intravenous furosemide administration is essential to perform ct urography ; we have also used this medication at the dose of 20 mg to adequately distend the non - dilated ureter . in addition , it causes no real additional cost as it has a very low price . this t2w - mru sequence , with its short acquisition time , permits repeated series of breath - hold images in several coronal oblique orientations , thus providing obvious improvement to the use of this sequence previously described by other investigators . our results attest that furosemide alone is sufficient to distend and visualize all parts . in identifying ureteral segments our results with t2w - mru were quite similar to those of ce - mru , except for the lower part of abdominal ureter . image quality was excellent in all patients , and , unlike other investigators , we never have difficulty in removing superimposed fluid - filled bowel loops by changing the orientation by rotations . a potential advantage of t2w - mru over ce - mru is that it can reveal an ovarian cyst or follicles which cause a deviation of the pelvic ureter . another advantage might be the evaluation of an inflammatory process , due to the superiority of t2-weighted sequences in revealing edema and a small amount of ascites . the ability to identify one or two different segment by rotation is an indicator for normal contractions of some parts of the ureter . this visualization of the peristalsis is a good indicator to affirm that there is no ureteral repercussion for instance of a pelvic mass . in contrary when using ce - mru , we have commonly found the entire column simulation mild obstruction . the size of ureter on ce - mru was larger than on t2w - mru with the same dose of diuretic . we suggest that the contrast medium itself is a significant factor to distend the ureter and produces rapidly an over distended bladder with a risk of false positive diagnosis of mild obstruction . we could not attribute this aspect to our protocol firstly because patients were asked to void their bladder just before the beginning of the mr examination , secondly because the two sequences were performed without any supplementary delay between them ( 5 min to obtain the excretory phase after gadolinium administration ) . in addition , t2w - mru has the advantage of obtaining high quality images in a few seconds , without need for multiplanar and mip reconstructions in the contrary of ce - mru . as breath - hold t2w - mru with intravenous diuretic and multiple rotations is sufficient to produce a qualitative study of non - dilated ureter with identification of the peristaltism , we can speculate that it could be an adequate sequence to detect an initial ureteral involvement before hydronephrosis occurs . the identification of the same missing segment on all rotations with a persistent non - dilated column above could reflect an abnormal peristaltism indicating a thorough analysis of this localization . this can be potentially very useful in the cases of extrinsic ureteral involvement such as in endometriosis which is progressive and clinically silent or can be confuse with other symptoms of the disease . the identification of the same segment on all rotations can be a clue for this initial diagnosis , before the classical appearance of persistent column with gradual tapering or filling defect at the level of obstruction . however , it needs further investigation to prove that t2-mru can detect initial obstruction , prior to clinical presentation of hydronephrosis . it could also be used to evaluate potential renal donors or in patients unable to receive gadolinium . even if our material has an important number of cases , this study has some limitations . first , there is no data regarding the relative performance of the techniques in a clinical context , but we would like to focus on the ability of t2w - mru to visualize the entire ureter . second , even if our field of view was large enough to obtain an evaluation of the whole ureter it does not permit a constant evaluation of the renal pelvic cavities , especially for our tallest patients . however a precise caliceal anatomy or pathology was not the purpose of our mr examinations . of course , equally to ce - mru , the major drawback of t2w - mru remains its poor sensitivity in detecting calcifications .", "the t2w - mru sequence performed with multiple coronal orientations and diuretic administration is sufficient to identify entirely the non - dilated ureter . our series was large enough to suggest that pelvic mr imaging combined with t2w - mru allows a complete work - up in a single imaging evaluation , especially if gadolinium injection is not necessary or contraindicated .", "the authors confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome .", "the authors confirm that any aspect of the work covered in this manuscript that has involved human patients has been conducted with the ethical approval of all relevant bodies and that such approvals are acknowledged within the manuscript ." ]

[ "the first case was described by cruveilhier in 1835 and termed as hamartoma . till date , less than 200 cases have been described with synonyms of brunner gland adenoma , brunner gland hamartoma ( bgh ) or brunneroma . there is no sex predilection and patients present in the fifth to sixth decades of life . they are usually seen as incidental findings on imaging with duodenal bulb as the most common location . however , it may present with a variety of clinical presentations depending on location and tumor size such as abdominal pain , dyspepsia , nausea , vomiting , upper gastrointestinal ( gi ) bleeding and occasionally obstructive symptoms with associated chronic pancreatitis . \n", "\n a 42-year - old man presented with history of pain epigastrium since one year and recurrent vomiting within half an hour of taking food since one month . a biopsy of duodenum was performed by the same procedure which was diagnosed as well differentiated adenocarcinoma . the patient was further subjected to abdominal computed tomography ( ct ) which showed circumferential thickening of the second part of the duodenum ( 15.2 mm maximum thickness ) abutting head of pancreas with loss of fat planes . laboratory investigations showed normal blood counts , liver , and kidney function tests but elevated serum amylase and lipase levels . this patient underwent radical surgery ( whipple s procedure ) at our institute with clinical diagnosis of carcinoma periampullary region and the specimen was sent for histopathological examination . gross examination of the specimen revealed circumferential thickening of the second part of the duodenum and bulky head of the pancreas . cut section revealed a diffuse grey white area measuring 4x4 cm with few cystic and hemorrhagic areas . six lymph nodes were isolated from the specimen and a single celiac node was examined which was received separately . microscopy showed a tumor composed of lobules of brunner s glands with a dilatation of few glands . these lobules were separated by fine fibrous septa and constituted > 50% of the thickness of the duodenal wall ( figures 1 , 2 and 3 ) . hyperplasia of brunner s glands spanning > 50% of wall thickness ( h&e stain , 4x ) lack of features of anaplasia in proliferating brunner s glands ( h&e stain , 20x ) smooth muscle actin stain highlighting the intervening smooth muscles ( ihc , 10x )", "\n brunner glands are branched acinotubular glands with submucosal location , found exclusively between the pyloric ring and the papillae of vater . their major role is the protection of duodenal epithelium from acidic gastric chyme by secretion of neutralizing alkaline fluid composed of viscous mucin . the pathogenesis of bgh has been linked to excessive local irritation from acidic gastric chyme , vagal stimuli , or unidentified antral hormones . hence , it is associated with conditions related to gastric hyperacidity and changes in gastric emptying such as esophagitis , hiatus hernia , and gastric - peptic ulcer . the association of this tumor with uremia and chronic pancreatitis has also been reported . in a study of patients undergoing pancreaticoduodenectomy , 76% of the patients had diffuse hyperplasia of brunner s gland , raising the question whether this is an adaptation to pancreatic insufficiency . an alternate suggestion for this hyperplasia is that these glands proliferate in reaction to inflammationas evidenced by presence of lymphocytes in the lesion . support to this hypothesis is not rationalized as lymphocytes may be present in normal submucosa of the gastrointestinal tract . in a study on the role of helicobacter pylori infection in the pathogenesis of bgh , 5 of 7 cases with bgh had concurrent helicobacter pylori infection . a definite relationship has not yet been proven because of the high prevalence of helicobacter pylori and rarity of this lesion in the general population . \n \n initially , the hyperplasia of brunner s gland can be focal , localized , and solitary usually involving a lobule . however , if stimulus persists then the lesion enlarges involving multiple lobules leading to formation of diffuse and annular lesion infiltrating into the wall and circumference of the duodenum . endoscopically these can be nodular , polypoid or diffuse with thickening of the duodenal wall . due to this presentation , these lesions can be misdiagnosed as gastrointestinal stromal tumor ( gist ) , lymphoma , carcinoid , peutz jeghers polyp , prolapsed pyloric mucosa , or aberrant pancreatic tissue . although surface biopsy of this lesion can be misleading as in our case , a deeper biopsy will be more diagnostic . microscopic diagnosis of hyperplasia requires presence of lobules of brunner s glands within the submucosa in at least 50% of the length of the biopsy showing no features of dysplasia . adenoma has been used for lesions > 1 cm and the term hamartoma if it contains mesenchymal elements also . conflicts regarding this terminology exist as some authors suggest that mere change in size indicate progression of hyperplasia and do not imply neoplastic behavior . ductal malignancy arising from brunner gland adenoma has been reported in one case which is a very rare circumstance ( 0.5% ) . brunner gland adenoma in the duodenal wall with a focus of atypical cells supported by mib-1 and p53 positivity has also been reported . \n \n the treatment of bgh is done with surgical or endoscopic removal in symptomatic patients taking into account the size and gross appearance . although endoscopic techniques of removal are more helpful , this procedure can be limited by difficult anatomical sites . recurrence in such a lesion is not known . \n \n even though diagnosis of bgh can be made by various imaging studies , in our case hence , these possibilities should be kept in mind while dealing with duodenal masses . \n", "", "" ]

[ "metastasis of transitional cell carcinoma ( tcc ) of the renal pelvis to the eye is a very rare event and has only been reported in one case in the literature . once diagnosed , these cases usually show widespread metastases and carry a very poor prognosis . we present a case report of a male patient with unilateral choroidal metastasis secondary to tcc of the renal pelvis . review of the relevant literature will be addressed to highlight the involvement of the choroid in cases of metastatic disease , the unusual presentation of tcc , diagnostic methods , and the various therapeutic modalities used to treat these cases .", "a 61-year - old male , who has a history of congestive heart failure , is a nonsmoker , and has no significant family history of malignancies , presented to the ophthalmology clinic with sudden - onset loss of vision of the left eye associated with intermittent left eye pain , relieved by analgesics . ophthalmic evaluation revealed a visual acuity of 20/40 in the right eye and 20/400 in the left eye . funduscopic examination of the left eye showed an amelanotic macular choroidal lesion associated with inferior exudative retinal detachment ( fig 1a ) . a b - scan ocular ultrasound showed a dome - shaped , 2.8-mm choroid thickening with variable internal reflectivity , associated with inferior exudative retinal detachment ( fig 1b ) . fluorescein fundus angiography revealed early choroid blockage ( fig 1c ) and late leakage associated with hot spots at tumor margin . all of these clinical features are suggestive of the clinical diagnosis of left choroidal metastasis . physical examination of the patient also revealed multiple suspicious chest wall nodules . in search for a primary malignancy , imaging studies were done , which revealed multiple pulmonary and liver metastasis , subcutaneous metastasis , multiple lymphadenopathies , and left renal upper pole mass ( fig 2 ) . ct - guided left renal mass biopsy and ultrasound - guided biopsy of the left chest wall subcutaneous mass were performed . they are negative for ck20 , pax2 , ttf-1 , synaphopysin , and s100 ( fig 3 ) . external beam radiation therapy ( ebrt ) is a common mode of treatment and will be considered if there is no improvement of visual symptoms with chemotherapy .", "choroidal metastasis is the most common intraocular malignancy , due to its rich vascular supply and high oxygen concentration [ 4 , 5 ] . less common primary cancers include the thyroid , prostate , kidney , testicles , pancreas , ovary , and liver . rarely is it seen in cases of tcc , as there have only been 5 previous reports in the literature involving choroidal metastasis , 3 from tumors involving the bladder , and 2 from other parts of the urinary tract . choroidal metastasis is commonly associated with disseminated disease and , unfortunately , carries a very poor prognosis . tcc usually metastasizes to the regional lymph nodes ( 78% ) , liver ( 38% ) , lung ( 36% ) , bone ( 27% ) , adrenal gland ( 21% ) , and intestine ( 13% ) . the heart , brain , kidney , spleen , pancreas , meninges , uterus , ovaries , prostate , and testes have been reported as unusual sites of presentation of tcc . other less common clinical presentations include flashes ( 5% ) and floaters ( 5% ) . diagnosis is based on clinical findings , which are typically seen as yellow , plateau - shaped lesions associated with subretinal detachment . a - scan and b - scan ultrasonography show irregular internal reflectivity and a solid plateau - shaped mass , respectively . fluorescein fundus angiography typically shows mottled hypofluorescence in early stage and leakage in late stage . the treatment of choroidal metastasis should be individually tailored , taking into consideration the patient 's systemic state , disease activity , and the presence of visual symptoms . its aim is palliation , to improve the quality of life of the patient and to restore or maintain visual function . local therapeutic regimens include ebrt , plaque radiotherapy , and complete enucleation for painful , blind eyes [ 13 , 14 ] . ebrt delivered at a dose of 40 gy over 20 fractions was effective in improving and maintaining vision in patients with choroidal metastasis [ 15 , 16 ] . in cases of widespread metastases , systemic chemotherapy alone or in combination with local therapy is an accepted therapeutic option . in patients with choroidal metastasis alone , local therapy has been reported to be safe , maintaining visual functions without the systemic side effects of chemotherapy .", "choroidal metastasis related to tcc of the renal pelvis is an extremely rare event and is associated with widespread disease and a very poor prognosis . systemic chemotherapy with carboplatin and gemcitabine is a palliative treatment option for this patient . ebrt offers local palliation and will be considered according to the patient 's visual response to chemotherapy .", "", "" ]

[ "chronic kidney disease ( ckd ) is defined as the slow and steady damage of kidney function in an irreversible manner , which ultimately results in end - stage renal disease ( esrd ) ( 1 , 2 ) . this chronic disorder is a serious health problem with a high prevalence rate in adults and children . it causes mortality and other health complications , and high costs are incurred due to the frequent medical diagnosis and poor prognosis of patients ( 1 , 2 ) . the causes of ckd are very different in children than in adults . in a recent north american pediatric renal transplant cooperative study ( naprtcs ) , congenital causes , including congenital anomalies of the kidney and urinary tract ( cakut ) ( 48% ) and hereditary nephropathies ( 10% ) , were the most common causes of ckd in the children ( 3 ) . based on previous studies , such a chronic disorder impairs the quality of life of children due to the development of various clinical symptoms , especially developmental disorders and psychiatric disorders ( 4 - 6 ) . ckd clinical manifestations in children are presented as edema , hypertension , hematuria , and proteinuria , and specifically during the neonatal period as weight gain , polyuric dehydration , and urinary tract infection ( 7 ) . in addition to the above mentioned symptoms , ckd may lead to a type of hyperactivity and central nervous system ( cns ) dysfunction ( especially affecting sympathetic function ) ( 8) . based on the literature , the prevalence of cognitive disorders such as memory disorder , and different psychiatric disorders including anxiety disorders ( ads ) , depression , and adjustment disorders in children with different levels of ckd was significantly higher compared to the group of healthy children and children with very early stages of ckd ( 9 - 12 ) . based on the available literature , patients with ckd who develop psychiatric and cognitive disorders face longer hospitalization times , more health complications , and greater mortality compared to other patients at the same stage without these disorders ( 11 , 12 ) . therefore , proper diagnosis of psychiatric and cognitive disorders in these patients is paramount . more specifically , one disorder whose relationship with ckd in children has not been adequately examined is obsessive - compulsive disorder ( ocd ) . obsessive - compulsive disorder ( ocd ) is a chronic disabling illness characterized by repetitive ritualistic behaviors over which the patients have little or no control ( 13 , 14 ) . a review of the literature revealed no study that assessed the association between ocd ( based on the obsessive compulsive inventory - child version ( oci - cv ) ) and ckd .", "the aim of this study was to investigate ocd in children with early stages of ckd and to compare it with healthy children .", "this case - control study was performed on 160 children in the age bracket of 7 to 17 years old who were referred to the pediatric clinic of amir kabir hospital in arak ( iran ) in 2015 . the ethics committee approved the study ( approval code : 93 - 162 - 1 , registration code : 1087 ) . eighty children with early stages of ckd ( stages 1 , 2 and 3 ) comprised the case group , and the control group consisted of 80 healthy children without ckd ; children were included in the study based on the inclusion criteria . the sample number was calculated with regards to the prevalence of cognitive disorders due to ckd ( = 0.05% , = 0.2% ) . ckd was defined as the presence of kidney damage ( for example , any structural or functional abnormality involving pathological , laboratory , or imaging findings ) for 3 months or a glomerular filtration rate ( gfr ) < 60 ml / minute/1.73 m for 3 months ( 2 ) . in this study , as per the ckd definition ( 2 ) , children with early stages of ckd ( stage 1 , 2 and 3 ckd ) who were diagnosed with ckd due to renal and urinary - genital tract anomalies , such as obstructive uropathy , renal dysplasia , or reflux nephropathy , were included in the case group . those children who were diagnosed with ckd due to reasons other than renal and urinary - genital tract anomalies , or those with stages 4 and 5 stage 1 , 2 , 3 and 4 ckd were defined as kidney damage with normal or increased gfr ( gfr 90 cc / minute/1.73 m ) , kidney damage with mild decreased gfr ( gfr = 60 - 89 cc / minute/1.73 m ) , kidney damage with moderately decreased gfr ( gfr = 30 - 59 cc / minute/1.73 m ) and kidney damage with severely decreased gfr ( gfr = 15 - 29 cc / minute/1.73 m ) , respectively ( 15 ) . esrd or stage 5 ckd was defined by the amounts of gfr < 15 cc / minute/1.73 m which are indicative of the start of dialysis ( 15 - 17 ) . we included children of both sexes in the age range of 7 to 17 years old , and children with stage 1 to 3 ckd ( for at least 6 months ) . we excluded patients with the following conditions or circumstances : a history of considerable psychiatric disorders ; intellectual disabilities , or nervous system disorders ; a history of any type of anxiety disorder before developing ckd ; congenital and chromosomal abnormalities ; a chronic medical condition ; a family history of major psychiatric disorders in first - degree relatives ; parents not consenting to participating in the study ; and not completing the questionnaire . intellectual disability was defined in terms of the intelligence quotient ( iq ) of 70 ( 18 ) . healthy children were selected from children who had been referred to the hospital as outpatients for minor conditions such as the common cold or abdominal pain . the matching method was used for selecting the healthy children , and children were matched in groups based on age , sex , and socioeconomic status . in this 3-month study ( april 2015 to july 2015 ) , a total of 40 children were excluded based on the inclusion and exclusion criteria . among 22 ( 100% ) patients who were excluded in the case group , 17 ( 77.27% ) and 5 ( 22.72% ) patients were excluded due to parental unwillingness to complete the oci - cv and a history of considerable psychiatric disorders ( anxiety disorders before the diagnosis of ckd ) , respectively . for the control group , the remaining children were excluded due to lack of parental consent . after obtaining informed consent from the children s parents , demographic , clinical , and perinatal data ( age , sex , residence , birth weight , current weight , height , body mass index ( bmi ) , mother s age at birth , gestational age , maternal education , household incomes , marital status , type of delivery , age at diagnosis of ckd , and duration of ckd ) were recorded . ocd in children was evaluated using the obsessive compulsive inventory - child version ( oci - cv ) by a psychologist ( consultant ) . this self - reporting questionnaire has been designed for people aged 7 to 17 years , containing 21 items and 6 subscales , including doubting / checking ( 5 phrase ) , obsessing ( 4 phrase ) , hoarding ( 3 phrase ) , washing ( 3 phrase ) , ordering ( 3 phrase ) , and neutralizing ( 3 phrase ) ( 19 - 21 ) . the subjects are supposed to indicate their degree of agreement or disagreement with each item through a 3-point likert scale ranging from never to always . the scoring options on this test were as follows : never = 0 , sometimes = 1 , and always = 2 . based on multiple sources of evidence , the oci - cv is considered to be a reliable and valid method for identifying children with ocd . the oci - cv was modestly correlated with obsessive compulsive symptom severity on the children s yale - brown obsessive compulsive scale ( cy - bocs ) , as well as with clinician - reported ocd severity ( 19 , 20 ) . the persian version of the oci - cv questionnaire was tested for reliability in a pilot study by the researchers with 30 patients in each of the case and control groups ; the cronbach s alpha was 0.89 . the collected data was analyzed with spss software ( statistical package for the social sciences , version 18.0 , spss inc . categorical data are expressed as numbers ( percentage ) and compared with a chi - square test .", "the mean weight , height , and bmi of the children were 38.15 6.8 kg , 127.61 8.5 cm , and 17.31 3.52 , respectively . of the 160 subjects , 86 ( 53.75% ) were boys and 74 ( 46.25% ) were girls . in the group comprised of children with ckd , the duration of the disease and the age of diagnosis were 1.48 2.12 and 6.9 5.4 years , respectively . abbreviations : bmi , body mass index ; ckd , chronic kidney disease ; cs , caesarean section ; nvd , normal vaginal delivery . household incomes : low mean monthly incomes < 5000000 rials ; moderate mean incomes between 5,000,000 and 10,000,000 rials ; high means incomes > 10,000,000 rials . the mean age ( p = 0.211 ) , weight ( p = 0.51 ) , height ( p = 0.113 ) , gender distribution ( p = 0.112 ) , average bmi ( 0.32 ) , mother s age at childbirth ( p = 0.66 ) , and the child s birth weight ( p = 0.08 ) were not significantly different between the children in the case and control groups . furthermore , the children in both groups were identically distributed in terms of residency status ( p = 0.3 ) , family s monthly income ( p = 0.376 ) , gestational age ( p = 0.12 ) , marital status ( p = 0.311 ) and type of delivery ( p = 456 ) . however , the maternal educational level was significantly different between the two groups ( p = 0.001 ) , so that 30 ( 37.5% ) and 37 ( 46.25% ) of mothers in the group of the children with ckd had college and high school education , respectively , whereas 16 ( 20% ) and 50 ( 62.5% ) of the mothers in the control group had college and high school education , respectively . the mean scores of doubting / checking ( case : 3.52 2.54 , control : 2.5 2.32 , p = 0.007 ) and ordering ( case : 2.59 1.81 , control : 1.5 2.56 , p = 0.005 ) of the children with ckd was significantly higher than the scores of the healthy ones . moreover , the mean total scores for the oci - cv of the children with ckd at 15.32 7.69 was significantly higher than those of the healthy ones at 11.12 2.54 ( p = 0.021 ) . nevertheless , the mean scores of obsessing ( p = 0.11 ) , hoarding ( p = 0.117 ) , washing ( p = 0.211 ) , and neutralizing ( p = 0.41 ) were not significantly different between the case and control groups ( table 2 and figure 1 ) . according to the results of spearman s test , there was significant correlation between the duration of ckd and doubting / checking ( p = 0.004 , correlation coefficient ( cc ) : 0.4 ) , obsessing ( p = 0.06 , cc : 0.02 ) , washing ( p = 0.031 , cc : 0.8 ) , ordering ( p = 0.001 , cc : 0.2 ) , and the total scores of the oci - cv questionnaire ( p = 0.04 , cc : 0.4 ) ( table 3 ) .", "according to the results of the current study , ocd and certain subscales of this disorder are more likely to occur in children with early stages of ckd than in healthy children . in this respect , doubting / checking and ordering had higher rates among children with ckd than among healthy children . furthermore , the results indicated that the duration of ckd was significantly correlated with the mean total scores of the oci - cv and certain subscales , such as doubting / checking , obsessing , washing , and ordering . on the basis of previous evidence , chronic disorders or diseases in adults and children can be associated with reduced quality of life and social , occupational and educational poor performance ( 22 ) . in fact , it has been revealed that such an association can in particular cases have a negative effect on the clinical course of the underlying disease ( 22 ) . according to relevant studies , the risk of mood and anxiety disorders occurring among people with chronic medical illnesses tends to be greater as compared to the corresponding occurrence rates among healthy people in the general population ( 22 ) . although there have been a number of studies assessing the psychiatric disorders in children with ckd , no study has been carried out on the relationship between ocd and ckd based on the oci - cv . ( 9 ) studied 19 children with ckd who did not need dialysis and 19 others with esrd in order to determine if any of them had a psychiatric disorder . the evidence indicated that 18.4% , 10.3% , 7.7% , 5.1% , and 2.6% of the subjects had adjustment disorder , depression , cognitive disorder , anxiety , and elimination disorder . the total prevalence of the above disorders were calculated at 68.4% and 36.8% in dialysis and non - dialysis groups , respectively . in another study on 30 children with both ckd and continuous peritoneal dialysis ( cpd ) , 30 children who had undergone renal implantation , and 33 healthy children , fukunishi and honda ( 10 ) revealed that there was a significant difference in the prevalence of adjustment disorder between the children in the three groups ; the dialysis and control groups had the highest and lowest prevalence of this disorder , respectively . with the objective of investigating cognitive function in children with ckd , slickers et al . studied 29 children aged 7 to 19 simulated through creatinine clearance ( crcl ) . the results showed that the severity of ckd was significantly correlated with decreased iq and impaired memory function . furthermore , it was revealed that a longer duration of ckd and its onset at a younger age were considerable risk factors for the development of cognitive disorders ( 11 ) . in another study on children with esrd and healthy children , fukunishi and kudo ( 23 ) concluded that the prevalence of anxiety and depression was significantly higher in children with esrd and peritoneal dialysis ( pd ) than in healthy children . ( 24 ) examined 15 children aged 8 to 16 with renal disease ( 8 children with esrd and 7 children with mild kidney disease as the control group ) and showed that the depression rate in the case group was significantly higher than that in the control group . in 2015 , yousefichaijan et al . ( 25 ) compared the occurrence of attention deficit / hyperactivity disorder ( adhd ) among 75 children aged 5 - 16 with ckd ( stages 1 to 3 ) and 75 healthy children . the results of this study indicated that the prevalence of adhd ( case group : 12% , control group : 16% , p = 0.664 ) was not significantly different between the two groups . in another study on adhd , yousefichaijan et al . ( 26 ) examined 100 children with esrd undergoing peritoneal dialysis and 100 healthy children . they found that the rates of attention deficit ( p = 0.01 ) and hyperactivity ( p = 0.002 ) among children with esrd were significantly higher than the corresponding rates among healthy children . according to previous studies , it can be argued that the risk of various psychiatric disorders in children with ckd at early stages and children with esrd tend to be significantly higher as compared to healthy children . moreover , such disorders can be found more frequently in children with esrd , particularly in those undergoing dialysis , than in patients experiencing the early stages of ckd . similarly , the results of this study were indicative of the higher prevalence of ocd as an anxiety disorder in children with ckd as compared to healthy children . although it can be concluded that psychiatric interventions , particularly in the case of anxiety disorders , can be beneficial for children with varying degrees of ckd , there is an insufficient number of studies exclusively focused on each of the psychiatric disorders ( such as ocd in our study ) and a lack of evidence concerning the clinical impact of these disorders on the clinical course of ckd . therefore , it is highly recommended that future studies be conducted so as to yield more definitive conclusions . one limitation of our study involved the lack of cooperation of some parents in completing the oci - cv questionnaire . although this criterion led to the exclusion of some otherwise eligible children , we tried to encourage the parents by describing the possible usefulness of the study and by offering to help them fill in the questionnaire . according to our findings , the risk of ocd among children with ckd is significantly higher than the risk among healthy children . although the results suggest that psychiatric interventions could be helpful in treating children with ckd , the limited number of studies on this particular issue suggests that further investigation into this medical condition is required so as to obtain more conclusive results ." ]

[ "problems with shoe wear have long been recognized as an endemic issue among the geriatric population with a prevalence rate of nearly 80% . women in particular are more susceptible to these problems than men . individuals having foot pathology are often severely physically impaired , making it increasingly difficult to perform activities of daily living . consequentially , this leads to physical inactivity , which is cited as one of the first signs of deterioration and the overall decrease in quality of life . moreover , some studies have linked physical inactivity to suicide , depression , and increased risk of cardiovascular - related problems . individuals with diabetes , chronic disease , nondiabetic neuropathy , and inflammatory conditions are at a severe disadvantage . further complications of foot pathology , which include cellulitis , ulcerations , and difficulty in maintaining balance , have increased the risk of serious injuries and fractures from falls . studies have shown that adults older than 65 years fall at least once per year on average , some of which are attributed to generalized thinning of skin and fat pad atrophy . the increased risk of falls may highlight the need to promote preventative measures to combat this issue . unfortunately , little current data or research focuses on the role of footwear in the prevention of inactivity in the elderly patients . common foot pathologies like corns , hallux valgus ( bunions ) , and hammertoes have been known to increase plantar pressure , cause discomfort , pain , and swelling . multiple etiologies have been noted to cause foot problems , and studies have indicated ill - fitting shoes as one of the major underlying cause . a prevailing hypothesis is that wearing the appropriate footwear will improve factors such as plantar pressure , thereby mitigating pathology while facilitating balance . this review aims to form a general basis of understanding footwear - associated foot pathologies pertaining to the elderly patients .", "elderly individuals with preexisting clinical conditions such as diabetes , neuropathies , and musculoskeletal disorders are at a higher risk of developing foot problems when compared to normal , healthy individuals . furthermore , factors such as exercise and living conditions contribute to the development of foot problems . finally , the uneven pressure provoked by ill - fitting footwear has been documented as a key factor that can cause , accelerate , or exacerbate foot - related conditions . some common foot pathologies include hallux valgus ( bunions ) , corns ( callus ) , and hammertoes . callus formations , or corns , result from continuous pressure from tight footwear , leading to hyperkeratosis of the skin . if left unattended , callus formations can lead to ulcerations , which are particularly troublesome for diabetic patients . \n the hallux valgus deformity , or bunion , is also frequently linked to ill - fitting ( ie , tight , narrow ) footwear . it is caused by the lateral deviation of the great toe , which causes a valgus deformity in the first metatarsophalangeal ( mtp ) joint . lesser toe deformities , such as hammertoes , are also associated with ill - fitting footwear . these deformities of the second , third , fourth , and fifth toe are caused by contracture of the phalangeal joints . clinical presentations include the deformity accompanied by pain over the dorsal surface of the foot , which tends to worsen with ill - fitting footwear . one of the unifying elements to these 3 common forms of foot pathology is their link with ill - fitting footwear and their tendency to go unnoticed in the elderly population .", "nonoperative approaches have been employed as the first line of treatment for the most common foot pathologies . measures are taken to adjust or alleviate pressure from the affected area . as a result , foot inserts ( ie , dr scholls ) are also used to facilitate balance by improving arch support , which can decrease the width of the foot during weight bearing maneuvers . furthermore , met pads and splits operative management is considered only when conservative measures fail to relieve progressed , advanced - stage pathology . usually , symptomatic relief can be achieved with more accommodating shoe wear after the soft tissue envelope around the foot is stabilized . this process of relieving the pressure areas and encouraging natural healing of ulcerations or soft tissue problems to heal before using modified shoe wear and orthotics often involves close follow - up in the office or wound care center . special consideration is given to diabetic patients , who have a higher risk of developing ulcerations due to peripheral neuropathy . therefore , routine physical examination is encouraged to detect and address these issues early before they lead to progressive deterioration . studies have shown that women are more susceptible than men to problems associated with inappropriate footwear in terms of the choice of footwear in the elderly patients , lord et al concluded that individuals had better balance when wearing shoes with higher collars than with lower ones . wide shoes are effective against bunions , and extra - depth shoes are more appropriate if the individual has hammertoes or mid - foot arthrosis . low - heeled footwear is known to reduce the risk of falls , likely due to the associated lowering of the center of gravity . while assessing different footwear , athletic and canvas shoes ( sneakers ) are associated with the lowest risk of a fall when compared to other types of shoes . moreover , some suggested that sole hardness have a little effect on overall balance in the absence of a specific condition , such as hallux rigidus , which may benefit from application of hard sole . as an extra preventative measure , better fitting footwear with slip - resistant soles is recommended both inside and outside the household to reduce the risk of falls . taken together , these studies seem to support the use of footwear with low heels , slip - resistant soles and wider frames to ensure comfort , better balance , and reduced risk of injury in the elderly population . in the event of multiple deformities , an individual may benefit from the combination of both extra - depth , wide , and low - heeled footwear to increase foot stability and facilitate balance .", "the use of appropriate shoe wear in the elderly patients is related to the socioeconomic factors . millions of elderly individuals live at or below the poverty level . in fact , women older than 75 years are 3 times more likely to become poor . without established financial programs such as social security , nearly half of all elderly individuals would be considered poor today . changes in living conditions ( ie , community homes , nursing homes , etc ) can often worsen the socioeconomic status of elderly patients . deteriorating health conditions followed by reduced physical activity can lead to a shift in clinical care , one that emphasizes the management of illness . in the face of such socioeconomic constraints , furthermore , retail stores often do not stock the customized shoes ( ie , extra wide ) needed for elderly customers , making availability an issue for elderly patients . the neglect of appropriate footwear in the elderly has been linked to early amputation , and in rare cases , early demise . \n to help alleviate the financial burden , elderly patients enrolled in medicare part b may benefit from the medical coverage of certain medical supplies and preventative services . eligible patients typically have diabetes or other severe foot diseases . under the medicare part b provision , such patients are entitled to a pair of custom - molded shoes , 3 pairs of inserts , or a pair of extra - depth shoes per calendar year . the patient will be held responsible for only 20% of the cost while medicare covers the rest .", ". some of these hazards such as wet pavements and bad weather conditions are capable of causing serious injury . these pathologies , in return , have contributed to the growing risk of even more serious injuries such as falls and fractures . ultimately , the noticeable decrease in physical activity in the elderly patients has been linked to depression and an overall decline health and quality of life . clinically , preventative measures such as appropriate footwear should be continually emphasized to stop or delay the progression of some foot problems . while recommending appropriate footwear is a cost - effective approach when compared to other options , it is often hindered by socioeconomic obstacles . nevertheless , studies have shown that it can be a vital step to ensure prolonged musculoskeletal and overall health among the elderly patients ." ]

[ "during vertebrate development , heart is formed by a highly stereotypic and organized process , and the expression of genes in these processes need be tightly regulated in a spatiotemporal manner ( barnett et al . , 2012 ; wang , 2012 ) . recent reports suggest that epigenetic regulation modulates genetic program during differentiation and development of various organs including heart and somitic lineages ( wamstad et al . , 2012 ) . for instance , modifications of histone substantially alter the accessibility and structure of chromatin , therefore modifying transcriptional activity of specific gene clusters . histones are known to undergo specific modification such as methylation and acetylation : while methylation and demethylation of histone are coordinated by histone methyltransferases ( hmts ) and demethylase ( hdms ) , acetylation of histone is modulated by acetyltransferases ( hat ) and deacetylases ( hdac ) ( bhaumik et al . , 2007 ; helin and dhanak , 2013 ) . due to their significance in regulating genetic program during development and diseases , recent efforts were directed to identify specific factors that mediate this process ( arrowsmith et al . , 2012 ; cayuso mas et al . , 2011 ; kim et al . , 2012a ; 2012b ) . in case of methylation , histone h3 lysine 4 residue ( h3k4 ) is sequentially modified through mono- , di- , and tri - methylation status , each of which step is mediated by specific enzymes . mlls ( mll15 ) , setdb1 , and setdb2 have broad roles for h3k4 methylation , and can methylate all type of h3k4 , such as non- , mono- , di - methylated h3k4 ( black et al . , 2012 ; sims and reinberg , 2004 ) . in contrast , smyd3 methylate mono- and di - methylated h3k4 , and setd7 can only catalase the non - methylated h3k4 to transit to mono - methylated h3k4 ( hamamoto et al . , 2004 ; wang et al . , 2001 ) set and mynd domain - containing proteins ( smyds ) , involving smyd15 , are high conserved protein family across plant , fungi , and animal as well as some protozoa , and have two functional protein domains , set and mynd domains ( del rizzo and trievel , 2011 ; dillon et al . , 2005 ) . especially , set domain is important for histone lysine methylation activity and mynd domain can mediate the protein - protein interaction and bind to dna motifs . smyd3 was originally reported as histone lysine methyltransferase ( hmt ) , which methlylates the mono-(h3k4me1 ) and di - methylated lysine 4 residue ( h3k4me2 ) to generate h3k4me3 , of histone h3 , but smyd3 also can bind to 5-ccctcc-3 motif on promoter region of nkx2.8 gene to induce its expression in cancer cells ( hamamoto et al . , 2004 ) . recent researches showed the global decrease of histone h4 lysine 5 ( h4k5 ) methylation in smyd3 knock - down condition and structural preference for histone h4 lysine 20 ( h4k20 ) of smyd3 , suggesting the substrate diversity of smyd3 ( foreman et al . it is known that smyd3 is highly expressed in a various cancers , including liver , prostate , rectal , and breast carcinomas , and stimulates the oncogenic activities , such as cell proliferation , adhesion and migration , of tumor cells ( frank et al . , 2006 ; hamamoto et al . , 2006 ; silva et al . , in addition , smyd3 is essential for myogenic differentiation through myod regulation ( proserpio et al . , actually , the deficient zebrafish embryos displayed the developmental cardiac and somite abnormalities , illuminating its functions in cardiac and somite muscle formation ( fujii et al . , 2011 ) . set domain containing protein 7 ( setd7 ) , also termed as set7/9 , is another type of histone lysine methyltransferase ( hmt ) and only have set domain for methyltransferase activity , but not mynd domain ( wang et al . , 2001 ) . it was initially discovered as a specific methyltransferase for only non - methylated histone h3 lysine 4 ( h3k4 ) that converted to mono - methylated histone h3 lysine 4 ( h3k4me1 ) ( wang et al . , 2001 ) . subsequently , recent accumulating evidence suggests that non - histone proteins , such as yap , dnmt1 , e2f1 , and stat3 , are also the substrates for setd7 ( estve et al . , 2009 ; oudhoff et al . , 2013 ; pradhan et al . , 2009 ; yang et al . , 2010 ) . the mouse knock out allele of setd7 is a viable and did not show any developmental and gross defect ( campaner et al . knock - down of setd7 showed the defects in skeletal muscle formation and myofibril structures ( tao et al . , 2011 ) . here , we examined the function of smyd3 and setd7 in heart development in zebrafish . collectively , our results demonstrate that smyd3 and setd7 function synergistically for skeletal muscle development and non - redundantly for heart morphogenesis .", "zebrafish ab strain as wild - type control for all experiments , expect the experiments using tg(cmlc2:egfp ) line . zebrafish strains were maintained as previously described ( westerfield , 1993 ) . to visualize the heart morphology in live embryos , tg(cmlc2:egfp ) control , smyd3 , and setd7 mos were designed by author and ordered to genetools company . mos were injected with 0.1% phenol - red solution into zebrafish embryos at 12 cell stages as desired concentrations . the sequences of mos used are control mo , 5-cctcttacctcagttacaatttata-3 ; smyd3 mo , 5-aagagctcttacctgacactgaacg-3 ; and setd7 mo , 5-agcttctcgtaccttccaccacttc-3. embryos injected mos were observed or collected at desired developmental stages for further experiment . to make digoxigenin - labelled antisense ribo - probe , zebrafish smyd1a ( nm_205540.1 ) , smyd2 ( nm_001013550.1 ) , smyd3 ( nm_001037400.1 ) and setd7 ( nm_001002456.1 ) full orfs were cloned into pgem - t vector ( promega ) after amplification of 24 hpf cdna . following primers were used for pcr reaction : smyd1a forward , 5-agcatgaccgtggagaagac-3 ; smyd1a reverse , 5-tgttcatgctttgatctgcac-3 ; smyd2 forward , 5-agca gcaggtcaatagcgatg-3 ; smyd2 reverse , 5-ttcctcccattatgtctgctc-3 ; smyd3 forward , 5-atggacggtcactg cagatga-3 ; smyd3 reverse , 5-ctagagcgtgttgagctc agc-3 ; setd7 forward , 5-aacatggacagcgatgatgac-3 , and setd7 reverse , 5-caggtgttcagaggagtc-3. the reaction producing ribo - probes were carried out according to manufacture protocol ( roche ) . whole mount in situ hybridization were routinely performed as previously described ( thisse and thisse , 2008 ) . as brief , to experiment , fixed embryos washed and digested with proteinase k solution as desired concentration and times . hybridization reaction was carried out at 65c , and the detection of hybridized rna probe was achieved by anti - digoxigenin fab fragment conjugated to alkaline phosphatase and bcip / nbt substrate ( roche ) . staining were stopped according to desired detection status and imaged in 75% glycerol solution for proper orientation . to generate the synthetic smyd3 and setd7 mrnas , we first cloned the full open reading frames ( orf ) of smyd3 and setd7 into pcs2 + expression vector , which have sp6 promoter sequence and sv40 poly - a tail sequence for in vitro transcription . these plasmids were cut by noti digestion to linearize , and capping mrna were produced using m7g(5)ppp(5)g ( roche , germany ) and sp6 rna polymerase ( roche , germany ) . synthetic mrna was diluted as 100 g / ml and phenol red was added prior to injection as 0.1% solution . injection was achieved at 12 cell stages as optimal concentrations ( about 100200 pg / embryo ) .", "zebrafish ab strain as wild - type control for all experiments , expect the experiments using tg(cmlc2:egfp ) line . zebrafish strains were maintained as previously described ( westerfield , 1993 ) . to visualize the heart morphology in live embryos , tg(cmlc2:egfp )", "control , smyd3 , and setd7 mos were designed by author and ordered to genetools company . mos were injected with 0.1% phenol - red solution into zebrafish embryos at 12 cell stages as desired concentrations . the sequences of mos used are control mo , 5-cctcttacctcagttacaatttata-3 ; smyd3 mo , 5-aagagctcttacctgacactgaacg-3 ; and setd7 mo , 5-agcttctcgtaccttccaccacttc-3. embryos injected mos were observed or collected at desired developmental stages for further experiment .", "to make digoxigenin - labelled antisense ribo - probe , zebrafish smyd1a ( nm_205540.1 ) , smyd2 ( nm_001013550.1 ) , smyd3 ( nm_001037400.1 ) and setd7 ( nm_001002456.1 ) full orfs were cloned into pgem - t vector ( promega ) after amplification of 24 hpf cdna . following primers were used for pcr reaction : smyd1a forward , 5-agcatgaccgtggagaagac-3 ; smyd1a reverse , 5-tgttcatgctttgatctgcac-3 ; smyd2 forward , 5-agca gcaggtcaatagcgatg-3 ; smyd2 reverse , 5-ttcctcccattatgtctgctc-3 ; smyd3 forward , 5-atggacggtcactg cagatga-3 ; smyd3 reverse , 5-ctagagcgtgttgagctc agc-3 ; setd7 forward , 5-aacatggacagcgatgatgac-3 , and setd7 reverse , 5-caggtgttcagaggagtc-3. the reaction producing ribo - probes were carried out according to manufacture protocol ( roche ) . whole mount in situ hybridization were routinely performed as previously described ( thisse and thisse , 2008 ) . as brief , zebrfish embryos were fixed by 4% paraformaldehyde ( pfa ) and stored in methanol . to experiment , fixed embryos washed and digested with proteinase k solution as desired concentration and times . hybridization reaction was carried out at 65c , and the detection of hybridized rna probe was achieved by anti - digoxigenin fab fragment conjugated to alkaline phosphatase and bcip / nbt substrate ( roche ) . staining were stopped according to desired detection status and imaged in 75% glycerol solution for proper orientation .", "to generate the synthetic smyd3 and setd7 mrnas , we first cloned the full open reading frames ( orf ) of smyd3 and setd7 into pcs2 + expression vector , which have sp6 promoter sequence and sv40 poly - a tail sequence for in vitro transcription . these plasmids were cut by noti digestion to linearize , and capping mrna were produced using m7g(5)ppp(5)g ( roche , germany ) and sp6 rna polymerase ( roche , germany ) . synthetic mrna was diluted as 100 g / ml and phenol red was added prior to injection as 0.1% solution . injection was achieved at 12 cell stages as optimal concentrations ( about 100200 pg / embryo ) .", "to delineate functions of hmts , such as smyd1a , smyd2 , smyd3 , and setd7 , during heart development , we have first analyzed the expression pattern of these genes by whole mount in situ hybridization analyses and conventional reverse - transcriptase ( rt ) pcr . transcripts of all hmts examined appear to be maternally deposited , and widely detected at early stages . at later stages , the expression becomes gradually restricted to several organs , including somite , brain and eye ( figs . 1 and 2 ) . while smyd1a and smyd2 showed higher expressions in developing somite region from 10 somite stage ( ss ) than smyd3 and setd7 , hinting their roles in somitogenesis ( figs . 1 and 2 ) ( ses et al . , 2013 ) , the developmental expression regions of smyd3 and setd7 were highly expressed within the heart forming region at 36 h post - fertilization ( hpf ) and 48 hpf , respectively ( red arrow heads in fig . 2 ) . these gene expression profiling data suggest functions of zygotic smyd3 and setd7 in heart development . to further delineate the roles of hmts in heart development , we carried out knock - down experiments of smyd3 and setd7 . first , to determine the functional requirement of smyd3 during zebrafish heart development , we knocked smyd3 down with a morpholino ( mo)-based gene targeting system . for this purpose , splicing blocking mo that binds to the junction of exon2 and intron2 of smyd3 pre - matured mrna was designed ( fig . 3a ) . to validate the mo efficacy , pcr primers were designed to detect normal and aberrant smyd3 transcripts in wild - type embryos and smyd3 morphants , and carried out conventional rt - pcr ( fig . 3b ) . in smyd3 morphant embryo , mature transcript that amplified by f+r2 primer was not presented , while aberrant transcript that produced by f+r1 primer was displayed ( fig . in addition , smyd3 expression was drastically reduced in smyd3 morphants at 22 ss , suggesting non - sense mediated decay of aberrant transcript caused by mo injection ( fig . 3c ) . at optimal concentration ( approximately 3.0 ng / embryo ) , gross morphology of smyd3 morphants was comparable to control embryos ( fig . 3d and data not shown ) . however , pronounced heart edema ( red arrow ) was apparent in smyd3 morphants at 48 hpf , suggesting that smyd3 function may be essential for cardiac development ( fig . mos targeting setd7 was designed in a way to target the junction of exon 1 and intron 1 of setd7 pre - mature mrna ( fig . expression of setd7 was noticeably decreased by mo injection at 22 ss , suggest that setd7 mo is a functional to block the normal transcription of setd7 gene ( fig . 4c ) . similar to smyd3 deficiency , the knock - down embryos of setd7 at optimal concentration ( 1.0 ng / embryo ) displayed cardiac edema ( red arrow ) without causing any obvious morphological defects ( fig . collectively , our results illustrate that the proper activities of h3k4 methyltransferases , specifically smyd3 and setd7 , are important for zebrafish heart formation . during histone h3 lysine 4 ( h3k4 ) methylation , setd7 can only methylate non - methylated h3k4 to produce mono - methylated h3k4 ( h3k4me1 ) , which then is further methylated by smyd3 to become h3k4me2 and h3k4me4 ( hamamoto et al . , 2004 ; wang et al . , individual roles of smyd3 and setd7 during cardiac and skeletal muscle development in zebrafish have been reported ( fujii et al . , 2011 ; tao et al . , 2011 ) . given that smyd3 and setd7 successively methylate h3k4 , we can assume that these two enzymes function synergistically during zebrafish development . to test this possibility , we examined whether co - injection of smyd3 and setd7 mos at a suboptimal concentration can give rise a synergistic effect for induction of developmental defects ( fig . the smyd3 and setd7 mos , by themselves at optimal concentrations ( 3.0 ng / embryo for smyd3 and 1.0 ng / embryo for setd7 ) , produced no discernible gross morphological defects except cardiac edema at 48 hpf ( figs . 3 and 4 ) . however , co - injection of these mos at optimal concentrations caused severe somitogenesis defects as indicated by the phenotype of shortened and curled body morphology ( fig . , the severity of the abnormalities was sharpened by co - injection of increased concentrations of these mos ( fig . as expected , these results suggest that smyd3 and setd7 have a synergistic function in zebrafish development . next , we delineated the synergistic roles of smyd3 and setd7 in zebrafish heart development . for this purpose , we used the heart - specific transgenic fish tg(cmlc2:egfp ) line to allow easy visualization of the heart morphology in live embryos ( fig . the injections of control , smyd3 , setd7 , and smyd3 + setd7 mos were carried out at the optimal concentrations ( 3.0 ng / embryo for smyd3 , 1.0 ng / embryo for setd7 ) . morphants without body morphological defects were selected for further analyses of heart development . at 48 hpf , heart tubes in the smyd3 and setd7 morphants displayed defects in the looping process and remained linear in shape ( fig . single knockdown of these genes caused a defect in heart morphogenesis , suggesting that each gene is indispensable for heart morphogenesis . interestingly , double knock - down of smyd3 and setd7 presented a more severe defect than single knockdown of either of those genes ( fig . however , the global expression level of clmc2 detected by whole mount in situ hybridization ( kim et al . , 2014 ) was not altered in these smyd3 , setd7 , and smyd3 + setd7 morphant embryos at 48 hpf ( data not shown ) , suggesting that specification and/or maintenance of cardiomyocytes were not compromised in those embryos . collectively , our results show that smyd3 and setd7 likely function synergistically in zebrafish development . moreover , our data suggest that heart development is more sensitive than somitogenesis to the decrease of smyd3 and setd7 levels . to test whether the developing heart defects in the smyd3-and setd7-deficient embryos are caused by the expressional changes of early cardiac muscle genes , we determined the early expressions of cmlc2 , vmhc , and amhc ( kim et al . , 2014 ) by whole mount in situ hybridization experiment . the expression level of these genes was not altered by the knock - down of smyd3 and/or setd7 and the gathered shape of cardiac precursor cells in these morphant embryos was also comparable to wild - type embryos ( fig . this finding suggests that smyd3 and setd7 are dispensable for proper expression of early cardiac muscle genes and differentiation of cardiac precursor cells . to investigate whether enforced expression of smyd3 and setd7 causes the cardiac defect in zebrafish , synthetic mrna encoding smyd3 or setd7 the co - injection of smyd3 and setd7 mrna as optimal dose ( each 100200 pg / embryo ) caused similar developmental defects as in embryos lack either smyd3 or setd7 , such as defective body axis formation . moreover , these embryos showed pronounced cardiac edema , indicating the defects in heart formation ( red arrow in fig . 7b ) . to better examine the defects caused by overexpression of these smyd3 and setd7 , we analyzed the expression of known cardiac markers , including cmlc2 , vmhc , and amhc . despite the obvious cardiac edema , the expression of these genes was unaltered in embryos overexpressing smyd3 and setd7 ( fig . therefore , it appears that excessive h3k4 methyltransferase activity can lead to defects in heart and skeletal muscle development in zebrafish . taken together , our results indicate that methylation can provide essential regulatory input during organ development in zebrafish . since similar developmental defects were observed in embryos with either excessive or reduced level of smdy3 and/or setd7 activity , it appears that appropriate level of histone methylation is critical to ensure the formation of cardiac and skeletal muscle in zebrafish .", "heart morphogenesis in vertebrate development is a highly complicated event that achieved by correct cell specification , migration , proliferation , apoptosis , and transition as well as controlled beating of heart myocytes ( barnett et al . , 2012 ) . in this process , proper gene expressions for structure of cardiac chamber at early developmental stages are critical for late heart forming event . our data presented here indicates that proper level of histone methylation , which is mediated by a series of enzymes commonly known as hmts , including smyd3 , and setd7 appears to be essential for heart development . during zebrafish development , the expression pattern of hmts indicates that transcripts encoding these proteins may be maternally deposited . in addition , considering their ubiquitous expression , these proteins may provide essential functions in development by regulating histone methylation for all types of cells ( figs . 1 and 2 ) . however , knock - down of smyd3 and setd7 in zebrafish embryos did not cause any obvious defects in early development , such as in gastrulation movement , germ layer formation , cell proliferation , or cell death . therefore , these genes may redundantly function to regulate early vertebrate development ( figs . 1 and 2 ) . in later developmental stages , the expressions of all hmts tested in this paper were gradually restricted and often increased in specific organs , including somite , brain , eye primordial cells , fin primordial region , and heart . for instance , smyd2 becomes highly expressed within eye primodium , while smyd3 and setd7 are enriched in developing heart , suggest that each hmts might have a non - redundant and organ - specific roles during late vertebrate developmental periods ( figs . 1 and 2 ) . it was report that knocked - down smyd3 and setd7 by mo led to developmental defects in heart and/or skeletal muscle ( fujii et al . consistent with previous reports , we also find that mo injections as high concentrations ( over 10 ng / embryo for smyd3 mo and 4.2 ng / embryo for setd7 mo ) cause the similar somite defects . however , the optimal injection conditions ( concentrations under 3.0 ng / embryo for smyd3 and 1.0 ng / embryo for setd7 mo ) morphant embryos showed only developmental heart abnormality without any somite defects , indicating that each organ may have different sensitivity to the fluctuation in smyd3 and setd7 level during organogenesis . interestingly , in skeletal muscle development , setd7 directly interacts with myod to induce myogenin and mef2 expression to generate differentiated myocytes ( tao et al . , 2011 ) . therefore , it is tempting to speculate that setd7 manipulation in zebrafish may lead to the alteration in expression of certain structural genes . however , we did not find any discernible change in the expression of cmlc2 , vmhc , and amhc in our smyd3 and setd7 double - deficient or overexpressed embryos , suggesting the presence of distinct mechanisms underlying setd7-mediated differentiation of the cardiac muscle and skeletal muscle cells . moreover , smyd3 and setd7 appear to be dispensable for the specification of cardiomyocytes , since the lack of smyd3 or setd7 did not abrogate the expression of cardiac markers ( figs . 6 and 7 ) . here , we have shown the knock - down and overexpression phenotypes of smyd3 and setd7 in zebrafish development . these findings extend our current knowledge of the roles of hmts and increase our understanding of the functional mechanism of hmts in heart development ." ]

[ "how does the pattern of retinal activity produced by looking at the image of halle berry ultimately lead to the perception of the actress rather than a violin ? how do the myriad different associations with halle berry ( actress , specific roles ) or violin ( inanimate object , music ) form through learning and memory ? they have been approached by a variety of techniques that fall into two broad categories , watching brain activity and disrupting brain function . in this review we will discuss the development of genetic techniques that bridge the divide between these two approaches and allow the targeting of molecular changes specifically to anatomically dispersed neural representations that are activated by discrete environmental stimuli . these new tools allow the establishment of causal relationships between the activation of sparsely distributed neural ensembles and changes at the behavioral level . the use of single unit recordings in awake behaving animals provides an exquisitely precise measure of the temporal activity of neurons . this has been used to extract information about how the brain encodes information by studying the correlation between neuronal activity and the presentation of specific sensory stimuli . the best - studied example is probably in the primate visual system where a hierarchical pathway has been defined ( van essen et al . , 1992 ) . neurons in the primary visual cortex ( v1 ) fire in response to very general visual features such as orientation whereas following processing through the ventral visual pathway , neurons in inferior temporal cortex respond to complex object features . this visual information is then relayed to the medial temporal lobes , which integrate multimodal sensory information and play a critical role in memory . single unit recording studies in the medial temporal lobe in humans have detected neurons with responses to highly defined categories . in the limit , units were found that responded to the presentation of a single individual in a variety of contexts ( quiroga et al . , 2005 ) . one neuron in the right anterior hippocampus responded to the actress halle berry , presented in a photograph , as a masked character ( catwoman ) , as a drawing , or as the letter string this level of response specificity shows that the cells are not tuned to general visual features common to images of halle berry but to the concept of the specific actress . the striking degree of responsive specificity of these units strongly suggests that they participate in the neural representation of specific individuals . however , these studies are still correlative in the sense that they allow us to watch neurons that fire in a manner suggesting a role in encoding specific information , but they do not allow us to disrupt these neurons specifically to test this hypothesis . historically much of what we know about the functional parceling of the brain has been obtained from lesion studies in experimental animals and in patients with damage to specific brain regions ( squire , 2004 ) . in learning and memory , studies of patient hm , who underwent a bilateral resection of the medial temporal lobe , have helped define this area , and in particular the hippocampal formation , as critical in the formation of long - lasting declarative memories . at a more molecular level both pharmacological and genetic manipulations have been used to test ideas about the cellular signaling mechanisms that underlie behavioral plasticity . however , each of these approaches is limited in that they act as sledgehammers , altering every neuron in a given brain region , when the electrophysiological studies suggest that it is really a very sparse group of neurons that is truly of interest in any given experimental context .", "one approach to circuit analysis is to describe the precise pattern of wiring within specific processing units like the hippocampus or a cortical column . for example , in the hippocampus there is the classic tri - synaptic circuit where information from the entorhinal cortex enters through the dentate gyrus , is relayed to ca3 neurons via the mossy fiber pathway and then to ca1 neurons via the schaffer collateral pathway and finally back out to the entorhinal cortex ( squire , 2004 ) . this connectivity diagram can be obtained to finer and finer levels of resolution and in principle an entire wiring diagram of a single brain at a single time point could be produced to the level of individual synaptic connections , similar to that obtained in c. elegans ( white et al . , 1986 ) . however , even if this were precisely defined down to the level of single synapses it is not likely that the mechanisms that give rise to a neural representation or memory trace would become apparent . as the example of the halle berry neuron indicates , these representations are likely to be quite sparse and embedded within a matrix of apparently identical neurons . the particular response patterns of an individual neuron are likely determined by the strength of specific synaptic connections that have been altered through experience . moreover , even if one could explain how these specific firing patterns arise through circuit plasticity , it would be difficult to experimentally establish the contribution of a particular ensemble of neural activity to an actual representation of the environment . an alternate view is that what defines a circuit is the environmental contingencies that lead to its activation ( figure 1 ) . in the mammalian brain this is generally referred to as an ensemble code or neural representation of the particular environmental stimulus . in the case of simple systems or reflex pathways , the wiring diagram often predicts the location of the neural ensembles that encode specific environmental information . for example , in the aplysia gill withdrawal reflex a group of sensory neurons are activated by tactile stimulation and synapse directly onto motor neurons to control withdrawal behavior ( kandel , 2001 ) . here the primary sensory neurons are defined by their enervation of the gill and their activation by tactile stimulation of the gill . the behavioral plasticity of the withdrawal reflex is controlled by synaptic plasticity within these sensory neurons . because of the uniformity and anatomical isolation of this group of cells , it has been possible to apply techniques for both watching and manipulating neurons within the context of a defined circuit ( representation ) in a behaving animal . the application of these convergent approaches has proven quite powerful in defining the cellular and molecular mechanisms that underlie behavioral plasticity in this system . the goal of this review is to discuss recent attempts to develop approaches that allow similar convergent molecular and physiological access to the more dispersed neural representations of the mammalian brain . the top panel shows a simplified version of the gill and siphon withdrawal circuit in aplysia . the sensory neuron cell bodies are located adjacent to each other in a cluster and possess similar biochemistry and response properties . the bottom panel represents the hippocampal circuit of the mammalian brain . the green circles represent neurons that are activated by a specific pattern of sensory stimulation and that when activated contribute to a specific behavioral response . the hippocampal circuit , like many other circuits in the brain , responds to sensory stimulation with activation patterns that can not be predicted from their wiring diagram . each of these neural ensembles involves a sparse subset of neurons that have an unpredictable spatial distribution . the aplysia neurons are primary sensory neurons and their response properties can be predicted by their physical location in the ganglion .", "one technique that has been used for many years to watch brain activity has taken advantage of a class of immediate early genes or iegs that are expressed in response to high - level neural firing ( sagar et al . , 1988 ) . the three most commonly used iegs for this purpose are cfos , arc , and zif268 . the expression of these genes is induced by action potential firing , and the -life of the gene products are relatively short . thus the expression pattern of iegs in brain sections from an animal provides a record of the neural activity from several hours prior to sacrifice and has been used extensively to map brain activation from a wide variety of environmental stimulation and in learning and memory relevant paradigms ( guzowski et al . , 2005 ) . one of the limitations of this approach is that it provides only a single time point record of activity patterns , making it difficult to determine how plasticity modulates activity or even how stable this pattern of gene expression is in relation to an identical stimulus . this problem was addressed using the expression of the ieg arc ( guzowski et al . , 1999 ) . by using fluorescent in situ hybridization to examine expression of arc mrna they were able to detect the pattern of arc expression at two separate time points in the same animal . they took advantage of the fact that they could detect the expression of the arc precursor rna while it was still in the nucleus as well as the mature mrna which was present in the cytoplasm and dendrites . the nuclear arc signal represented very recent and ongoing expression reflecting neural activity several minutes prior to sacrifice of the animal , while the cytoplasmic signal reflected activity that had occurred 30 min or more prior . they used this approach to examine the consistency of activation of the hippocampus when an animal was repeatedly exposed to the same environment . they found that when animals were allowed to explore the same environment , they re - expressed arc in many of the same neurons that had also expressed it on the first exposure . this is a critical result in that it demonstrated for the first time that ieg expression could be used to consistently reflect patterns of activity associated with a discrete representation and provided results that were qualitatively and quantitatively similar to results obtained with electrophysiological recordings of the hippocampus . the temporal information regarding neural activity that can be obtained using ieg expression is clearly limited relative to electrophysiological recordings . for example , while it is clear that high - level firing induces expression , it is not clear what the precise threshold is and how this might vary among different neuronal cell types . one advantage is that large brain regions can be surveyed and precise anatomical information can be obtained . a second advantage is that the promoter regulatory elements that confer neural activity dependence can , in theory , be used to drive expression of any linked heterologous transgene . 1992 ) using the cfos promoter to drive activity dependent expression of e. coli -galactosidase . more recently , an axonally targeted -galactosidase was expressed from the cfos promoter , providing the potential to trace the projections of specific active neuronal populations ( wilson et al . , 2002 ) . the use of these promoter elements is general and provides the potential to introduce functional effector molecules directly into activated neural ensembles to allow their molecular manipulation . the use of ieg promoters as tools for both watching and potentially manipulating functional neural circuits is limited in a number of ways . for example , the direct introduction of toxins or other molecular regulators via the cfos promoter could be complicated by developmental effects of their expression . in addition , it would be useful for many studies to allow the molecular change introduced into the activated neurons to be maintained for more prolonged periods than the short ( minutes to hours ) times afforded by the promoters themselves . we therefore set out in a recent study to develop a genetic system with the following features . ( 1 ) the expression of any transgene of interest should be linked to neural activity only during a specific experimenter controlled time window . ( 2 ) the transgene expressed in those active neurons should be maintained for a prolonged period , but no further labeling of active cells should occur following closure of the permissive time window . we achieved activity dependent regulation of transgene expression with these two features by combining elements of the tetracycline system for gene regulation with the cfos promoter as shown in figure 2 ( reijmers et al . the first uses the cfos promoter to drive expression of the tetracycline transactivator ( tta or tet - off ) . in mice carrying only this transgene high - level neural activity will result in the induction tta , which is a transcription factor that can be blocked by the antibiotic doxycycline ( dox ) . in the absence of dox tta drives expression of genes linked to a teto - promoter sequence . the second transgene incorporates both a teto - linked reporter ( in this case the somato - axonal marker taulacz ) as well as a transcriptional feedback loop to maintain teto - linked gene expression indefinitely once it is activated . the tta ( tta * ) in this construct was made dox insensitive by introduction of a point mutation in the tet binding domain . in the presence of dox the teto - linked reporter is not activated even in those neurons in which the cfos - linked tta is expressed . however , if dox is withdrawn then both taulacz and tta * are expressed , but only in those neurons that were active to a high enough level to induce the cfos - linked tta . once activated , the tta * sets up a transcriptional feedback loop that can be maintained even in the presence of added dox . in this manner discrete time windows for genetic tagging of active neurons can be opened and closed through the use of dox . in the absence of dox , any neuron that has sufficient induction of cfos - linked tta to activate the feedback loop will persistently activate the taulacz reporter , as well as any other teto - linked transgene that is introduced into the mouse ( aiba and nakao , 2007 ) . this expression will be maintained even when the time window for sampling active neurons is closed by the readministration of dox . in this way a persistent record of neurons that were active during the off - dox period can be maintained . we called this the tettag mouse , which stands for tetracycline transactivator controlled genetic tagging of active neural circuits . the tettag mouse . mice carrying two transgenes were used . the first transgene uses the cfos promoter to drive expression of the tetracycline transactivator ( tta ) . tta activates the teto promoter in the absence but not presence of doxycycline ( dox ) . the second transgene uses the teto promoter to drive expression of a dox insensitive tta ( tta * ) , which , once expressed , sets up a positive feedback loop that continuously drives expression of a -galactosidase reporter coupled to the tau protein ( taulacz ) . neurons activated during fear conditioning ( while off dox ) were tagged with long - lasting expression of taulacz ( lac ; red circle ) . mice were put back on food with doxycycline and a retrieval test was done 3 days later , followed by analysis of the brains 1 h after retrieval for expression of lacz and zif268 . neurons activated during learning expressed lacz and those active during retrieval expressed zif268 ( zif ; green circle ) . the number of neurons in the amygdala that expressed both lac and zif , indicating that they were activate during both learning and retrieval , was positively correlated with the strength of the fear memory that the animal displayed . we used the tettag mouse to examine the neural circuit that mediates fear memory ( reijmers et al . , 2007 ) . we asked whether neurons that are stimulated during learning in a pavlovian fear - conditioning paradigm were reactivated during retrieval of the memory as shown in figure 2 . we subjected tettag mice to a learning trial consisting of paired presentations of a tone ( cs ) and a foot - shock ( us ) . this results in a long - lasting fear memory for both the tone ( cued conditioning ) and the conditioning box in which the animals were shocked ( context conditioning ) . the learning trials took place during a time window in which the animals were free from dox , allowing activated neurons to be tagged with long - lasting expression of taulacz . the animals were then returned to dox to prevent further tagging of activate neurons , tested for retention of the memory in a retrieval trial , and sacrificed after 1 h for analysis using the endogenous ieg zif268 as a measure of recent neural activity . by comparing the expression of lacz ( activity during learning ) and zif268 ( activity during memory retrieval ) we could determine the degree of circuit reactivation . we found that the number of reactivated neurons in the amygdala , a region critical in fear conditioning ( ledoux , 2007 ) , correlated with the retrieval of the fear memory . we hypothesized that these reactivated neurons represent a component of the memory trace for conditioned fear . to test this idea we weakened the strength of the memory by extinction training ; giving repeated cs presentations without the shock us . animals were first fear conditioned while free from dox to tag the learning activated neurons . the extinction training then took place following re - exposure to dox ( to prevent further labeling ) and a memory retrieval trial was conducted 1 h prior to analysis . we found that there was a significant correlation between the strength of the remaining fear response and the degree of circuit reactivation ; animals with a high fear response during retrieval showed strong reactivation of the learning circuit while those with low fear responses showed a low degree of reactivation . in addition , we found some specificity in the anatomy of the responses so that reactivation in the basal amygdala was correlated with context fear while reactivation in the lateral amygdala was associated with the strength of the cued ( tone ) fear memory . these results are consistent with the known role of these subdivisions of the amygdala with the two different forms of fear memory ( quirk et al . , 1995 ; these results demonstrate that memory retrieval results in a reactivation of some of the same neurons that were active during the initial learning . we suggested that neurons activated by the us ( shock ) but also receiving weak cs inputs were altered during learning such that the cs alone could now activate them . in this way presentation of the cs alone after learning would recapitulate a portion of the aversive us leading to downstream fear responses . this approach represents a somewhat elaborate way of simply watching neural activity and the results are still purely correlative . however , by using a regulatable and binary genetic system , it should be relatively easy to introduce additional effector transgenes into the mice to control the activity or biochemistry of these neurons and directly test their role in memory . one recent study by josselyn and coworkers has achieved this direct manipulation of neural ensembles associated with a specific memory trace using a somewhat different approach .", "the use of neural activity to introduce genetic alterations into neurons offers the possibility of obtaining direct molecular control over the neurons that participate in a specific neural representation or memory trace . an alternate approach that realized this goal took advantage of the finding that certain molecular manipulations could recruit neurons to participate in control of a specific memory . in one recent study , ( han et al . , 2007 ) , it was found that over expression of the transcription factor creb in neurons resulted in their preferential recruitment into a fear memory trace . in this study , neurons in the amygdala were randomly infected with a viral vector that over expressed creb and the animals were then trained in fear conditioning . they then performed a retrieval trial and examined the expression of the ieg arc in the amygdala . they found that the creb over expressing neurons were more likely to be activated during the memory retrieval . this result suggests that these creb over expressing neurons were predisposed to participate in the memory trace . the mechanism by which these neurons are preferentially recruited is unclear but it does demonstrate that , at least in the amygdala , there is a good deal of flexibility in which neurons can be used to encode a specific memory . the system is not hard wired at the level of individual neurons but there is a sort of competition , with creb over expression favoring a neuron 's recruitment into the memory trace . josselyn and coworkers went on to take advantage of this creb priming trick to directly manipulate a specific fear memory trace ( han et al . , study the viral vector that delivered creb to amygdala neurons also carried a gene that allowed for the expression of the diphtheria toxin receptor ( dtr ) ( figure 3 ) . the creb over expression recruited the neurons to the memory trace and expression of the dtr allowed for the selective ablation of these specific neurons with diphtheria toxin ( dt ) . ablation of the creb over expressing neurons disrupted the fear memory while ablation of a similar number of random neurons in the amygdala did not . the memory effect was long lasting and specific ( the same animals could learn a second fear association ) demonstrating that this limited group of neurons played a critical role in the specific memory encoded during the creb expression time window . this is the first example of the disruption of a specific memory within a distributed network . disrupting a specific memory in the mouse . a viral vector expressing both creb and cre recombinase was injected into the amygdala of idtr mice leading to the expression of both cre and creb in a random subset of neurons ( circles with creb / cre ) . the cre recombinase removed a transcriptional stop sequence and allowed for expression of dtr in these creb expressing neurons . an earlier study from the same authors ( han et al . , 2007 ) demonstrated that the creb expressing neurons participate in the storage of the fear memory ( green circles symbolize neurons that participate in the storage of the memory ) . after fear conditioning , mice were injected with diphtheria toxin ( dt ) , which killed the creb expressing neurons which participated in the encoding of that memory ( red circles ) . this caused a significant reduction in the strength of the fear memory measured during retrieval .", "while the ability of creb over expression to recruit neurons to participate in a specific memory is interesting in its own right , it would be useful to have an approach to manipulate neurons that were naturally activated by any general environmental stimulus . this has recently been accomplished using a technique in which neurons expressing -galactosidase can be specifically disrupted with a pharmacological agent ( koya et al . , 2009 ) . the study used rats that carry a cfos - promoter driven -galactosidase to label activated neurons . to manipulate the neurons they use a drug that is inactive in the absence of -galactosidase ( daun02 ) but can be hydrolyzed to a compound that can reduce ca dependent action potentials ( santone et al . , 1986 ) . they examined context specific sensitization to cocaine , which is an associative paradigm where the response to a drug of abuse is potentiated when it is administered in the same environment in which it has been repeatedly taken . animals were given repeated injections of cocaine over 1 week in context a to produce the context specific sensitization ( measured as increased locomotor response to the drug ) . following the training , a final sensitization trial to induce -galactosidase was given and 90 min later daun02 was injected into the nucleus accumbens to disrupt the -galactosidase expressing neurons . previous studies had suggested that the nucleus accumbens was a critical site of plasticity mediating this behavior ( mattson et al . , 2008 ) . the injected animals showed a reduction in the context specific component of the sensitization but retained normal responses to cocaine in a novel context b when tested several days later . like the results with the creb over expression , this study suggests that a specific associative representation ( context a + cocaine ) is being interfered with selectively . while the behavioral results in this study are intriguing and the linkage to cfos based expression provides a potentially general approach for manipulating discrete neural representations , there are a number of important questions that remain to be addressed with this technique . first , the electrophysiological effect of the daun02 treatment was not examined directly in the neurons but inferred from studies in cell lines . whether the effects are mediated by suppression ca dependent action potentials or some other effect of the time course of any neural excitability or ca channel changes is also a critical parameter . the effect of the daun02 treatment was examined 3 days after the initial injection of the compound and it is unclear whether the observed effect was due to ongoing suppression of activity or to a persistent effect manifest during the initial treatment . nevertheless , it demonstrates the general principles of this approach , which could be combined with the host of recently developed genetic regulators of neural activity .", "while neural representations are encoded in the specific ensemble of neurons that are activated in response to a stimulus , the plasticity that molds these patterns of activation is thought to occur at the synapse . it has been known for some time that long - term memory lasting 24 h requires new gene expression initiated at the time of learning , while short - term memory lasting a few hours lacks this requirement ( davis and squire , 1984 ) . since the short and long - term memories for the same event presumably involve the same pattern of synaptic changes , it raises the question of how the required gene products exert their effects selectively on the appropriate synapses . a potential answer to this question was suggested by frey and morris in studies of long - term potentiation ( ltp ) , a form of synaptic plasticity thought by many to underlie memory ( frey and morris , 1997 , 1998 ) . they found that synaptic activity could produce a sort of molecular tag at a synapse that would allow it to utilize newly expressed gene products to maintain ltp for long periods . we recently used the cfos based genetic approach to demonstrate a similar mechanism in behavioral learning and memory . a number of studies have implicated the regulated trafficking of the glutamate receptor glur1 to synapses as an important mechanism in both ltp and fear learning ( kessels and malinow , 2009 ) . in addition to allowing the genetic tagging of activated neurons , the ieg promoters like cfos show a very rapid onset and offset of expression , making them useful for cellular trafficking and turnover studies . we took advantage of this property to examine the trafficking of glur1 following learning in the fear - conditioning paradigm ( matsuo et al . , 2008 ) . mice carrying both a cfos - tta and teto - gfpglur1 fusion transgene were used in this study ( figure 4 ) . in the absence of dox neural activity will induce a pulse of expression of the gfp tagged glur1 and the distribution to synapses of this newly synthesized receptor can be followed histologically . animals were fear conditioned in the absence of dox to both induce a contextual fear memory and to induce synthesis of the gfp tagged receptor in activated neurons . the distribution of the receptor to dendritic spines ( the site of most excitatory synapses ) in the hippocampus was examined 24 h after the conditioning . we found that the receptor was not evenly distributed but present in only about 50% of spines , even in controls . in the fear conditioned animals we found a similar distribution except that there was an increase in trafficking to one morphological type of spine , the mushroom spines . this preferential trafficking only happened when the conditioned stimulus ( cs : novel box ) and the unconditioned stimulus ( us : foot shock ) were paired , but not when cs or us were presented separately . the newly synthesized glur1 requires 2 h to begin to reach the dendritic spines , yet is somehow preferentially recruited to a specialized class of spine based on the associative conditioning that occurred 2 h prior to its arrival . this is indicative of a synaptic tagging event acting in behavioral memory similar to that described for ltp ( frey and morris , 1997 ) . learning regulated targeting of glutamate receptors . the first transgene is identical to the one described in figure 2 and uses the cfos promoter to drive expression of a tetracycline transactivator ( tta ) . the second transgene was a teto - promoter gfp tagged glutamate receptor subunit ( glur1-gfp ) . animals were fear conditioned in the absence of dox to produce both a fear memory and a pulse of gfp - glur1 expression in active neural ensembles in the hippocampus . the distribution of gfp - glur1 in dendritic spines was analyzed 24 h following the conditioning using dii to label all spines on a given neuron . fear conditioning led to an increase in trafficking of the receptor specifically to mushroom type spines . this experiment demonstrates how genetic tools can be used to image a specific molecular event selectively within an activated neural circuit .", "the ability to genetically manipulate activated neuronal ensembles or neurons participating in a sparsely encoded memory trace offers a number of advantages that are only beginning to be realized . a parallel line of technological development has focused on generating genetic tools for manipulating neuronal activity . the light regulated channelrhodopsin chr , developed by deisseroth and colleagues ( zhang et al . , 2006 ) , allows for the very precise light regulated control of action potential firing in neurons expressing the channel . there is an expanding tool box of genetic effectors like chr that are light or ligand controlled and can be used to either stimulate or suppress neural activity ( luo et al . , 2008 ) . in addition , there are a number of similar effectors that can be used to regulate second messenger signaling pathways when expressed in heterologous cells ( isiegas et al . , 2008 ; the combination of these new tools with activity based genetic delivery and multi time point brain activity mapping at cellular resolution will open up a variety of new questions to experimental analysis . to return to the initial discussion of neural representations , the fusion of these approaches should allow one to address the question of what neural firing patterns mean to the animal . to take the example of the halle berry neurons ( actually the equivalent in genetically accessible animal models ) one could ask what would be the consequences of silencing this specific group of neurons . what fraction of neurons in the representation need to be silenced in order to impair recognition ? how does the silencing of a specific group of neurons in one brain region affect the activation patterns in downstream areas ? an alternate approach is to ask whether a representation can be built by experimenter driven stimulation of the appropriate neurons . in one recent study , svoboda and colleagues delivered the chr2 molecule to random populations of neurons in the somatosensory cortex . they found that stimulating as few as 300 neurons could be detected by the animals and used to alter behavior in a conditioning task ( huber et al . , 2008 ) . however , the coordinated activation of those neurons presumably does not form any natural representation . suppose that instead of a random group of neurons one could activate a subset of neurons that responded to a natural stimulus , say the tone cs in a fear - conditioning task . the artificial stimulation of those neurons paired with a foot shock would presumably lead to a conditioned fear of the extrinsic stimulation cs . if so would all the features of this sensory representation be maintained , for example frequency selectivity ? with what fidelity does artificial stimulation of the tone representation in one brain region recapitulate the brain activity patterns produced by the natural tone itself ? this type of approach should now be achievable and allow direct functional investigation of the structure of neural representations . finally , the ability to genetically alter activated neural ensembles provides an entre into a more specific biochemistry of the brain . in the glur1 trafficking studies discussed above , the gfp tagged receptor provides not only a signal to watch molecular movements , but also a tag for specific biochemical analysis . for example , one could ask how the synapses from activated neurons that received new receptor differ biochemically from those that did not by using the antibodies to the gfp tag to affinity purify positive from negative material . a similar approach could be used to tag specific cellular compartments or molecular complexes so that biochemical studies can be limited to just the activated neurons , sparsely embedded in a matrix of inactive neurons and glia . it should also be possible to improve the specificity of the genetic modifications using a variety of genetic tricks such that only neurons active in one brain region or active at time point 1 but not time point 2 would be tagged . the increased specificity along with the new genetic tools and biochemical tagging should provide a new level of circuit analysis in the brain and break down the barrier between watching neural firing and manipulating neural function .", "the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest ." ]

[ "angiogenesis , the formation of new blood vessels by sprouting of pre - existing ones , is a main mechanism of vascularization during embryonic development , growth , formation of the corpus luteum and endometrium , regeneration and wound healing . however , deregulated , abnormal angiogenesis is involved in many pathological processes [ 1 , 2 ] . the complex sequence of events involved in angiogenesis is related to changes in endothelial cell biosignalling . the relationships of angiogenesis with cancer have special relevance , since angiogenesis has been described as one of the hallmarks of cancer , playing an essential role in tumour growth , invasion , and metastasis . since tumour blood vessels show many differences from normal vessels and are not genetically unstable , they are potential targets for therapy of all types of cancer . due to the pivotal role played by endothelial cells in tumour angiogenesis , most previous efforts were devoted to the development of agents that could block their activation by an angiogenic signal ( mainly vegf ) , or to inhibit one or several specific functions of activated endothelial cells ( proliferation , adhesion to extracellular matrix , proteolytic activities , migration , invasion or differentiation ) . national cancer institute database showed that in august 1999 a total of 20 angiogenesis inhibitors were being tested in clinical trials . remarkably , most of them were monotherapies with the antiangiogenic agent , and those compounds that had then reached the phase iii , including several inhibitors of matrix metalloproteinases , were discontinued due to their lack of activity or the appearance of undesirable toxicities . in spite of the great number of angiogenesis inhibitors described so far ( estimated to be > 300 drug candidates in 2001 ) , and the interesting results obtained in experimental models , even showing complete tumour regressions in pre - clinical studies , modest or even negative results emerged from the first generation of compounds entered in clinical trials . nevertheless , there is no reason for premature pessimism , as revealed by current ongoing trials and the clinical developmental status of anti - angiogenic drugs .", "a critical analysis of the disappointing results obtained in previous clinical trials points to different reasons for this failure . these include flaws in the methods used to select these inhibitors and in the design of the clinical trials to test their effects , as well as an oversimplified view of tumour vasculature pathophysiology . angiogenesis inhibitors are initially selected by means of in vitro assays that make use of endothelial cells from different sources . the results obtained in this primary screening can be dependent on the type of endothelial cell . afterwards , the antiangiogenic activity of the selected compounds is usually tested with several in vivo assays ( for a review , see ) . although useful , these have limitations . some of them do not take into account the tumour microenvi ronment ( this is the case of vascularization assays in the chicken chorioallantoic membrane and in the mouse cornea ) . other assays make use of rapidly growing tumours and/or animals that do not fit into the clinical reality . most of the tumour systems used with laboratory animals show angiogenic responses much higher than those induced by human tumours . furthermore , the effectiveness of an angiogenesis inhibitor can be hampered by the intrinsic heterogeneity of human tumour angiogenesis . animal and preliminary clinical trials have revealed that different tumours respond very differently to antiangiogenic therapy . in fact , the responses have been extremely heterogeneous , most probably due to the randomized design of the trials . a clinical challenge in antiangiogenesis is the finding of biological markers that help to identify subsets of patients more likely to respond to a given antiangiogenic therapy , as well as to determine optimal dosing of therapy , to detect early clinical benefit or emerging resistances and to decide whether to change therapy in second - line treatments . , microvessel density has been proven to be a useful prognostic indicator but , at the same time , does not seem to be a good direct indicator of antiangiogenic treatment efficacy . surrogate biomarkers could include those related to the various steps of the angiogenic process , including variations in endothelial - cell survival , alterations in the endothelial - cell signaling , and variations in the number of circulating endothelial progenitor cells [ 3 , 13 , 15 , 16 ] . another possibilityis the fractal analysis of the vascular network in tumour biopsies . however , these approaches are far from an ideal biomarker for clinical practice . although some authors consider taking biopsies repeatedly to be feasible , most physicians consider this to be very cumbersome for patients . since tumour angiogenesis produces interstitial hypertension in tumours , the determination of interstitial pressure of tumours can be considered an alternative surrogate biomarker . easier to determine and less invasive approaches include the measurements of circulating levels of several angiogenic factors , but so far no growth factor has been validated for predicting response to antiangiogenic therapy , as well as high resolution image analysis that requires expensive instrumentation that could not be available in all institutions . on the other hand , the clinical end - points for dose - defining trials ( phase i ) and efficacy trials ( phase ii ) should be reconsidered . the expected good tolerability and low toxicity of well selected antiangiogenic compounds give little relevance to the determination of maximum tolerated doses ( mtd ) and dose - limiting toxicity ( dlt ) , which could be replaced by the determination of optimal biological dose ( obd ) in phase i trials . more and better - designed pharmacokinetic studies are required not only to determine the obd , but also to determine the optimal schedule of drug administration . some years ago , trials with this antiangiogenic compound were discontinued due to low responses and toxicity . however , an improvement in the treatment regimen yielded enhanced response with decreased toxicity in phase i and ii trials . probably , a readjustment of doses and/or schedule could contribute to diminish or even abolish some of the side effects produced by other previously tested antiangiogenic compounds . in phase ii trials , objective responses ( i.e. the degree of tumour regression ) alternative parameters such as disease stabilization , progression - free survival and time to progression should be used . however , these parameters are more difficult to be evaluated properly and they require larger patient samples and more prolonged treatments . validation and standardization of monitoring techniques for antiangiogenic therapy are urgently required . the fact that tumour vasculature has been understood in an oversimplified fashion is another explanation for the poor results obtained in the first generation clinical trials . it is now known that different tumour types may acquire their blood supply by different mechanisms . tumour vasculature is not necessarily derived from endothelial cell sprouting;instead cancer tissue can acquire its vasculature by a number of alternative mechanisms that could be the basis for developing effective clinical modalities using antivascular therapy of cancer . the recruitment of circulating endothelial progenitor cells , mainly from bone marrow origin , can contribute to the tumoural neovascularization by vasculogenesis , a process that was initially thought to be limited to embryonic development . in fact , as recently reviewed , there is accumulating evidence that progenitor cells as well as other stromal cells are actively recruited into tumours and that this recruitment is essential for the proangiogenic environment of tumours . another possibility is the co - option of vessels during early growth of tumours in the absence of angiogenesis [ 11 , 28 ] . on the other hand , vascular mimicry , the generation of microvascular channels by genetically deregulated and aggressive tumour cells , is an alternative way to provide blood supply to tumours and is independent of angiogenesis . the potential of mono - cytes / macrophages to contribute to neovascularization has recently come into focus . some experimental evidences indicate that infiltrating mononuclear cells may incorporate in the lumen microvessels and that peritoneal macrophages may form capillary - like lumens and branching patterns in vitro[31 , 32 ] . pathological angiogenesis is characterized by structurally and functionally abnormal vessels and lymphatic vessels [ 1 , 2 ] . these abnormalities result from an imbalance between levels of pro- and antian - giogenic molecules . as a result , this , in turn , compromises the delivery and effectiveness of conventional therapies , as well as molecular targeted therapies . finally , in this brief analysis of the rationale behind the failure of the first generation of antiangiogenic agents , concern should be given to the way in which scientists communicate their findings to society . angiogenesis research is an especially competitive area in which the promising preclinical results have been very often prematurely amplified by mass - media releases . the high prevalence of cancer and the extremely high sensitivity of society towards this primary medical problem facilitate that great expectations could lead to deep disappointment .", "a second generation of antiangiogenic trials is beginning to show highly significant potential [ 8 , 35 ] . the first study showing phase iii data validating an antiangiogenesis strategy for treating human cancers was obtained with bevacizumab , a humanized recombinant monoclonal antibody that neutralizes the biologically active forms of vegf that interact with vegf receptors 1 and 2 . in a communication that received much attention in the 2003 asco meeting , the authors reported that the beva - cizumab / ifl ( irinotecan / fluorouracil / leucovorin ) combination led to significantly prolonged survival and had a better ability to shrink tumours than standard ifl alone , without statistically significant increases in adverse events in patients with metastatic colorectal cancer . the results were based on 412 patients in the ifl / placebo arm and 403 patients in the ifl / beva - cizumab arm . the presence of bevacizumab in the treatment produced remarkable and statistically very significant increases in all the four determined survival and response parameters : median survival , progression - free survival , objective response , and duration of response . these impressive results led the fda to approve the use of bevacizumab in patients with metastatic colorectal cancer and they have been finally published in the form of a research article in the new england journal of medicine . however , as stated in the accompanying editorial , although it is tempting to attribute the effect of bevacizumab to a direct antiangiogenic mechanism , the validity of this assumption is presently uncertain . once more , mass - media releases have led to unrealistically high expectations . as commented in the aforementioned editorial , patients need to be informed that beva - cizumab does not cure metastatic colorectal cancer and that there is no evidence as yet that the antibody has antitumour activity when administered as a single agent for this disease. although hurwitz et al . did not measure surrogate markers of antiangiogenesis , they mention that bevacizumab may have altered tumour vasculature and decreased elevated interstitial pressures in tumours , thereby enhancing the intracellular delivery of chemotherapy agents . recently , bevacizumab plus folfox4 ( oxaliplatin/5-fu / leucovorin ) treatment was approved for second - line metastatic colorectal cancer . in october 2006 , bevacizumab in combination with paclitaxel and carboplatin treatment was approved for the first - line treatment of patients with non - small cell lung cancer . currently , more than 100 clinical trials with bevacizumab are ongoing , including phase iii trials in kidney , breast , prostate and ovarian cancer , among others . in fact , successful clinical trials of multitargeted compounds have yielded two significant fda approvals . in december 2005 , sorafenib ( bat 43 - 9006 ) , an inhibitor of the faf / mek / erk and the vegfr and pdgfr signaling pathways , received fda approval for the treatment of renal cell carcinoma . sunitinib ( su11248 ) , an oral inhibitor of vegfr2 , pdgfr , flt-3 and c - kit , received fda approval in january 2006 for patients with gastrointestinal stromal tumours ( gist ) and advanced kidney cancer , being the first time the agency had approved a new oncology product for two indications simultaneously [ 42 , 43 ] . it seems that , after a period of flawing interest , antiangiogenic compounds have regained their place in the centre of anticancer treatment trials , as shown by the eastern cooperative oncology group portfolio of clinical trials . from the results obtained so far in clinical trials , it can be concluded that the future clinical success of angiogenesis inhibitors could be related to their use in combination with chemotherapy or radiotherapy . since abnormal angiogenic vessels compromise the delivery of drugs targeting tumour cells , the normalization of tumour vasculature with antiangiogenic therapy has emerged as a new paradigm for combination therapy [ 19 , 45 ] . synergic effects can be expected , since judiciously applied antiangiogenic therapy can increase the penetrability of chemotherapeutic agents , as well as the radiosensibility of tumour cells . a detailed analysis of how antiangiogenic compounds reduce vessel density shows that these drugs reduce vascular permeability , destroy immature vessels and increase the recruitment of pericytes to stabilize other vessels . this transient stabilization has been termed the normalization window , defined as a period of time where tumour blood flow and oxygenation increases , thus providing an opportunity to better deliver chemotherapeutic drugs and radiation therapy . as previously mentioned , the heterogeneity of blood vessel growth , the fact that angiogenesis differs among tumour types is a basis for the observed differences in response to antiangiogenic therapy in both animal and clinical trials . therefore , a multidrug approach might be more successful than monotherapy . the combined used of several antiangiogenic compounds targeting different steps of angiogenesis should be explored .", "as stated before , reliable biomarkers are strongly needed to validate the efficacy of antiangiogenic therapy , to identify responsive patients and optimal doses , to predict efficacy of regimens that include anti - angiogenic agents , and to detect and prevent tumour escape . the lack of reliability of measurements of circulating levels of angiogenic factors has made the search for new biomarkers to shift away from measuring their levels to measuring their effects , such as the recruitment of endothelial progenitor cells from the bone marrow to the tumour where they contribute to neovascularization . preclinical studies have shown that circulating endothelial cells , which are probably derived from blood vessel wall turnover , and circulating endothelial progenitors kinetics correlate well with several standard laboratory assays , that can not be used in humans . the initial suggestion that variation in the levels of circulating endothelial progenitor cells could be a useful surrogate marker to monitor angiogenesis has been confirmed and extended in an outstanding report published in cancer cell . this report provides evidence that the levels of circulating endothelial progenitor cells are genetically predetermined and regulated by regulators of angiogenesis , including vegf , tie-2 and thrombospondin-1 . moreover , antiangiogenic therapy can be optimized by monitoring the levels of both circulating endothelial cells and circulating endothelial progenitor cells [ 13 , 25 ] . therefore , the kinetics of these cells in peripheral blood is suggested to be useful surrogate markers of pathological angiogenesis with potential application for the monitoring of antian - giogenic therapy response .", "there are clear signs that during the last year antian - giogenesis research has entered a new age . table 1 tries to summarize the trials and errors in past failures and possible solutions to them . antiangiogenic cancer therapy : past and future ; clinical trial lessons the development of new and better models for the in vivo assay of potential inhibitors of human angio - genesis should be considered a priority in this field of research . there is increasing concern that by using approaches based on traditional end - points , potentially interesting angiogenic modulators might be rejected prematurely . consequently , the extensive use of correlative studies in the early phases of drug development to establish surrogate biomarkers for use in efficacy trials is strongly recommended . methods of imaging will be helpful to assess the efficacy of treatment [ 12 , 13 , 20 ] . a careful selection of the clinical setting for the investigation ( for example , tumour type and stage of disease ) and innovative statistical designs to optimize the selection of patients must be carried out before expensive , definitive phase iii clinical trials . an example is the randomized discontinuation trial design ( rdtd ) , aimed to select a subset of enrolled patients who are more homogeneous with respect to important prognostic factors than the group of patients that would otherwise be randomized in the trial . frequent administration of chemotherapy at low doses , ranging from one - tenth to one - third of the mtd , significantly increases the antiangiogenic effect . metronomic scheduling has shown impressive antitumour activity in animal models and is now being tested either alone or in combination with other antiangiogenic agents in clinical trials [ 35 , 47 , 48 ] . furthermore , this metronomic approach is also used with radiation therapy , when administered at lower than normal doses , known as hyper - fractionated radiation. the concept of vascular targeting is related to antiangiogenesis but involves a different approach . juliana denekamp outlined the concepts behind vascular targeting for cancer treatment in the early 1980s , showing that physical occlusion of the blood supply to tumours in rodents led to tumour regressions . vascular targeting agents exert their primary action on the pre - existing blood vessels of solid tumours . vascular targeting therapies would share the advantages of antiangiogenic therapies and could offer some additional advantages . first , blood flow is a defined surrogate marker of biological activity that can be measured in the clinic . second , temporary effects on vascular functioning may be sufficient . and third , unlike angiogenesis inhibitors , vascular targeting agents should require only intermittent administration to synergize with conventional treatments rather than chronic administration . the clinical studies completed to date with vascular targeting agents are encouraging . progression into combination studies has begun . recently , concerns have been raised on the possibility of resistance to antiangiogenic therapy [ 13 , 21 ] . in fact , an effective antiangiogenic therapy could select for resistant and aggressive cancer cells during therapy - induced tumour regression . ideally , the most effective therapy would suppress all cancer cells , avoiding relapse . on the other hand , hypoxia is common in tumours , despite the increase in their vascularization , because of a poor perfusion caused by aberrant vessels . strategies that target hypoxic cells may therefore synergize with antiangiogenic treatments . the contribution of inflammatory cells to tumour angiogenesis the inhibitors of this pathway exhibit high tolerability and they can be administered chronically . their performance in clinical studies is currently being tested . however , the recent problems with cycloxygenase inhibitors in cancer prevention treatment raise serious concerns for their use [ 55 , 56 ] . inhibitors of angiogenesis could slow the progression of premalignant lesions and reduce the risk of developing invasive tumours . they have the potential to be used in primary , secondary or tertiary cancer prevention settings . in fact , monotherapies with antiangiogenic compounds could be useful as adjuvant treatments in situations of minimal residual disease following either cytore - ductive surgery or cytotoxic treatment . however , in spite of the fact that past clinical studies have shown that many angiogenesis inhibitors can be given safely to patients , more long - term toxicity studies are needed . how much antiangiogenic therapy will be incorporated in the future to the treatment of cancer patients depends on further advances in the understanding of the molecular mechanisms involved in tumour angio - genesis , the development of standardized methods to assess surrogate predictive markers of response , and the capability of performing a new generation of appropriately designed clinical studies . a convergence of the efforts carried out in basic , applied and clinical research would contribute to achieve these goals . this knowledge will be applied not only to cancer treatment but also to other diseases characterized by abnormal vasculature such as hemangiomas , diabetic retinopathy , macular degeneration and psoriasis , among others for which antiangiogenic approaches have already shown benefits [ 1 , 2 ] ." ]

[ "regulation of gene expression is one of the most enigmatic facets of molecular genetics that results in intricate appearance of a biological entity . scientists have been attempting to elucidate the regulatory mechanisms of gene expression for long and the radical discovery of regulatory function of endogenous small noncoding rnas is overwhelming the scientific community with their ever increasing potentials . small noncoding rnas of 1840 nucleotides ( nt ) in size have been proved to play a vital role in a remarkably wide range of biological processes , including cell proliferation , developmental timing and patterning , chromatin modification , genome rearrangement , and stress response in plants and animals . small rnas regulate a variety of biological processes in plants by interfering with messenger rna ( mrna ) translation , directing mrna cleavage or promoting the formation of compact , transcriptionally inactive chromatin . several distinct classes of small rnas have been reported so far including micrornas ( mirnas ) , small interfering rnas ( sirnas ) , repeat - associated small interfering rnas ( ra - sirnas ) , piwi interacting rnas ( pirnas ) , natural antisense transcript derived small interfering rnas ( nat - sirnas ) , transacting small interfering rnas ( ta - sirnas ) , heterochromatic small interfering rnas ( hc - sirnas ) , secondary transitive small interfering rnas , primary small interfering rnas , competing endogenous rnas ( cernas ) , and long small interfering rnas [ 1113 ] . it has only been a few years since it was appreciated that micrornas provide an unanticipated level of gene regulation in both plants and metazoans . mirnas are well differentiated due to some of their particular characteristics ; they are derived from distinct genomic loci and processed from transcripts that can form local rna hairpin structures , and usually mirna sequences are nearly always conserved in related organisms [ 13 , 14 ] . most mirnas are transcribed by rna polymerase ii which folds into a stable , usually imperfect , hairpin structure ; pri - mirna transcript is cleaved to pre - mirna by rnaseiii - type dicer - like 1 ( dcl1 ) protein to produce a distinctive ~21 nt , double - stranded rna . this duplex is exported into the cytoplasm by hasty and methylated at the 3 end by hen1 . a cytoplasmic helicase unwinds the translocated duplex into a single - stranded mature mirna , which is finally incorporated into rna - induced silencing complex ( risc ) [ 5 , 17 , 18 ] . a mature mirna sequence can range from 19 to 24 nucleotides ( nt ) in length and act as a regulatory molecule in posttranscriptional gene silencing by base pairing with target mrnas . within the risc complex , mirnas function in the direct cleavage of 3 untranslated region of protein - coding genes or translational repression depending on its perfect or imperfect match with the targets . the same mature mirna can also be present as several length variants ; these populations of mirna variants are called isomirnas , which are isoforms of micrornas caused by an imprecise or alternative cleavage of dicer during pre - mirna processing . several mirnas have been identified in plants , and they have been characterized in a wide variety of metabolic and biological processes with important functions . the first plant mirnas were described in arabidopsis thaliana ; currently the latest mirbase release ( v20 , june 2013 ) contains 24,521 microrna loci from 206 species , processed to produce 30,424 mature microrna products . earlier , mirnas have been identified through either bioinformatics analysis or sequencing ; various methods have been used to identify mirnas in rice , wheat , tomato , and maize . besides the mirnas that are highly conserved in different species , there are species - specific mirnas originating from recently evolved mirna genes [ 28 , 29 ] . the expression of these species - specific mirnas is often low and can therefore be difficult to detect by traditional methods . in recent times , high - throughput sequencing platforms are showing significant promise for small rna discovery and genome - wide transcriptome analysis at single - base pair resolution [ 23 , 31 ] . sequencing techniques such as the solexa platform , solid , and 454 technology as well as other massively parallel sequencing strategies have been successfully applied in order to identify mirnas in many plant species , such as rice , alfalfa , grape , tomato , orange , soybean , peanut , poplar , and black gram . in comparison with microarray , deep sequencing has several advantages , the major one being its application in comprehensive identification and profiling of small previously unknown rna populations . nevertheless , analyses of these data are not perfect , especially in the absence of native genome sequence . jute ( corchorus olitorius and corchorus capsularis ) is a bast fibre , like flax and hemp . cultivation of this environmentally friendly as well as the most affordable fibre producing plant is concentrated around the ganges delta region of bangladesh and india where the warm , wet climate during the monsoon season provides ideal growing conditions . in terms of usage , production , and global requirement , jute is second only to cotton . jute plants are easy to grow , have a high yield per acre , and , unlike cotton , have little need for pesticides and fertilizers . the leaves and roots left after harvest enrich the soil with micronutrients , maintaining soil fertility . when used as a geotextile , it puts nutrients back in the soil when it decomposes . this rain - fed crop during its growth helps to clean the air by assimilating three times more co2 than an average tree , converting the co2 into oxygen . despite its great agronomic importance , research on jute at the molecular level so far only 1,210 sequences are found in the genbank with no deposits of any mirna sequences in mirbase database . within this context , the current study has employed the deep sequencing strategy in an attempt to effectively identify conserved and novel jute mirnas . quantitative real - time pcr ( qrt - pcr ) has been performed to determine the expression of these mirnas . for mapping the identified mirnas , genome of vitis vinifera", "seeds of farmer popular o-9897 variety of tossa jute ( corchorus olitorius ) were collected from bangladesh jute research institute ( bjri ) . they were surface sterilized with 70% ethanol , subsequently washed in distilled water , and allowed to germinate on sterile petri dishes containing 3 mm moist filter paper ( whatman ) at 30 1c and 65% relative humidity . seeds were allowed to grow for 4 days under the specified conditions . on the fourth day of germination , seedlings were collected and immediately snap - frozen in liquid nitrogen and stored at 80c for subsequent use . rna was isolated from collected seedlings using trizol reagent ( invitrogen , usa ) by following the manufacturer 's instructions . later , rna samples were sent to beijing genome institute ( bgi , shenzhen , china ) for deep sequencing of small rna by illumina hiseq high - throughput sequencing platform . in short , after ligation with 5 and 3 adaptors , the short rnas so obtained were reverse - transcribed to cdna according to the illumina protocol . the resulting small rna library was then sequenced following sbs method ( sequencing by synthesis ) by illumina hiseq high - throughput sequencing . raw data obtained from illumina hiseq high - throughput sequencing was at first filtered by removing contaminants which include low quality reads , reads with 5 primer contaminants , reads without 3 primer , reads without the insert tag , reads with poly a , and reads shorter than 18 nt . after cleaning , clean reads fully matching other rnas , including mrna , rrna , trna , snrna , snorna , and repeat rna , were excluded by using blastn - short alignment ( blast2.2.26 + , ftp://ftp.ncbi.nih.gov/blast/executables/blast+/2.2.26/ ) and aligning against sanger rna family database ( rfam 11.0 , ftp://ftp.sanger.ac.uk/pub/databases/rfam ) . the remaining unique sequences were further aligned against mirbase - v20 allowing up to 3 mismatches to identify known mirnas present in c. olitorius . mature mirnas present within a genome encoding identical or nearly identical sequences were then grouped together into a family . prediction of novel mirna was done using prediction software , mireap ( http://sourceforge.net/projects/mireap/ ) , developed by bgi by taking into consideration secondary structure , cleavage position of dicer protein , and minimum free energy of the unannotated small rna tags . strategic conditions for selecting unique mirna are as follows : ( i ) the tags which are to be used to predict novel mirna should be from unannotated tags which can match reference genome ( vitis vinifera ) , from the tags which align to introns as well as antisense exons ; ( ii ) genes , whose sequences satisfy the above standards and their secondary structures which allow hairpin mirnas to fold within them and the presence of mature mirnas in one arm of the hairpin precursors , are considered as candidate genes for mirna ; ( iii ) the possible candidate mature mirna strand contains 2-nucleotide 3 overhang ; ( iv ) hairpin precursors of the candidate mirnas are devoid of large internal loops or bulges ; ( v ) secondary structures of the hairpins are stable , with the minimum folding energy ( mfe ) lower than or equal to 20 kcal / mol . the rules used for target prediction in plants are based on those suggested by allen et al . and by schwab et al . these are ( i ) presence of maximum 4 mismatches between small rna and target ( g - u bases count as 0.5 mismatches ) , ( ii ) not more than 2 contiguous mismatches in the mirna / target duplex , ( iii ) no end - to - end mismatches at the 5 of mirna from 2 to 12 positions of the mirna / target duplex , ( iv ) no mismatches in positions 10 - 11 of mirna / target duplex , ( v ) a maximum of 2.5 mismatches in positions 112 of the mirna / target duplex from 5 region of mirna , and ( vi ) minimum free energy ( mfe ) of the mirna / target duplex which should be 74% of the mfe of the mirna bound to its perfect complement . the targets of mirnas were further validated by a well - recognized mirna - target prediction tool : psrna target . in addition to potential target prediction for known and novel mirnas , pathways which include the corresponding target genes as well as the biological function of such genes are taken into consideration by using the grape genome as a reference . gene ontology ( go ) is an international standard classification system for gene function , which provides a set of controlled vocabulary to comprehensively describe the property of genes and gene products . there are 3 ontologies in go : biological process , cellular component , and molecular function , containing lists of biological functions that illustrate each gene and its product ( http://www.geneontology.org/ ) . each category defines precise participation of a given gene within an organism . as for pathway identification , kegg ( http://www.genome.jp/kegg/ ) kegg analyses reveal the main pathways with which the target gene candidates are involved . to verify the identified known and potential novel mirna candidates in jute , stem - loop reverse transcription - pcr was performed . stem - loop primers were designed according to the method described by chen et al . . this primer binds to specific mirna at the 3 region owing to the precision conferred by the primer with the exact reverse complement of six nucleotides from the 3 end of each particular mirna sequence , which is reverse - transcribed by the rt enzyme . two thousand nanograms of total rna were used to perform the rt reaction with superscript iii first strand synthesis system ( invitrogen , usa ) according to the protocol of varkonyi - gasic et al . , which has been further standardized for jute . for a reaction volume of 20 l , 0.5 l of 10 mm dntps was at first taken together with an appropriate amount of rna and an adjusted amount of depc - treated h2o followed by heating the mixture for five minutes at 65c and then immediately transferring the same on ice . while keeping on ice for approximately 2 minutes , 4 l of 5x fs buffer , 2 l of 0.1 m dtt , 1.2 l of 1 m stem - loop primer , 0.25 l of m - mlv superscript iii rt ( 200 u/l ) , and 0.1 l rnaseout ( 40 u/l ) were added to the reaction mix . the rt reaction was carried out in a thermal cycler ( mastercycler , eppendorf , germany ) , followed by a pulse rt cycle starting from incubation at 16c for 30 minutes , then 60 cycles of 30 sec . at 30 , 30 sec . at 42c , and 1 sec at 50c . this step was followed by another incubation step of 5 mins at 85c to inactivate the rt enzyme . end point pcr was then conducted with mirna specific forward primer and a universal reverse primer to check the presence of the specific mirnas . pcr products were electrophoresed on 3% agarose gel in 1x tae and stained with ethidium bromide before visualization under a transilluminator . we further conducted quantitative real - time pcr for confirming the expression of some selected known and novel mirnas using a 32-well plate roche lightcycler nano system and the roche sybr green master i ( roche diagnostics , germany ) . briefly , equal amount of cdna was taken in a reaction volume of 7.5 l in triplicate with 0.1875 l of each primer ( forward and universal reverse ) and 3.75 l of sybr green master i. thermo cycling conditions were set at an initial polymerase activation step for 600 seconds at 95c , followed by 45 cycles of 5 sec at 95c for template denaturation , 10 sec at 60c for annealing , and 1 sec at 72c for extension and fluorescence measurement . later , a dissociation protocol with a gradient from 50c to 95c was used for each primer pair to verify the specificity of the rt - qpcr reaction and the absence of primer dimers .", "in order to identify micrornas in jute , rna was isolated from the total tissue of four - day seedlings and subjected to illumina hiseq high - throughput sequencing by synthesis ( sbs ) technology . among a total of 16912862 raw reads , the details of tag cleaning are summarized in table 1 , which shows that a total of 16644324 clean reads were obtained by removing 3 adapter null , insert null , 5 adapter contaminants , sequences smaller than 18 nt , and poly a. this comprises about 99% of the high quality reads . distribution of these clean reads that contain a pool of small rnas ranging from 18 to 30 nucleotides is shown in figure 1 . however , the sizes of small rnas were not found to be uniform ; majority ( 92.69% ) of the srnas are 2024 nt in size , with 21 nt being the most abundant ( 42.19% ) , followed by 24 nt ( 22.95% ) and 20 nt ( 14.25% ) , respectively . these sequences were then aligned to rfam 11.0 and genbank database ( blastn ) to identify common srnas other than mirnas , as well as to remove mrnas ( see supplementary file-1 in supplementary material available online at http://dx.doi.org/10.1155/2015/125048 ) . the remaining sequences were matched against mirbase-20 database for the prediction of mirnas , revealing 33433 unique and 8994892 redundant reads , which were finally used to identify known mirnas . the novel mirnas in jute were identified from unannotated tags by using mireap , software developed by bgi ( described in section 2 ) . in the absence of the complete genome sequence and with practically no information on mirna of jute in mirbase , clean reads were aligned to the mirna precursor / mature mirna of all plants in mirbase allowing up to three mismatches or free gaps to identify known mirnas . the expression of mirna is generated by summing the count of tags which can align to the temporary mirna database , generated by choosing the most expressive mirna of each mature mirna family . a total of 227 known mirnas were identified in this study , of which 164 belong to 23 conserved and 63 to 58 nonconserved families . conservancy of mirna families found in jute showed high homology with their respective homologs in other model plants ( figure 2 ) . however , the number of family members of conserved mirnas was highly variable , with mir156 being the largest family , consisting of 26 members , whereas mir403 , mir394 , mir827 , mir477 , and mir2111 were the smallest among the families , comprising only one member . mir166 and mir169 were the second largest with each having 18 members and mir171 was the third largest family with 14 members ( figure 3 ) . most of the conserved families contain both 5p and 3p mature mirna sequences , attaching a high confidence to the data set . highly variable reads numbers were also found among the families , even in members of the same family , indicating different expression levels of these mirnas . among them , col - mir157a had the highest level of expression , having 5531609 counts , and the other mirnas like col - mir156a , col - mir166a , and col - mir167h also had relatively high reads numbers , counting more than 150000 . several conserved mirnas ( like mir171 , mir398 , and mir159 ) and , as expected , most of the nonconserved mirnas had relatively low copy numbers . interestingly , a mirna named col - mir3954 from an undefined family had very high level of expression , having 868222 reads , third highest of all mirnas found in jute . high expression frequency of mirnas derived from the 3 arm of some pre - mirnas , like col - mir166h-3p , col - mir166g-3p , col - mir166j-3p , col - mir165a-3p , col - mir396b-3p , col - mir396e-3p , and so forth , compared to their corresponding 5 arm - mirnas , supports the observations of functional activity of both arms of pre - mirna hairpins [ 55 , 56 ] . the mirna hairpins are mostly located in intergenic regions , introns , or reverse repeat sequence of coding sequences . thus tags belonging to these regions characteristic hairpin structure of mirna precursor was used to predict novel mirna with prediction software mireap ( http://sourceforge.net/projects/mireap/ ) by exploring the secondary structure , the dicer cleavage site , and the minimum free energy of the unannotated small rna tags which could be mapped to the reference vitis vinifera genome . predicted secondary structures were further validated by mfold ( supplementary file-3 ) and novel mirnas were identified based on the selection criteria described in section 2 . 17 potential novel mirnas have been identified in this study of which 9 mirnas were derived from 3 arm of the pre - mirna sequences and 8 from the 5 arm ( table 3 ) . average length of the pre - mirnas sequences ranged from 78 to 349 nt , similar to those found in maize and rice ; minimum folding energy ( mfe ) for jute mirnas was observed to be within a range from 21 to 105.3 kcal / mol , similar to the range observed in cucumber ( supplementary file-4 ) . expression of novel mirna was determined by summing the count of such mirnas which have no more than 3 mismatches on either the 5 or 3 ends and with no mismatch in the middle . novel mirnas usually have lower levels of expression than the conserved mirnas , as evident from findings of several plant species like soybean , brassica napus , maize , arabidopsis , and wheat [ 59 , 6164 ] . for a precise elucidation of the role of mirnas , target identification and determination of their biological functions it is now evident that cleavage or translational repression site of most known plant mirnas is located in the cds ( coding sequence ) region of their target mrna with perfect or nearly perfect sequence complementarity , making it feasible to identify plant mirna targets [ 4 , 21 , 66 ] . in this study , target genes of mirnas were identified by blastn against the genome sequence of vitis vinifera , following methods described by allen et al . and schwab et al . [ 9 , 45 ] . among a total of 79 identified mirna ( both conserved and nonconserved ) families , 116 potential target genes were predicted for 39 families ( supplementary file-5 ) . a total of 46 genes from this prediction overlapped with targets identified by psrna target which found 99 target genes for 19 mirna families ( supplementary file-9 ) . highest number ( 16 ) of targets was identified for mir397 family , all of which are laccase , an enzyme , involved in plant cell wall lignification . most of the other families targeted only a single gene . for the novel jute mirnas , a total of 11 targets were predicted for 4 among the 17 identified mirnas ( table 4 , details in supplementary file-6 ) , with a maximum number of target genes ( 6 ) recognized for col - mirn7 . most of col - mirn7 targets are nb - arc domain containing protein , which is a resistance ( r ) protein , involved in pathogen recognition and subsequent activation of innate immune responses . to better understand the functions of mirnas , target genes were analyzed by gene ontology ( go ) level 3 to divulge the regulatory network of mirnas and target genes . such analysis demonstrates that for jute 133 predicted target genes ( both for known and novel mirnas ) can be classified into 20 having biological , 5 cellular , and 5 molecular functions . same gene was found to be involved in multiple processes with the reverse being also true ( figure 4 and supplementary file-7 ) . as illustrated by kegg pathway analysis ( supplementary file-8 ) , the predicted target genes of jute mirnas were found to be involved in 42 different pathways . some of the mirnas identified through deep sequencing were verified by the standard stem - loop rt - pcr method followed by end point pcr and qrt - pcr . the stem - loop primers were designed with a 3 specificity for a particular mirna which hybridizes to the same and is reverse - transcribed by the rt enzyme . these primers increase the sensitivity of the reactions such that this method can significantly distinguish two mirnas with only one single nucleotide change . the rt product is then subjected to end point and qrt - pcr . forward primers were precisely designed from the first 15 bases of each mirna with 5 extension of random gc rich sequence to increase the melting temperature as mentioned by varkonyi - gasic et al . in 2007 while the reverse primer is a universal sequence that is designed from the 5 region of the stem - loop rt primer . a set of 11 randomly selected conserved mirnas as well as 9 novel mirnas were used for verification . in this study , the stem - loop primer used was 50 bp long together with 5 ( forward primer ) and 3 extensions , and the end point pcr product size ranged from 60 to 70 bp . amplification of the product gave a sharp band for each of the selected known and novel mirnas ( shown in figure 5 ) . cdnas were further amplified by qrt - pcr in technical triplicates from which log 2 values of cq were calculated for each of the mirnas and average of these values was compared with the log 2 value of read counts obtained from deep sequencing . most of the qrt - pcr results acceded with the sequencing data ; however , in some cases , discrepancy was observed ( figure 6 ) .", "widespread discovery of mirnas and their critical role in gene regulation has made it ever important to recognize them in different species . while a large amount of mirnas are reported and deposited in databases from different plants , mirna associated research in jute is still to be instigated . without the genome sequence of jute at hand , identification of mirna and their targets in jute by deep sequencing of small rnas use of closely related species ' genomes as proxy references can facilitate mirna identification in nonmodel species like jute for which genome sequence is not available . we have used the grape genome as the background because of sequence similarity between these two species . . analyses of size distribution patterns of the reads show that the most abundant srnas in jute are 21 nt in size which is about 42.19% , consistent with recent identification of srnas in different plant [ 34 , 62 , 64 ] . sequencing frequencies for mirnas in a library can be used as an index for estimating the relative abundance of mirnas . numerous small rna sequences , engendered from illumina hiseq high - throughput sequencing platform , show the presence of different mirna families and are even able to differentiate between distinct members of a given family . mir156 family which is highly conserved across the species was found to be the largest family in jute seedlings with the highest expression of col - mir157a followed by col - mir156a . two other members of the same family , namely , col - mir156c and col - mir156k , also show significant levels of expression . during shoot development , mir156 regulates the transition of plants from juvenile to adult phase by targeting spl genes . in arabidopsis , mir156 is strongly expressed during seedling development and shows weak expression in mature tissues . this could explain the relative abundance of the members of mir156 family since rna used in sequencing was extracted from jute seedlings . deep sequencing technology allows distinguishing and measuring mirna sequences with only a few nucleotide changes . for example , the abundance of mir156 family varied from 1 read ( col - mir156p ) to 5531609 reads ( col - mir157a ) . this was also the case for some other mirna families , such as col - mir166 ( from 3 to 215636 reads ) and col - mir167 ( from 12 to 154973 reads ) . presence of a prevailing member in a mirna family may indicate the dominant role of this member during the growth phase at which the samples were collected . it is also to be noted that most of the conserved mirna families consist of more than one member , whereas nonconserved mirnas identified in this study are mostly represented by a single mir ( mirna ) gene . it has been hypothesized that mir genes originate by gene duplication events followed by random mutation processes to evolve in multiples of imperfectly paired hairpins [ 77 , 78 ] . consequently , ancient evolutionarily conserved mirnas are represented by multiple mir genes whereas nonconserved mirnas ( believed to be evolutionarily recent ) generally originate from a single locus . it is plausible that the conserved mirnas are responsible for control of basic cellular and developmental pathways common to most eukaryotes whereas nonconserved mirnas are involved in regulation of species - specific pathways and functions . species - specific mirnas are believed to have recently evolved and , in general , expressed at levels lower than those of strictly conserved mirnas [ 34 , 77 ] . data acquired from sequencing frequencies of conserved and nonconserved mirnas fits well with this extrapolation , where the nonconserved and species - specific mirnas show residual accumulation in the tested tissue . however , one mir-3954 , a single member of an undefined family , appears to be expressed in significantly high levels . its only homolog deposited in mirbase v20 is in c. sinensis , showing high frequency of reads . 17 new jute specific mirnas identified in this study show a size anticipated for srnas derived from dcl1 processing , although sequence variants that possess shortened or extended 5 or 3 ends were also found . ten among the seventeen new col - mirnas are 21 nt in size , consistent with canonical dcl1 products . however length variation was also found . two , col - mir2 and col - mir9 , are 20 nt in size ; three , col - mir5 , col - mir6 , and col - mir14 , are 22 nt long . col - mir13 was found to be 23 nt in size which can probably be explained by the fact that diverse mirna families are also independently processed by dcl3 to generate a new class of bona fide ( 2325 nt ) mirnas with no canonical size , called long mirnas . a total of 20 mirnas of both conserved and species - specific origin were corroborated by stem - loop rt - pcr and their expression pattern was assessed by qpcr to validate the data obtained from deep sequencing . discrepancies in the expression pattern of some mirnas found by deep sequencing and qpcr can be attributed to practical differences between the sensitivity and specificity of these two techniques . the sensitivity and large dynamic range of next generation sequencing ( ngs ) , along with its consistent prediction of fold changes when compared with gold - standard qpcr , support its use for discovery - oriented and exploratory mirna profiling experiments [ 84 , 85 ] . to evaluate and outline a putative function for a mirna in plants , we have predicted target genes for known and potential new mirnas identified in this study using the genome of vitis vinifera as a reference . most of the target genes for conserved mirna families predicted in jute have already been confirmed in model plants , as target genes are commonly conserved [ 78 , 80 ] . mir156/157-squamosa promoter - binding protein , mir166-homeodomain leucine zipper protein iii ( hd - zip iii ) , mir167-auxin response factor ( arf ) , mir164-nac domain protein , mir172-transcription factor apetala2 , mir159-myb transcription factor , mir171-gras family transcription factor , mir394-f - box family protein , and mir395-atp sulfurylase well characterized mirna - target pairs in other plants have been found in jute . however , a number of widely studied mirna - target pairs , such as mir398-copper superoxide dismutase , mir399-e2 ubiquitin conjugating protein , and mir162-dicer - like 1(dcl1 ) , were not found in this study . this could possibly be due to the fact that the jute genome sequence is not available to be used as a reference . however , conserved mirnas with their nonconserved targets , including mir167-peroxidase29 , mir396-eukaryotic translation initiation factor 2c , mir168-nac domain containing protein , mir164-growth regulating factor 1 , mir390-ap domain containing transcription factor , mir160-myb transcription factor , and mir393-gtp - binding protein alpha subunit , were also found to be present in jute , allowing presumption of nonconserved targets for conserved mirnas . highest number of target genes were identified for mir397 , which is laccase , a well - studied enzyme , encoded by multigene families in poplar , arabidopsis , rice , and liriodendron tulipifera , reported to be involved in lignin biosynthesis of plants [ 99101 ] . toughness of this biopolymer also poses a major obstacle to pulping , forage digestibility , and biofuel production . it has been reported that transgenic p. trichocarpa plants overexpressing ptr - mir397a result in a reduction of klason lignin content , supporting the idea that use of mir397 would be an attractive means for reducing lignin - related problems . future experiments including in - depth studies of mir397-laccase pair may help in producing quality products from jute .", "this set of experimentations for identification of mirnas and their potential targets can initiate further study on understanding the mechanisms of regulation of jute mirna ." ]

[ "the use of laparoscopy in gynecology has developed into a vital tool for the evaluation and treatment of pelvic pathology . the predominant method of entry in gynecologic surgery worldwide remains a closed technique , with or without pneumoperitoneum . this approach unfortunately has been demonstrated in multiple studies to have the potential for visceral and vascular injury due to the blind insertion of veress needles or trocars . complications reported in the literature from the closed entry technique range from 0.05% to 0.67% , and include vascular injury , enterotomy , urinary tract injury , subcutaneous emphysema , and gas embolism . these complications arise due to the normal anatomic relationship of the periumbilical site to the underlying great vessels and viscera and are particularly problematic in patients with adhesions or at extremes of body weight . additionally , delay in diagnosis of bowel injuries frequently sited with this closed technique accounts for significant morbidity and mortality . this technique consists of creating a small umbilical incision under direct visualization to enter the abdominal cavity followed by the introduction of a blunt trocar . hasson proposed its potential benefits to be the avoidance of blind insertion of the veress needle and bladed trocar , prevention of preperitoneal insufflation and gas embolism , guaranteed pneumoperitoneum , and a more anatomical repair of the abdominal wall . since that time , hasson and others have corroborated these proposed benefits with data obtained from large case series . this large chart review was undertaken to examine our experience with open laparoscopy and to determine whether preoperative characteristics can predict open laparoscopic entry complication .", "we completed a retrospective chart review of all patients who underwent laparoscopy via an open technique at the mayo clinic arizona in the department of gynecology ( 8 surgeons ) from january 1 , 1998 through december 31 , 2006 . data were extracted from a computer - generated search , and each electronic medical record was individually reviewed to ensure inclusion criteria and obtain end points . all charts were reviewed from the point of surgery through the remainder of their care at our institution . intraoperative complications ( bowel and vascular injury , failure to enter ) , postoperative complications ( hernia , hematoma , cellulitis , abscess , cosmetic issues ) , body mass index ( bmi ) , number of previous abdominal surgeries , and length of follow - up were extracted from charts . statistical analysis was performed using jmp 6.0 for windows ( sas institute , cary , nc ) . the open laparoscopic technique used in this series is similar to that originally described by hasson and is as follows : the umbilicus is held and everted with 2 allis forceps . a vertical skin incision , 10-mm to 15-mm long , is created at the deepest portion of the umbilicus . the underlying fascia is grasped with 2 kocher forceps , elevated , and incised in a vertical midline fashion . if the peritoneal cavity is still intact , it is grasped with kocher or allis forceps and entered with metzenbaum scissors . finally , the laparoscope is introduced and the organs below the entry site are inspected as is the remainder of the abdominal cavity . closure of the umbilical entry site is performed with direct visualization and identification of the fascial layer . a 0-polyglactin suture on a # 2 urologic needle is used for the fascial layer , and a 40 polyglactin suture is used for skin approximation . an occlusive dressing is removed from the site in the ensuing 24 hours to 36 hours .", "mean patient bmi was 26.5 ( range , 14 to 57 ) , mean previous abdominal surgery was 1.3 ( range , 0 to 18 ) , and mean follow - up was 340 days ( range , 0 to 3028 ) . at the time of entry with the open technique , we experienced 2 ( 0.1% ) instances of enterotomy and 3 ( 0.1% ) cases of failed entry . of the enterotomies , in one patient dense adhesions from a previous debulking laparotomy ( requiring over 3 hours of laparoscopic adhesiolysis ) were present with jejunum adherent immediately under the umbilicus . the second patient had the transverse colon adherent to the umbilicus ; she had had 4 previous laparotomies . of the failed entries , the first patient had dense umbilical adhesions from 2 prior laparotomies , and the procedure was aborted . in the second patient , surgeons were unable to reach the peritoneal cavity due to the patient 's pannus ( bmi 43 ) , and a left upper quadrant entry was chosen . the third patient had dense periumbilical adhesions from a previous colectomy , so the procedure was converted to a minilaparotomy . patients with umbilical infection were subclassified based on whether the diagnosis was made over the phone due to patient complaints of erythema or drainage , by examination and diagnosis of cellulitis , or an umbilical abscess requiring evacuation . patients with subsequent umbilical hernias were subclassified into symptomatic ( noted by a physician or patient ) or asymptomatic ( discovered serendipitously at a subsequent surgery ) ( table 1 ) . fisher 's exact test was used to determine the association between each of the recorded complications and the number of previous abdominal surgeries and obesity ( bmi>30 ) ( table 2 ) . a significant association was noted between umbilical infection and previous abdominal surgery ( p=0.049 ) , and between umbilical hernia and obesity ( p=0.024 ) .", "this chart review confirms previous findings of case series , literature reviews , and meta - analyses of the complications associated with open laparoscopic entry . a large dutch review of 12,444 cases of open laparoscopy culled from 6 previously published case series found a 0.048% rate of enterotomy and no cases of vascular injury or gas embolism . these rates contrast with an enterotomy rate of 0.083% and vascular injury rate of 0.075% from 489,335 cases of closed laparoscopic entries in this same review . similarly , an australian meta - analysis examined 22,465 cases of one published open laparoscopy series and 760,890 patients from 22 published closed laparoscopy series . they noted an enterotomy rate of 0.049% ( 11 patients ) in open cases and 0.067% ( 515 patients ) in closed cases ( nonsignificant difference ) . no cases occurred of vascular injury in open cases , while the rate of vascular injury in closed cases was 0.044% ( 336 patients ) , which was statistically significant ( p=0.003 ) . similar rates of umbilical infection and hernia occurred with both techniques . to date , there are no reports of fatal vascular injuries and only 2 instances of nonfatal major vascular injuries in the literature associated with the use of the open technique . the first case occurred during the skin incision , when the scalpel directly entered the aorta ( the hasson trocar had not been used ) . the second case involved a damaged metal hasson cannula with a protruding spike that caused an aortic laceration . the lack of fatal vascular injuries noted in this and other large series is of utmost significance . data from litigious allegations related to 135 laparoscopic procedures over a 19-year period revealed a disproportionate percentage of the cases involving vascular injuries . additionally , these injuries are associated with a significant mortality risk , with 5 of 9 vascular injuries at closed laparoscopic entry reported to the medical defense union resulting in death . moreover , the morbidity incurred with major vascular injury includes transfusions , prolonged hospitalization , loss of limb , or other long - term sequelae . visceral injuries , in particular small bowel and colon , are also life - threatening , and because they are commonly missed during a closed entry , and injury may later be recognized only when symptoms of peritonitis develop . although intestinal injuries occurred in this case series , a major advantage of the open technique is the immediate recognition and repair of the enterotomy . neither of the 2 patients in this series suffered any long - term complications related to enterotomy . previous abdominal surgery and obesity are therefore not contraindications to an open laparoscopic entry , and in fact the open approach may offer some advantage over the closed technique in these patients . several series have reported lower hernia formation with the open technique , perhaps because of the ability to easily identify the fascial layer during closure . the fact that none of the hernias occurred prior to 6 weeks postoperatively also suggests that a good primary reappoximation was achieved at surgery . although this is a retrospective review of patients presenting to a referral center with somewhat limited follow - up , the large number of procedures performed via a consistent technique enhances the strength of this study . additionally , each case was hand reviewed for complications rather than using icd-9 codes to assess for rate of complications . finally , when available , patients charts for the entire time they received care at our institution , in some cases up to 8 years , were reviewed .", "the use of an open laparoscopic entry is advocated because it is a safe , simple means of accessing the peritoneal cavity . this case series confirms previous reports of the low risk of enterotomy , absence of fatal vascular injury , and comparable rates of umbilical infection / hernia associated with an open entry technique . the rapid recognition of enterotomy with this entry technique , and the utility of this technique in obese patients or those with previous abdominal procedures are additional advantages ." ]

[ "pulmonary hypertension ( ph ) is predominantly defined by a mean pulmonary artery pressure at rest greater than or equal to 25 mm hg . it is an enigmatic vascular disease and the pathogenesis of ph is multifactorial of origin and , hence , is categorized as idiopathic type [ 14 ] . as ph develops in a wide variety of clinical circumstances and is associated with diverse histological manifestations , a classification system is developed [ 5 , 6 ] . the dana point expert group has published a consensus of ph classification based on pathology , survival , natural history / epidemiology , etiology , and response to the treatment . among them , pah specifies that the disease primarily restricted to the pulmonary arterioles , a typical characteristic which shows an elevated pulmonary arterial pressure [ 2 , 3 ] . the pathological consequence of pah is the structural remodeling of pulmonary arteries ( pa ) , where increased proliferation of pulmonary artery smooth muscle cells ( pasmc ) and dysfunction of pulmonary artery endothelial cells ( paec ) occur in the vascular bed [ 79 ] . the morphological changes consist of hypertrophy of the tunica media , multicellular vascular lesions which obstruct and obliterate pulmonary arterioles leading to intimal thickening . the obstructed vessels limit the blood flow via pa and increase right ventricular afterload leading to right ventricular hypertrophy ( rvh ) and rv dysfunction [ 1012 ] . at molecular level , it is believed that the remodeling events in ph demand the participation of all cell - types present in the pulmonary arteries and that influence the pathological manifestation in the pulmonary vessel wall . the contributing factors that influence the remodeling process are hypoxic state , inflammation , vessel injury , and oxidative stress in the pulmonary vessels . as all forms of ph have in common an altered production of various endothelial vasoactive mediators , such as nitric oxide , prostacyclin , or endothelin- ( et- ) 1 , to establish the correct balance between vasoconstriction and vasodilatation [ 1316 ] . currently , the management for ph is aimed at optimizing cardiopulmonary interactions by targeting prostacyclin , endothelin , and nitric oxide signaling pathways . the most commonly used treatment regimen of ph is the use of prostacyclin analogues ( alprostadil , epoprostenol , treprostinil , and iloprost ) , endothelin receptor antagonists ( bosentan , ambrisentan ) , and inhaled no . in addition , phosphodiesterases ( pdes ) inhibitors ; pde-3 inhibitors ( e.g. , milrinone and enoximone ) , and pde-5 inhibitors ( e.g. , sildenafil and tadalafil ) are used to treat ph . they were used as an alternative therapeutic strategy which targets downstream components of the no signaling pathway by inhibiting pde-5 , the enzyme that catalyzes the conversion of cgmp to gmp . despite the advancement of modern surgery or ph - specific therapy , although ph ( or pah ) is well - studied encompassing both cardiac and vascular boundaries , the precise cellular and molecular mechanism of initiation and progression of ph are not completely understood and are still being explored . there is no cure of this disease and current therapies are limited to reverse the vascular remodeling . evolving evidence indicates that dysregulation of micrornas ( mirna or mir ) contributes to ph pathogenesis [ 1924 ] . indeed , an emerging body of evidence demonstrates that a fine balance in mirna levels seems to be a fundamental to maintaining homeostasis in the pulmonary vasculature and an imbalance with mirna level playing a critical role in the pathogenesis of ph by regulating a set of targeted genes . this review will collate vascular remodeling during ph , mirna biogenesis , recent advances on mirna modulation in ph , therapeutic opportunity , and conclusion .", "the mirna(s ) are composed of a vast family of short , noncoding rnas ( ~22 nucleotide long ) . mirnas are found from a single cell organism to plant and higher animals and even viruses [ 3739 ] . in humans , the biogenesis ( canonical ) pathway of mammalian mirnas is a two - step enzymatic process initiated in the nucleus and then transported to cytoplasm ( figure 1 ) . the transcription of mirnas generally processed by rna polymerase ii ( less frequently by rna polymerase iii ) and are typically capped and polyadenylated [ 41 , 42 ] . as a result , a long primary mirna transcripts ( pri - mirna ) containing a stem - loop structure is developed which is recognized by a large protein complex , called the microprocessor , the main components of which are the rnase iii drosha and digeorge syndrome critical region 8 ( dgcr8 ) [ 4348 ] ( figure 1 ) . for most pri - mirna , drosha is contributing the cleavage process called cropping with the assistance of its binding partner protein , dgcr8 [ 41 , 48 , 49 ] . the resultant product is a ~60 nucleotides long , hairpin - structured , called precursor mirna ( pre - mirna ) . dgcr8 directly interacts with the pri - mirna stem and flanking segments , a crucial measurement for one end of the mature mirna . in addition to the canonical pathway of mirna biogenesis described above , an alternative pathway also exists that are independent of drosha . in alternative pathways , the mirna can be released as mitrons ( pre - mirna like introns ) from pri - mirna and proceed for mirna processing unit without assistance of microprocessor [ 50 , 51 ] . the pre - mirnas are actively transported from the nucleus into the cytoplasm by exportin-5 ( exp-5 ) ( figure 1 ) . in the cytoplasm , another rnase iii enzyme , called dicer , catalyzing the process with the assistance of partner molecules argonaute ( ago2 ) , hiv-1 transactivation response rna - binding protein ( trbp ) and/or protein activator of pkr kinase ( pact ) , producing a short dsrna duplex , mirna / mirna of approximately 22-nucleotide length [ 44 , 5356 ] . the mirna / mirna duplexes are then incorporated into a ribonucleoprotein ( rnp ) complex called mirisc ( rna - induced silencing complex)that plays a critical role in the mirna - mediated mechanism of gene regulation . during assembly process of the mirisc , the mirna / mirna duplex is loaded into the ago , and the strands ( called the passenger strand ) are released and degraded . eventually , the bound mirna strand ( called the guide strand ) dictates mirisc to interact with partially complementary sequences in target transcripts ( localized within the 3utr ) and primarily triggers mrna deadenylation and degradation .", "micrornas ( mirnas ) are small , endogenously expressed noncoding rnas that regulate gene expression at posttranscriptional level , via degradation or translational inhibition of their target mrnas [ 37 , 59 ] . mirnas are ~22 nucleotides in length which bind to the 3 untranslated region of specific target genes and thereby suppress / inhibit the translation of target genes [ 60 , 61 ] . mirnas are key regulators of a wide range of cellular processes and play a pivotal role in vascular inflammation and cardiovascular pathologies inclusive of ph . from extensive studies from the past few years , it has become apparent that mirnas are expressed in a cell- and tissue - specific manner and are critically involved in various biological processes . emerging evidence indicates that mirnas contribute an important role in the maintenance of pulmonary vascular homeostasis and in the pathogenesis of ph . in the following part , a discussion of the roles of mirnas in ph - related signaling pathways is provided . mir-21 is a ubiquitously expressed mirna that is traditionally considered to be an oncogenic mirna ( oncomir ) . the two - channel microarray was performed to quantify mirna expression in whole lung extracts during the development of ph or pah caused by chronic hypoxia or monocrotaline in rats . it was suggested that mir-21 expression was downregulated in mct - induced ph model , but not in chronic hypoxia rats . mir-21 showed a similar expression level in both normoxic hypoxic cells , whereas tgf-1 , an important regulator of pulmonary vascular remodeling in ph , reduced expression of mir-21 . this suggests that although hypoxia- and monocrotaline - induced ph shares some common cellular processes driving the characteristic vascular remodeling , the different pathobiology induced by hypoxia and monocrotaline may lead to the different regulation of mir-21 expression . furthermore , the downregulation of mir-21 was confirmed in human lung tissue and serum from patients with idiopathic ph . since bmps induce smooth muscle cell differentiation through upregulating the expression of mir-21 , the downregulation of mir-21 in this setting may relate to the reduced bmp signaling and contribute to the alteration of smooth muscle cell phenotype in ph . however , yang and colleagues reported that mir-21 expression was increased in distal small pulmonary arteries of hypoxia - exposed pah mice and levels of bmpr2 , wwp1 , satb1 , and yod1 , the putative mir-21 targets , were decreased in the same tissue . transfection of mir-21 mimics also led to the reduced expression of bmpr2 , satb1 , and yod1 in pasmcs . the crucial role of mir-21 in vascular pathology has been evidenced by the results that the blockade of mir-21 impeded the development of intimal hyperplasia after acute vascular injury and bleomycin - induced pulmonary fibrosis [ 28 , 64 ] . yang et al . also found that inhibition of mir-21 alleviated chronic hypoxia - induced ph and attenuated pulmonary vascular remodeling . in human pasmcs , overexpression of mir-21 promoted , whereas sequestration of mir-21 abrogated cell proliferation and the expression of cell proliferation - associated proteins . the mir-21 null mice showed an exaggerated ph response to hypoxia , suggesting a major role of mir-21 in the pathogenesis of chronic hypoxia - induced pulmonary vascular remodeling and ph . although an association between mir-21 and ph ( pah ) is identified , the function of mir-21 in the development of ph is inconsistent in many experimental models . therefore , further investigations are required to clarify the role of mir-21 in the pathogenesis of ph . the mir-204 appears to be the first mirna that showed a mechanistic link between pulmonary arterial remodeling and cellular function . mir-204 was reported to be decreased in rodent lungs with hypoxia- and mct - induced pah and lung specimens from patients with pah . the reports suggested that stat3 activation contributed a crucial role in regulating mir-204 in pasmc . it was further demonstrated that mir-204 inhibition increased expression of shp2 , triggered the activation of src kinase , stat3 , and nuclear factor of activated t cells ( nfat ) , and thereby reduced apoptosis and was promoted proliferation of pasmcs . finally , delivery of synthetic mir-204 mimic to the lungs lowered pulmonary artery pressure , reduced medial wall thickness , normalized levels of mir-204 , shp2 , and stat3 , and alleviated the disease severity . this study may indicate the safe and effective use of mimic delivery to the pulmonary vasculature for future therapeutic purpose . interestingly , wei et al . have identified reduced level of mir-204 in the buffy coat of human subjects may correlate with pah severity and might serve as a circulatory biomarker for ph . expression of mir-143/145 was driven by tgf- and bmp4 and induced contractile gene expression through downregulating klf4 and myocardin [ 31 , 68 , 69 ] . the elevated levels of mir-145 were observed in primary pasmcs cultured from patients with bmpr2 mutations and also in the lungs of bmpr2-deficient mice , suggesting a role of bmpr2 signaling in modulating mir-145 expression . caruso and colleagues also found that mir-145 was downregulated in patient samples obtained from idiopathic and congenital ph but upregulated in plexiform lesions . the role of mir-145 in pah was further explored and elevated expression of mir-145 was shown in the wild type mice exposed to hypoxia . mir-145 deficiency and a locked nucleic acid anti - mir-145 resulting in significant protection from hypoxia - induced ph may represent a potential therapeutic target . the mir-17 - 92 cluster encodes seven related mirnas , which result from the transcription of a single pre - mirna and is further processed and cleaved to the mature mirnas . mir-17 - 92 cluster was retrieved as potential modulators of bmpr2 signaling by performing a computational algorithm on the bmpr2 gene . overexpression of mir-17 - 92 resulted in a marked reduction of bmpr2 protein level , and bmpr2 was proved to be directly targeted by mir-17 and mir-20a by using a bmpr2 reporter in hek293 cells . stat3 was found to be involved in il-6 signaling - mediated upregulation of mir-17 - 92 cluster and the subsequent downregulation of bmpr2 , since a highly conserved stat3-binding site exists in the promoter region of mir-17 - 92 gene . the role of a cholesterol - modified antagomir to mir-20a in hypoxia - induced ph was explored by brock et al . mir-20a restored functional bmpr2 signaling in human pasmcs and intraperitoneal administration of anti - mir-20a increased bmpr2 levels and alleviated vascular remodeling in lung tissue of hypoxic mice . antagomirs to mir-17 and mir-92a reduced muscularization of pulmonary arteries in the hypoxic mouse and monocrotaline rat models of ph , but only anti - mir-17 decreased rvsp and parameters of right heart dysfunction . bertero and colleagues identified mir-130/301 family as a master regulator of cellular proliferation in ph by constructing in silico a network of genes and interactions based on curated seed genes with known importance in ph . mir-130/301 expression was found to be increased in lungs of mice suffering from ph induced by su5416 administration with chronic hypoxia , in lungs of rats with mct - induced ph and in lungs of juvenile sheep with shunt - induced ph . in both human paecs and pasmcs , multiple ph inducers , including hypoxia , il-1 , and il-6 , increased mir-130/301 expression and hypoxia was found to upregulate mir-130/301 via a dependence on hif-2 and pou5f1/oct4 . it was further validated that mir-130/301 modulated apelin - mir-424/503-fgf2 signaling in paecs and mir-130/301-ppar- axis controlled proliferation of pasmcs by increasing stat3 expression and activity and repressing mir-204 expression . in hypoxia - induced ph mouse model , induction of mir-130/301 was promoted while mir-130/301 inhibition prevented ph pathogenesis .", "ph is broadly considered to be a vascular disease as vasoconstriction in the pulmonary artery is a prime cause for the development of ph . the hyperproliferation of pulmonary vascular cells ( mainly vsmc ) and subsequent neointima formation in the small pas are hallmark in ph . on the other hand , pulmonary arterial endothelial cells ( paecs ) further contribute a critical role in vascular homeostatic balance in the pulmonary vascular bed by orchestrating the vessel tone , leucocytes trafficking , and so forth [ 74 , 75 ] . proliferation of fibroblasts is reported in hypoxia - induced ph that plays a role in adventitial thickening [ 77 , 78 ] . under pathological situation like ph ( pah ) , the endothelium becomes dysfunctional which allows the penetration of infiltrating molecules , increased leucocytes adhesion , getting resistant to apoptosis that result in severe remodeling in the pulmonary vessels . these infiltrating molecules invade the barrier of neighboring cells like smooth muscle cells and activating the resident of adventitious fibroblasts . the remodeling process is basically an interaction between these cell - types present in the pulmonary arterial layers that resulting in a marked histological change in the pulmonary vasculature . an emerging body of evidences suggests that the mirnas play a pivotal role in the vascular cell integrity and plasticity during the progression of ph . here , although identification of mirnas in vascular diseases like ph is relatively new , but the earliest association connecting vsmc remodeling with mirnas was reported in 2007 . described the mirna signature in the vascular wall of balloon - injured carotid artery rat model . they showed that mir-21 was upregulated and the cellular effects were targeted by pten and bcl2 for neointimal lesion formation . the initial studies of mir-21 in vascular remodeling were followed by series of milestone works that include mir-143/145 cluster [ 24 , 31 , 7983 ] . two of the key mirnas involved in regulation of the phenotype of smcs are mir-143 and mir-145 . mir-145 is transcribed bicistronically along with mir-143 from human chromosome 5 . with relevance to pah , downregulation of mir-145 downregulation of target genes klf4 and klf5 by activation of mir-143/mir-145 upregulates smc - specific genes such as sma , calponin , and sm22- , triggering differentiation and lowering the proliferation rates in vascular smcs [ 31 , 82 ] . another mirna , mir-221/222 that triggers pdgf signaling in vsmc is thought to contribute in neointimal proliferation [ 35 , 84 , 85 ] . it is demonstrated that inhibition of mir-221 prevented pdgf induced proliferation , while forced expression of mir-221 increased proliferation and reduced the expression of vsmc marker [ 35 , 84 ] . in addition to the above mirnas , other mirnas are reported to regulate smc gene expression in hypoxia - induced ph . it is demonstrated that mir-210 that acts as a hypoxia - inducible mirna both in vitro and in vivo , inhibits pasmc apoptosis in hypoxia by specifically repressing e2f3 . a cell - based high throughput screening of a human mirna library with the nfat luciferase reporter system identified mir-124 as a new candidate in ph . it showed decreased nfat reporter activity and , decreased dephosphorylation and the nuclear translocation of nfat . an elegant study by courboulin et al . demonstrated that mir-204 downregulation correlates with ph severity and responsible for proliferative and antiapoptotic phenotypes of ph - pasmcs targeting stat3 and nfatc . interestingly , a recent study showed that a serine / threonine kinase mst1 , a modulator of cell death , appears to be a target of mir-138 . the authors suggest that mir-138 may be a negative regulator of pasmc apoptosis in hypoxic mediated pah . a panel of mirnas which are shown to modulate smc fate and modulation are listed in table 1 [ 2636 ] .", "therapeutic target which defines the treatment of a disease by means of a well - defined biological molecule . in this tiny rna molecule , have become an important gene modulator of various biochemical , physiological , and cellular functions . as we observed that either deficiency or abundance of a specific mirna or cluster of mirnas contributed a pathological state of many cardiovascular diseases including ph , it is reasonable to accept those mirnas as therapeutic target for diagnosis and therapeutic intervention . this is because we can utilize ability of a single mirna to control the expression of multiple ( hundreds ) proteins suggest that changes of a single mirna may influence several signaling pathways associated with pathological disease state . while each target is regulated subtly , the additive effect of coordinated regulation of a large suite of transcripts is believed to result in strong phenotypic outputs . several animal models in diverse disease pathology showed usefulness of mirnas as target molecules for therapeutic benefit ; this review will focus on vascular remodeling associated with ph . at therapeutic strategic stand - point , the aberrant mirna expression can be modulated or restored to normal by two main approaches : an anti - mirna ( anti - mir ) and mirna mimic . the former can be applicable to those mirnas whose expressions were increased in disease pathology and , therefore , silencing or inhibiting will be beneficial . to modulate mirna expression , the current strategy utilized chemically modified , cholesterol - conjugated single - stranded rna analogues complementary to the mature mirnas for antagomir or mimics , respectively . an elegant work demonstrated by liu et al . in a balloon - injury model represents a successful knocking down of mir-221/222 that suppressed vsmc proliferation and neointimal lesion formation . recently , brock et al . have shown that treatment with antagomir-20a restores the levels of bmpr2 in pulmonary arteries and prevents vascular remodeling in hypoxia - induced pah . for downregulated mirnas , a mimic approach is generally undertaken which basically rescued the underexpressed mirna in the tissue under pathological situation . this strategy is sometimes referred to as mirna replacement as it reintroduced the similar depleted mirna those were downregulated during diseases progression . therefore , by introducing the mirna mimic , the cells can restore the function as the mirna mimic is expected to target the same set of mrnas that is also regulated by the endogenous mirna . in a very comprehensive study by courboulin et al . , they demonstrated that reestablishing of mir-204 level by delivery of synthetic mir-204 into the lungs of experimentally - induced pah animals significantly reduced the disease pathology . although the mirna - based intervention is attractive and seems to be feasible , however , several challenges are noted . first , the critical mirnas responsible for ph must be confirmed definitively in well - accepted animal models along with explanted human pah samples , biopsies , and so forth , second , the cellular , molecular and physiological function of these mirnas should be performed in diseases progression and prevention ; third , the delivery route should be precisely targeted or restricted to lung vascular cells , for example , paec , pasmc , or fibroblasts . finally , the dose of antagomir or mimic delivery to the vascular bed should be carefully monitored further to avoid off - target effects . therefore , our future direction should be focus of vascular cell - specific delivery of mirna mimic or inhibitors . additionally , a regulated release of mirnas by conjugating nanoparticle will be considered for long - term use . finally , mirna - mediated therapeutics can be achieved by inhalation which may reduce the potential off - target issues to the other organs .", "biomarker generally refers to measurable substance in biological state . at clinical stand - point , it is extremely important as it predicts the medical outcome of a disease and its progression . over the past few years , it has been demonstrated that mirnas are found in the blood , plasma , urine , platelets , and saliva in a surprisingly stable form [ 9197 ] . the stability of mirna in the extracellular environment offers a great opportunity to consider as biomarker at clinical settings . using mirna as a biomarker offers many advantages like early prediction of diseases , differential expression during disease progression / pathologies , high degree of specificity , and sensitivity and importantly having longer half - life in the system . accumulating evidence suggested that the stability of mirna in extracellular environment illustrated by packed with lipid vesicles , wrapped in protein or lipoprotein complexes [ 98100 ] . in the context of cardiac diseases that include myocardial infarction , hypertrophy and heart failure ; several mirnas are identified and predicted to be considered as biomarker [ 101104 ] . the mirnas are circulating freely in the mammalian blood and can be predicted as biomarker for early diagnosis of cardiovascular diseases in humans [ 102 , 105108 ] . several evidences indicate that mirnas are secreted as micro vesicles or exosome and apoptotic bodies that may be responsible for release the mirnas into the circulation [ 109112 ] and are extremely stable in the blood or serum . rhodes and colleagues measured plasma levels of mirnas in eight patients newly diagnosed with pah and eight healthy controls by use of microarray analysis . fifty - eight mirnas showed differences in plasma concentration between the two groups and mir-150 was largest downregulated in pah . reduced expression of mir-150 predicted 2-year survival and correlated with disease severity . the mir-150 level was also found to be significantly reduced in circulating microvesicles and lymphocytes from patients with pah . courboulin et al . reported that downregulation of mir-204 in buffy - coat cells correlated with pah severity and might serve as a circulatory biomarker of pah . on the contrary , increasing higher plasma levels of mir-130/301 family members were observed in patients with increasing hemodynamic severity of ph . our previous study detected the pattern of mirnas in buffy - coat samples from mild - to - severe human ph subjects compared with the control subjects by mirna array . our study revealed that moderate to severe ph in human subjects are associated with significant downregulation of plasma levels of circulatory mir-1 , mir-26a , and mir-29c . the kinetics of the changes differed from moderate to severe ph human subjects as we observed further downregulation of the above mirnas in severe ph . mir-21 , mir-23b , mir-130a , mir-491 , and mir-1246 were moderately upregulated in moderate ph subjects and were more pronounced in severe ph . another set of mirnas that include mir-133b , mir-204 , and mir-208b were significantly upregulated in all moderate ph subjects and showed further increment in severe ph subjects . our study provided the evidence for the first time that circulating mirnas in the setting of ph may be used as early detection parameters for ph . nevertheless , the data presented were based on limited study population and the results need to be confirmed in a larger cohort study . furthermore , the dysregulation mirna in ph remains to be tested whether these circulatory mirnas have any influence in the target genes . circulatory mirnas are considered to be novel promising biomarkers for detection of ph . however , at current stage , using circulatory mirna as diagnostic tool for detecting ph is still in its infancy . the key advantage of circulating mirnas is a noninvasive testing procedure and relies on the stability of mirnas in the circulation and their storage condition . we give credit to the concept of quick determination of secretory molecules ( mirnas ) in the blood ; but the importance of mirna must be reevaluated at the clinical setting or model at multiple laboratories . another limitation is the small number of cohort study which appears to be heterogeneous clinical sample - type . these points may consider as preliminary - type of observation and need further confirmatory studies for considering mirnas as biomarkers for clinical use .", "understanding of mirnas role and function in the pulmonary circulation will offer and contribute a great potential to the pathogenesis of ph or pah . the mirna signature can be used as a diagnostic tool for the development of therapeutic strategy . the characterization of these mirnas under various experimental settings ( in vitro and in vivo ) should be performed along with the human subjects . the outcome will validate both physiological and pathological roles of these mirnas during the development and progression of ph prior to consider them for therapeutic intervention or future clinical trials . furthermore , use of disease specific mouse models , conditional transgenic , and knockouts are necessary to elucidate the functional role in vivo and verify its therapeutic potential , both of which remain largely unexplored . more novel approaches such as combination of argonaute protein immunoprecipitation , deep sequencing , or proteomic profiling approaches are required to identify its potential bona fide targets responsible for its functional impact ." ]

[ "pasteurella multocida was first described by revolee in 1877 and further characterized by pasteur in 1880 . pasteurella species usually have low virulence in humans , but serious manifestations sometimes occur and can lead to bacteremia . we present a patient who developed septic shock as a result of p. multocida bacteremia , presumptively from an intra - abdominal infection .", "a 52-year - old african american female was brought to the emergency department for generalized abdominal discomfort , altered mental status , diarrhea , fevers and chills for 4 - 5 days . her past medical history was significant for alcohol abuse and alcohol - related liver cirrhosis . in the emergency department , vital signs were as follows : blood pressure of 74/42 mmhg , heart rate of 112 beats / min , respiratory rate of 20 breaths / min and temperature of 98.6 f. remarkable findings on physical examination included jaundice as well as abdominal distention and tenderness to palpation , especially in the right upper quadrant . as per family , she did not have recent travels or exposure to sick people . they denied having domestic animals at home or any contact to the patient with pets . admission diagnosis was systemic inflammatory response syndrome with sepsis and septic shock with multi - organic dysfunction syndrome , presumptively secondary to community - acquired pneumonia , possible biliary tract infection ( ascending cholangitis ) , probable spontaneous bacterial peritonitis ( sbp ) and alcoholic hepatitis . empiric intravenous antimicrobial therapy , composed by piperacillin / tazobactam ( 2.25 g every 8 h ) and azithromycin ( 500 mg every 24 h ) , was initiated . laboratory results on admission were : a complete blood cell count of 21,000/mm(90% neutrophils ) , a platelet count of 51,000/mm , a sodium level of 127 meq / l , a potassium level of 5.3 meq / l , a bicarbonate level of 15 meq / l and a creatinine level of 6.3 mg / dl . alkaline phosphatase level was 290 iu / l , gamma - glutamyl transpeptidase level was 290 u / l and lactate dehydrogenase level was 482 total bilirubin level was 14.5 mg / dl , with a direct bilirubin level of 10.1 mg / dl . arterial blood gases showed a ph of 7.15 , a pco2 of 23 , and a po2 of 132 while the patient was receiving mechanical ventilation with fio2 of 60% . after 24 h of admission , patient remained in critical condition and requiring maximum doses of vasopressors , despite of what it seems to be the appropriate empiric antimicrobial therapy . blood culture system bact - alert 3d was used for the recovery of the organism . no minimal inhibitory concentrations for these antimicrobials were reported . in spite of all medical efforts ,", "p. multocida is a small gram - negative non - spore forming facultative anaerobe organism that is a natural inhabitant of the normal gastrointestinal flora and the upper respiratory tract of wild and domestic animals , especially cats and dogs . it is easily overgrowing by other flora in the sputum and might be regularly missed , as it resembles haemophilus influenzae , francisella tularensis and yersinia pestis . the five species that cause the majority of pasteurellosis are : multocida , septica , canis , stomatis , and dagmatis . human p. multocida infections have been reported to occur with and without animal exposure , the former mostly associated with the domestic cat and dog bites or scratches . most of the infections involve the skin and soft - tissues and they can be complicated by abscess formation , osteomyelitis or septic arthritis . the respiratory tract is the second most common site of infection with a wide spectrum of diseases that includes rhinosinusitis , tracheobronchitis , epiglottitis , pneumonia , empyema and lung abscesses . the majority of p. multocida pneumonia cases occur in elderly patients with underlying chronic pulmonary disease . invasive forms of p. multocida infection usually occur in immunocompromised patients , such as those with advanced age , chronic renal failure , solid tumors , hematological malignancies , diabetes mellitus or liver cirrhosis . p. multocida causes a wide variety of disease , including abdominal and pelvic infections , endocarditis , meningitis and endophtalmitis . von graevenitz et al . reviewed 21 proven cases of p. multocida bacteremia . a remarkable number of those cases involved patients with liver diseases , including liver cirrhosis of any etiology , hepatitis and infiltrating tumors . impaired function of the reticuloendothelial system and the presence of portosystemic shunts in patients with liver disease seem to play a major role in the development of bacteremia . this case seems peculiar in that no direct contact with a domestic animal could be documented . our patient had a history of alcohol - induced liver cirrhosis , which predisposed her to the development of bacteremia . however , retrospectively , it is difficult to establish the primary source of infection in this case . the findings of the abdominal examination may suggest that the primary source was the intra - abdominal cavity . unfortunately , the medical team was not able to performed paracentesis to obtain ascitic fluid for cytology and cultures due to patient s medical condition . no abdominal imaging was available . on the other hand , the chest radiography findings of infiltrate and pleural effusion may point the respiratory tract as the primary source of infection . we believed that , given the history of symptoms on admission and the presence of underlying liver disease , the intra - abdominal cavity was the most likely primary source of infection in this patient . nasopharyngeal colonization with p. multocida with transient bacteremia and seeding of the peritoneal cavity in immunosupressed cat owners could play an important role in the development of sbp . ten of these patients were exposed to animals , nine had positive blood cultures and four patients died . however , penicillin - resistant strains in human infections have been described . in these cases , 2 and 3 generation cephalosporins , macrolides , fluoroquinolones , tetracyclines , and trimethoprim - sulfamethoxazole outcome is associated with severity of the infection , the extent of the underlying disease and the early initiation of appropriate therapy .", "although cases of bacteremic p. multocida infections has been infrequently reported in the literature , clinicians should considered this organism as an important and potentially lethal pathogen in humans , where it can cause life - threatening infections . p. multocida should be included in the microbiologic differential diagnosis in patients with underlying chronic liver diseases who presents with possible intra - abdominal infection , even without history of exposure to domestic animals ." ]

[ "coronary artery bypass grafting ( cabg ) is considered to be the gold standard in patients with multivessel disease and remains the treatment of choice for patients with severe coronary artery disease , including three - vessel or left main coronary artery disease . the use of cabg , as compared with both percutaneous coronary intervention ( pci ) and medical therapy , is superior with regard to long - term symptom relief , major adverse cardiac or cerebrovascular events and survival benefit [ 14 ] . however , because of the use of cardiopulmonary bypass and median sternotomy , cabg is associated with significant surgical trauma leading to a long rehabilitation period and delayed postoperative improvement of quality of life . an alternative hybrid approach to multivessel coronary artery disease combines surgical left internal thoracic artery ( lita ) to left anterior descending coronary artery ( lad ) bypass grafting and percutaneous coronary intervention of the remaining lesions [ 3 , 68 ] . ideally , the lita to lad bypass graft is performed in a minimally invasive fashion through minimally invasive direct coronary artery bypass grafting ( midcab ) . this hybrid approach takes advantage of the survival benefit of the lita to lad bypass , while minimizing invasiveness and lowering morbidity by avoiding median sternotomy , rib retraction , aortic manipulation , and cardiopulmonary bypass [ 3 , 8 , 1014 ] . the purpose of the hybrid approach is to achieve complete coronary revascularization with outcomes equivalent to conventional coronary artery bypass grafting , while ensuring faster patient recovery , shorter hospital stays , and earlier return to work due to lower morbidity and mortality rates . angelini and colleagues reported the first hybrid coronary revascularization ( hcr ) procedure in 1996 , and several patient series using hybrid coronary revascularization have been published since then . these series support the above - mentioned presumptions and indicate that the hybrid approach is a feasible option for the treatment of selected patients with multivessel coronary artery disease involving the left main . moreover , the introduction of drug - eluting stents ( dess ) with lower rates of restenosis and better clinical outcomes may make hybrid coronary revascularization a more sustainable and feasible option than previously reported [ 9 , 15 ] . nevertheless , this hybrid approach has not been widely adopted because practical and logistical concerns have been expressed . these concerns implicate the need for close cooperation between surgeon and interventional cardiologist , logistical issues regarding sequencing and timing of the procedures , and the use of aggressive anticoagulant therapy for percutaneous coronary intervention that may worsen bleeding in the surgical patient [ 7 , 14 , 16 ] . this review aims to clarify the place of hybrid coronary revascularization in the current therapeutic armamentarium against multivessel coronary artery disease . second , the results of previous patient series using the hybrid approach are summarized and interpreted .", "the medline / pubmed database was searched in january 2012 using the medical subject headings ( mesh ) for coronary artery disease and angioplasty , balloon , coronary combined with the following free - text keywords : multivessel coronary artery disease , minimally invasive coronary artery bypass , percutaneous coronary intervention , and hybrid coronary revascularization . one hundred seventy - seven articles matching these search criteria were found , and the search for additional papers was continued by analysing the reference lists of relevant articles . randomized controlled trials , nonrandomized prospective and retrospective ( comparative ) studies were selected for inclusion . letters , editorials , ( multi)case reports , reviews , and small studies ( n < 15 ) were also excluded . studies examining the hcr procedure for multivessel coronary disease were included , while studies investigating the hcr procedure for left main coronary stenosis were excluded . authors and medical centres with two or more published studies were carefully evaluated and were represented by their most recent publication to avoid multiple reporting of the same patients . a total of eighteen included studies remained eligible for analysis after applying these in- and exclusion criteria ( figure 1 ) . the primary outcome measures were in - hospital major adverse cardiac and cerebrovascular events ( macces ) , packed red blood cells ( prbcs ) transfusion rate , lita patency , hospital length of stay ( los ) , 30-day mortality , survival , and target vessel revascularization ( tvr ) . secondary outcome measures were intensive care unit ( icu ) los and intubation time , as only a limited number of studies reported these outcome measures . in addition , the period of time between pci and lita to lad bypass grafting and the cost effectiveness of hcr were examined . the long - term lita patency was not included as an outcome measure , since only a limited number of studies report this outcome measure in a clear and concise manner . in - hospital major adverse cardiac and cerebrovascular events were defined as postoperative stroke , myocardial infarction ( mi ) , or death during hospital stay . only the fitzgibbon patency class a ( widely patent ) was considered as a patent lita to lad bypass graft , while the fitzgibbon patency class b ( flow limiting ) and c ( occluded ) were defined as a nonpatent lita to lad bypass graft . hospital los was defined as the number of days spent in hospital from operation to discharge . if the need for repeated revascularization involved a coronary artery initially treated with either bypass grafting or pci , this repeated revascularization was considered to be target vessel revascularization . one observer extracted all available outcome measures of each article and a second observer checked and supervised the first observer thoroughly . when an article did not disclose one or more of these outcome measures or reported medians and ranges as central tendency instead of means and standard deviations , the study was excluded from the analysis of that particular variable . the results were analysed using ibm spss statistics 19 software ( ibm inc . , armonk , continuous data were presented as mean and standard deviation ( sd ) , while categorical data were expressed as numbers and percentages .", "nine hundred seventy patients undergoing hcr procedures were included for analysis ( tables 1 and 2 ) [ 6 , 7 , 1114 , 1728 ] . the most important findings are reported below . the classical indication for an hcr procedure is multivessel coronary artery disease involving lad lesion judged suitable for minimally invasive lita to lad bypass grafting but unsuitable for pci ( type c ) , and ( a ) non - lad lesion(s ) ( most of the time right coronary artery ( rca ) and/or circumflex coronary artery ( cx ) lesions ) amenable to pci ( type a or b ) [ 7 , 11 , 12 , 14 , 17 , 18 , 20 , 22 , 23 , 2628 ] . high - risk patients especially with severe concomitant diseases ( e.g. , diabetes mellitus , malignancies , significant carotid disease , severely impaired lv function , and neurological diseases ) , who are more prone to develop complications after cardiopulmonary bypass and sternotomy , might benefit from the circumvention of cpb and sternotomy [ 11 , 18 , 20 , 2224 ] . exclusion criteria for hcr consist of contraindications to minimally invasive lita to lad bypass grafting or pci . lita to lad bypass grafting in a minimally invasive fashion requires single - lung ventilation and chest cavity insufflation . therefore , hcr procedures are contraindicated in patients with a compromised pulmonary function ( i.e. , forced expiratory volume in one second less than 50% of predicted ) and a small intrathoracic cavity space [ 14 , 27 , 28 ] . moreover , patients with a nongraftable or a buried intramyocardial lad , history of left subclavian artery and/or lita stenosis , morbid obesity ( bmi > 40 kg / m ) , and previous left chest surgery are not well suited for minimally invasive lita tot lad bypass grafting [ 14 , 20 , 22 , 27 , 28 ] . conditions rendering pci unsuitable include peripheral vascular disease precluding vascular access , coronary vessel diameter smaller than 1.5 mm , tortuous calcified coronary vessels , fresh thrombotic lesions , chronic totally occluded coronary arteries , extensive coronary involvement , chronic renal insufficiency ( serum creatinine 200 mol / l ) , and allergy to radiographic contrast [ 7 , 14 , 18 , 20 , 22 , 27 , 28 ] . finally , haemodynamic instability , need for a concomitant operation ( e.g. , valve repair or replacement ) , and decompensated congestive heart failure are regarded as exclusion criteria [ 7 , 17 , 20 , 22 , 27 , 28 ] . three hcr strategies can be distinguished : ( i ) performing pci first , followed by lita to lad bypass grafting or ( ii ) vice versa ; ( iii ) combining lita to lad bypass grafting and pci in the same setting in a hybrid operative suite . in the included studies , staged hcr procedures ( i and ii ) were applied much more frequently than simultaneous procedures ( iii ) . in a staged procedure , in which pci and lita to lad bypass grafting are carried out at separate locations and/or different days , both interventions can be performed under ideal circumstances ( in a modern catheterization laboratory and modern operating room , resp . ) [ 11 , 18 , 29 ] . however , patients have to undergo 2 procedures , while they remain incompletely revascularized and at risk for cardiovascular events for an extended period of time [ 14 , 29 ] . when pci is performed first , a staged procedure takes place with an unprotected anterior wall , which could pose serious health risks in case the lad lesion is considered the culprit lesion . in addition , lita to lad bypass grafting is performed after aggressive platelet inhibition for prevention of acute ( stent ) thrombosis , which might lead to unnecessary postponement of following operation or may cause a higher than expected rate of bleeding [ 12 , 13 , 21 , 29 ] . moreover , stent thrombosis is risked after reversal of surgical anticoagulation and is related to the inflammatory reaction after cardiac surgery . furthermore , the opportunity for quality control of the lita to lad bypass graft and anastomosis by a coronary angiogram is lost and , therefore , this strategy requires a reangiography [ 12 , 13 ] . these repeat control angiograms increase overall healthcare costs unnecessarily and decrease cost effectiveness . first , revascularization of non - lad vessels provides an optimized overall coronary flow reserve , thereby minimizing the potential risk of ischemia and myocardial infarction during the lad occlusion for lita to lad bypass grafting [ 6 , 12 ] . second , it is possible for the interventional cardiologist to fall back on conventional cabg in case of a suboptimal pci result or major pci complications . however , failure of pci leading to emergency conventional cabg has become extremely rare with decreasing incidence since the introduction of coronary artery stenting [ 12 , 20 , 2932 ] . furthermore , this strategy allows hcr in patients with the immediate need for pci in a non - lad target and no immediate possibility for emergency bypass surgery [ 11 , 24 ] . critical stenosis in the right coronary artery ( rca ) or the left circumflex coronary artery ( lcx ) or difficult pci targets are considered as clear indications for a pci first approach because these patients can undergo conventional cabg in case of pci failure . when the lita to lad bypass graft is performed first , antiplatelet therapy is routinely started after surgery to prevent antiplatelet - related bleeding complications during surgery and is present at time of pci [ 6 , 13 , 27 ] . these antiplatelet agents can be administered long term , which is mandatory for preventing stent thrombosis . moreover , the quality control of the lita to lad bypass graft and anastomosis can be performed simultaneously without a further angiogram [ 6 , 12 , 13 , 18 , 20 , 23 , 25 , 26 , 29 ] . in addition , pci is performed in a protective environment with a revascularized anteroseptal wall , which probably reduces the procedural risks and gives the interventional cardiologist the ability to approach lesions that would be quite challenging without a revascularized lad [ 13 , 20 , 25 , 26 , 29 ] . however , patients undergoing this strategy could require a second , much higher - risk , surgical intervention due to complications of the pci [ 13 , 23 , 25 ] . finally , the cardiac surgeon has to be aware of possible intraoperative ischemia during this hcr strategy because the collateral , non - lad vessels are unprotected . nevertheless , combining the two procedures in one stage under general anaesthesia in a specific hybrid - operating room , which combines the potential of catheterization and cardiac surgery , has advantages compared with staged hcr procedures [ 7 , 14 , 25 , 28 ] . this simultaneous approach represents a single procedure that achieves complete revascularization , while minimizing patient discomfort and reducing the need for anaesthetics [ 12 , 14 , 18 , 20 , 28 ] . this approach eliminates logistic concerns about timing and sequence of two separate procedures and maximizes patient satisfaction [ 7 , 14 , 25 , 28 ] . moreover , the quality of the lita to lad bypass graft and anastomosis can be confirmed immediately by an intraoperative angiogram , which enables direct revision of the lita to lad bypass graft [ 18 , 25 ] . complications and difficulties during pci or midcab can be dealt with immediately in the same setting by conversion to conventional , open - chest cabg . perioperative haemorrhage can become a problem because full antiplatelet therapy and incomplete heparin reversal are necessary instantly after midcab to prevent a transient rebound increase in thrombin formation associated with stent thrombosis and ensure an optimal intraoperative des placement [ 7 , 14 , 18 ] . besides , off - pump surgery may give rise to hypercoagulability and increased platelet activation during the early postoperative period , which is associated with an increased risk of stent thrombosis . therefore , a modified antiplatelet protocol and careful patient selection seem appropriate , especially in one - stop hcr , in order to minimize the risk of stent thrombosis without increasing perioperative bleeding risk . a tried and tested protocol of dual antiplatelet therapy ( dapt ) includes continuous use of aspirin ( 100 mg / day ) until the operation day and intraoperative administration of a loading dose clopidogrel ( 300 mg ) via a nasogastric tube after confirming lita graft patency , followed by a maintenance dose of 75 mg / day for 12 months . however , caution is required when using dapt , since reversal agents for clopidogrel and aspirin are not available . moreover , newer more potent antiplatelet agents , like prasugrel and ticagrelor , should be reserved exclusively for selected cases ( high risk of stent thrombosis ) and managed with even more care , since the clinical experience with these newer antiplatelet agents is limited in cardiac surgery and the bleeding risk may be increased . furthermore , intraoperative collaboration and communication among cardiac surgeons , interventional cardiologists , and anaesthesiologists should be outstanding and ongoing to optimize continuity of care [ 11 , 14 ] . currently , this simultaneous procedure is used in only a few centres , and some authors state that this might be caused by the need to possess catheterization laboratories outfitted to accommodate cardiac surgery or hybrid operating rooms equipped with a mobile coronary angiography c - arm or permanent fluoroscopic equipment [ 7 , 13 ] . the latter is reflected in the small number of patients undergoing a simultaneous procedure in our sample of included studies [ 7 , 13 , 14 , 18 , 24 , 25 , 28 ] . expansion of other percutaneous and hybrid procedures like hybrid af ablation may help to make these hybrid , multipurpose operating rooms more common in the future . however , staged hcr procedures could offer a more realistic alternative for many institutions without a so - called hybrid operating room , and this is supported by the fact that staged hcr procedures are applied much more frequently than simultaneous procedures in the included studies [ 6 , 1113 , 1724 , 26 , 27 ] . tables 3 and 4 present the period of time between both procedures in a staged hcr strategy , and this period of time varied notably from 0 to 180 days . therefore , some patients remained incompletely revascularized and were in theory at risk for cardiovascular events for a considerable length of time , while complete myocardial revascularization should be the main goal of treatment in patients with multivessel coronary artery disease . moreover , delhaye et al . found that pci with clopidogrel preloading can be performed within 48 hours of lita to lad bypass grafting without increasing the bleeding risk . in addition , zenati et al . performed pci zero to four days after lita to lad bypass grafting without increasing the prbc transfusion requirements , while lowering the hospital length of stay ( 2.7 1.0 days ) . the mean hospital length of stay was 5.5 1.8 days ( range : from 2.7 to 8.2 days ) , and hospital length of stay seems not to be influenced by the hcr strategy used ( table 2 ) . as shown in table 1 , the surgical techniques for lita to lad bypass grafting have evolved continuously since the introduction of the hcr procedure in 1996 by angelini et al . most of the initial patient series performed the lita to lad bypass graft in a minimally invasive fashion carrying out a mini - thoracotomy on the anterolateral chest wall in imitation of angelini et al . [ 3 , 7 , 12 , 1719 ] . in this so - called minimally invasive direct coronary artery bypass ( midcab ) approach the anastomosis to the lad is performed with 8 - 0 or 4 - 0 prolene sutures on the beating heart ( without cpb ) with the help of mechanical stabilizers . in more recent patient series , the lita was identified and harvested thoracoscopically or robotically , which decreased rib retraction , chest wall deformity , and trauma [ 11 , 14 , 21 , 22 , 27 ] . this approach significantly minimizes the typical thoracotomy - type incisional pain and wound complications of conventional midcab , while optimizing graft length and retaining the reliability of manually sewn lita to lad anastomosis [ 21 , 22 ] . some teams prefer to place the lita bypass graft to the lad through a ministernotomy ( inversed l - shaped or reversed j - shaped ) , which makes it possible to switch to full sternotomy in case complications may occur during the original operation [ 20 , 23 , 28 ] . nevertheless , this surgical technique increases surgical trauma and , therefore , may raise morbidity and mortality . in addition , some centres even decided to perform the lita to lad bypass graft through a full sternotomy on the beating heart ( off - pump cabg ) , thereby further increasing invasiveness [ 6 , 25 , 26 ] . if the lita bypass graft is placed on the lad through a sternotomy on the arrested heart ( on - pump cabg ) , circumvention of cpb is lost too [ 6 , 25 , 26 ] . thus , both on - pump and off - pump cabg can be seen as suboptimal procedures to carry out the lita to lad bypass graft . this might explain the higher macce rates found by zhao et al . and delhaye et al . and the high 30-day mortality discovered by zhao et al . and gilard et al . , who decided to place the lita to lad bypass graft on the arrested heart through full sternotomy in the majority of the patients [ 6 , 25 , 26 ] . lastly , some authors prefer to perform the lita to lad bypass graft in a totally endoscopic , port - only fashion using totally endoscopic coronary artery bypass grafting ( tecab ) [ 13 , 24 ] . this most challenging form of lita to lad bypass grafting using robotic telemanipulation techniques was initially performed on the arrested heart with the use of peripherally introduced cardiopulmonary bypass with intraaortic balloon occlusion and cardioplegic arrest [ 13 , 24 ] . a major disadvantage of this approach is the use of the heart lung machine , which increases the risk of stroke , bleeding , and an inflammatory response to surgery . the latter can be solved by using beating heart tecab ( bh - tecab ) , in which cpb and its considerable drawbacks are avoided . total endoscopic completion of the lita to lad bypass graft on the beating heart requires an additional port subxiphoidally to place a specially designed endoscopic stabilizer , which stabilizes the heart to optimize the quality of the anastomosis . this so - called beating heart totally endoscopic coronary artery bypass ( bh tecab ) procedure might be the least invasive approach for coronary bypass surgery without making concessions to graft patency [ 24 , 3538 ] . however , the tecab procedure is an extremely challenging and a potentially expensive procedure with an extensive learning curve , which may raise concerns about widespread adoption and application . the postoperative lita patency seemed to be independent of the surgical technique of lita to lad bypass grafting , since lita patency has shown to be approximately equal for all surgical techniques ( table 2 ) . the postoperative lita patency varied between 93.0% and 100.0% ( mean : 98.8% 2.3% ) . the mean in - hospital macce rate was 1.3% 1.9% ( range : from 0,0% to 5.6% ) with relatively high macce rates shown by katz et al . strikingly , three of these authors ( katz et al . , zhao et al . , and delhaye et al . ) performed lita to lad placement on the arrested heart [ 13 , 25 , 26 ] . the percentage of patients requiring prbc transfusion varied considerably between 0.0% and 35.4% ( mean : 13.6% 11.7% ) . the surgical technique or hcr strategy ( staged versus simultaneous ) used did not appear to affect the percentage of patients requiring prbc transfusion . overall , the 30-day mortality rate was 0.4% 0.8% ( range : from 0.0% to 2.6% ) . interestingly , higher than expected 30-day mortality rates were found in studies ( gilard et al . and zhao et al . ) using on - pump cabg to perform the lita to lad bypass graft in the majority of patients [ 6 , 25 ] . finally , the mean overall survival rate in hybrid treated patients was 98.1% 4.7% ( range : from 84.8% to 100.0% ) . besides the technical improvements of lita to lad bypass grafting , innovations occurred in the field of pci . this development was supported by the increased rate of des implantation in later patient series compared to earlier patient series , which used percutaneous transluminal coronary angioplasty ( ptca ) only or ptca in combination with bms implantation . application of drug - eluting stents should lower the restenosis rate , but their potentially beneficial effect on the target vessel revascularization ( tvr ) is not supported by data from the included studies ( table 2 ) . however , the ( early and late ) patency rate of new generation drug - eluting stents in non - lad lesions , provided that proper dapt is applied , may already be superior to that of saphenous vein grafts . hard evidence is however lacking , since a head - to - head comparison of ( early and late ) patency rates between des ( in non - lad lesions ) and saphenous vein grafts is not available . finally , the introduction of bioresorbable scaffold ( brs ) technology may improve sustainability , safety and feasibility of future hcr interventions . the application of brs technology can make long - term dapt redundant reducing bleeding complications without increasing the risk of stent thrombosis and may allow future reinterventions or reoperations on the same vessel if necessary due to its bioresorbable features . a relatively small number of studies in our sample ( table 5 ) compared the hcr procedure using minimally invasive lita to lad bypass grafting with conventional cabg or off - pump coronary artery bypass ( opcab ) [ 7 , 12 , 27 , 28 ] . all four of these studies selected matched controls who had undergone elective cabg or opcab with lita and saphenous vein grafts through median sternotomy during the same period using propensity score matching [ 7 , 12 , 27 , 28 ] . hu et al . found that patients in the hybrid group had a statistically significant shorter hospital length of stay , icu length of stay , and intubation time compared with opcab , while de cannire et al . reported that hospital and icu length of stay was statistically shorter in hybrid treated patients compared with patients treated with cabg [ 7 , 12 , 28 ] . showed that intubation time , icu , and hospital length of stay were similar between the hybrid and opcab group . moreover , these studies revealed that prbc transfusion requirements were reduced by the hybrid approach [ 12 , 27 , 28 ] . lastly , the in - hospital macce rates were considerably lower in the hybrid groups compared with both the cabg and the opcab groups . currently , only a few studies have explicitly explored the costs associated with hybrid coronary revascularization . de cannire and colleagues were the first to quantify costs associated with hcr and to compare these costs with costs involved in conventional double cabg . costs were calculated using six major expenditure categories : costs of hospital admission ( including intensive care unit and postsurgical cardiac ward cost as well as costs associated with delayed repeat procedures ) , pharmaceutical costs , surgical costs , pci - related costs , costs of blood products , and other miscellaneous fees ( including physiotherapy and consultants ) . the extra cost associated with pci ( including stents ) in the hybrid group in comparison with the cabg group ( 2.517 288 versus 0 0 ) , which uses autologous grafts to treat non - lad lesions , counterbalanced the cost savings on all other expenditure categories , which resulted in a nonsignificant cost difference at 2 years between both groups ( 10.622 1329 versus 9699 2500 ; not statistically significant ) . it is worth mentioning that the reduced icu and hospital length of stay due to faster recovery were largely responsible for the cost reduction in the hybrid group compared with the cabg group ( 3.033 499 versus 4.156 1.413 ) . showed that shorter intubation times , shorter icu and hospital length of stay , and less prbc transfusions resulted in a significant reduction in costs for hybrid treated patients in the postoperative period . conversely , intraoperative costs were statistically significant higher in patients undergoing hcr compared with opcab , largely because of longer operative times and the use of coated stents ( des ) rather than autologous grafts ( $ 14.691 2.967 versus $ 9.819 2.229 ; p < 0.001 ) . in conclusion , the difference in intraoperative costs was almost completely outweighed by the lower postoperative costs in the hybrid group . this resulted in slightly , but not significantly , higher overall costs in the hybrid group . the nonhealthcare costs after hcr will presumably be lower than after cabg or opcab because both kon et al . and de cannire et al . showed that return to work was significantly faster in the hybrid group , leading to a marked reduction in absenteeism from work in hybrid treated patients [ 7 , 12 ] . this difference in nonhealthcare costs should be able to compensate the opposite difference in healthcare costs , resulting in a negligible difference in total societal costs . moreover , the emergency of simultaneous hybrid procedures in especially designed multipurpose operating rooms combining the potential of catheter - based procedures and cardiac surgery will reduce the unnecessary costs incurred by staged hcr procedures [ 12 , 25 ] . lastly , more experience with minimally invasive cardiac surgery will shorten operative times , which might help reduce total healthcare costs .", "this review is the largest and most comprehensive report to date comparing the clinical outcomes of patients who underwent either hybrid coronary revascularization or conventional on- or off - pump cabg for multivessel coronary artery disease . three principal findings were revealed as follows : ( 1 ) hybrid treated patients showed a significantly faster recovery with lower prbc transfusion requirements and less in - hospital major adverse cardiac and cerebrovascular events than patients treated by on- or off - pump cabg ; ( 2 ) staged procedures were associated with considerable period of times between both procedures , leaving patients incompletely revascularized and in theory at risk for cardiovascular events for a considerable length of time ; and ( 3 ) the invasiveness of surgical lita to lad bypass grafting appeared to influence the clinical outcome , with higher macce and 30-day mortality rates in patients treated by more invasive surgical techniques using cpb and/or median sternotomy . as with any review , this report shares the limitations of the original studies . first , the initial reports especially included a relatively small number of patients , which may have resulted in biased results due to outliers . furthermore , almost all studies were performed retrospectively with inherent patient selection bias , since the decision to perform the hcr procedure was taken on an individual and highly selective basis according to cardiac surgeon and interventional cardiologist discretion . likewise , the inclusion and exclusion criteria used to select high - risk patients for the hcr procedure differed notably between the included studies , yielding a very heterogenic population . in addition , the used surgical techniques to perform the lita to lad bypass graft varied considerably , with learning curve issues and different levels of expertise and equipment . all these factors potentially contribute to heterogeneity , which may reduce the certainty of the evidence presented in this review . moreover , the mean length of followup was generally short , almost never exceeding two years , which made it difficult to assess long - term clinical outcomes of hybrid treated patients . therefore , this review relies mainly on in - hospital and short - term outcomes to assess the safety and feasibility of the hcr procedure . another limitation was the lack of long - term systematic and routine angiographic followup of graft and stent patency in the majority of studies included in the present review , which precluded any conclusions about the graft and stent longevity of the hcr procedure . furthermore , the comparative studies lacked randomization and nonblinded assessment of outcome , which might have led to selection bias and might have influenced outcome measures by preconceived notions about the superiority of the hcr procedure . finally , postoperative pain , which might be higher in patients treated with conventional midcab , was not included as outcome measure in the present review , because only a limited number of studies assessed this outcome measure . notwithstanding these weaknesses and limitations , this review selected the best evidence currently available to give a broad and comprehensive overview of the preliminary results of the hcr procedure . larger , multicenter , prospective , randomized trials with long - term clinical and angiographic followup and cost analysis comparing hcr with both conventional on - pump and off - pump cabg or multivessel pci will be necessary to further evaluate whether this hybrid approach is associated with similar promising long - term results . in the meantime , the first prospective , randomized pilot trial to compare hcr with conventional cabg in patients with multivessel coronary artery disease has been started . these data are also needed to identify patient populations that would benefit most from this hybrid approach . furthermore , more insights in the different surgical techniques for lita to lad bypass grafting and their clinical outcomes are necessary . therefore , the different surgical techniques for lita to lad bypass grafting in the hcr procedure should be integrated in these large , multicenter hcr studies in order to determine the best way of lita to lad bypass grafting in hcr . moreover , different hcr strategies ( staged versus simultaneous ) should be compared to decide which strategy will serve which patients best . finally , the advantages and disadvantages of a hybrid operative suite need to be explored further .", "the large variability in hcr techniques makes it difficult to draw firm conclusions from the currently available evidence , but hcr appears to be a promising and cost - effective alternative for cabg in the treatment of multivessel coronary artery disease in a selected patient population . the hcr procedure was associated with short hospital stays ( including icu stay and intubation time ) , low macce and 30-day mortality rates , low prbc transfusion requirements and tvr , high postoperative lita patency rates , and high survival rates . these promising early outcomes warrant further research with larger sample size , multicenter rcts to determine the definite place of hcr in the current therapeutic armamentarium against coronary artery disease . until then , this review justifies the continued use of the hybrid approach , but careful patient selection and close cooperation between cardiac surgeons and interventional cardiologists will determine the clinical outcomes to a significant extent ." ]

[ "globally four million deaths occur every year in the first month of life . almost all ( 99% ) neonatal deaths arise in low - income and middle - income countries [ 1 , 2 ] . in india alone , around one million babies die each year before they complete their first month of life , contributing to one - fourth of the global burden [ 1 , 3 ] . the neonatal mortality rate in india was 32 per 1000 live births in the year 2010 , a high rate that has not declined much in the last decade [ 4 , 5 ] . india 's neonatal mortality rate dropped significantly , that is , by 25% , from 69 per 1,000 live births in 1980 to 53 per 1,000 live births in 1990 followed by a 15% , decline from 51 to 44 per 1,000 live births between 1991 and 2000 . in recent years the nmr has dropped by 15% that is , from 40 per 1000 live births in 2001 to 34 per 1000 live births in 2009 . urban - rural differences in neonatal mortality exist with the mortality rates higher by 50% in rural ( 42.5/1000 live births ) compared to urban ( 28.5/1000 live births ) areas , as per the national family health survey ( nfhs-3 ) . the common causes of neonatal deaths in india include infections , birth asphyxia , and prematurity which contribute to 32.8% , 22.3% , and 16.8% of the total neonatal deaths , respectively [ 7 , 8 ] . india is one of the ten countries , along with china , democratic republic of congo , pakistan , nigeria , bangladesh , ethiopia , indonesia , afghanistan , and tanzania , that account for more than 65% of all intrapartum related neonatal deaths . despite the recognition of neonatal survival as a key to child survival , poor progress in neonatal survival in india poses concern regarding attainment of the fourth millennium development goal ( mdg ) target , that is , to reduce under-5 child mortality by two - thirds by 2015 .", "despite having a comparatively higher neonatal mortality rate , rural india is tackling with the problem of ill equipped public health facilities . the numbers of existing peripheral health facilities fall short of what has been recommended by the government of india . the healthcare in rural areas has been developed as a three - tier structure based on predetermined population norms . the subcenter is the most peripheral institution and the first contact point between the primary healthcare system and the community . primary health centers ( phcs ) comprise the second tier in rural healthcare structure envisaged to provide integrated curative and preventive healthcare to the rural population . community health centers ( chcs ) form the uppermost tier and their function is mainly to provide specialized obstetric and child care . a situational analysis done by the neonatal health research initiative ( nhri ) , indiaclen from 20072009 , in 24 centers of the country , suggested that less than 20% of the chcs / phcs provide essential newborn care services . also , the availability of a neonatal resuscitation area was relatively low in chcs ( 46% ) and phcs ( 14% ) . as per the district level health survey ( dlhs-3 ) , newborn care equipment was available in only 27.9% phcs . also , while around 76% of the community health centres had newborn care management facilities , just 35.1% had facilities for managing low birth weight babies . these findings underscore the critical condition of the public health facilities that are meant to cater to the health problems of the newborns in rural india . rural public health facilities across the country are having a difficult time attracting , retaining , and ensuring regular presence of highly trained medical personnel especially the gynecologists and pediatricians that are epochal in ensuring and promoting newborn health . statistics for 2010 suggest a shortfall of 10.3% for doctors at primary health centers ( phcs ) . the condition of 4535 community health centers supposed to provide specialized medical care is even more appalling . as compared to requirements for an existing infrastructure , there was a shortfall of 62.6% of specialists at the chcs , 55.2% of obstetricians and gynecologists and 69.5% of pediatricians . according to the dlhs facility survey ( 2003 ) , healthcare facilities with newborn care staff and a medical officer trained in newborn care were 59% , 45.0% , and 34% at district hospital , first referral units ( frus ) and chcs , respectively . as on march 2010 , the overall shortfall in the posts of health worker ( female)/auxiliary nurse midwife ( anm ) was 8.8% of the total requirement . similarly , in case of health worker ( male ) , there was a shortfall of 64.1% of the requirement . in case of health assistant ( female ) , the shortfall was 31.9% and that of health assistant ( male ) was 44% . the lack of qualified child care specialists results in a majority of rural households receiving care for their ill babies from private providers , many of whom are less than fully qualified .", "the government of india has launched various initiatives envisaging a high priority action with regard to neonatal health . under national rural health mission ( nrhm ) , accredited social health activists ( ashas ) are being deployed and assigned the responsibility to create awareness in the community regarding maternal and child health issues . they are further expected to mobilize the community and help them in accessing healthcare services . a safe motherhood intervention named janani suraksha yojana ( jsy ) has been implemented under the nrhm to increase the institutional delivery rates and provide skilled care at birth for the newborn . under the reproductive and child health program ( rch - ii ) , the quality and reach of antenatal care is planned to be expanded and home - based newborn care using integrated management of neonatal and childhood illness ( imnci ) protocols is envisaged . the imnci strategy encompasses a range of interventions to prevent and manage the commonest major childhood and neonatal illnesses that cause death , that is , acute respiratory infections , diarrhoea , measles , malaria , and malnutrition . the imnci package is planned to be implemented at the level of household and subcentres ( through anms ) and primary health centres ( through medical officers , nurses , and lady health visitors ) . till october 2011 facility - based care of neonates ( f - imnci ) is proposed through strengthening of infrastructure , provision of extra nurses , and skills upgradation of physicians and nurses . the government , with the help of unicef , has started setting up special care newborn units ( scnus ) for managing sick newborns [ 17 , 19 , 20 ] . these units have been established at district hospitals and are expected to have a minimum of 12 to 16 beds manned by 3 physicians , 10 nurses , and 4 support staff . further , newborn stabilization units ( nbsus ) are being set up in first referral units ( frus ) and community health centers ( chcs ) and they aim to provide care to sick newborns referred from peripheral health facilities . as of october 2011 , 1134 nbsus have been set up . a total of 8582 new born care corners ( nbccs ) , which are special corners within the labour room where resuscitation , infection control , and early breast feeding can be commenced , have been set up , as of 2011 . janani shishu suraksha karyakram ( jssk ) was launched on 1 june , 2011 with the aim to promote institutional delivery , eliminate out - of - pocket expenses , and facilitate prompt referral through free transport . a program on basic newborn care and resuscitation , named navjaat shishu suraksha karyakram ( nssk ) , is being launched to address important interventions at the time of birth that is , prevention of hypothermia and infections , early initiation of breastfeeding , and basic newborn resuscitation . the objective is to have one person trained in basic newborn care and resuscitation at every delivery . this training is being imparted to medical officers , staff nurses , and anms at chc / frus and 24 7 phcs where deliveries are taking place . provision of comprehensive emergency obstetric and new born care ( cemonc ) services and basic emergency obstetric and newborn care ( bemonc ) at various levels has also been given due importance . neonatal health is seemingly one of the priority issues in the agenda of the government which gets reflected in the various programs devised and implemented . the worrisome issue is the fact that improving health systems through facility upgradation and ensuring availability of trained manpower and logistics comprise essential prerequisites for the success of these programs / initiatives . the reluctance of trained manpower , especially doctors , to serve in rural areas has become a major impediment in the government 's ability to provide quality health services .", "the main obstacles to improving newborn survival are that many babies are born at home without being attended by skilled personnel , faulty home - based newborn care practices are widespread , lack of awareness among care givers limits care - seeking for neonatal illness and even if that is taken care of , lack of trained health workforce adds to the problem . this deficiency in skilled manpower undermines the initiatives by the government to improve neonatal health . another set of dilemma exists in bringing the neonates and the health system closer to each other . there are broadly two ways of doing so , either bring the health system closer to the neonate or bring the neonate closer to the health system . both of these are feasible and hold the promise to yield positive results but the real challenge lies in their reproduction and sustainment at the national level .", "in order to ensure the availability of trained medical personnel in rural areas , we first need to understand the reasons behind the observed shortage . recruiting trained doctors by all means is one of the essential components towards providing quality maternal and neonatal care services . a recent report documents that out of the 264 paediatricians ( including both postgraduates and diploma holders ) that are produced annually in india , only around half of them ( i.e. , 158 ) are available for public sector service , a large chunk either emigrate or get attracted towards private sector jobs in urban setups . the predominant reasons for preference to work in urban areas include adequate infrastructural facilities , high salary , and a decent standard of living [ 24 , 25 ] . further , in the recent years , there has been substantial emigration of trained doctors to developed countries , much of it coming from lower and middle income countries [ 2628 ] . among the developing countries , india is the biggest exporter of trained physicians with india - trained physicians accounting for about 10.9% of british physicians and 4.9% of american physicians . a report of the national commission on macroeconomics and health documented that around 10% of the obstetrician(s ) and paediatrician(s ) that the country produces eventually emigrate . although the recipient nations and the physicians that emigrate benefit from this migration , the home country loses its important health potentialities . there is no clear - cut solution to the problem of lack of doctors in rural setup . interventions in education and financial incentives along with professional support probably have the potential to ease out the problem , as had been seen in rural australia where the gprip continuing medical education grants and locum grants designed to assist rural general practitioners to maintain and increase their skills in areas relevant to rural practice helped in their retention in rural areas . the provision of better financial incentives oriented specifically to doctors working in the rural areas might be crucial to attract and retain more doctors in these areas . in canada , the distribution of doctors was positively influenced by raising fees in rural and underserved areas and reducing fees in areas , but in the philippines , rural incentives had an unintended negative impact due to the fact that local governments were unable to hire healthcare professionals at the high salary levels specified [ 3133 ] . thus , the experience with paying direct financial incentives , such as rural allowances , has been variable and usually depends on the affordability of resources but this should not undermine the potential it might offer to increase the influx of doctors in rural areas . other key initiatives could include establishing rural doctor networks , mentorship programmes , and giving rural practitioners preference in admissions in specialty programs . exposure to rural areas as part of the training of medical graduates , so they can understand the working conditions and acquire rural clinical skills , is essential and has the potential to yield positive results . this has been documented in thailand where a majority of graduates continued in rural practice after completing a compulsory rural residency . to prevent brain drain , international scholar exchange programmes could be thought of as an option besides improving healthcare infrastructure and creating an enabling work environment . certain care practices can be deleterious to the health of the baby like applying ghee / oil on cord , early bathing , avoidance of colostrum feeding ( considering it as harmful for the baby ) and not practicing exclusive breast feeding . realizing the presence of such traditions in the community and formulating intensive information , education , and communication ( iec ) campaigns to address these is required . there is a need to develop programs where there is a collective involvement of the communities in order to identify problems and their solutions . several such programs have been implemented in other parts of the globe and have yielded positive results . bolivia 's warmi program where the key highlight was participatory planning at the community level , with an emphasis on women 's participation to identify obstetric and perinatal health problems and their potential solutions . as a result of the intervention , neonatal mortality decreased from 120 per 1000 live births to 40 per 1000 live births . in rural nepal , a cluster randomized trial suggested that women 's groups facilitated by a local female community worker could reduce neonatal mortality rates by about 30% . in eastern india , the ekjut trial ( 20052008 ) evaluated the impact of community mobilization on birth outcomes in three districts of jharkhand and orissa . these studies offer evidence to encourage community involvement and leverage the community resources to bring about improvements in neonatal health . further , there is a need to make an effort to integrate community mobilization with health system strengthening . in a review of the evidence - based , cost - effective interventions for reduction of neonatal mortality , darmstadt et al . documented that a combination of outreach and home - based newborn care at 90% coverage could avert 1837% neonatal deaths . home - based newborn care could be explicated as a family as well as community oriented services that involve community mobilization and the empowerment of care givers to demand quality services for their sick newborns . hbnc mainly aims at reducing the neonatal deaths by preventing or treating morbidities such as infections , asphyxia or hypothermia which largely form the preventable causes of mortality . moreover , they are the underlying causes of nearly 55% of the neonatal deaths in india and addressing them could drastically cut down on the mortality rate . have documented that community - based pneumonia case management can lead to a 27% decrease in all - cause neonatal mortality , which indeed is a very high achievement . the most convincing example was set out by bang et al . in rural gadchiroli where female village health workers were selected from the local population and they were also trained to manage neonatal sepsis by providing parenteral antibiotic treatment to sick neonates . in the three years of intervention , there was a 71% reduction in perinatal mortality and a 62% reduction in neonatal mortality compared with the control area . in another example from sirur , a periurban area near pune , maharashtra , india , forty female village health workers were trained to serve a population of 47,000 . the village worker identified high - risk cases that required treatment by herself and the nurse , under the supervision of the field medical officer . she also made 3 home visits : on day 1 or soon after delivery and on days 8 and 29 . as a result of the intervention , a decline in the neonatal mortality rate of 25% from 51.9 to 38.8 per 1,000 live births was recorded . other successful examples include trials of home - based care in north india , bangladesh , pakistan , and nepal [ 4649 ] . in addition to creating awareness among community members and care givers in the family through information , education , and communication ( iec ) activities , a prerequisite for implementation of home - based care is the development of simple and easily comprehensible standard management guidelines . further , it would be a challenging task to upscale the home care newborn package to the most vulnerable states such as uttar pradesh , bihar , jharkhand , madhya pradesh , orissa , and rajasthan with a high neonatal mortality rate . in rural india , most of the births ( 53% ) occur at home largely unattended by skilled personnel . the lack of a trained personnel predisposes the newborn to a variety of birth related complications mainly birth asphyxia , birth injuries , and infections . moreover , most of the neonatal deaths occur in the first week of life with a majority of them dying on the first day of birth , thus reflecting the poor intrapartum care that the mother receives [ 1 , 50 , 51 ] . with the shortage of trained personnel , nonavailability of adequate healthcare facilities , poor connectivity to a health facility , and lack of transport facilities , providing care at home through training of midwives / traditional birth attendants ( tbas ) would probably be a better option . they can be a vital link between women and the health system , giving advice , encouraging women to go to the clinic to deliver , and accompanying mothers to provide moral support . one such successful case study is from indonesia [ 52 , 53 ] . in 2003 , nearly half of all newborn deaths in the cirebon district of indonesia were due to birth asphyxia . in order to address this situation in cirebon , program for appropriate technology in health ( path ) supported by saving newborn lives / save the children began for training community midwives ( bidan di desas ) . these midwives were taught a series of initial steps for assessing and managing a newborn 's condition , including the use of a locally produced tube and mask resuscitation device that could be used in home birth settings . one year after the training , it was found that newborn deaths due to birth asphyxia dropped by 47 percent in the district , at a cost of only $ 42 per asphyxia death averted . in zambia , midwife training programs significantly decreased the seven - day neonatal death rate in community health clinics . the midwives were given training in essential newborn care ( enc ) and in neonatal resuscitation . after training , the all - cause , 7-day neonatal mortality rate decreased from 11.5 deaths per 1000 live births to 6.8 deaths per 1000 live births . the perinatal mortality rate decreased from 18.3 deaths per 1000 births to 12.9 deaths per 1000 births . similar examples providing evidence for up scaling of trained midwives in order to lower down the neonatal mortality can be drawn from sri lanka , thailand , malaysia , and pakistan [ 5559 ] . infant mortality rates ( reflecting neonatal mortality as well ) are one of the most important indicators of the differentials in health and socioeconomic condition in a community . a substantial progress in lowering down the high burden of neonatal mortality is unlikely unless ways can be found to enhance the economic wellbeing of the lower socioeconomic groups . a pertinent example is that of kerala , a southern state of india , where the state 's achievement of stabilizing population growth , attaining high levels of literacy , and life expectancy have led to a significant decline in the infant mortality rates [ 60 , 61 ] . in a study done in rural haryana to document the determinants of neonatal deaths , it was found that the occurrence of deaths was a multifactorial process with involvement of factors at community level , family level ( socioeconomic ) , and biological level and that the socioeconomic determinants explained a large proportion of neonatal deaths . further , rahman et al . in their study in qatar found that low - cost , community - based interventions , on the background of socioeconomic development , had a stronger impact on neonatal and perinatal survival as compared to high - cost institutional interventions . similar findings documenting the importance of socioeconomic development in reducing the burden of neonatal deaths have been reported from studies done in chile , malaysia , malawi , and arab countries [ 6468 ] . neonatal care in rural india is largely provided by a large number of unqualified healthcare providers [ 6971 ] . they are the early providers of neonatal care and often attract a large number of ill newborns because of their easier access and comparatively cheaper treatment that they offer . there is a wide range of quality of services provided by these doctors and it would be useful to standardize their services by providing support in the form of training and technical support . though it does not qualify as a paragon solution , but this concept would probably score well , given the limited resources the country has . in alignment with what had been advocated by yadav et al . let best not be the enemy of the good , it would be beneficial to engage these local healthcare providers and equip them with necessary skills to provide acceptable standards of neonatal care until constraints on the supply of qualified and motivated healthcare providers into the system can be alleviated . they could further be involved in promoting key newborn essential care practices as they are popular and acceptable in the community . in china , rural healthcare is provided by village doctors who are trained in preventive and curative medicine of both traditional chinese and allopathic schools . the skills acquired are regularly upgraded by apprenticeship and in - service courses [ 73 , 74 ] . another example is from usa where the shortage of physicians in the 1960s paved the way for the emergence of physician assistants who were licensed to practice medicine under the supervision of physicians . they made a considerable contribution by working in rural areas which otherwise would not have received any care at all [ 75 , 76 ] . successful examples of providing quality healthcare through involvement of local healthcare practitioners can also be seen in ghana , mexico , and bangladesh [ 77 , 78 ] . providing a degree of bachelor of rural medicine and surgery ( brms ) after three - and - a - half years of training , as opposed to five and - a - half years of training for a usual medical graduate , has recently been discussed as one of the possible options to cater to the need of quality healthcare in rural india . the government of india , in consultation with the medical council of india ( mci ) , is planning to introduce this course in medical schools proposed to be established at district hospitals . the concept of a new degree course of a comparatively shorter duration is to encourage students from rural areas to take up medicine and subsequently provide services in their respective rural areas . the potential impact of selecting medical students of rural origin has been documented by rabinowitz et al . in a longitudinal study that evaluated the impact of the physician shortage area program ( psap ) in the usa . on multivariate analysis , rural origin was the single variable most strongly associated with rural practice . studies done in south africa , southern australia , and canada have also substantiated that the doctors with rural background have more tendency to work in rural areas [ 8183 ] . students enrolled in the proposed brms course will be taught preclinical as well as clinical subjects with more focus on paediatrics and obstetrics / gynaecology . further , it is envisaged to impart special training in care of the newborn and vaccination . chhattisgarh , a state in central india , has come up with the concept of awarding a degree named rural medical assistants ( rmas ) . this three - year course was a response to a major crisis in human resources for health that the state faced . three colleges were inaugurated in 2001 and were situated in rural / tribal districts , but with access to a large government hospital ( usually the district hospital ) to make it possible for clinical teaching and internship . there has been overwhelming positive response to recruitment of rmas to the most rural and tribal phc postings , where previously no trained physician existed . it will certainly improve health care delivery in rural , remote , and tribal areas by providing qualified practitioners but the training of these rural healthcare practitioners will be a major area of concern . it is doubtful as to how overworked , poorly staffed , ill - equipped district hospitals , which cater to thousands of patients , can become quality training grounds for healthcare practitioners . ensuring that these graduates would practice only in rural areas and not shift to urban setup further , there is a need to document the difference in the quality of care provided by the new cadre of healthcare professional and mbbs graduates . the use of mobile phones to improve the quality of care and enhance efficiency of service delivery within healthcare systems is known as mobile health , or m - health . who defines m - health as the provision of health services and information via mobile technologies such as mobile phones and personal digital assistants ( pdas ) . m - health tools have shown promise in providing greater access to healthcare to populations in developing countries , as well as creating cost efficiencies and improving the capacity of health systems to provide quality healthcare . studies done in kenya , sierra leone and zanzibar unleash the immense potentialities this innovative concept holds in addressing a wide variety of healthcare challenges [ 8688 ] . as earlier discussed , in rural setup , access to healthcare professionals and medical facilities is limited . although much work has not been done in context of m - health in india , yet efforts are required to be made to implement this in the indian context based on the initial success in other developing countries . the feasibility does not seem to be highly questionable considering the recent increase in the number of mobile phone users in rural areas . according to the press release by the telecom regularity authority of india ( trai ) , the number of telephone subscribers in india increased to 943.49 million at the end of february 2012 . the share of urban subscribers had been 65.59% whereas share of rural subscribers had reached 34.41% . subscription in rural areas had increased from 320.29 million in january 2012 to 324.68 million in february 2012 , an increase of 4.39 million in just one month . now with the recent initiative by the government to provide a subsidy of 20 percent on bills of less than rs 300 a month to mobile users in rural india , the increase in the number of mobile users could further be expected . mobile telephone short - message service ( sms ) can be used for delivering health behaviour change interventions . this service has wide population reach , can be individually tailored , and allows instant delivery , suggesting potential as a delivery channel for health behavior interventions . researchers in korea , croatia , new zealand , and united kingdom have used sms to deliver information pertaining to diabetes and asthma self - management , smoking cessation , and increasing physical activity and this has proved to be beneficial byincreasing awareness and bringing about the desired behaviour change [ 9194 ] . mobile technology can also be involved in better training of community health workers in using cellular short messages ( sms ) to encode and transmit basic health information such as vital signs and health symptoms to a monitoring computer . algorithms on the monitoring computer could recognize emergent conditions and send system - generated notification informing the community health worker of the appropriate management of the baby related to the inputted vital signs and symptoms . given the volume of neonatal care services that are being sought through the private sector in rural areas , one can not hope to reduce neonatal mortality through public sector interventions alone [ 6971 ] . because the private sector does not operate within the restrictive confines of a government bureaucracy the advantage with such a partnership could be the wider coverage and increased service utilization . also , using strengths and skills of each partner enhances efficiency . successful examples of improving maternal and neonatal health through public private partnerships have been documented in the literature [ 9598 ] . one such example is of pampers / unicef collaboration to eliminate neonatal tetanus . through this collaboration , over 300 million tetanus vaccines , protecting over 100 million mothers and their babies in 25 of the world 's poorest countries , have been provided . in even the poorest countries , the private sector is a major provider of goods , services , and information for maternal and child health . there could be different ways to involve the private sector , depending on the resources available and the need of services . one of the strategies could be to use microfinance to allow private sector doctors and other healthcare providers to provide quality practices . one such innovative scheme in india is the chiranjeevi scheme in gujarat [ 98 , 100 ] . it is an innovative health financing scheme covered through public - private partnership for emergency obstetric care and emergency transport services , for women belonging to below poverty line ( bpl ) category . have published an evaluation of a pay for performance ( p4p ) scheme implemented in rwanda . p4p scheme involves for - profit organizations who are provided incentives based on improvements in utilization and quality of care . statistically significant improvements were observed in the maternal and neonatal health ( mnh ) indicators of institutional delivery and quality of prenatal care which increased by 21% , and 7.6% , respectively over baseline in the p4p districts [ 101 , 102 ] . introducing public - private partnerships to improve the quality of maternal and child health services is not new in india . key examples include vande mataram scheme in west bengal which involves private sector for provision of safe motherhood and family planning services , janani express yojna in madhya pradesh for transportation in case of obstetric emergencies , and use of vouchers in uttar pradesh where reproductive and child health services for below poverty line ( bpl ) women and children are provided through private practitioners [ 103 , 104 ] . under national rural health mission ( nrhm ) , several initiatives based on public - private partnerships have been or / are planned to be implemented . the key issues include sustainability of such initiatives and ensuring that quality services are being provided .", "to conclude , the neonatal mortality rate in india is still high and skewed towards rural areas . much of the problem lies in the nonavailability of trained manpower and this in turn influences the quality of care the neonates receive . bringing qualified health professionals to rural , remote , and underserved areas is a challenging task which needs to be addressed urgently to avert neonatal deaths . other options such as training of local rural healthcare providers and traditional midwives , promoting home - based newborn care , creating community awareness and community mobilization along with strengthening public - private partnerships should be explored further , as evidence generated from previous studies and large scale projects support these strategies as a way to improve neonatal health . more research should be directed towards upcoming innovations such as m - health in order to exploit the potential they offer in terms of enhancing the quality of care .", "while the focus should be on devising strategies to recruit and retain trained manpower in rural areas , alternative strategies such as community mobilization , upscaling of home - based newborn care , imparting training and subsequent involvement of local rural healthcare providers and midwives should be attempted as well . more research is required to reveal the potential that innovations such as m - health , telemedicine , and public - private partnership hold in context to improving the quality of care in rural india ." ]

[ "the hypersensitivity syndrome , described as drug rash with eosinophilia and systemic symptoms ( dress ) syndrome is a severe , acute , drug reaction , defined by the presence of fever , cutaneous eruption , and systemic findings including enlarged lymph nodes , hepatitis , or hematologic abnormalities with eosinophilia and atypical lymphocytes1,2 ) . the syndrome can involve several sites , leading to findings such as pneumonitis , renal failure , myocarditis , thyroiditis , or neurologic symptoms , but the liver is the most commonly affected internal organ3 ) . this reaction can be life threatening , with a mortality rate of approximately 10% , most commonly secondary to liver failure1 ) . here we report the first pediatric case of liver transplantation for the treatment of acute liver failure caused by vancomycin - induced dress syndrome in korea .", "a 14-year - old girl was referred to asan medical center children 's hospital on the basis of test results that indicated abnormal liver function . she had been injured by a traffic accident 10 years earlier , and had undergone plastic surgery twice to heal wounds on her forehead . thereafter she had suffered from methicillin - resistant staphylococcus aureus ( mrsa ) osteomyelitis , which had been treated with intravenous vancomycin over five weeks . following this treatment , she developed fever and whole - body pruritic erythema , and abnormal liver function tests 6 days prior to her transfer . on admission , she presented with fever , nausea , vomiting , and abdominal discomfort . a generalized erythematous rash with variously sized , discrete lesions was noted on the face , trunk , and extremities . the patient reported an itching and heating sensation , which was aggravated after vancomycin injection . laboratory tests showed a total eosinophil count of 3,150/mm ( normal , < 500 mm ) , c - reactive protein level of 15.1 mg / dl ( normal , < 0.6 mg / dl ) , creatinine level of 2.5 mg / dl ( normal , 0.7 to 1.4 mg / dl ) , aspartate aminotransferase ( ast ) level of 320 iu / l ( normal , < 5 to 40 iu / l ) , alanine aminotransferase ( alt ) level of 263 iu / l ( normal , < 5 to 40 iu / l ) , alkaline phosphatase level of 440 iu / l ( normal , 40 to 120 iu / l ) , gamma glutamyl transpeptidase level of 321 iu / l ( normal , 8 to 35 iu / l ) , lactate dehydrogenase level of 2,437 iu / l ( normal , 120 to 250 iu / l ) , total bilirubin level of 3.3 mg / dl ( normal , 0.2 to 1.2 mg / dl ) , direct bilirubin level of 1.8 mg / dl ( normal , < 0.5 mg / dl ) , prothrombin time ( pt ) international normalized ratio ( inr ) of 1.68 ( normal , 0.8 to 1.3 ) , and activated partial thromboplastin time of 34.5 ( normal , 25 to 35 ) . serologic tests for hepatitis a , b , and c , as well as cytomegalovirus , epstein - barr virus , and autoimmune hepatitis were all negative . the vancomycin level was 26.9 mg / l ( normal , 20 to 40 mg / l ) . treatment with vancomycin was stopped , and replaced with ciprofloxacin for the treatment of osteomyelitis . intravenous delivery of a high dose of methylprednisolone was initiated upon an initial suspicion of dress syndrome . however , her liver function worsened progressively on hospital day 7 , with an ast level of 1,285 iu / l , alt level of 1,077 iu / l , total bilirubin level of 15.5 mg / dl , direct bilirubin level of 8.0 mg / dl , and pt inr of 4.8 , all suggesting acute liver failure . given the presence of aggravated hepatic encephalopathy , azotemia , and that the patient was refractory to medical treatments , she received a living - donor liver transplantation from her aunt on hospital day 9 . the detailed care of preliver and postliver transplantation and care of acute liver failure in our program was described elsewhere4,5 ) . after liver transplantation , the skin rash disappeared , with normalization of the eosinophil count and scores in liver and renal function tests . no severe clinical and surgical complications developed postoperatively . serial liver biopsies showed no evidence of acute rejection . over the course of a 25-month follow - up period , there has not been any definite recurrence of dress syndrome , with the exception of persistently elevated levels of liver enzymes and intermittent eosinophilia . the patient is currently suffering from iatrogenic cushing 's syndrome owing to the high dose of steroids administered to control her elevated levels of liver enzymes .", "we here report a pediatric case of acute liver failure resulting from dress syndrome , which was treated by liver transplantation . most reported cases of dress syndrome show that a cure can be achieved by the immediate withdrawal of the causative agent and the administration of methylprednisolone6 ) . liver involvement in dress syndrome is common and may range from a transitory increase in liver enzymes to liver necrosis with acute liver failure . to our knowledge , acute liver failure caused by dress syndrome has been reported at least twice in other european countries8,9 ) . in one of these cases9 ) , the patient died waiting for a liver transplant , whereas in the other case8 ) , fatal recurrence occurred after liver transplantation despite potent immunosuppression and cessation of the precipitating factors . the pathogenesis of dress syndrome is not fully understood , and may be multifactorial10 ) . although it is most commonly associated with antiepileptic drugs , dress syndrome has also been reported after exposure to a range of medications , including sulfasalazine , doxycycline , allopurinol , linezolid , and vancomycin1,8,10 ) . the differential diagnosis includes stevens - johnson syndrome ( sjs ) , life - threatening , cutaneous adverse reaction . the two syndromes overlap clinically , but have different characteristics , treatments and prognoses11 ) . therefore , a high index of suspicion and rapid diagnosis may be necessary to save the patient 's life . the only undisputed way to treat the use of systemic corticosteroids is common , although evidence regarding their effectiveness is scant12 ) . in our case , we replaced vancomycin with ciprofloxacin approximately 6 days after symptoms of dress syndrome were evident , and immediately began corticosteroid treatments after the switch to ciprofloxacin therapy . although ciprofloxacin is also known to cause dress13 ) , we finally chose that agent to control infection because other several antibiotics aggravated skin rash as well . these measures were ineffective in preventing liver failure . to our knowledge , this is the first pediatric case report to describe vancomycin - induced acute liver failure occurring as a component of the dress syndrome in korea . we highlight the need for awareness of the association between drugs , dress syndrome and liver failure . given the absence of reports describing the outcomes of liver transplantation in patients with dress syndrome , particular attention should be devoted to identification of its possible recurrence after liver transplantation ." ]

[ "despite the implementation of clinical guidelines and improvements in the critical care of patients with traumatic brain injury ( tbi ) , the prognosis of patients with severe tbi has remained poor during the last decade.16 ) the mortality rate of severe tbi is known to be about 32% to 52% , and about 6% to 11% of patients become vegetative or are severely disabled.313 ) it has been reported that dysfunction of at least one non - neurologic organ system develop in about 89% of patients with severe tbi . among them , the incidence of renal failure in tbi has been reported as 0.45% to 1.9%.1217 ) acute renal failure ( arf ) may occur by ischemic renal injury , sepsis or administration of nephrotoxic agents in tbi patients , and can be fatal to these patients . therefore , renal replacement therapy is required for tbi patients with arf to decrease post - traumatic mortality . among treatment modalities for arf , continuous renal replacement therapy ( crrt ) has shown to have a much lesser effect on intracranial pressure ( icp ) than intermittent hemodialysis ( ihd).6 ) additionally , some authors have reported that crrt have an effect on lowering icp.89 ) regarding hemodynamics , crrt can be a better treatment modality for arf compared to ihd in severe tbi patients . however , little has been reported about the treatment results of crrt on tbi patients with arf . we aimed to investigate the outcomes of crrt on tbi patients in whom arf developed after trauma , and to study relevant factors which influence patients survival .", "between april 2011 and june 2015 , 1,190 tbi patients were treated at our institution . among them , 29 patients underwent renal replacement therapy for arf . we retrospectively reviewed the laboratory , clinical , and radiological data of those 29 patients . initial glasgow coma scale ( gcs ) score was checked when the patient was arrived to the emergency room . injury severity score ( iss ) has six categories to head and neck , face , chest , abdomen or pelvic contents , extremities or pelvic girdle , and external . iss scores ranges from 1 to 75 ( iss scores of 5 for each category ) . the mean age at admission was 60.2 years ( range , 16 - 84 ) . the mean initial gcs score was 9.2 ( range , 3 - 15 ) , and the mean iss was 24 ( range , 9 - 59 ) . brain computed tomography ( ct ) scan showed epidural hematoma ( edh ) , subdural hematoma ( sdh ) , intracerebral hemorrhage ( ich ) , and traumatic subarachnoid hemorrhage ( t - sah ) in 5 , 25 , 7 , and 17 patients , respectively . eighteen out of 29 patients underwent surgical intervention for treatment of tbi ( table 1 ) . all 29 patients had normal kidney function on admission . however , 3 patients had a past history of chronic kidney disease . when the patients urine output decreased , we checked the serum creatinine level and calculated the glomerular filtration rate . crrt was started when the serum creatinine level increased to more than twice of the basal level , and the urine output decreased to less than 30 ml per hour despite hydration and diuretics . the mean serum creatinine level was 1.03 mg / dl ( range , 0.6 - 5.0 ) at admission and 2.79 mg / dl ( range , 1.6 - 7.9 ) before starting crrt . crrt was maintained until serum creatinine level was normalized ( 0.7 - 1.2 mg / dl ) . the median starting date of crrt was 3 days ( range , 0 - 34 ) after tbi . if the patients ' vital sign was stable , more than 100 ml / hour ( > 2400 ml / day ) of dialysis volume was removed . we investigated survival time of 29 tbi patients who underwent crrt and analyzed the relationship between survival time and the laboratory , clinical , and radiological factors . survival factors were categorized as follows ; age ( 65 vs. < 65 ) , 24-hours urine output ( 500 ml vs. < 500 ml ) before receiving crrt , iss ( < 25 vs. 25 ) , the presence of sdh , initial gcs score ( < 9 vs. 9 ) , pupil size at admission ( isocoria vs. anisocoria ) , cranial surgery ( yes vs. no ) , and intracranial hemorrhage ( edh , sdh , ich , t - sah absent vs. present ) . statistical analysis was performed using spss version 12.0 ( spss inc . , kaplan - meier analysis was used and survival outcome was compared using the log - rank sum test for categorical variables .", "between april 2011 and june 2015 , 1,190 tbi patients were treated at our institution . among them , 29 patients underwent renal replacement therapy for arf . we retrospectively reviewed the laboratory , clinical , and radiological data of those 29 patients . initial glasgow coma scale ( gcs ) score was checked when the patient was arrived to the emergency room . injury severity score ( iss ) has six categories to head and neck , face , chest , abdomen or pelvic contents , extremities or pelvic girdle , and external . iss scores ranges from 1 to 75 ( iss scores of 5 for each category ) . the mean age at admission was 60.2 years ( range , 16 - 84 ) . the mean initial gcs score was 9.2 ( range , 3 - 15 ) , and the mean iss was 24 ( range , 9 - 59 ) . brain computed tomography ( ct ) scan showed epidural hematoma ( edh ) , subdural hematoma ( sdh ) , intracerebral hemorrhage ( ich ) , and traumatic subarachnoid hemorrhage ( t - sah ) in 5 , 25 , 7 , and 17 patients , respectively . eighteen out of 29 patients underwent surgical intervention for treatment of tbi ( table 1 ) .", "all 29 patients had normal kidney function on admission . however , 3 patients had a past history of chronic kidney disease . when the patients urine output decreased , we checked the serum creatinine level and calculated the glomerular filtration rate . crrt was started when the serum creatinine level increased to more than twice of the basal level , and the urine output decreased to less than 30 ml per hour despite hydration and diuretics . the mean serum creatinine level was 1.03 mg / dl ( range , 0.6 - 5.0 ) at admission and 2.79 mg / dl ( range , 1.6 - 7.9 ) before starting crrt . crrt was maintained until serum creatinine level was normalized ( 0.7 - 1.2 mg / dl ) . the median starting date of crrt was 3 days ( range , 0 - 34 ) after tbi . if the patients ' vital sign was stable , more than 100 ml / hour ( > 2400 ml / day ) of dialysis volume was removed .", "we investigated survival time of 29 tbi patients who underwent crrt and analyzed the relationship between survival time and the laboratory , clinical , and radiological factors . survival factors were categorized as follows ; age ( 65 vs. < 65 ) , 24-hours urine output ( 500 ml vs. < 500 ml ) before receiving crrt , iss ( < 25 vs. 25 ) , the presence of sdh , initial gcs score ( < 9 vs. 9 ) , pupil size at admission ( isocoria vs. anisocoria ) , cranial surgery ( yes vs. no ) , and intracranial hemorrhage ( edh , sdh , ich , t - sah absent vs. present ) . statistical analysis was performed using spss version 12.0 ( spss inc . , kaplan - meier analysis was used and survival outcome was compared using the log - rank sum test for categorical variables .", "the actuarial median survival time of the 29 patients was 163 days after trauma ( range , 3 - 317 ) . among the 29 , 22 patients died with a median survival time of 8 days ( range , 3 - 55 ) ( figure 1a ) . the causes of death were tbi - related in 8 , sepsis due to pneumonia or acute respiratory distress syndrome ( ards ) in 4 , and multi - organ failure in 10 . among the various factors related to survival , 24-hr urine output before receiving crrt ( 500 ml vs. < 500 ml ) ( figure 1b ) , the presence of sdh ( figure 1c ) , and iss ( < 25 vs. 25 ) were significant after univariate analysis ( figure 1d , table 2 ) . median survival time was 10 days ( range , 3 - 136 ) when the patients urine output was less than 500 ml per day before receiving crrt , whereas more than half of the patients survived when the urine output was 500 ml per day or more ( p=0.015 ) . of the nine patients who had a 24-hours urine output of more than 500 ml per day before receiving crrt , 4 ( 44.4% ) patients died ( tbi - related brain herniation in 1 , multi - organ failure in 3 ) , with a median survival time of 146 days ( range , 4 - 317 ) . of the 20 patients who had a 24-hours urine output of less than 500 ml per day before receiving crrt , 18 ( 90.0% ) patients died ( tbi - related brain herniation in 7 , multi - organ failure in 11 ) , with a median survival time of 10 days ( range , 3 - 136 days ) ( figure 1b ) . for the patient with sdh , the median survival time was 102 days , whereas more than half of the patients survived when sdh was absent ( p=0.030 ) ( figure 1c ) . median survival time was 2529 days when the iss score was less than 25 , whereas it was 73 days when the iss score was 25 or more ( p=0.047 ) ( figure 1d ) . in multivariate analysis , only 24-hours urine output before receiving crrt ( 500 ml vs. < 500 ml ) remained as a significant factor ( p=0.026 ) ( table 2 ) .", "the actuarial median survival time of the 29 patients was 163 days after trauma ( range , 3 - 317 ) . among the 29 , 22 patients died with a median survival time of 8 days ( range , 3 - 55 ) ( figure 1a ) . the causes of death were tbi - related in 8 , sepsis due to pneumonia or acute respiratory distress syndrome ( ards ) in 4 , and multi - organ failure in 10 .", "among the various factors related to survival , 24-hr urine output before receiving crrt ( 500 ml vs. < 500 ml ) ( figure 1b ) , the presence of sdh ( figure 1c ) , and iss ( < 25 vs. 25 ) were significant after univariate analysis ( figure 1d , table 2 ) . median survival time was 10 days ( range , 3 - 136 ) when the patients urine output was less than 500 ml per day before receiving crrt , whereas more than half of the patients survived when the urine output was 500 ml per day or more ( p=0.015 ) . of the nine patients who had a 24-hours urine output of more than 500 ml per day before receiving crrt , 4 ( 44.4% ) patients died ( tbi - related brain herniation in 1 , multi - organ failure in 3 ) , with a median survival time of 146 days ( range , 4 - 317 ) . of the 20 patients who had a 24-hours urine output of less than 500 ml per day before receiving crrt , 18 ( 90.0% ) patients died ( tbi - related brain herniation in 7 , multi - organ failure in 11 ) , with a median survival time of 10 days ( range , 3 - 136 days ) ( figure 1b ) . for the patient with sdh , the median survival time was 102 days , whereas more than half of the patients survived when sdh was absent ( p=0.030 ) ( figure 1c ) . median survival time was 2529 days when the iss score was less than 25 , whereas it was 73 days when the iss score was 25 or more ( p=0.047 ) ( figure 1d ) . in multivariate analysis , only 24-hours urine output before receiving crrt ( 500 ml vs. < 500 ml ) remained as a significant factor ( p=0.026 ) ( table 2 ) .", "tbi patients are often exposed to multiple trauma , sepsis , and many nephrotoxic drugs . therefore , arf can occur after hypotensive acute tubular necrosis ( atn ) , vasomotor atn , and toxic atn after tbi.5 ) urea and other solutes are increased in arf patients and these solutes can pass into the brain because the blood - brain barrier ( bbb ) breaks down in patients with tbi . , this compensation mechanism is disrupted in tbi , thereby cerebral edema can become more worse.1211 ) additionally , arf can alter the concentration of neurotransmitters or circulating cytokines , acid - base balance , hemostasis , and drug metabolism . increased circulating cytokines can lead to disruption of the bbb , allowing increased access to cytotoxic inflammatory cells , cytokines , complement , amino acids , and organic osmolytes.14 ) therefore , it has been reported that tbi patients with arf have a higher incidence of poor outcome when compared with tbi patients without renal dysfunction.12 ) renal replacement therapy for arf in the patient with tbi is challenging , because conventional ihd is known to lead to an increase in brain water content , even in non - tbi patients undergoing regular hemodialysis.5 ) compared to standard ihd , crrt has been shown to result in greater intracranial stability.615 ) during treatment with crrt , changes in osmolality and changes of urea and bicarbonate levels were much less than those during ihd.5 ) this intracranial stability can be achieved by improved cardiovascular stability , because various modes of crrt can be selected according to cardiovascular volume status.4 ) therefore , it can be stated that crrt has many advantages over conventional ihd in the treatment of arf for patients with tbi . the acute kidney injury network working group summarized the available evidence and presented absolute indications for initiation of crrt as follows ; a serum urea concentration > 224 mg / dl ( blood urea nitrogen [ bun ] > 100 mg / dl ) , hyperkalemia ( > 6 meq / l and electrocardiogram abnormalities ) , hypermagnesemia ( > 8 meq / l ) , severe acidosis ( ph < 7.15 ) , lactic acidosis related to metformin use and anuria with diuretic resistant volume overload.710 ) however , to our knowledge , there are no previous reports regarding indications for initiation of crrt for arf in patients with tbi . in our study , it was found that starting early crrt before when the urine output is reduced to less than 500 ml per day significantly prolonged survival time of tbi patients with post - traumatic arf . our result suggests that starting crrt in the early stage of arf is required to improve the survival rate of tbi patients who have worse outcomes compared to those without post - traumatic arf . this study has some limitations of a retrospective design , a small number of patients , and a non - comparative study . however , to the best of our knowledge , this is the first analysis of outcomes of crrt in tbi patients with post - traumatic arf . further studies with a prospective design , many more cases , and control group with conventional ihd are necessary to prove the beneficial therapeutic effects of crrt compared to conventional ihd , and the best timing of initiation of crrt in the tbi patients with post - traumatic arf .", "according to our results , we suggest that early intervention with crrt before urine output is reduced to less than 500 ml per day may be beneficial in the treatment of tbi patients with impending arf . to define the therapeutic advantages of early crrt in the tbi patients with arf , a well - designed and controlled study with more cases" ]

[ "systemic lupus erythematosus ( sle ) is an autoimmune disease of connective tissue involving multiple organs . it is currently accepted that there are several genetic , environmental , and hormonal factors responsible for complex immunological disorders contributing to its development . recent studies have shown that abnormal stimulation of innate immunity may have a great influence on the immunopathogenesis of sle . hence , the receptors for pathogen - associated molecular patterns ( pamps ) have been the source of much recent attention . one of the representatives of this group is toll - like receptors ( tlrs ) . they are associated with innate immunity insofar as they are agents in the pathogenesis of sle and lupus - like syndromes . tlr3 , tlr7 , and tlr9 seem to be involved in the development of autoimmune diseases . the ligation of a tlr activates a chain of proteins which transmit a signal to the nucleus , which in turn leads to increased production of proinflammatory cytokines , the expression of major histocompatibility complex ( mhc ) class i and ii antigens , and costimulatory molecules , which effectively activate antigen presentation and acquired immunity [ 5 , 6 ] . intracellular tlrs , apart from pathogen recognition and initiation of innate immunity , are capable of recognizing endogenous ligands . in sle patients , impaired apoptosis and invalid cell debris clearance lead to increased concentration of serum nucleic acids ( ssrna , dsrna , and dna ) , which are well - known ligands for tlr3 , tlr7 , and tlr9 . nucleic acid - dependent activation of endosomal tlr is mediated by bcr receptor on lymphocytes b and fc , binding immunologic complexes and inducing their endocytosis . the activation of these receptors by specific ligands is thought to initiate autoimmune processes , which has been confirmed by studies on animal sle model . tlr stimulation leads to increased expression of proinflammatory cytokines ( il-6 , ifn , and tnf ) , which may reflect the intensity of the disease . on the other hand , synthetic oligodna with tlr receptor inhibitory properties causes the opposite effect , leading to a clinical improvement being observed in animal sle models . the aim of our study was to assess the tlr3 , tlr7 , and tlr9 expression on peripheral blood mononuclear cells ( pbmcs ) , including cd3 t lymphocytes and their cd4 and cd8 subpopulations , and cd19 b lymphocytes , in patients with sle , compared to healthy controls . the original results of this study serve as the first presentation of a simultaneous analysis of the relationship between the expression of the studied tlrs and disease activity , the degree of organ damage , several clinical and laboratory parameters , and the influence of immunosuppressive treatment . moreover , a correlation between the expression of tlrs and gender as well as pre- and postmenopausal period was evaluated .", "thirty - five sle patients , diagnosed as having met at least 4 criteria according to the acr , were included in the study . all of the patients had been treated at the department of dermatology and venereology , medical university of lodz and did not present symptoms of active infection or neoplastic disease at the time of the study . the study group comprised 30 women and 5 men aged from 25 to 65 years . the average duration of sle was 7 years , ranging from 3 months to 21 years . disease activity was assessed according to the slam ( systemic lupus activity measure ) scale . patients who reached 10 and more points were diagnosed as having active sle . during the study , organ damage was then assessed with the slicc / acr ( systematic lupus international collaborating clinics / american college of rheumatology ) damage index . however , 22 patients received 1 point and 8 of them 2 points , indicating severe organ damage . pbmcs were isolated by gradient centrifugation using ficoll - histopaque-1077 ( paa laboratories , pasching , austria ) . briefly , blood was precisely applied on the surface of the gradient and centrifuged at 1600 rpm for 20 min . the obtained buffy coat at the interphase was collected and dispersed in 5 ml of hank 's medium ( biomed , lublin , poland ) and centrifuged at 1600 rpm for 10 min . the supernatant was collected and cells were washed twice with rpmi 1640 medium ( paa laboratories , pasching , austria ) at 1100 rpm for 5 min . each time . isolated pbmcs were divided into 1 10 cells per tube ( each 100 l of pbs ) and incubated with surface monoclonal antibodies against cd3 , cd4 , cd8 , and cd19 conjugated with the fluorochromes allophycocyanin ( apc ) , peridinin chlorophyll protein ( per - cp ) , and phycoerythrin - cy7 ( pe - cy7 ) ( all from bd pharmingen , san diego , ca , usa ) at a concentration of 20 l/1 10 cells , in darkness at room temperature for 30 min . the cells were then fixed and permeabilized using an intracellular tlr staining kit according to the producer 's protocol ( imgenex , san diego , ca , usa ) . the cells were then incubated with monoclonal antibodies against tlr3 , tlr7 , and tlr9 conjugated with fluorescein isothiocyanate ( fitc ) and phycoerythrin ( pe ) and their corresponding isotype controls ( invivogen , san diego , ca , usa ) , at a concentration of 4 l/1 10 cells , in darkness , at room temperature for 30 min . six - color , two laser flow cytometry measurements were performed using the facs canto ii cytometer , equipped with bd facs diva software ( all becton dickinson , san jose , ca , usa ) as previously reported . the cell fluorescence was estimated using standard fluorescence filters : fl1 ( 313 nm 10 ) , fl2 ( 264 nm 10 ) , fl3 ( 374 nm 10 ) and fl4 ( 467 nm 10 ) , fl5 ( 355 nm 10 ) , and fl6 ( 653 nm 10 ) . for each sample , the lymphocyte population was discriminated from pbmcs by forward scatter ( fsc ) versus side scatter ( ssc ) distribution . then , the percentages of cd3 , cd4 , cd8 , and cd19 expressing tlr3 , tlr7 or tlr9 were assessed . finally , the ratios of tlr3 , tlr7 , and tlr9 in the whole population of pmbcs were calculated . representative dot plots from flow cytometry measurements of tlr3 and tlr9 expression on t- and b - cells in patients and healthy controls ( panel b ) are presented in figure 1(a ) . representative dot plots from flow cytometry measurements of tlr7 expression on b - cells in patients and healthy controls are presented in figure 1(b ) . for measurable characteristics , minimum and maximum values were shown ; average values were calculated : the arithmetic mean , median , and mode were calculated as were the parameters describing the internal differentiation ( standard deviation ) . the interquartile range was also calculated as the distance between the third and the first quartiles . for quality characteristics , the percentage of occurrence of the categories was determined . to determine the pattern of distribution of the quantitative variables , the shapiro - wilk test was used . the mann - whitney test was used to assess the significance of any differences in average values between two groups , as the distribution pattern was not normal , and the anova rank test and kruskal - wallis test , followed by a post hoc test of multiple comparisons of average ranks ( dunn test ) , were performed , to evaluate the differences in average values in several groups . the assessment of the relationship between the measurable variables was based on the spearman rank correlation coefficient . in all comparisons , the level of significance was p 0.05 .", "significantly higher percentages of tlr3- and tlr9-positive pbmcs and cd3 t lymphocytes , including those positive for cd4 and cd8 antigens , as well as cd19 b lymphocytes were observed among patients with sle , compared to healthy controls ( figures 2 and 3 ) . a higher percentage of cd19 b lymphocytes expressing tlr7 was found in patients with sle than in healthy subjects ( p < 0.006 ) ( figure 4 ) . with regard to pbmcs and both subpopulations of t lymphocytes , tlr7 expression did not differ between patients and healthy controls ( table 2 ) . there were no significant correlations between the proportions of various cell subsets expressing the studied tlrs and disease activity ( table 2 ) . no statistically significant correlation was observed between the expression of any of the studied types of tlr among the given cell subpopulations and the degree of organ damage according to the slicc / arc damage index . however , subjects with severe organ dysfunction presented a higher percentage of tlr9-positive pbmcs , cd4 and cd8 t lymphocytes , and cd19 b lymphocytes ( table 3 ) . a significant mutual correlation was seen to exist between the expression of tlr3 and tlr9 in pbmcs ( p < 0.00001 ) . no statistically significant correlation was observed between the expression of studied tlrs and the patient 's gender . a significantly higher percentage of cd19 b lymphocytes expressing tlr7 was found in premenopausal women with sle than in postmenopausal women ( 3.52% 6.46 versus 0.12% 0.17 resp . , p < 0.03 ) . a significantly lower count of cd4 cells with tlr9 was observed in patients with lymphopenia , compared with patients with a normal lymphocyte count ( > 1000/mm ) in the peripheral blood ( 4.59% 5.83 versus 6.86% 6.83 resp . , p < 0.005 ) . in this subpopulation of cells , there was a significantly higher count of cells among patients with hypogammaglobulinemia , representing less than 12% of all proteins in the proteinogram analysis , compared to subjects with normal concentrations of gammaglobulins ( 32.12% 13.78 versus 11.46% 12.05 , resp . , p < 0.05 ) . among patients with anaemia , there was a higher percentage of tlr7-positive cd3 ( 4.19% 5.45 ) , cd4 ( 4.19% 5.45 ) , and cd19 cells ( 5.87% 8.71 ) , compared to patients with haemoglobin concentration > 12 g / dl ( 0.55% 1.08 , p < 0.05 ; 0.55% 1.08 , p < 0.03 ; 0.85% 2.24 , p < 0.02 , resp . ) . moreover , an erythrocyte sedimentation rate ( esr ) of more than 25 was significantly more frequent in subjects with lower counts of tlr3-positive , cd19 b lymphocytes compared to esr 25 ( 2.55% 2.85 versus 5.10% 3.37 , resp . , p < 0.03 ) . a review of the clinical findings reveals that only patients with joint symptoms have lower tlr9-positive cd19 b lymphocyte counts , compared to subjects with no joint symptoms . no statistically significant correlation was observed between the expression of studied tlrs and immunosuppressive treatment .", "despite intensive research in many centres , the pathogenesis of sle remains poorly understood , and hence , the condition lacks targeted therapy . however , the discovery of tlrs in humans opened a new field in the studies of lupus , and our study of tlr3 , tlr7 , and tlr9 confirms their potential influence on the disease . tlr expression has been studied on the molecular level ( mrna ) , as well as the protein level , and involves many subsets of peripheral blood cells [ 1520 ] . higher expression of tlr9 has been shown in sle patients , compared to healthy individuals , which is consistent with our findings . however , the results of studies concerning tlr3 and tlr7 expression are inconsistent . most of them concentrate on tlr9 expression in b lymphocytes , probably due to the fact that these cells are the main source of pathological antibodies responsible for the propagation of the disease [ 16 , 18 , 20 ] . a higher count of cd19 b and cd3 t lymphocytes expressing tlr9 were seen in our study group , compared to healthy controls . this observation is similar to those obtained by wu et al . , who assessed patients with newly diagnosed , untreated sle . on the other hand , papadimitraki et al . observed a higher percentage of tlr9-positive cd19 b lymphocytes in a group of patients with active disease , compared to those with inactive disease . what is more , they observed a decrease in tlr9 expression on b cells of as much as 50% when the patient entered remission . it is plausible that in remission , stimulation of b lymphocytes through tlr9 is less intense , and as a result of this phenomenon , the autoimmune inflammation subsides . a potential confirmation of this hypothesis is a study by wong et al . , who demonstrated a positive correlation between the concentrations of proinflammatory cytokines and chemokines produced after tlr9 stimulation and disease activity . however , in our study , no difference was observed between active and inactive sles in terms of the percentage of tlr9-positive cd19 b lymphocytes . this discrepancy between our and other centres may stem from the use of different criteria for patient selection . patients with lupus nephritis dominated in the study by papadimitraki et al . , constituting 36% of the whole study group , whereas they only constituted 3% of our group . other research demonstrates greater tlr9 expression in the glomeruli of patients with lupus nephritis , and that the stimulation of glomeruli with endogenous tlr9 ligands augments inflammatory reactions in the kidneys . wong et al . analysed tlr3 , tlr7 , and tlr9 expression in cd19 b lymphocytes and cd4 and cd8 t lymphocytes among 16 chinese women . this is the only available publication which addressed the same markers as the present study . the results regarding tlr3 and tlr9 expression in cd19 b lymphocytes and cd4 and cd8 t lymphocytes obtained in by both the present study and that of wong et al . . however , these results need to be confirmed by rt pcr on t cells as was done on b cells by nakano et al . . differences between those results concerned only tlr7 population . while our report shows a markedly higher count of tlr7-positive lymphocytes b cd19 in sle patients than in healthy subjects , wong et al . did not find any difference in tlr7 expression for any cell subset between patients and healthy controls . however , after tlr7 stimulation , they observed an increase in the production of the chemokines cxcl10 and ccl5 by pbmcs from patients with sle . the observed inconsistence of the results may be due to heterogeneous nature of the study groups used by the two studies or their different genetic background . similar to our results , although obtained via molecular techniques , are the findings by komatsuda et al . , who report that the concentrations of mrna for tlr7 and tlr9 in pbmcs are significantly higher among patients than in healthy controls . there are several publications regarding correlations between the expression of tlrs with sle activity , but the conclusions are contradictory . wong et al . do not report any such correlation in terms of tlr3 , tlr7 , or tlr9 expression . the lack of any relationship was probably due to the relative predominance of subjects with an inactive disease , according to sledai scale ( sle disease activity index ) . in addition , no such significant relationship was demonstrated , although the majority of patients ( 63% ) presented with active sle . nakano et al . studied 19 subjects in the active sle phase and identified a positive relationship between tlr9 mfi ( mean fluorescence intensity ) in b lymphocytes and sledai score . analysed a group of 35 newly diagnosed patients and found a negative correlation between the percentage of tlr9-positive b cells and sle activity . they pointed to a possible protective role of tlr9 in the development and propagation of sle . the discrepancy of published data may be explained by heterogeneous study groups in terms of clinical and therapeutic parameters . the presence of anti - dsdna antibodies and tlr expression was also noted in the present study . this type of antibody is a pathognomonic marker of sle , specific for renal involvement . available publications concerning the relationship between the expression of tlr and the presence of anti - dsdna antibodies are inconsistent . there was a positive correlation between the concentration of anti - dsdna antibodies with the percentage of tlr9-positive cd19 b lymphocytes from patients with active sle . however , other studies , as well as our own results , do not reveal any significant relationships between these parameters [ 17 , 21 ] . on the contrary , komatsuda et al . observed a negative correlation between anti - dsdna autoantibodies and the tlr9 mrna content in cells . this may be explained by the heterogeneity of studied populations in terms of clinical presentation , accompanying diseases , treatment modalities , and occult infections , in particular . , unlike other researchers , evaluated an entire pbmc population , including b and t lymphocytes and monocytes , and subjects included in the study were untreated . moreover , while the authors assessed the concentration of anti - dsdna antibodies , the others only noted their presence . a significant part of our study was the assessment of tlr expression with characteristic clinical and laboratory parameters . in the present study , a lower percentage of cd4 cells expressing tlr9 was seen in patients with lymphopenia , compared to those with lymphocyte counts above 1000/l . to our knowledge , there has been only one publication evaluating the relationship between lymphocyte count and tlr expression so far . did not find any relationship between the amounts of tlr2 - 5 , tlr7 , and tlr9 mrna in pbmcs and leukocyte , lymphocyte , neutrophil , and platelet counts , although 18 of 21 subjects presented with hematological abnormalities . it may be plausible that the decreased lymphocyte count of cd4 t lymphocytes coexpressing tlr9 may be related to immunosuppressive treatment in this group of patients . , who report that methylprednisolone inhibits the survival of activated cd4 lymphocytes activated by specific tlr3 and tlr9 ligands in vitro but has no effect on their expression . there were 8 subjects ( 23% ) with hypogammaglobulinemia in our group and half of them were receiving immunosuppressants . they presented with a significantly higher percentage of cd4 tlr9-positive cells , compared to individuals with gammaglobulin levels above 12% . it may be that the treatment with glucocorticosteroids and/or cytostatic agents led to a decrease of gammaglobulins but did not diminish the number of cd4 t lymphocytes expressing tlr9 . this may be due to a low number of subjects with hypogammaglobulinemia with and without immunosuppressive treatment . however , it can not be excluded that the differences in tlr9 expression between these two subgroups ( lymphopenia and hypogammaglobulinemia ) may be caused by the different numbers of patients receiving immunosuppressive drugs , the number being lower in the case of hypogammaglobulinemia . our findings warrant further studies on tlr expression in t lymphocytes from patients with sle , as they may lead to a better understanding of the complex interactions between innate and acquired immunity in the pathogenesis of sle . one profitable course of action would be to inquire into the molecular level of the cell cycle using rt - pcr . the results of the present study note a lower count of cd19 b lymphocytes with tlr3 in patients with esr > 25 . glucocorticosteroids have a strong anti - inflammatory potential , caused by the inhibition of cytokine biosynthesis at the genome level treatment with this group of drugs may have led to a decrease in cytokines in the sera of patients with high esr , resulting in a lower percentage of b cells expressing tlr3 . despite this , the treatment did not quench the inflammatory process and , therefore , did not lower the increased esr . the only available article by nakano et al . , where the authors evaluated the correlation between tlr9s in lymphocytes b and t with increased esr , does not confirm any significant relationship . a higher count of cd3 , cd4 , and cd19 cells coexpressing tlr7 was found in patients with anemia compared to subjects with hemoglobin above 12 g / dl . in sle , anemia may stem from autoimmune hemolysis or chronic inflammatory process ( anemia of chronic diseases , acd ) . in our group , this type of anaemia develops due to a chronic inflammatory reaction , characterized by increased concentrations of tnf- , il-1 , or ifn - gamma , which inhibit the secretion of erythropoietin and availability of iron , essential for efficient erythropoiesis . as a result of tlr activation , numerous proinflammatory cytokines it was indicated that proinflammatory cytokines may regulate tlr expression . moreover , the proinflammatory cytokine - dependent expression of tlr , adaptor proteins , and kinases participating in signal transduction towards the cell interior has been proved . an increased concentration of ifn- in the serum of sle patients , combined with raised ifn - type i dependent gene expression in the mononuclear cells of peripheral blood cells , has been characterized as interferon signature . the continuous , tlr - mediated biosynthesis of ifn- by nucleic acids containing immunologic complexes may be responsible for the interferon signature phenomenon . moreover , it has been revealed that the level of ifn alpha - dependent gene expression is correlated with sle activity and more detrimental clinical disease forms , associated with damage to the kidneys , bone marrow , or cells of the central nervous system [ 29 , 30 ] . increased inf - alpha concentration is regarded as the response to the continuous activation of tlr pathways . however , komatsuda et al . did not confirm any correlation between tlr and ifn - alpha induced ly6e ( lymphocyte antigen 6 complex , locus e ) gene expression . the observed higher percentage of cd3 , cd4 , and cd19 cells with tlr7 among subjects with anemia may reflect the presence of chronic inflammation and increased proinflammatory cytokines . in our study group , a higher count of tlr7-positive b and t cells was seen although 78% of patients received immunosuppressive drugs . this may be due to the majority of patients experiencing active sle ( 89% ) . exacerbation of sle may be induced by the usage of oestrogen - based anticontraceptive pills , that may also elevate the risk of a more severe disease course . however , during menopause sle tends to become milder , which is probably due to a decrease in oestrogen levels in peripheral blood . in our study , we demonstrated a statistically significant higher percentage of b lymphocytes cd19 expressing tlr7 in premenopausal women , compared to females after menopause . these observations suggest the influence of female sex hormones on tlr7 expression on lymphocytes b. this effect has been confirmed in other studies . young et al . ( 2011 ) indicated the increased in vitro expression of endosomal tlrs , including tlr7 , on pbmc cells from normal women ader estradiol stimulation , with no effect after treatment with testosteron . in another study , 17-estradiol treatment of normal postmenopausal women enhanced tlr7/9 pdc production of ifn . however , secretion of ifn by plasmacytoid dendritic cells after tlr7 activation was lower in postmenopausal than in premenopausal females . furthermore , stimulation of tlr7 with a synthetic agonist in lupus - prone mice lacking the alpha oestrogen receptor led to a lower il-6 synthesis by lymphocytes b than in wild type animals . when the clinical symptoms were analyzed , a significantly lower count of lymphocytes b cd19 with tlr9 was found in patients with joint symptoms ( 75% of subjects ) than in patients with no joint symptoms ( 25% ) . some publications describe the expression of tlrs in rheumatoid arthritis [ 38 , 39 ] and note that patients demonstrate higher expression of tlr2 , 3 , and 4 on fibroblasts from the synovial tissue . the synovial fluid contains various tlr ligands such as peptidoglycan , dsrna released from necrotic cells , lipopolysaccharides , and cpg - rich nucleic acid . their presence stimulates the synthesis of many proinflammatory cytokines and chemokines , which sustain inflammation in joints [ 38 , 39 ] . the lower percentage of cd19 b lymphocytes expressing tlr9 in patients with joint symptoms in our study group may be related to the presence of immunosuppressive treatment . however , immunosuppressive therapy was found to have no influence on tlr expression in our study , which is consistent with the results of other researchers [ 16 , 17 ] . reports a significant decrease of mfi for tlr9 in cd20 b lymphocytes in 8 out of 11 patients with sle . no correlation was observed between tlr expression and the degree of organ damage , according to slicc / acr . the lack of any relationship may be explained by the fact that the organ damage reflects the final outcome of the inflammatory process . a significant positive correlation was recorded between tlr3 and tlr9 expression in pbmcs ( p < 0.00001 ) . however , it is probable that the significant correlation of tlr3 and tlr9 , but not tlr7 , stems from the higher lability of ssrna ( the ligand for tlr7 ) , which undergoes rapid degradation by ribonucleases and is quickly removed from circulation . tlrs are able to recognise endogenous antigens which are released upon cell damage or stress and have been shown to play a key role in numerous autoimmune diseases [ 40 , 41 ] . these tlr ligands bind tlrs , possibly initiate intracellular signaling pathways , and may initiate autoimmunity processes . tlrs act on the monocyte - macrophage system and activate dendritic cells , which then engage self - antigens , as the first step for the induction of autoimmunity . tlr9 activation induces the expression of membrane - bound b - cell activating factor ( baff ) on human b cells and leads to increased proliferation in response to both soluble and membrane - bound baff . a sizable body of evidence suggests that the endolysosome - restricted nucleic acid sensing subset of tlrs ( na - tlrs ) plays an important role in the production of antinuclear autoantibodies . recently , koh et al . documented that na - tlrs promote the induction of antinuclear abs in sle . their data indicates that the presence of na - tlrs in b cells is necessary to drive the initial autoimmune response and to promote the activation and escape of tolerance of self - reactive b cells . in addition , overexpression of tlr7 within the b cell compartment was found to enhance b cell tlr7 expression , permit the specific development of anti - rna autoantibody production , and exacerbate sle disease in an animal model . moreover , the inhibition of both tlr7 and tlr9 reduces autoimmune pathology in experimental sle [ 48 , 49 ] . this observation suggests that the aberrant activation of a number of tlr pathways may lead to the initiation and/or perpetuation of sle and may indicate the direction for more specific therapy of this disease . in conclusion , our results suggest that tlrs exert an influence on sle development and describe the potential roles played by tlrs in the involvement of specific organs in this disease . even so , more targeted studies concerning the biology and function of tlrs are warranted and may lead to the development of a new class of drugs ." ]

[ "neuroretinitis ( nr ) is considered to be an inflammatory condition which is characterized by optic disc edema and , as a result , formation of a macular star figure . nr is an atypical presentation of toxoplasmosis infection , and such cases are quite rare .", "a 13-year - old girl presented with painless subacute visual loss in her right eye for a week at khatam - al - anbia eye hospital in mashhad , iran .", "although toxoplasmosis nr is rare , it should be considered in the differential diagnoses of nr .", "neuroretinitis ( nr ) is considered an inflammatory condition which is characterized by optic disc edema and , as a result , formation of a macular star figure ( 1 ) . this disorder is possibly caused by an infectious process affecting the disc ; in other instances , a post - viral or autoimmune mechanism seems to be a more probable cause ( 1 ) . furthermore , some instances of nr have been reported to be accompanied by a wide spectrum of infectious pathogens ( 1 ) . the most common case reported so far is the result of cat scratch disease ( csd ) , accounting for two - thirds of cases in one study ( 2 ) . however , there are other infectious etiologies of neuroretinitis , including rubeola , toxoplasmosis , herpes simplex , varicella , tuberculosis , lyme disease , leptospirosis , syphilis , various fungi , and multiple viral illnesses ( 1 ) . additionally , sporadic cases of nr may occur owing to noninfectious forms of uveitis , such as sarcoidosis and periarteritis nodosa ( 3 , 4 ) . optic disc edema with a macular star may also occur as a result of other factors such as diabetic papillopathy , hypertensive neuropathy , and anterior ischemic optic neuropathy papilledema ( 1 ) . to determine the relevant history of patients with nr , the practitioner needs to concentrate on plausible risk factors for specific infectious parameters , such as travelling to areas where lyme disease or tuberculosis ( for example ) are endemic , exposure to waste material ( e.g. , leptospirosis ) , animal exposure ( especially cats ) , and sexual contact that may have resulted in the contraction of syphilis . the clinician should investigate patients for systemic symptoms such as lymphadenopathy , headache , fever , and skin rash . laboratory tests ought to be customized for the individuals on the basis of information from both their reported history and the examination . serologic tests for most cases may include the fluorescent treponemal antibody absorption test ( fta - abs ) , cat scratch titers ( bartonella species ) , and a tuberculosis skin test ( purified protein derivative , or ppd ) ( 1 ) . in this study , we present a case of unilateral neuroretinitis in which the serology result was apparently negative for acute infection of toxoplasmosis ( negative igm titer for toxoplasmosis , but positive for igg ) . based on anti - toxoplasma treatment that was administered for the positive igg levels , the condition responded well with near optimal visual recovery .", "a 13-year - old girl was referred to the ophthalmic emergency department of khatam - al - anbia eye hospital , mashhad , iran , which is affiliated with mashhad university of medical sciences ( mums ) , in may of 2013 with painless subacute visual loss in her right eye for one week . she denied any focal neurologic condition , pain on eye movement or ophthalmodynia , or any other systemic symptom . she also stated that she had not engaged in outdoor camping or any other related activity . furthermore , she had no remarkable past medical and ophthalmologic history , and she had no contact or proximity to pets , especially cats . upon examination , the best corrected visual acuity ( bcva ) was 20/250 in the right eye and 20/20 in the left eye . moreover , the patient had a grade 2 + relative afferent papillary defect ( rapd ) in the right eye . an examination of the anterior segment demonstrated 2 + cells in the anterior chamber ( a / c ) and 1 + vitreous reaction with normal intraocular pressure ( iop ) in the right eye . the investigation of the right fundus revealed optic disc swelling , macular star lipid ( hard exudates ) deposition , and peripapillary vascular sheathing ( figure 1 ) . many tests were performed for the patient , including the erythrocyte sedimentation rate ( esr ) , complete blood count differential ( cbc diff . ) , c - reactive protein ( crp ) , toxoplasmosis serology , purified protein derivative ( ppd ) , angiotensin converting enzyme ( ace ) , fta - abs , brain and orbital mri , chest x - ray , and infectious and rheumatologic consultation . bartonella henselae serology was not available in our ophthalmology center , nor in any other nearby neighborhood . based on our clinical results , neuroretinitis etiology revealed csd as the most common infectious etiology , along with the negative systemic and ocular history , and empirical treatment commenced including azithromycin 500 mg daily , trimethoprim / sulfamethoxazole , and the application of a topical steroid . after 72 hours , all the above - mentioned laboratory indices returned to the normal level , except for the toxoplasmosis serology . furthermore , the serologic test for toxoplasmosis was negative for igm antibody whereas the igg antibody titer was > 100 iu / ml . relying on the positive igg toxoplasmosis serology and the absence of any response to initial treatment , we embarked on shifting to classic anti - toxoplasmosis treatment , i.e. , sulfadiazine 500 mg qid ( four times a day ) , pyrimethamine 50 mg / day , and folinic acid . after 72 hours had passed , prednisolone 1mg / kg / day was added and thereafter tapered during the treatment . the disc edema diminished over the course of a week . also , visual acuity gradually improved and the a / c inflammatory reaction faded away . after the decrease in disc swelling , a very small hyperpigmented focus appeared in the juxtapapillary position . gradually , over the next two months , the patient s visual acuity improved to 9/10 and the optic disc swelling and macular exudates resolved ( figure 2 ) .", "the clinical syndrome of idiopathic stellate maculopathy accompanied by optic nerve edema was first identified by leber in 1916 ( 5 ) . later on , this syndrome was renamed as leber s stellate neuroretinitis . as is now known , the most common form of infectious nr results from csd ( 1 ) . the engagement of the optic nerve suggests the need for urgent intervention . in spite of the lack of evidence either for animal exposure or any other systemic condition for this case , empirical csd treatment was instituted as lab tests were pending and given that cat scratch serology was not accessible . toxoplasmosic retinochoroiditis is rendered as one of the most frequent causes of posterior uveitis , specifically in young patients ( 6 ) . however , toxoplasmic optic neuropathy is an infrequent condition and is often characterized by subacute visual loss and optic nerve swelling , and is sometimes associated with a macular star ( neuroretinitis ) ( 7 ) . the engagement of the optic nerve most commonly encountered in ocular toxoplasmosis is optic nerve edema with a simultaneous distant active lesion . other types of optic nerve involvement include pure papillitis presented as optic disc swelling , associated with peripapillary vascular sheathing close to the healed lesion , and neuroretinitis , i.e. , optic disc swelling with macular hard exudate deposition . additionally , monocular involvement has been observed in most cases with favorable visual prognosis ( 8) . considering the positive toxoplasmosis serology in this patient , toxoplasmosis neuroretinitis was suspected , hence prompting the specified treatment . this case disclosed optic disc involvement and neuroretinitis owing to juxtapapillary chorioretinitis reactivation . in some cases , the juxtapapillary chorioretinal scar can not be found in the acute stage of disease due to disc and peripapillary edema . although toxoplasmosis neuroretinitis is rare , it should be still considered in any suspected case of neuroretinitis . this case re - emphasizes the necessity of considering toxoplasmosis in the differential diagnosis of neuroretinitis . awareness of this potential cause and prompt treatment after positive testing may therefore result in a good visual outcome ." ]

[ "we grew the budding yeast saccharomyces cerevisiae in 200 l batch culture on bd falcon 96-well microtest plates at 30.7 c ( 0.2c , standard deviation ) using synthetic media ( yeast nitrogen bases + nitrogen , complete supplement mixture ) supplemented with 2% sucrose . cultures were maintained in a well - mixed condition by growing on a shaker at 825 r.p.m . serial dilutions were performed daily ( 23 hours of growth ) with variable dilution factors . population densities were recorded each day before the serial dilution by measuring optical density at 600 nm using a thermo scientific varioskan flash multimode reader . the calibration between optical density and cell density was based on the previous characterization of this system . in the group of connected populations , for each dilution factor there were 4 replicate arrays each consisting of 10 patches . populations were connected by controlled dispersal between nearest neighbors ( dispersal rate d=0.5 , which is defined as the fraction of population going out of a patch ) . reflecting boundary conditions were adopted , meaning that a population on the edge would have 75% of its cells remaining in the patch during the dispersal step . in the group of isolated populations , the experiment was performed in a similar spatial setting except that there was no dispersal ( d=0 ) ; for each dilution factor there were 4 arrays each consisting of 5 patches , thus a total of 20 replicate populations isolated from each other . the dilution factors for the data presented in fig . 2 are 500 , 1000 , 1200 , 1400 and 1600 . in the experiment to measure recovery length , populations were connected by nearest - neighbor dispersal ( d=0.5 , reflecting boundary conditions ) . the dilution factor for the bad region ( 1 patch ) was 2500 ; the dilution factor for the good region ( 5 patches ) was varied as the environmental driver . the dilution factors for the data presented in fig . 4 are 500 , 750 , 1000 , 1133 , 1200 , 1266 , 1350 and 1400 . statistical indicators for the connected populations were calculated among 10 populations in one array on each day and averaged over a span of at least 5 days , after the populations stabilized . the mean value of 4 replicate arrays and sem ( n=4 ) are shown in figure 2 . for the isolated populations , statistical indicators were calculated on each day among 20 populations over 5 days . we used bootstrap to compute ses of the indicators by resampling 1000 times the ensemble of replicate populations ( for the coefficient of variation and the temporal correlation ) or arrays ( for the spatial correlation ) . the coefficient of variation ( cv ) was calculated as the sample standard deviation ( supplementary fig . 3b ) divided by the sample mean . because the local populations in our experiment were grown in a homogeneous environment , in principle they could all be treated as replicates . assuming the system is ergodic , the cv calculated over an ensemble of replicates can be interpreted either as spatial cv of many populations at one time point or temporal cv of a single population over many time points . the temporal correlation , defined as the lag-1 autocorrelation , was estimated by the pearson s correlation coefficient between the population densities at subsequent days . to correct for negative bias in small samples n is a fixed number for different dilution factors , so using the modified estimators would not affect the trend of indicators . the spatial correlation , defined as the two - point correlation between all neighboring pairs , was estimated by the moran s coefficient . the expectation of moran s coefficient is 1n1 in the absence of spatial correlation ; we used a modified estimator with an additional term 1n1 so that the expectation is 0 . in this case , the sample size n is the number of patches in an array : n=10 for connected populations ; n=5 for isolated populations . for detailed formula of the statistical indicators , see supplementary note 4 . in the analysis we ensured environmental homogeneity by removing a linear gradient of population density observed in connected populations the small gradient is presumably caused by some heterogeneity in experimental conditions ( temperature , dilution errors , etc . ) across the plate . removing gradient - type spatial heterogeneity before statistical analysis is similar to the detrending procedure commonly used in time - series analysis ; it prevents spurious signals such as positive spatial correlation ( supplementary fig . after the recovery profile stabilized , we tracked the population density profiles of at least 6 replicates over several days . the half - point recovery length l was estimated by performing a shape - preserving interpolation ( matlab function pchip , piecewise cubic hermite interpolating polynomial ) to the recovery profile and then locating the position of half recovery at which n(x = lhalf)=12n(x=5 ) . the population density of the bad region ( dilution factor 2500 ) in our experiment was close to 0 ( fig . 4 and supplementary fig . 7 ) . in the more general scenario with a sharp boundary between two regions of different quality ( supplementary fig . 6 and 8) , the position of half recovery can be defined as the midpoint between the equilibrium population density of the region of interest and the population density at the boundary . the exponential recovery length was estimated by fitting an exponential function with three parameters c1 exp(x / l ) + c2 to the recovery profile n(x ) . the data points used for exponential fitting are from position 1 to 5 ( except for dilution factor 500 , the data for fitting are from position 0 to 5 ) . we note that our definition of exponential recovery length is phenomenological , because : 1 ) the deviation is expected to be exponential only close enough to the equilibrium ; 2 ) at higher dilution factors the profile can deviate from an exponential form ( supplementary fig . 4c ) may be due to the limited data points used in fitting or experimental errors . for both the half - point recovery length and the exponential recovery length , we used bootstrap to compute ses for the indicators by resampling the ensemble of steady - state profiles 100 times and fitting the average recovery profile .", "we grew the budding yeast saccharomyces cerevisiae in 200 l batch culture on bd falcon 96-well microtest plates at 30.7 c ( 0.2c , standard deviation ) using synthetic media ( yeast nitrogen bases + nitrogen , complete supplement mixture ) supplemented with 2% sucrose . cultures were maintained in a well - mixed condition by growing on a shaker at 825 r.p.m . serial dilutions were performed daily ( 23 hours of growth ) with variable dilution factors . population densities were recorded each day before the serial dilution by measuring optical density at 600 nm using a thermo scientific varioskan flash multimode reader . the calibration between optical density and cell density was based on the previous characterization of this system . in the group of connected populations , for each dilution factor there were 4 replicate arrays each consisting of 10 patches . populations were connected by controlled dispersal between nearest neighbors ( dispersal rate d=0.5 , which is defined as the fraction of population going out of a patch ) . reflecting boundary conditions were adopted , meaning that a population on the edge would have 75% of its cells remaining in the patch during the dispersal step . in the group of isolated populations , the experiment was performed in a similar spatial setting except that there was no dispersal ( d=0 ) ; for each dilution factor there were 4 arrays each consisting of 5 patches , thus a total of 20 replicate populations isolated from each other . the dilution factors for the data presented in fig . 2 are 500 , 1000 , 1200 , 1400 and 1600 . in the experiment to measure recovery length , populations were connected by nearest - neighbor dispersal ( d=0.5 , reflecting boundary conditions ) . the dilution factor for the bad region ( 1 patch ) was 2500 ; the dilution factor for the good region ( 5 patches ) was varied as the environmental driver . 4 are 500 , 750 , 1000 , 1133 , 1200 , 1266 , 1350 and 1400 .", "statistical indicators for the connected populations were calculated among 10 populations in one array on each day and averaged over a span of at least 5 days , after the populations stabilized . the mean value of 4 replicate arrays and sem ( n=4 ) are shown in figure 2 . for the isolated populations , statistical indicators were calculated on each day among 20 populations over 5 days . we used bootstrap to compute ses of the indicators by resampling 1000 times the ensemble of replicate populations ( for the coefficient of variation and the temporal correlation ) or arrays ( for the spatial correlation ) . the coefficient of variation ( cv ) was calculated as the sample standard deviation ( supplementary fig . 3b ) divided by the sample mean . because the local populations in our experiment were grown in a homogeneous environment , in principle they could all be treated as replicates . assuming the system is ergodic , the cv calculated over an ensemble of replicates can be interpreted either as spatial cv of many populations at one time point or temporal cv of a single population over many time points . the temporal correlation , defined as the lag-1 autocorrelation , was estimated by the pearson s correlation coefficient between the population densities at subsequent days . to correct for negative bias in small samples n is a fixed number for different dilution factors , so using the modified estimators would not affect the trend of indicators . the spatial correlation , defined as the two - point correlation between all neighboring pairs , was estimated by the moran s coefficient . the expectation of moran s coefficient is 1n1 in the absence of spatial correlation ; we used a modified estimator with an additional term 1n1 so that the expectation is 0 . in this case , the sample size n is the number of patches in an array : n=10 for connected populations ; n=5 for isolated populations . for detailed formula of the statistical indicators , see supplementary note 4 . in the analysis we ensured environmental homogeneity by removing a linear gradient of population density observed in connected populations . the small gradient is presumably caused by some heterogeneity in experimental conditions ( temperature , dilution errors , etc . ) across the plate . removing gradient - type spatial heterogeneity before statistical analysis is similar to the detrending procedure commonly used in time - series analysis ; it prevents spurious signals such as positive spatial correlation ( supplementary fig .", "after the recovery profile stabilized , we tracked the population density profiles of at least 6 replicates over several days . the half - point recovery length l was estimated by performing a shape - preserving interpolation ( matlab function pchip , piecewise cubic hermite interpolating polynomial ) to the recovery profile and then locating the position of half recovery at which n(x = lhalf)=12n(x=5 ) . the population density of the bad region ( dilution factor 2500 ) in our experiment was close to 0 ( fig . 4 and supplementary fig . 7 ) . in the more general scenario with a sharp boundary between two regions of different quality ( supplementary fig . 6 and 8) , the position of half recovery can be defined as the midpoint between the equilibrium population density of the region of interest and the population density at the boundary . the exponential recovery length was estimated by fitting an exponential function with three parameters c1 exp(x / l ) + c2 to the recovery profile n(x ) . the data points used for exponential fitting are from position 1 to 5 ( except for dilution factor 500 , the data for fitting are from position 0 to 5 ) . we note that our definition of exponential recovery length is phenomenological , because : 1 ) the deviation is expected to be exponential only close enough to the equilibrium ; 2 ) at higher dilution factors the profile can deviate from an exponential form ( supplementary fig . 4c ) may be due to the limited data points used in fitting or experimental errors . for both the half - point recovery length and the exponential recovery length , we used bootstrap to compute ses for the indicators by resampling the ensemble of steady - state profiles 100 times and fitting the average recovery profile", "" ]

[ "the prediction of a patient 's growth potential is of clinical significance because it determines the optimal timing of starting or stopping bracing and the timing of growth - guidance surgery with an expandable prosthesis.1 \n 2 \n 3 \n 4 many clinical and radiologic methods are available to predict a patient 's growth potential including the age at menarche , difference in body height growth and arm span , risser sign , and tanner and whitehouse ( tw3 ) digital skeletal age.5 \n 6 \n 7 \n 8 however , most of these measures have disadvantages in the current clinical practice.9 clinical measurements of standing height and arm span require serial measurements to determine growth trends , and its retrospective nature thus likely misses the period of peak growth spurt . the menarche age is not consistent in determining the termination of peak growth as some patients develop delayed menarche and its presence marks a stage too late for any meaningful bracing.10 similar to menarche , the risser sign has been shown by several studies to be a poor indicator of skeletal maturity , cessation of growth , and more importantly the risk of curve progression.11 \n 12 \n 13 \n 14 \n 15 up to 75.2% of patients may have persistent growth despite capping of the iliac apophysis.13 in addition , the progression of iliac apophysis has been shown to proceed in a reverse direction ( posteromedially to anterolaterally ) or in fragments.15 the digital skeletal age assessment using the tw36 \n 7 \n 8 or the greulich and pyle16 methods are more accurate in determining different growth phases , but these techniques are complex and far too difficult to apply in the clinical setting . sanders et al showed that according to the tw3 , the finger epiphysis maturation is closely related to the peak height velocity.17 the peak height velocity is noted to occur during early adolescence , and menarche and the risser sign appear after the peak growth spurt.18 \n in response to the limitations possessed by the previous methods in assessing skeletal maturity , the distal radius and ulna ( dru ) classification was created and reported by luk et al.19 this classification , which included 11 radius grades ( r1 to r11 ) and 9 ulna grades ( u1 to u9 ) , was found to accurately determine the peak height velocity ( r7 and u5 ) and cessation of growth ( r10 and u9 ) . however , the reliability of the classification has not been assessed . as such , the following study aimed to test the classification 's reliability .", "a prospective radiographic study assessing patients with adolescent idiopathic scoliosis ( ais ) undergoing bracing during the month of july 2014 was performed to assess the reliability of the dru classification.19 ethics approval was obtained from our institutional review board . the study was performed at the duchess of kent children 's hospital , pokfulam , hong kong , a tertiary referral center for spinal disorders . for the dru classification to be practical clinically , the reliability testing should be based on a wide range of patient ages , as commonly seen in our clinics . in addition , the aim of this classification was to correctly identify patients in the peak growth spurt so that timely interventions can be applied . thus , choosing braced patients would likely encompass a larger range of grades and avoid reliability assessment of only a certain age group or dru grade . the follow - up left - hand radiographs during the study month were obtained in all subjects , and data was extracted regarding the age at which the radiograph was taken and the sex of the patient . these anteroposterior radiographs of the hand and wrist were taken with 42 peak kilovoltage and 1.6 ma - seconds of x - ray energy . k.d .- k.l . ) , one junior and one senior consultant spine surgeon , for grading . there was no discussion between the two examiners about how to classify each grade prior to the initiation or during the study . radiographs were accessed by a dicom - based radworks 5.1 ( applicare medical imaging bv , zeist , the netherlands ) computer software program . all data was coded and entered on a spreadsheet and was blinded to the examiners until the end of reliability measurements . spss version 20 ( chicago , illinois , united states ) was utilized to perform the statistical analysis . the reliability assessment was based on intraclass correlation ( icc ) , which was an appropriate statistical tool for this analysis.20 icc could be interpreted based on the following values : 0 to 0.29 indicated poor agreement ; 0.30 to 0.49 indicated fair agreement ; 0.50 to 0.69 indicated moderate agreement ; 0.70 to 0.80 indicated strong agreement ; and > 0.80 indicated almost perfect agreement.21 \n 22 the 95% confidence interval ( ci ) bounds were assessed for precision . a p value of < 0.05 was considered significant .", "all data was coded and entered on a spreadsheet and was blinded to the examiners until the end of reliability measurements . spss version 20 ( chicago , illinois , united states ) was utilized to perform the statistical analysis . the reliability assessment was based on intraclass correlation ( icc ) , which was an appropriate statistical tool for this analysis.20 icc could be interpreted based on the following values : 0 to 0.29 indicated poor agreement ; 0.30 to 0.49 indicated fair agreement ; 0.50 to 0.69 indicated moderate agreement ; 0.70 to 0.80 indicated strong agreement ; and > 0.80 indicated almost perfect agreement.21 \n 22 the 95% confidence interval ( ci ) bounds were assessed for precision . a p value of < 0.05 was considered significant .", "one hundred sixty - one subjects ( 124 females and 37 males ) satisfied our inclusion criteria and were recruited for this study . the mean age was 13.3 ( standard deviation : 1.5 ; range : 8 to 18 ) . the interobserver reliability was icc = 0.97 ( 95% ci : 0.96 to 0.98 ) and icc = 0.97 ( 95% ci : 0.96 to 0.98 ) for the radius and ulna , respectively . was icc = 0.93 ( 95% ci : 0.91 to 0.95 ) for the radius and icc = 0.95 ( 95% ci : 0.930.96 ) for the ulna ; for k.d .- k.l . , icc = 0.94 ( 95% ci : 0.91 to 0.95 ) for the radius and icc = 0.95 ( 95% ci : 0.93 to 0.96 ) for the ulna . , there were 18 one - grade disagreements ( 11.1% ) and 1 two - grade disagreement ( 0.6% ) . for the ulna , there were 39 one - grade disagreements ( 24.1% ) and 1 two - grade disagreement ( 0.6% ) . was as follows : for the radius , there were 28 one - grade disagreements ( 17.3% ) and 5 two - grade disagreements ( 3.1% ) . for the ulna , there were 42 one - grade disagreements ( 25.9% ) and 2 two - grade disagreements ( 1.2% ) . the intraobserver disagreement for k.d .- k.l . was as follows : for the radius , there were 26 one - grade disagreements ( 16.0% ) and 4 two - grade disagreements ( 2.5% ) . for the ulna , there were 40 one - grade disagreements ( 24.7% ) and 5 two - grade disagreements ( 3.1% ) .", "for the radius , there were 18 one - grade disagreements ( 11.1% ) and 1 two - grade disagreement ( 0.6% ) . for the ulna , there were 39 one - grade disagreements ( 24.1% ) and 1 two - grade disagreement ( 0.6% ) . the intraobserver disagreement for j.p.y.c . was as follows : for the radius , there were 28 one - grade disagreements ( 17.3% ) and 5 two - grade disagreements ( 3.1% ) . for the ulna , there were 42 one - grade disagreements ( 25.9% ) and 2 two - grade disagreements ( 1.2% ) . the intraobserver disagreement for k.d .- k.l . was as follows : for the radius , there were 26 one - grade disagreements ( 16.0% ) and 4 two - grade disagreements ( 2.5% ) . for the ulna , there were 40 one - grade disagreements ( 24.7% ) and 5 two - grade disagreements ( 3.1% ) .", "from this study , we found that the dru classification had excellent reliability among a wide range of radius and ulna grades.19 although we did not have patients young enough to present with radius stages r1 to r4 , interventions for ais such as bracing are only applicable during the peak growth spurt . the r6 to r9 and u5 to u8 were important grades because they represented the period of peak growth spurt , and these grades were well represented in our testing . after all measurements were made through this vigorous process of testing , the two examiners thoroughly discussed each grade description and were able to form a consensus about which aspects of the classification were more representative and easier to apply during reading . the difficulties encountered by both readers during testing mainly arose from the grading of r7 to r9 and u5 to u8 . both reviewers agreed that the grading of r7 to r9 and u5 to u8 were the most difficult because they were based on descriptions of multiple parameters . each clinician might focus on something different , such as the appearance of the physis narrowing , the definition of capping , and when the epiphysis acquired the same width as the metaphysis , among others . disagreements in the ulna grades appear larger than the radius grades , likely due to a larger range of ulna grades measured as compared with the radius . for the radius , the main identifying factor between r7 and r8 was the appearance of capping over the medial side in r7 ( fig . 1 ) and over the lateral side in r8 ( fig . 2 ) . both readers agreed that capping was defined as a hornlike structure at the ends of the epiphysis like a sharp bony outgrowth . a simple step to confirm this observation on the radius was to trace the proximal border of the epiphysis to find any dipping of the line toward the metaphysis at the medial or lateral ends . for r9 ( fig . 3 ) , strong capping was not always a consistent finding , thus narrowing of the physis was the main determinant description for this grade . \n examiners should focus on the medial side capping and the absence of lateral side capping ( red arrow ) . note the hooklike structure / sharp outgrowth at the medial physis facing the metaphysis that deviates from the physeal line . this radiograph also represents the u6 grade with the appearance of an epiphysis and metaphysis of the same width but no narrowing of the medial physeal plate ( white arrow ) . \n examiners should focus on the lateral side capping ( red arrow ) . note the hooklike structure / sharp outgrowth at the lateral physis facing the metaphysis that deviates from the physeal line . \n , the ulna is graded u7 as there is narrowing of the medial physeal plate ( black arrow ) but incomplete fusion . generally , grading of u1 to u4 was not difficult due to some distinct features such as the size of the epiphysis for u3 and the appearance of the styloid for u4 . the difficulties were noted for descriptions in u5 to u8 . for u5 ( fig . 4 ) , the appearance of a denser ulna head was not easily observed and so flattening of the radial half of the ulnar epiphysis was the main determinant . for u6 ( fig . 1 ) , we found that the overlapping of the metaphysis with the epiphysis at the center third was inconsistent in most radiographs , and thus we often relied on the appearance of the epiphysis being as wide as the metaphysis . to differentiate u7 ( fig . 5 ) , we usually used narrowing of the medial ulnar physis ( u7 ) and fusion of the medial ulnar physis ( u8 ) as the determining factor . the descriptions of the smooth curve line articulation in u7 might not be very easily understood . \n examiners should focus on the flattening of the radial epiphysis ( white arrow ) and should note that the epiphysis is not as wide as the metaphysis , indicated by the vertical white line touching the metaphysis but not the epiphysis . \n interestingly , the interobserver reliability was somewhat better than the intraobserver reliability , which suggests that the readers may become more competent in the measuring skills with increased grading experience from measuring these 161 radiographs , demonstrating a learning curve effect . hence , this classification scheme should be useful as a guide for decision making and as a communication tool between clinicians . despite not having any prior discussions or training into how each grade should be correctly assessed , the results showed that both examiners used similar grading methods in approaching each radiograph . thus , the difficulties and suggestions raised during the consensus meeting were important to help less experienced examiners with this classification scheme . the dru has been shown to be strongly associated with the peak growth phase and growth cessation,19 which can help clinicians decide between bracing and surgery . the results from this study suggested that the dru classification was a reliable method to assess skeletal growth . although the most appropriate time to get these radiographs still requires further investigation , the authors suggest a yearly radiograph with consideration of radiographs every 6 to 9 months during the period of peak growth spurt . in the future , prospective studies are being developed to apply this classification scheme on patients with ais to determine its sensitivity and specificity in identifying the peak growth spurt , predicting curve deterioration , as well as detecting skeletal maturity . in addition , it can be used to help decide the appropriate timing for initiation and cessation of bracing , when to use growth guidance surgery , and when to recommend fusion . by more accurately predicting a child 's remaining growth potential , we can potentially narrow the duration of effective bracing without jeopardizing the deformity control , thereby reducing any unnecessary psychological burden.23 this knowledge could also guide the duration and interval of follow - up and the number of x - rays needed , thus reducing radiation exposure . additional studies are needed to validate this dru classification in other ethnicities and populations and to further directly compare it with other new and established skeletal maturity schemes.24 \n 25 \n 26 \n 27 \n this study has shown that the dru classification is a reliable tool for measuring skeletal growth . further prospective studies are required to test its predictability of peak growth spurt and growth cessation and to compare it with other skeletal growth measures such as the risser sign , tw3 grading , and age of menarche ." ]

[ "\n neospora caninum is an apicomplexan protozoan , infecting a large range of mammals . in cattle , several studies focussing on the specific immune response to n. caninum in cattle have shown that the time of gestation is important with regard to the outcome of the infection . proinflammatory cytokines , produced by lymphocytes , are crucial for controlling a variety of intracellular pathogens , including n. caninum . these cytokines are produced by natural killer ( nk ) cells , as well as by cd4 t - cells and cd8 t - cells . cd4 cells mediate the humoral response and their involvement is associated with increased igg1 levels , whereas cd8 cells are involved in the cellular immune response , which is characterized by increased production of igg2a . n. caninum is an intracellular parasite and resides within a specialized compartment , a parasitophorous vacuole , surrounded by a parasitophorous vacuole membrane ( pvm ) . following egress from a host cell , these parasites immediately search for a new host cell to invade , and the direct accessibility for components of the immune system to the parasite within the circulation is rather limited . thus , extracellular tachyzoites have a low chance to be detected by t - helper-2-(th2- ) type cells . a humoral immune response is therefore not sufficient to clear a n. caninum infection . for more efficient protection against intracellular pathogens such as n. caninum , the host usually generates a cellular , t - helper-1-(th1- ) type response [ 6 , 7 ] . this is possible since secretory parasite molecules pass through the pvm into the host cell cytoplasm , where they interact with and manipulate host cell functions . once in the cytoplasm these molecules can be processed and corresponding peptides are presented on the host cell surface via major histocompatibility complex ( mhc ) class i molecules . cd8 lymphocytes will recognize the peptides presented on these mhc - i molecules and activate th1 cells , which then produce cytokines such as interleukin-12 ( il-12 ) , interferon gamma ( ifn- ) , and tumour necrosis factor ( tnf- ) . the production of these cytokines leads to the activation of pathways that generate free oxygen radicals and nitric oxide ( no ) and its metabolites among other factors , which are potentially lethal for many protozoa . however , these processes can also be deleterious to the fetoplacental interface and potentially induce abortion and/or fetal resorption , especially in the first trimester of pregnancy , when levels of pregnancy hormones , such as progesterone , which counteracts these effects , are relatively low . as a result , there is no or only little th2 cytokine polarization and the dam will generate a th1 immune response , which also affects the placental and foetal tissue . since a strong th1 response is incompatible with successful pregnancy , the infection can lead to the loss of the unborn foetus . progesterone promotes th2 cell proliferation the production of il-4 , il-5 , and il-10 is known to inhibit no and tnf- production and impairs nk cell activity [ 1113 ] . however , since the immune response is not capable of controlling the parasite , the risk of transplacental transmission of n. caninum is relatively high . thus , resistance to n. caninum seems to rely , at least partially , on a functional th1 response . in this study , inbred balb / c , cba / ca , and c57bl/6 strains of mice ( with different mhc - i haplotypes ) were compared for their capacity to cope with a n. caninum infection . in addition , we show that cba / ca mice were the by far most resistant , and protection correlated with the induction of a th1-biased response . the results demonstrate that a meaningful evaluation of the efficacy of , for example , vaccine candidates and/or chemotherapeutically interesting compounds against experimental infection with n. caninum in mice requires standardization with regard to the mouse strains used for such experiments .", "female balb / c , cba / ca , and c57bl/6 mice aged 812 weeks were obtained from harlan ( horst , the netherlands ) . mice were randomly divided into groups as soon as they came to hand and marked individually . the conditions in the animal housing facilities were negative air pressure , air exchange of 300 m / hr , temperature of 22c 2c , and artificial day / night cycle with 12 hrs light per day . five mice were housed per group in macrolon type iii cages ( 820 cm ) enriched with polyvinyl chloride tubes and tissues . mice received ad libitum standard rodent feed ( rmhb2118 , ab - diets woerden , the netherlands ) . water was supplied in bottles and refreshed at least three times a week . prior to challenge , mice were given an acclimatization period of 7 days , during which they were observed daily for normal behaviour , wellness , food and water intake , and posture . \n n. caninum tachyzoites ( nc - liv isolate ) were maintained by continuous passages in vero cells grown in rpmi 1640 medium supplemented with 10% foetal bovine serum ( fbs ) , 2 mm l - glutamine and 25 mg / l gentamycin sulphate . cultures were grown in a humidified incubator at 37c with 5% co2 . to liberate the tachyzoites from the vero cells , the culture was taken up through a 20 g needle and extruded through a 26 g needle . for the preparation of parasites for infection , tachyzoites were purified on a pd10 column ( ge healthcare , diegem , belgium ) centrifuged and resuspended in sterile 0.04 m isotonic pbs . tachyzoites were counted in a neubauer chamber . in order to prepare n. caninum tachyzoite antigen lysate , purified tachyzoites were subjected to one freeze - thaw cycle followed by sonication on a branson sonifier s-250a ( branson ultrasonics , danbury , usa ) for 60 sec , output 4 , and a cycle duty of 50% . mice were challenged by intraperitoneal injection of 1 10 , 5 10 , or 25 10 live nc - liv tachyzoites in 0.5 ml 0.04 m isotonic pbs . mice were then closely monitored daily for neosporosis disease symptoms according to a clinical scoring system that included hunched back , rough hair coat , impaired movement , and spinning . for each feature the average clinical score per group was determined by dividing the total score per group by the amount of mice in that group . as soon as the health status of the animals deteriorated , the frequency of observations was increased to two times a day . blood samples were collected by orbital puncture at day 0 and day 34 postinfection ( at the time of euthanasia ) or , whenever possible , at the time of inter - current death . latest at day 34 postinfection ( pi ) , surviving mice were sacrificed by cervical dislocation and brains and spleens were harvested . for each mouse , sections of 3.5 m thickness were cut longitudinally from nervus i to medulla oblongata with cerebrum , hippocampus , and cerebellum on a brand microm h355 s microtome ( adamas , rhenen , the netherlands ) , and were placed onto glass slides . serum antibody levels were analysed using a commercially available elisa kit based on antigens of n. caninum ( idexx neospora ab test , idexx laboratories , westbrook , usa ) according to the instructions of the manufacturer . on each plate , sera were tested in 2log titrations , and negative and positive sera were tested in octaploid . sera of nave swiss outbred mice ( charles river , sulzfeld , germany ) were used as a negative control . as a positive control for igg1 and igg2a detection , sera of balb / c mice vaccinated with the chimeric antigen r - mic3 - 1 and challenged with n. caninum ( nc-1 isolate ) sera of c57bl/6 mice taken 34 days after infection with 5 10 nc - liv tachyzoites were used as a positive control for igg2c analysis . horseradish - peroxidase-(hpr- ) conjugated goat antimouse igg1 , igg2a , and igg2c were obtained from southern biotechnology ( birmingham , usa ) and diluted in enzyme immune assay ( eia ) buffer containing 0.05% polysorbate 80 . results were analysed on a tecan sunrise device ( breda , the netherlands ) using xfluor4 software at 650 nm . antibody titres were determined using caspex software ; abendvertical version 0.11 v1 ( msd animal health , proprietary software ) . the cutoff in antibody end titres was defined at bmin2 , where bmin is the negative control . spleens from surviving mice were washed twice in rpmi 1640 medium supplemented with 100.000 iu penicillin / streptavidin , 1 mm sodium - pyruvate , and 2 mm l - glutamine and dissociated using the gentlemacs dissociator ( miltenyi biotec , leiden , the netherlands ) according to the instructions of the manufacturer . suspensions were filtered through a 100 m cell strainer and centrifuged at 300 g for 10 min at 4c . samples were depleted of erythrocytes with erythrocyte lysis solution containing 829 g / l ammonium chloride , 100 g / l sodium hydrogen carbonate , and 3.7 g / l disodium edetate . splenocytes from mice of the same group were pooled in a 1 : 1 ratio and a total of 1 10 splenocytes were cultured for 72 hrs in 100 l complete rpmi medium containing 10% fbs in a humidified incubator at 37c with 5% co2 . 100 l of an antigenic extract of either 2 10 , 1 10 , or 5 10 nc - liv tachyzoites prepared as described above was added to the cells . complete rpmi medium and a phorbolmyristateacetate ( pma)/ionomycin mixture ( 9.74 ng / ml/0.26 mm ) were used as negative and positive controls , respectively . cytokine levels in supernatants of stimulated splenocytes were analyzed using the bendermedsystems flowcytomix mouse / rat basic kit combined with the cytokine mouse simplex kits for ifn- ( bendermedsystems , vienna , austria ) . the samples of the cytomix assay were interpolated in the standard curve by selecting the 5p logistic fit function : the bendermedsystems flowcytomix pro 2.4 software fits the best curve according to y = d + ( ( a d)/(1 + ( x / c ) ) ) . the mean fluorescence intensity ( mfi ) of each standard point is blank - corrected by division of the blank - mfi ( b_b0 = mfi / mfi of blank100 ) . the maximum acceptable bias , which displays the variation for the ideal standard curve defined by the theoretical standard concentrations , was set at 30% . \n neospora - specific quantitative real - time pcr to determine the number of tachyzoites that has reached the cerebral tissue was performed as previously described [ 2 , 3 , 15 ] . dna extraction from brain tissue was performed using the dneasy blood & tissue kit ( qiagen , hilden , germany ) according to the manufacturer recommendations . the dna concentration in each sample was determined by uv spectrophotometry ( nanodrop , thermo scientific , delaware , usa ) and was adjusted to 5 ng/l with sterile dnase - free water . quantitative real - time pcr was performed using the light cycler instrument ( roche diagnostic , basel , switzerland ) . the parasite counts were calculated by interpolation from a standard curve with dna equivalents from 1000 , 100 , and 10 parasites included in each run . mice survival was analyzed according to kaplan - meier and the survival curves between groups were compared with the logrank test followed by regression coefficient analysis . the weight of the survivors , cerebral parasite burden , lesion score , and the serological data were compared using kruskal - wallis one - way anova followed by the kruskal - wallis multiple comparison z - value test . the p value between two significantly different groups was calculated by mann - whitney u test . the proportion of animals with clinical signs , proportion of animals with brain lesions and data on n. caninum - dna - positive animals were organized in a contingency table and compared by a chi - square test .", "balb / c mice ( figures 1(a ) and 1(b ) ) challenged with 1 10 tachyzoites were not affected , with no decrease in body weight following infection . however , challenge with 5 10 nc - liv tachyzoites resulted in neosporosis symptoms in balb / c mice , starting on day 18 pi . hunched back , ruffled coat , and weight loss of ~25% were observed in these mice . two mice reached the point where severe neurobiological symptoms made it necessary to get them euthanized on day 23 pi , while the other three mice , although exhibiting clinical symptoms , but less severe , survived throughout the observation period of 34 days ( figures 1(a ) and 1(b ) ) . balb / c mice were not able to control the highest infectious dose of 25 10 tachyzoites , resulting in death of all five animals within that group between days 7 and 12 pi . cba / ca mice ( figures 1(c ) and 1(d ) ) exhibited a high degree of resistance against n. caninum at an infection dose of 1 10 and 5 10 tachyzoites , with no mortality and no body weight changes during the entire experimental period ( figures 2 and 1(d ) ) . however , none of the cba / ca mice could control the infection at the highest dose and they all were euthanized within 9 days pi ( figures 1(c ) and 1(d ) ) . in comparison to the other two mouse strains , c57bl/6 mice ( figures 1(e ) and 1(f ) ) , all mice started to exhibit clinical symptoms including weight loss of ~10% and two out of five mice had to be euthanized at day 29 pi ( figure 2 ) . strong variations were observed in the group of c57bl/6 mice challenged with 5 10 tachyzoites . three mice of this group demonstrated severe symptoms of neosporosis and were euthanized on days 7 , 8 and 13 pi , while the other two mice showed only very mild symptoms on day 8 pi and returned to being clinically normal 9 days after the challenge . shortly after a challenge with 25 10 tachyzoites , c57bl/6 mice showed a hunched back and ruffled hair coat ( figures 1(e ) and 1(f ) ) . symptoms progressed to include impaired movements and spinning when picked up by the tail , requiring euthanasia of four of these mice on day 8 pi . the remaining mouse of this group completely recovered by day 11 pi and survived till the end of the experiment . statistically , cba / ca mice exhibited a significantly higher survival rate than c57bl/6 mice at an infection dose of 1 10 tachyzoites ( p < 0.001 , cox regression analysis ) and than c57bl/6 and balb / c mice at an infection dose of 5 10 tachyzoites ( p < 0.001 , cox regression analysis ) . the mean weight of the survivors of the balb / c mice infected with 1 10 tachyzoites was slightly higher than those infected with 5 10 parasites , although the difference was not significant ( kruskall - wallis multiple comparison test ) . the number of symptomatic mice in the cba / ca mice at an infection dose of 5 10 tachyzoites was significantly lower than in the two other groups ( p < 0.01 , chi - square test ) while no significant differences between strains were observed for the other infection doses ( table 2 ) . the number of mice presenting brain lesions as well as the lesion score per group at an infection dose of 5 10 parasites was significantly lower in the cba / ca mice ( p < 0.01 , chi - square test and p < 0.001 , mann - whitney u - test , resp . ) than in the two other groups ( table 2 ) . no significant difference was observed regarding the overall lesion score per strain ( table 2 ) . the surviving balb / c mice infected with 1 10 tachyzoites ( n = 5 ) and cba / ca mice infected with 5 10 tachyzoites ( n = 5 ) were challenged again with 25 10 n. caninum tachyzoites at day 34 . however , they all succumbed to infection within 24 hrs , indicating that they had not built up any form of protective immunity . in order to obtain information on the type of immune response induced by n. caninum infection , the production of igg1-type antibodies is primarily induced by a humoral immune response , whereas igg2a subclasses indicate the involvement of a cellular immune response . since c57bl/6 mice poorly produce igg2a antibodies , the igg2c levels were measured in serum of these mice . in none of the groups n. caninum - specific antibodies experimental infection of balb / c mice with 1 10 or 5 10 tachyzoites resulted in high igg2a and igg1 serum levels , indicating the presence of a mixed cellular and humoral immune response ( table 1 ) . in cba / ca mice challenged with 1 10 or 5 10 tachyzoites , predominantly igg2a antibodies were detected . igg2a and igg1 were undetectable in cba / ca mice challenged with 25 10 tachyzoites , most likely because mice succumbed to the infection before a humoral response was mounted ( table 1 ) . in general , sera of mice surviving until day 34 pi contained higher levels of igg2c compared to sera of their group mates that died at earlier time points ( table 1 ) . the difference igg2a / c - igg1 postinfection was highly significantly higher in the cba / ca mice than in the two other strains ( p < 0.01 , mann - whitney u - test ) and significantly higher in the c57/bl6 mice than in the balb / c mice ( p < 0.05 , mann - whitney u - test ) at an infection dose of 1 10 parasites . at an infection dose of 5 10 tachyzoites , both cba / ca and c57/bl6 mice had a significantly higher igg2a / c - igg1 difference than the balb / c mice ( p < 0.01 , and p < 0.05 respectively , mann - whitney u - test ) , and cba / ca mice also had a higher igg2a / c - igg1 difference than c57/bl6 , although not significant ( table 2 and figure 4 ) . the production of ifn- , indicating the occurrence of a cellular immune response , was measured in the supernatants of spleen cells derived from survivors of balb / c mice ( infected with 5 10 tachyzoites ) , cba / ca mice ( infected with 1 10 tachyzoites ) , and all surviving c57bl/6 mice . splenocytes were stimulated with either n. caninum antigens or pma / ionomycin for 72 hrs , or were left unstimulated . stimulation with a lysate corresponding to 2 10 , 1 10 or 5 10 purified nc - liv tachyzoites resulted in increased production of ifn-. ifn- production was a dose - dependent stimulus in all groups ( figure 3 ) . the strongest ifn- response was observed for the cba / ca mice , as was especially evident using low and medium tachyzoite stimulus doses . a lower ifn- production was observed for the balb / c mice , while splenocytes from c57bl/6 mice demonstrated large variations in ifn- production upon antigen stimulation . data from balb / c mice and cba / ca mice infected with 25 10 parasites is missing , since these mice succumbed to infection prior to 34 days postinfection . spleen cells of balb / c mice infected with 1 10 tachyzoites ( n = 5 ) and cba / ca mice infected with 5 10 tachyzoites ( n = 5 ) , which were used for rechallenge , were not assessed . the cerebral parasite load in all groups was measured by real - time pcr and compared with histopathological findings . corresponding results are summarized in table 2 . in three out of five balb / c mice challenged with 1 10 tachyzoites , no parasite dna could be detected by pcr , while high numbers were detected in the other two samples . however , in only one of these mice a brain lesion was detected by histology . challenge of balb / c mice with 5 10 tachyzoites resulted in high cerebral parasite load in all mice , and brain lesions were detected upon histopathological inspection in four out of five mice . after infection of balb / c mice with 25 10 tachyzoites , parasites were detected at moderate to high numbers in all brain samples investigated , but histopathology failed to detect any lesions at all . real - time pcr detected n. caninum dna in one of 10 brain samples of cba / ca mice challenged with 1 10 and 5 10 tachyzoites . in contrast , lesions were observed by histopathological means in two mice infected with 1 10 tachyzoites . however , these were samples that were not positive by real - time pcr . in the cba / ca mice infected with 25 10 tachyzoites , lesions in the central nervous system ( cns ) were observed by histopathology for three out of five animals , and real - time pcr showed high numbers of parasite load in all 5 members of the group , which all died between days 8 and 9 pi . in three c57bl/6 mice challenged with 1 10 tachyzoite lesions in the brain were observed . in two of them infection with 5 10 tachyzoites resulted in parasites and lesions in three mice on days 13 and 34 pi . in the c57bl/6 mice infected with 25 10 tachyzoites , four out of five mice died 8 days after challenge , with real - time pcr - positive brain tissue but no lesions detectable upon histopathology . one mouse survived until day 34 pi , and this mouse exhibited a high cerebral parasite load and also brain lesions . thus , a high cerebral parasite load as determined by real - time pcr did not always correspond with the detection of lesions in the brain and did also not always match with the occurrence of clinical signs of neosporosis . at an infection dose of 1 10 and 25 10 tachyzoites , no significant difference in the number of pcr positive mice between the groups was observed ( chi - square test ) . at an infection dose of 5 10 tachyzoites , the number of pcr - positive mice was significantly lower in the cba / ca mice than in the balb / c and c57/bl6 mice ( p < 0.01 and p < 0.05 , respectively , chi - square test ) . no significant difference regarding the parasite load of infected mice was observed between the groups ( kruskal - wallis multiple comparison test ) .", "in this study , we compared the capacities of three inbred mouse strains expressing different mhc - i molecules to cope with a n. caninum infection . significant differences in the responses to n. caninum infection in inbred and outbred mice have been demonstrated previously , and these differences have been postulated to be due to varying abilities to present n. caninum antigens on their mhc molecules . when using mouse models to investigate the outcome and immunological parameters of infection , the selection of the n. caninum isolate as well as inoculum doses are important variables that will influence the results . the present study showed high variations in morbidity and mortality in the three inbred mouse strains balb / c , cba / ca , and c57bl/6 after inoculation with different numbers of n. caninum ( nc - liv ) tachyzoites , demonstrating that the choice of mouse strain plays a crucial role for the assessment of experimental infections and agents that could potentially interfere therein . while balb / c mice did not suffer from clinical signs of neosporosis after a challenge dose of 1 10 n. caninum tachyzoites , infection with 5 10 tachyzoites resulted in severe clinical symptoms starting at day 20 pi . this is in accordance with several vaccination studies , which have used the balb / c and c57bl/6 models to investigate the protective effects of vaccine candidates ( reviewed in [ 2 , 3 ] ) . in contrast , only limited research has been performed using cba / ca mice as a model to investigate the immune responses against n. caninum infection . our investigations demonstrated that this mouse strain is clearly more resistant against neosporosis compared to balb / c and c57bl/6 mice . previously , these features of cba / ca mice with regard to n. caninum infection had been highlighted by rettigner et al . , who described this mouse strain to be resistant against an infection of 5 10 tachyzoites of the nc-1 isolate of n. caninum following treatment with immunosuppressing agents and suitable for the generation of cerebral tissue cysts . this is surprising , since the nc-1 isolate , in contrast to nc - liv , does hardly convert into bradyzoites under in vitro conditions such as high ph treatment or incubation with sodium nitroprusside [ 19 , 20 ] . with respect to c57bl/6 mice , ramamoorthy et al . observed no clinical signs of neosporosis throughout an observation period of 21 days after an infection with 5 10 nc-1 tachyzoites but also showed these mice to be highly susceptible to an infection with 2 10 nc-1 tachyzoites . others failed to observe clinical signs in c57bl/6 mice after a challenge with 5 10 nc-1 tachyzoites as long as 44 days pi . in this study , after a challenge dose of 1 10 nc - liv tachyzoites disease symptoms occurred after 26 days but only after 8 days when 5 10 tachyzoites were inoculated , with three out of five mice succumbing to infection . this illustrates that , besides the mouse strain used , the selection of the neospora isolate also makes a big difference . in any case , an infection dose of 25 10 tachyzoites of the nc - liv isolate was far too high for any of the mouse strains to cope with , requiring euthanasia of all but one mouse between days 712 pi . since n. caninum is an intracellular parasite , the protective immune response is likely to involve cell - mediated immunity ; thus th1-mediated responses are important . however , in balb / c mice it has been demonstrated that besides cellular immunity also humoral immune responses are crucial in controlling n. caninum infection and limiting the pathological changes caused by an excessive proinflammatory response [ 23 , 24 ] . teixeira et al . described that intraperitoneal injection of n. caninum tachyzoites ( nc-1 isolate ) in balb / c mice induced a parasite - specific , nonpolyclonal , b - cell response . a strong bias towards a th2-type response resulted in increased susceptibility to n. caninum and enhanced the corresponding pathologic effects . however , vaccination of mice with recombinant rhoptry antigen rop2 emulsified in saponin adjuvants induced an igg1-biased humoral immune response , while formulating the same antigen in freund 's incomplete adjuvants resulted in an igg2a - biased antibody response , and both were protective in the nonpregnant balb / c mouse model . protection associated with an igg2a - biased humoral immune response was also demonstrated for recombinant protein disulfide isomerase applied for vaccination intranasally [ 2729 ] . vaccination of mice with native ncsrs2 induced a th2-biased protective response that reduced congenital infection of offspring balb / c mice . reduction in congenital infection , also associated with a th2-biased immune response , was demonstrated by vaccination experiments in balb / c mice employing combined ncmic1 , ncmic3 , and ncrop2 antigens . taken together , it is probably rather a suitable balance in th1/th2-type immune responses than a strict th1 response that allows the host to deal with n. caninum infection . similar to earlier studies in balb / c mice [ 25 , 26 ] , levels of igg1 antibodies in animals infected with 1 10 and 5 10 tachyzoites were generally high , and it is interesting to note that a challenge dose of 5 10 tachyzoites in balb / c mice resulted in more pronounced igg1 responses compared to the igg1 antibody levels found in mice inoculated with 1 10 tachyzoites , while igg2a titres were basically equal in both groups . in the balb / c mice inoculated with 5 10 tachyzoites , titres for igg1 and igg2a were similar , which is in agreement with previous observations reported by lundn et al . . in addition , we found that splenocytes obtained from balb / c mice inoculated with 5 10 tachyzoites and stimulated with n. caninum antigen produced ifn- , albeit at a lower level compared to cba / ca mice . the results of rettigner et al . on n. caninum infection in cba / ca mice showed no evidence of differential isotype secretion but a high expression of ifn- , while in our experiments sera of infected cba / ca mice exhibited a high igg2a response , and splenocytes of cba / ca mice infected with 1 10 n. caninum tachyzoites also secreted high levels of ifn-. this indicated that the induction of a cellular immune response is a prerequisite for successful dealing with the infection . the importance of the humoral immune response during n. caninum infection was demonstrated by eperon et al . who showed increased susceptibility to n. caninum ( nc-1 isolate ) infection in mt b - cell - deficient mice when compared to wild - type c57bl/6 mice . they also described the predominant presence of parasite - specific igg2a isotypes and an absence of igg1 isotypes in c57bl/6 mice at various time points . , we did not observe any igg2a antibody titres in c57bl/6 mice and analysed the igg2c antibody isotype levels instead . our results showed the major parasite - specific igg isotype to be igg2c . in agreement with a shift from a th1- to a th2-biased response , increasing titres of n. caninum - specific igg1 were detected in mice sera collected at later time points after infection . while n. caninum obviously induces differential humoral and cellular immune responses in different mouse strains during the acute phase of infection , one could imagine that chronic infection would be characterized by the production of tissue cysts . therefore , in order to mimic the infection in cattle , a mouse model able to form tissue cysts , and also exhibiting recrudescence during pregnancy , is required . however , only few reports have actually been published on the production of n. caninum tissue cysts in mice . tissue cyst production had been demonstrated in outbred mice and immunosuppressed cba / ca mice inoculated with nc-1 or nc - liv tachyzoites but not all of the mice investigated harboured cerebral tissue cysts and rettigner et al . showed that immunosuppressed female cba / ca mice given 5 10 nc-1 tachyzoites were able to survive and to consistently develop cerebral tissue cysts . more recently , tissue cyst production , marked by positive staining with an antibody directed against the toxoplasma bradyzoite antigen bag5 , has also been demonstrated in an experimentally infected carnivorous marsupial , the fat - tailed dunnart sminthopsis crassicaudata . it is also very likely that n. caninum tissue cysts are not necessarily formed predominantly in the cns but also at other locations such as muscle tissue [ 34 , 35 ] , and this should be taken into account in future studies . in conclusion , we comparatively assessed balb / c , cba / ca , and c57bl/6 mice as models for n. caninum infection and demonstrated that cba / ca mice exhibited the highest degree of resistance at low and medium infection doses but rapidly developed acute signs of neosporosis when challenged with the highest dose of n. caninum nc - liv tachyzoites . this mice strain has previously been shown to be suitable for tissue cyst production , which renders them an interesting model for investigations on protective effects and potential tools for vaccination . in contrast , the use of balb / c mice , although extensively employed , is debatable due to their inherent th2 bias , resulting in increased susceptibility to n. caninum . thus , there is clearly a need for a standardisation of experimental murine infection models for n. caninum in order to achieve comparable results between research groups that work with different tools for immunoprevention and chemotherapy ." ]

[ "", "the 16s r rna gene sequences were deposited in genbank under the following accession numbers : bacillus dakarensis marseille - p3515 ( lt671589 ) , bacillus sinesaloumensis marseille - p3516 ( lt671591 ) , halobacillus massiliensis marseille - p3554 ( lt671593 ) , lentibacillus massiliensis marseille - p3089 ( lt598549 ) , oceanobacillus senegalensis marseille - p3587 ( lt714172 ) oceanobacillus timonensis marseille - p3532 ( lt671597 ) , virgibacillus dakarensis marseille - p3469 ( lt631544 ) and virgibacillus marseillensis marseille - p3610 ( lt714170 ) .", "the strains were deposited in the collection de souches de lunit des rickettsies ( csur , wdcm 875 ) under numbers bacillus dakarensis marseille - p3515 ( p3515 ) , bacillus sinesaloumensis marseille - p3516 ( p3516 ) , gracilibacillus timonensis marseille - p2481 ( p2248 ) , halobacillus massiliensis p3554 ( p3354 ) , lentibacillus massiliensis marseille - p3089 ( p3089 ) , virgibacillus dakarensis marseille - p3469 ( p3469 ) and virgibacillus marseillensis marseille - p3610 ( p3610 ) ." ]

[ "cardiovascular disease is the predominant cause for mortality in patients with type-2 diabetes mellitus , accounting for 5060% of deaths . metabolic syndrome ( mets ) refers to a conglomeration of cardiovascular risk factors that identifies a cohort of individuals with increased risk of cardiovascular morbidity . asian indians are ethnically predisposed to increase cardiovascular morbidity . increased adiposity ( overall and especially visceral ) in spite of lower body mass index ( bmi ) , as compared to other ethnic groups ( like caucasians ) may contribute to this increased risk . bmi , waist circumference ( wc ) , waist - hip ratio , and waist - height ratio ( whtr ) are simple anthropometric indices to measure this generalized and central adiposity . specific challenges associated with diabetes in india , the single most important contributor to cardiovascular morbidity and mortality , is the high prevalence of diabetes ( 9% ) and also prediabetes ( 1214% ) , young age of disease onset , with significantly higher rates of disease progression ( prediabetes to diabetes conversion rate in india , china , finland , and the usa being ( 1418% , 11% , 6% , and 2.5% , respectively ) . hence , there is an urgent need of easy to measure anthropometric marker , specific to the indian population , which is a good marker for early diagnosis of this high - risk cohort of individuals , who are predisposed to mets , diabetes , and cardiovascular morbidity . few studies have suggested that subcutaneous adipose tissue in the upper body distribution ( upper body obesity ) , to have an independent role in predicting cardiovascular morbidity . neck circumference ( nc ) has been validated to be a simple measure of upper body subcutaneous fat deposition and being a predictor of cardiovascular risk factors . data on the evaluation of nc as a predictor of cardiovascular risk factors in scant from india . furthermore , the predictive potential of nc based indices for predicting mets and its components , as compared to the traditional anthropometric indices ( bmi , wc , and whtr ) among indians are not known . neck height ratio ( nhtr ) has also been suggested to be a measure of upper body adiposity like nc . nhtr has the advantage over nc , as it adjusts for the difference in nc attributable to differences in heights . nhtr has not been previously evaluated as an index for predicting mets and other cardiovascular risk factors . hence , the aim of this study was to evaluate the role of nc and nhtr as independent predictors of mets and its components among asian indians as compared to traditional anthropometric indices ( bmi , wc , and whtr ) .", "individual attending the biannual health camps conducted by the department of endocrinology and metabolism across the city of calcutta were considered . screening of apparently healthy individuals that included family members of patients with diabetes was done during these camps by either fasting blood glucose ( fbg ) if they came after an overnight fast , or a non - fbg using a glucometer ( accu - chek active ; roche , mumbai , india ) . individuals , 3080 years age , without any co - morbid states , were considered for the study . patients not metabolically stable with uncontrolled blood glucose values on glucometer screening , viz . fasting / random blood glucose > 300 mg / dl were excluded . for those patients having blood glucose values in the diabetes range , only those who were recently diagnosed with diabetes ( within last 6 months ) patients on medications that can interfere with body composition and lipids such as anti - depressants , glucocorticoids , and anti - lipid medications were excluded . pregnant women were not considered for the study . the study protocol was explained , and only those who gave informed written consent included individuals attended the camp again the next day after 12 h fast , underwent anthropometric assessment , blood samples collected , serum separated which was transported to the department in cold chain and stored at 80c . height ( to 0.1 cm ) was measured in all individuals using a charder hm200pw wall - mounted stadiometer ( calibrated using a 36 calibration rod [ perspective enterprise , portage , michigan , usa ] ) , and body weight ( to 100 g ) measured using an electronic calibrated scale ( tantia , japan , model - ha521 , lot number-860525 ) . bmi was calculated as weight in kilograms divided by the square of height in meters ( kg / m ) . nc was measured using a calibrated plastic tape , with the head positioned along the frankfurt plane , at mid - neck height , between the mid - cervical spine and mid - anterior neck , to within 1 mm . in men with a laryngeal prominence , wc was measured at the end of a gentle expiration midway between the lower rib margin and iliac crest with the patient standing with feet 2330 cm apart . the collected blood samples were used for estimation of fbg , glycosylated hemoglobin ( hba1c% ) and fasting lipid profile using clinical chemistry analyzer ( daytona , serial number-58260536 , furuno electric , nishnomeya , japan ) . the presence of mets was ascertained using the modified national cholesterol education program adult treatment panel ( ncep atp ) iii criteria ( ethnic - specific cutoffs for wc viz . > 90 cm in males and > 80 cm in females ) with the presence of three or more considered diagnostic . the prevalence of mets in india has been reported to be 31.6% . keeping a power of 80% and type - i error at 5% , it was calculated that we needed to evaluate at least 235 individuals with mets in our study . anova with post hoc analysis and kruskal wallis nonparametric anova with dunn 's postcorrection were performed for normally and nonnormally distributed variables , respectively . pearson 's or spearman 's correlation coefficient was calculated for normally and nonnormally distributed variables , respectively . for categorical data , frequencies , and percentages the associations between metabolic risk factors and anthropometric parameters were assessed using partial correlation analysis . receiver operating characteristic ( roc ) analyses were performed to assess the accuracy of the anthropometric parameters as diagnostic tests for detecting mets and determine optimal sex - specific nc cut - offs in relation to mets . the youden index , defined as ( sensitivity + specificity)-1 was used to determine the optimal cut - off points . spss version 16 ( chicago , il , usa ) was used for statistical analysis .", "anova with post hoc analysis and kruskal wallis nonparametric anova with dunn 's postcorrection were performed for normally and nonnormally distributed variables , respectively . pearson 's or spearman 's correlation coefficient was calculated for normally and nonnormally distributed variables , respectively . for categorical data , frequencies , and percentages the associations between metabolic risk factors and anthropometric parameters were assessed using partial correlation analysis . receiver operating characteristic ( roc ) analyses were performed to assess the accuracy of the anthropometric parameters as diagnostic tests for detecting mets and determine optimal sex - specific nc cut - offs in relation to mets . the youden index , defined as ( sensitivity + specificity)-1 was used to determine the optimal cut - off points . spss version 16 ( chicago , il , usa ) was used for statistical analysis .", "a total of 867 individuals were screened of which 451 individual who fulfilled all inclusion and exclusion criteria , gave informed consent , and came for detailed anthropometric and biochemical evaluation were included in the study and analyzed [ figure 1 ] . in our study , 228 , 55 and 168 individuals were classified as having normoglycemia , prediabetes , and diabetes respectively as per fbg and hba1c values . males had significantly lower bmi , but higher nc as compared to females [ table 1 ] . the occurrence of hypertension , dysglycemia ( prediabetes or newly diagnosed diabetes ) was significantly higher in males [ table 1 ] . total cholesterol and low - density lipoprotein cholesterol ( ldl - c ) were significantly higher in females [ table 1 ] . patients with mets in both the sexes had significantly higher nc , nhtr , hba1c , fbg , and dyslipidemia ( higher triglycerides , total cholesterol / high - density lipoprotein cholesterol ( hdl - c ) ratio , ldl - c / hdl - c ratio , and lower hdl - c ) , as compared to those without mets [ table 1 ] . ckd : chronic kidney disease ; cld : chronic liver disease ; cva : cerebrovascular accident ; tia : transient ischemic attack ; cad : coronary artery disease baseline characteristics of the study population with regards to sex distribution and occurrence of metabolic syndrome ( n=451 ) males and females were divided into subgroups based on nc tertile [ table 2 ] . in both the sexes , individuals in the highest tertile of nc had significantly greater central obesity , significantly more likely to have bmi in the overweight and obese range , lower hdl - c and significantly higher occurrence of mets [ table 2 ] . males , but not females in the highest tertile of nc had significantly higher occurrence of hypertriglyceridemia [ table 2 ] . nc had a strong significantly positive correlation with other evaluated anthropometric parameters ( wc , bmi , and nhtr ) in both the sexes after adjusting for age [ table 3 ] . nc had significant positive correlation with serum total cholesterol and triglycerides in males [ table 4 ] . a significant inverse correlation was observed between nc and hdl - c in both the sexes [ table 4 ] . wc , bmi , and nhtr had significant positive correlation with total cholesterol , triglycerides , and ldl - c in males [ table 4 ] . anthropometric and biochemical characteristics of the study subjects as per the distribution of neck circumference relation between anthropometric indices after adjusting for age in males and females correlation between anthropometric indices and cardio - metabolic risk factors after adjusting for age the areas under the roc curves ( area under the curves [ aucs ] ) were constructed to evaluate the predictive values of anthropometric indices for mets and its components [ table 5 ] . the auc for nc for predicting mets in males and females was 0.753 and 0.768 respectively , which was statistically significant [ table 5 ] . this auc for nc was lower than that for wc but higher than that for bmi with regards to predicting mets in both the sexes [ table 5 ] . nhtr had a higher auc than nc for predicting mets in males ( 0.77 and 0.753 , respectively ) [ table 5 ] . among all the 4 anthropometric parameters evaluated , nhtr had the highest auc with regards to predicting mets , hypertension , hypertriglyceridemia , and low hdl - c in males [ table 5 ] . in females , wc had the highest auc with regards to predicting mets and hypertension , whereas nc had the highest auc with regards to predicting low hdl - c . area under the roc by different anthropometric indices as predictor of metabolic syndrome and cardio - metabolic risk factors ( n=451 ) a nc of > 34.9 cm ( sensitivity 78.6% ; specificity 59.3% ) for men and > 31.25 cm ( sensitivity 72.3% ; specificity 64.4% ) for women were the best values of combined sensitivity and specificity in identifying mets . a logistic regression analysis , using mets as the dependent variable , showed that the relationship between nc and mets after adjusting for sex and age was statistically significant ( odds ratio 1.52 [ 95% confidence interval [ ci ] : 1.371.68 ] ; p < 0.001 ) . similarly a nhtr of > 21.17 cm / m ( sensitivity 80.7% and specificity 64.6% ) for men and > 20.48 cm / m ( sensitivity 80.4% and specificity 60% ) for women were the best values of combined sensitivity and specificity in identifying mets . a logistic regression analysis , using mets as the dependent variable , showed that the relationship between nhtr and mets after adjusting for sex and age was statistically significant ( odds ratio 1.96 [ 95% ci : 1.672.29 ] ; p < 0.001 ) . twenty - four out of 258 males ( 9.3% ) and 8 out of 148 females ( 5.4% ) with mets had normal wc . ten ( 41.66% ) and 18 ( 75% ) of these 24 males had nc > 24.9 cm and nhtr > 21.17 cm / m , respectively . three ( 37.5% ) and 4 ( 50% ) of these 8 females had nc > 31.25 cm and nhtr > 20.48 cm / m , respectively .", "wc a measure of abdominal subcutaneous and visceral fat traditionally has served as the standard index to identify patients with mets . however , it has been observed that a significant proportion of patients with cardiovascular disease with normal wc , thereby highlighting its shortcomings . studies have suggested that upper body subcutaneous fat is responsible for a much larger proportion of systemic free fatty acid release than visceral fat , as it is lipolytically more active than lower body adipose tissue . hence , this fat may have a significant contribution to genesis of insulin resistance and dyslipidemia . nc has been demonstrated to be an index of upper body subcutaneous fat , with correlation with indices of obesity and individual cardiovascular risk factors . in a longitudinal follow - up study , it was shown that the change is nc correlated with changes in cardiovascular risk factors and that using wc alone was a simplification and did not account for all the changes in risk factors . followed up participants from the framingham heart study over a period of 10 years prospectively and noted that nc correlated with development of multiple cardiovascular risk factors . the same group also estimated visceral adiposity by computed tomography along with anthropometry and metabolic parameters and demonstrated that nc was associated with cvd risk factors even after adjustment for vat and bmi , thus , suggesting that upper body subcutaneous fat may be a unique pathogenic fat depot . in our study , patients in the highest tertile of nc had significantly higher occurrence of mets , central obesity , and dyslipidemia parameters that are established predictors of cardiovascular morbidity . nc had a strong positive correlation with traditional anthropometric indices of central obesity ( bmi and wc ) , which are also established predictors of cardiovascular risk . nc had significant positive correlation with total cholesterol , triglycerides , and significant negative correlation with hdl - c in males . , wc had the largest auc for predicting mets in both males and females , followed by nhtr , nc , and bmi . nhtr had the largest auc for predicting hypertriglyceridemia and low hdl - c in males . nc , in contrast , had the largest auc for predicting low hdl - c in females . our study demonstrated that among south asians , an nc of > 34.9 cm for men and > 31.25 cm for women were the best predictors for identifying mets . these cut - offs are lower than that previously reported in literature from other parts of the globe . suggested cut - offs of 39 cm and 35 cm for diagnosing mets among turkish population . the same cut - off was proposed by yang et al . for diagnosing mets in the chinese population . zhou et al . in another study from a different part of china suggested lower cut - offs for 37 cm and 33 cm for identifying mets in males and females , respectively . the lower cut - offs observed in our study in part can be explained by the lower median nc among our study subjects . different ethnicity and body composition of different populations evaluated in different studies may explain this difference . our study proposed for the 1 time cut - offs for nhtr in predicting mets . a nhtr of > 21.17 cm / m for men and > 20.48 cm / m for women were the best predictors for identifying mets . overall nhtr had a better odds ratio for predicting mets as compared to nc . among patients of mets with normal wc , 75% and 50% of males and females respectively had nhtr above the suggested cut - offs , highlighting that nhtr may be a better predictor of mets . limitations of this study include the cross - sectional design , which prevents us from establishing causality . nc was used as surrogate for upper body subcutaneous tissue , which however was not quantified by an imaging modality in our study . the higher prevalence of mets observed in our study , as compared to previously reported literature may in part be explained by the opportunistic screening done during patient recruitment . to summarize it may be said that our study reinforced the previous observation of nc being a good predictor of mets and other related cardiovascular risk factors in asian indians . our study demonstrated for the 1 time the reliability of nhtr as a predictor of mets and its components and highlighted that nhtr is perhaps an even better index than nc with regards to cardiovascular risk prediction . this study was funded by a research grant from research society of study of diabetes in india , west bengal chapter .", "this study was funded by a research grant from research society of study of diabetes in india , west bengal chapter .", "" ]

[ "pityriasis rosea is considered to be a benign cutaneous condition [ 14 ] . however , in the setting of pregnancy , adverse effects on the newborn may be observed . craniosynostosis is a congenital abnormality , which can occur as an isolated finding or as part of a syndrome with other associated features . a woman who developed pityriasis rosea during her first trimester of pregnancy and who subsequently delivered a healthy baby with craniosynostosis is described , and observations of infants born to women who are diagnosed with pityriasis rosea during their gestation are summarized .", "a 28-year - old healthy woman presented at 10 weeks gestation with a skin rash . two weeks earlier , at eight weeks gestation , she had noticed an initial skin lesion on her abdomen ( figure 1 ) . her husband , a 29-year - old man , had presented two months earlier with similar - appearing annular lesions on his neck ( figure 2 ) . a larger lesion had initially appeared on his left neck . within the next five days , additional lesions appeared on the remainder of his neck and subsequently , a few lesions appeared on his distal upper extremities . a diagnosis of inverse pityriasis rosea was established based on the clinical history and lesion morphology . his lesions resolved over the next two weeks after treatment with triamcinolone 0.1% cream twice daily . cutaneous examination of the patient s lesions showed multiple annular plaques with peripheral scaling on the abdomen and back ( figure 1 ) . a diagnosis of pityriasis rosea was established based on the appearance of the lesions and history . cetaphil cream was applied twice daily to the patient s lesions ; the lesions resolved during the next eight weeks . she was classified as a high - risk pregnancy and was followed closely for the remainder of her gestation . after 15 hours of labor , the baby had not descended into the pelvis ; there was no fetal distress , and a decision for c - section was made . his apgar scores at one and five minutes were 9/9 , weight 3827.1 g , and height 50.8 cm . prior to discharge from the hospital , it was noted that the infant had a curved head . neurosurgery consultation confirmed the diagnosis ; at 9 weeks postpartum , endoscopic repair was performed successfully with no adverse sequelae .", "pityriasis rosea classically presents as annular plaques with peripheral scale , typically located between the neck and the groin , and may be seasonal in occurrence . less commonly , it can present with lesions on the neck and extremities ( inverse pityriasis rosea ) [ 1011 ] or during pregnancy . however , associations with human herpes virus ( hsv)-6 and hsv-7 have been observed [ 1418 ] . several studies have found that patients with pityriasis rosea have higher levels of hsv-6 and hsv-7 detected in their skin , suggesting that infection by these viruses may have a causal effect on the development of pityriasis rosea . occasionally , pityriasis rosea has been documented in siblings or in spouses ( table 1 ) [ 1921 ] . in these circumstances , our patient s husband developed and cleared inverse pityriasis rosea two months prior to his wife developing classic pityriasis rosea . similar to our patient , in the majority of cases of pityriasis rosea occurring in couples , the lesions appeared in the husband prior to the wife ( table 1 ) . the interval between onset of pityriasis rosea in the wife after occurrence in the husband ranged from seven days to one year ( median : 2 months ) . our review of the literature , including the patient in this report , discovered 54 women who developed pityriasis rosea during their pregnancy ( table 2 and table 3 [ 6,1214,23,24 ] ) . the onset of pityriasis rosea ranged from week 8 of gestation ( 3 patients : cases 7 and 8 in table 2 and case 14 in table 3 ) to week 32 ( 1 patient : case 7 in table 3 ) . the median number of weeks of pregnancy at the onset of pityriasis rosea was 19 . twenty - five women ages 24 to 34 ( median age 29)had no prior pregnancies . however , pityriasis rosea occurred during either the second ( 20 women ) or the third pregnancy ( 6 women ) for the other women . most of the women ( 66% , n=35 ) developed pityriasis rosea during the second trimester of gestation ( 1328 weeks ) . nineteen percent ( 10 women ) had the onset of their dermatosis during the first trimester ( 012 weeks ) . only 10% ( 5 women ) experienced it in the third trimester ( 2940 weeks ) . several retrospective studies have observed adverse events affecting the newborn in women who develop pityriasis rosea during pregnancy ( table 2 ) . in these individuals , the adverse events predominantly included stillbirth at 11 to 28 weeks ( median 16 weeks ) , premature delivery ( < 37 weeks ) , hypotonia , weak motion , and low birth weight . less common adverse effects were hydramnios and foramen ovale . some investigators have discovered that pityriasis rosea occurring earlier in pregnancy , such as in the first trimester , have been more often associated with a poorer prognosis , compared to women who developed the dermatosis during the second or third trimesters . however , our review of the literature showed that the majority of women ( 16/25 , 64% ) who experienced adverse events had the onset of pityriasis rosea that occurred during the second trimester . the onset of pityriasis rosea occurred during the first trimester in 9 women ( 36% ) and none in the third trimester . additional studies looking at the association between hsv-6 and hsv-7 dna and the occurrence of pityriasis rosea in pregnancy have also been performed . some of the studies found reactivation of hsv-6 during pregnancy . however , a positive correlation between viral infection and clinical features of pityriasis rosea was not established . individual case reports , including the patient in this report , have described 29 women who developed pityriasis rosea during pregnancy and have delivered healthy newborns ( table 3 ) [ 6,1214,23,24 ] . indeed , the literature shows a ratio of 6:5 with regards to healthy newborns versus newborns with adverse events being delivered to women with gestational pityriasis rosea . however , the number of publications regarding gestational pityriasis rosea on the outcome of the newborn may not accurately reflect the incidence of normal newborns whose mothers had gestational pityriasis rosea , since clinicians may not report these women or journals may elect not to publish the papers . it can occur as an isolated incidental event or as part of syndrome ( table 4 ) . as an isolated incidental finding , sagittal craniosynostosis , as observed in our patient s newborn , is the most common form . if left unrepaired , craniosynostosis may lead to a deformed skull , elevation of intracranial pressure , and cognitive impairment . our patient s infant was evaluated shortly after delivery and had repair of the sagittal craniosynostosis at 9 weeks , with no complications and subsequent normal development . the incidence of sagittal craniosynostosis is about 1 in 5,000 live births . to the best of our knowledge , isolated craniosynostosis has not been observed in newborns of women who developed pityriasis rosea during their gestation . indeed , the occurrence in our patient s child may merely be a coincidence and not associated with her episode of pityriasis rosea during her first trimester .", "however , the true incidence is not known , since gestational pityriasis rosea is not frequently reported . some researchers noted that pityriasis rosea occurring earlier in pregnancy had a greater probability of resulting in adverse events for the fetus , including stillbirth , low gestational weight , hypotonia , and/or premature delivery . however , there are a similar number of reports of women who developed pityriasis rosea during their gestation and delivered normal newborns . our patient developed pityriasis rosea during her first trimester beginning at 8 weeks gestation and lasting through 18 weeks . her son was carried to term and delivered at 40 weeks and 6 days with apgar scores , weight , and height in normal range . whether the presence of craniosynostosis was associated with our patient s development of pityriasis rosea during her pregnancy remains to be determined ." ]

[ "bile cast syndrome ( bcs ) is a complication of orthotopic liver transplantation ( olt ) . we describe 2 patients with bcs who were successfully diagnosed with endoscopic retrograde cholangio pancreatography ( ercp ) and one of whom was successfully treated endoscopically .", "a 65-year - old female underwent olt 6 months prior to presentation for chronic hepatitis c and hepatocellular carcinoma . her intraoperative course during liver transplantation was complicated by a significant amount of blood loss from hepatic veins . she had a complicated postoperative course including hemorrhagic shock , abdominal compartment syndrome and acute renal failure which required hospitalization for 2 months after the transplantation . she developed an anastomotic bile leak which was treated with placement of a 10 fr stent . at 4 weeks and 8 weeks after initial ercp , the cbd stents were exchanged and secondary sclerosing cholangitis was suspected based on cholangiographic appearance . her immunosuppression regimen included tacrolimus 9 mg bid and mycofenolic acid 180 mg bid . laboratory data showed bilirubin of 1.1 mg / dl ( reference range : 0.2 mg / dl-1.3 mg / dl ) , alanine aminotransferase of 53 iu / l ( reference range : 13 iu / l-69 iu / l ) , aspartate aminotransferase of 60 iu / l ( reference range : 15 iu / l-46 iu / l ) , alkaline phosphatase of 257 iu / l ( reference range : 42 iu / l-140 iu / l ) , -glutamyl transferase of 466 iu / l ( reference range : 5 iu / l-55 balloon sweeps yielded a small amount of debris , but cholangiographic findings persisted that were concerning for filling defects or marked ductal irregularity in the region of the common hepatic duct and bifurcation . cholangioscopy ( fig . 2 ) was performed with the spyglass single operator biliary visualization system ( boston scientific , natick , ma , usa ) . a tubular biliary cast , in the shape of the bile duct , was seen in the donor bile duct extending from the anastomosis into the intrahepatic ducts . attempts to extract the cast with a snare , basket , mechanical lithotripter , balloon , spybite(boston scientific , natick , ma , usa ) biopsy forceps , and standard egd forceps were all unsuccessful due to inability to grasp the cast and tethering of the cast to intrahepatic extension . a 10 fr 12-cm cbd stent was placed . in subsequent months , it appears to contain filling defects which retrospectively are related to presence of biliary cast material in the right and left main hepatic ducts . \n a 58-year - old male with a history of hepatitis c and cirrhosis presented with fevers , chills , and a headache . the patient s immunosuppression regimen included mycophenolate 360 mg bid , prednisone 5 mg daily and tacrolimus 8 mg bid . laboratory data showed bilirubin of 2.2 mg / dl ( reference range : 0.2 mg / dl-1.3 mg / dl ) , alanine aminotransferase of 162 iu / l ( reference range : 13 iu / l-69 iu / l ) , aspartate aminotransferase of 159 iu / l ( reference range : 15 iu / l-46 iu / l ) , alkaline phosphatase of 1277 iu / l ( reference range : 42 iu / l-140 iu / l ) and white blood cell count of 8.17 10 cells / l . the left main hepatic duct was dilated with a 4-mm dilating balloon to facilitate clearance of the filling defects . cholangiogram prior to intervention shows diffuse stricturing of the left and right hepatic ductal system in patient 2 . b cholangiogram after removal of the biliary cast showing a widely patent left hepatic duct in patient 2 . \n subsequent to removal of the biliary cast , endoscopic treatment for the anastomotic stricture resulted in resolution of the stricture .", "a 65-year - old female underwent olt 6 months prior to presentation for chronic hepatitis c and hepatocellular carcinoma . her intraoperative course during liver transplantation was complicated by a significant amount of blood loss from hepatic veins . she had a complicated postoperative course including hemorrhagic shock , abdominal compartment syndrome and acute renal failure which required hospitalization for 2 months after the transplantation . she developed an anastomotic bile leak which was treated with placement of a 10 fr stent . at 4 weeks and 8 weeks after initial ercp , the cbd stents were exchanged and secondary sclerosing cholangitis was suspected based on cholangiographic appearance . her immunosuppression regimen included tacrolimus 9 mg bid and mycofenolic acid 180 mg bid . laboratory data showed bilirubin of 1.1 mg / dl ( reference range : 0.2 mg / dl-1.3 mg / dl ) , alanine aminotransferase of 53 iu / l ( reference range : 13 iu / l-69 iu / l ) , aspartate aminotransferase of 60 iu / l ( reference range : 15 iu / l-46 iu / l ) , alkaline phosphatase of 257 iu / l ( reference range : 42 iu / l-140 iu / l ) , -glutamyl transferase of 466 iu / l ( reference range : 5 iu / l-55 balloon sweeps yielded a small amount of debris , but cholangiographic findings persisted that were concerning for filling defects or marked ductal irregularity in the region of the common hepatic duct and bifurcation . cholangioscopy ( fig . 2 ) was performed with the spyglass single operator biliary visualization system ( boston scientific , natick , ma , usa ) . a tubular biliary cast , in the shape of the bile duct , was seen in the donor bile duct extending from the anastomosis into the intrahepatic ducts . attempts to extract the cast with a snare , basket , mechanical lithotripter , balloon , spybite(boston scientific , natick , ma , usa ) biopsy forceps , and standard egd forceps were all unsuccessful due to inability to grasp the cast and tethering of the cast to intrahepatic extension . a 10 fr 12-cm cbd stent was placed . in subsequent months , it appears to contain filling defects which retrospectively are related to presence of biliary cast material in the right and left main hepatic ducts . \n", "a 58-year - old male with a history of hepatitis c and cirrhosis presented with fevers , chills , and a headache . he underwent deceased donor liver and kidney transplantation two months prior to his presentation . the patient s immunosuppression regimen included mycophenolate 360 mg bid , prednisone 5 mg daily and tacrolimus 8 mg bid . laboratory data showed bilirubin of 2.2 mg / dl ( reference range : 0.2 mg / dl-1.3 mg / dl ) , alanine aminotransferase of 162 iu / l ( reference range : 13 iu / l-69 iu / l ) , aspartate aminotransferase of 159 iu / l ( reference range : 15 iu / l-46 iu / l ) , alkaline phosphatase of 1277 iu / l ( reference range : 42 iu / l-140 iu / l ) and white blood cell count of 8.17 10 cells / l . the left main hepatic duct was dilated with a 4-mm dilating balloon to facilitate clearance of the filling defects . 4 ) . following extraction , cholangiograms revealed a widely patent left main duct ( fig . cholangiogram prior to intervention shows diffuse stricturing of the left and right hepatic ductal system in patient 2 . b cholangiogram after removal of the biliary cast showing a widely patent left hepatic duct in patient 2 . \n subsequent to removal of the biliary cast , endoscopic treatment for the anastomotic stricture resulted in resolution of the stricture .", "bile casts occur in 4 % to 18 % of olt recipients 1 . more than 70 % of casts are identified in the first 16 weeks after transplant 1 . bile casts are dark and hard in appearance and formed by lithogenic material confined to the bile duct dimensions 3 \n 4 . although the exact mechanism of injury is unknown , ischemic injury to the biliary epithelium is most common 5 \n 6 \n 7 . other mechanisms including anastomotic and non - anastomotic strictures leading to accumulation of biliary sludge can lead to formation of casts 5 . bacterial infection and stones are also predisposing factors for biliary ductal injury and cast formation 6 . microcirculatory dysfunction is also suggested , given the higher prevalence of renal failure and need for renal replacement therapy in patients with bcs 7 . a combination of endoscopic interventions is considered including sphincterotomy , balloon and basket extraction , lithotripsy and stent placement . in one study , re - transplantation and 12-month mortality rates were significantly higher with bcs ( 30 % vs. 7 % ) and bcs patients also required a longer intensive care unit stay post - olt 7 . success rates with endoscopic therapy for bcs have been reported to be 70 % recently 7 . previously in a large study of post - olt adverse events , biliary cast syndrome occurred in 4/260 patients and a median of 3.5 ercp treatments attempted over a median of 21 weeks achieved a success rate of 25 % 8 . percutaneous drainage has also been advocated , especially in the setting of cholangitis or with roux - en - y choledochojejunostomy . percutaneous transhepatic choledocoscopy via a tract created with radiographic assistance is a novel approach used for management of bcs . when there is intrahepatic biliary system involvement , management becomes even more challenging . retransplantation is considered when complete removal is not feasible and if the patient s clinical condition deteriorates 2 \n 7 . a combined endoscopic and percutaneous method was able to successfully clear the casts in 60 % of patients in one study 9 . surgical intervention including hepaticojejunostomy , choledochojejunostomy and revision of choledochocholedocostomy reportedly has had success rates above 85 % 10 ." ]

[ "a 27-year - old malay male patient presented two weeks \n after alkali splashed into his left eye while working . prior to \n reporting to us , the patient consulted a general practitioner \n who prescribed some topical eye drops ( details of the \n treatment were not available ) and the eye condition worsened . \n anterior segment examination revealed a central corneal \n ulcer ( 1 1 mm ) with stromal infiltration ( 4 6 mm ) , limbal \n ischemia , normal anterior chamber depth and visual acuity \n of counting fingers . a gentle b - scan ruled out any posterior \n segment abnormality . a clinical diagnosis of left corneal ulcer with limbal ischemia \n secondary to alkali injury with secondary infection was made . \n culture from corneal scraping was negative . despite intensive \n therapy with oral ( tab ciprobay 500 mg twice daily , bayer ) \n and topical ciprofloxacin ( gutt ciloxan 0.3% hourly , alcon ) \n ( no topical steroids used ) with other supportive therapy ( tab \n vitamin c 1 g daily , pharmaniaga ) , further deterioration led \n to formation of central corneal perforation with surrounding \n stromal thinning and flat anterior chamber . we performed a gunderson conjunctival flap under local \n anesthesia ( la ) , which retracted and failed after one week . \n slit - lamp examination revealed a large central corneal perforation \n ( 4 5 mm ) with surrounding stromal thinning involving two- \n thirds of the cornea without any signs of infection . in order to maintain the shape of the eyeball and due to the \n problem of non - availability of donor cornea , we decided to use \n processed bp xenograft ( lyolemb ) supplied by national tissue \n bank , universiti sains malaysia after obtaining the consent of the \n patient . a 360 peritomy was done and the graft was sutured with \n 7 - 0 polyglycolic and polylactic acids ( vicryl ) to the perilimbal \n area with conjunctival advancement . topical ( gutt ciloxan 0.3% \n hourly , alcon ) and oral ciprofloxacin ( tab ciprobay 500 mg \n twice daily , bayer ) was prescribed in the postoperative period \n [ figure 1 ] . nine months follow - up \n showed a well - taken graft without any exposure / dehiscence and \n minimal inflammation [ figure 2 ] . slit - lamp examination could \n not reveal the details of the anterior chamber and beyond due to \n conjunctival advancement and vision was noted as perception \n of light with inadequate projection of rays .", "conjunctival flap is unlikely to seal large corneal perforations \n associated with corneal melting following chemical injury . \n efficacy of amt alone or with glue in treating small corneal \n perforation has been well established but found unable to \n promote corneal stability in patients with severe corneal \n thinning and therefore , an additional urgent tectonic procedure \n has to be undertaken.3 non - availability of donor cornea poses \n a considerable problem in some countries . processed bp is a tough lyophilised collagen sheet used \n as a surgical armamentarium in wrapping orbital implants.4,5 it renders the material antigenically inert and adds to its \n strength , pliability and longevity.5 it has been shown to be \n well tolerated with minimal inflammation in animal studies.4 \n although theoretic concerns about prion disease can be raised , \n no evidence of this problem has been reported from over \n 90,000 human implantations.6 devron reported no significant \n ophthalmic reactions due to this xenograft in his cases of orbital \n implants.7 at the national tissue bank , universiti sains malaysia , \n the procured pericardium sheet was washed using chemical \n agent ( sterile phosphate buffer at ph 7.4 ) and frozen to 40c . \n it was then freeze - dried and packaged in double - layer plastic \n bag . the processed bp was radio - sterilized at 2.5 mega rads \n using gamma radiation and was then ready for use.8 we used \n this locally processed bp in our case . its use in complicated ocular surface \n diseases such as large corneal perforation with corneal melting \n can be promising . its use needs to be further studied more \n extensively as an alternative graft material in large corneal \n perforations ." ]

[ "the desynchronization of central and peripheral circadian systems contributes to the decline in optimal functioning of bodily systems . this includes changes in neuroendocrine circadian rhythms , insulin sensitivity , altered thermoregulation and acceleration of tumor growth ( cincotta et al . , 1993 ; touitou and haus , 2000 ; hastings et al . , 2003 ; straub and mocchegiani , 2004 ; cretenet et al . , 2010 ; heller et al . , 2011 these are complex interactions and dysfunction can be due to targeted disruption of neurons , neurotransmitter or neuropeptide production , transport or secretion . it is reasonable to expect that the neuronal activity or expression of circadian clock genes be reduced or rhythms phase shifted ( kolker et al . , 2003 ) . additionally , the connections between central and peripheral oscillators may be degraded or less functional ( morales et al . , 1987 ) . any one or a combination of these abnormalities may result in the decoupling of the circadian oscillators and the ensuing pathologies . the master central pacemaker is the suprachiasmatic nucleus ( scn ) which controls circadian rhythmicity . rhythmicity can dampen and/or elongate / shift with age but the number or cell size of neurons in the scn does not change . , 1993 ; madeira et al . , 1995 ) . additionally , glucose uptake decreases in the scn in aged animals ( wise et al . , 1988 ) and the expression of neuropeptides also diminishes . vasoactive intestinal polypeptide ( vip ) expressing neurons of the scn are retinorecipient and vasopressin ( avp ) expressing neurons of the scn modulate rhythmicity ( wu et al . , 2007 ) . in aged male humans , the number of scn neurons that express vip decreases and in aged female rats the expression becomes arrhythmic ( zhou et al . both genders also express less avp protein , less mrna ( roozendaal et al . , 1987 ; zhou et al . , 1995 ) and the normal daytime peak of avp is reduced ( hofman and swaab , 1994 ; liu et al . , 2000 ) . decreases in the neuroendocrine output of the scn may directly or indirectly affect the coupling of central and peripheral oscillators . in the scn , clock genes constitute the core clock mechanism of the mammalian timekeeping system . though the system is fairly complex , for brevity the simplest model is described . bmal1 and clock proteins dimerize and induce the transcription of period ( per ) and cryptochrome ( cry ) genes . in a negative feedback loop , levels of per and cry proteins increase and at a certain threshold form heterodimers which turn off the clock bmal1 regulated transcription of per and cry genes . this process takes roughly 24 h ( ko and takahashi , 2006 ) . in aged animals the expression of certain clock genes changes in the scn . per1 , per2 , and cry1 expression does not change significantly with age , but the normal photic stimulation of per1 expression is reduced ( asai et al . . additionally , the free - running period of per1luc rhythmicity is shortened in aged animals ( yamazaki et al . , 2002 ) and the amplitude of clock and bmal1 expression is decreased ( kolker et al . , 2003 ) . changes in clock gene expression in peripheral tissues do not always reflect what is seen in the scn ( yamazaki et al . , 2000 ) , and may result from a disruption of signals to these tissues or the tissues themselves , which are more susceptible to the aging process . furthermore , projections to and from the scn including peripheral oscillators may change with age . in motoneurons , aging results in a shortening in delay of spike potentials between axon and soma , as well as decreases in axon conduction velocity and increases in input resistance ( morales et al . the output from circadian and peripheral oscillators do not only influence the sleep / wake cycle , but regulates metabolism and reproduction . studies indicate that some tissues retain the ability to oscillate , even if connections from the master pacemaker have been degraded . peripheral tissues in vitro that have become arrhythmic can be chemically induced to oscillate ( yamazaki et al . however , in aged animals exhibiting a decreased photic response , retinal projections to the scn are not degraded and must be related to either the retina or scn clock functions ( zhang et al . , it is increasingly evident that determining the source of age - related sleep / wake or circadian dysfunctions is rather complex .", "another source of sleep / wake changes can likely be attributed to age - related neuronal dysfunction in the arousal and sleep promoting areas of the brain . the scn directly or indirectly communicates with the sleep and wake promoting systems ( abrahamson et al . , 2001 ; aston - jones et al . , 2001 orexinergic ( or hypocretinergic ) neurons are known to stabilize or maintain wake ( saper et al . , 2001 , 2005 ) . these cells receive input from the scn via the dorsomedial hypothalamus ( dmh ) and are localized in the perifornical and lateral hypothalamus ( lh ) . there are two forms of the neuropeptide orexin / hypocretin ( a and b ) and two receptors . though the neurons are limited to a discrete area , both orexinergic fibers and receptors are widely distributed throughout the brain . in diurnal and nocturnal rodents , orexinergic neurons are most active during the active phase ( martinez et al . , 2002 ) . hypothalamic microdialysate analysis shows orexin-1 levels increase during wake and rem in adult animals ( kiyashchenko et al . , 2002 ) . mammals with no orexin or dysfunctional orexin / hypocretin receptors have disrupted sleep / wake cycles and narcoleptic symptoms . when orexin is decreased , the circadian rhythm of the sleep / wake cycle is disrupted . the flip flop model of sleep / wake control suggests that there is a mutual inhibition between the areas that control sleep and the areas that control the wake state ( saper et al . , 2001 ) . in short , the ventrolateral preoptic area ( vlpo ) controls sleep and the brainstem cholinergic and monoaminergic systems control waking . flipping weight between these areas blocking or destroying these neurons or the orexin receptor 2 may flip the animal s state quickly from waking to sleep and vice versa , such as what occurs in narcoleptic individuals , orexin knockout mice , and canine narcolepsy ( chemelli et al . , 1999 ; lin et al . , 1999 ; peyron et al . , 2000 ; a disruption in orexin function or a reduction in orexin levels leads to less stable sleep / wake cycles such as that seen in many elderly patients with sleep disorders ( porkka - heiskanen , 2003 ) . additionally , decreases in excitatory orexin innervation to the noradrenergic locus coeruleus ( lc ) is thought to be a contributing factor of poor sleep / wake quality in aged cats ( zhang et al . orexin b immunoreactive ( -ir ) axon density was determined to be significantly lower in the lc of aged macaques than that observed in the young or adult animals ( 43 and 35% decrease respectively ; downs et al . , 2007 ) . real time pcr studies showed that preprohypocretin mrna does not change in the aged hypothalamus ( terao et al . , 2002 ) but in situ hybridization studies show that at the single cell level , preproorexin gene expression does decrease in cell count and optical density ( porkka - heiskanen et al . , 2004 ) . furthermore , orexin a and b protein expression as measured by radioimmunoassay was decreased in the lh ( porkka - heiskanen et al . , the number of orexinergic - ir neurons as well as the optical density of respective fibers in the lh is reduced in aged animals ( brownell and conti , 2010 ; sawai et al . , 2010 ) . it is interesting to note that orexinergic innervation of the cholinergic basal forebrain , which modulates wake and rem sleep , is reduced in aged guinea pigs ( zhang et al . , 2005 ) . hypocretin receptor 1 mrna is reduced in the hippocampus and hypocretin receptor 2 mrna is significantly reduced in thalamic areas , hippocampus , and the brainstem ( terao et al . , 2002 ) . neural activity measured by c - fos immunoreactivity is reduced in orexinergic neurons of mice at 24 months ( naidoo et al . , 2011 ) . changes are also seen in the cholinergic and monoaminergic wake active areas of aged animals . nicotinic and muscarinic receptors of the acetylcholinergic system decrease in the scn with age ( van der zee et al . , 1991 ) . in young animals , the noradrenergic neurons of the lc are important in wake promotion , receive direct input from the scn and follow a circadian pattern of activation ( aston - jones et al . , 2001 ) . in aged rats , lc projections to the frontal cortex and dentate gyrus decrease but axonal branching increases depending on the target and age . this is suggested to be a compensatory mechanism ( shirokawa et al . , 2000 ) . in the ventral periaqueductal gray ( vpag ) the wake active dopaminergic neurons we have recently reported a reduction in the neural activity of these dopaminergic neurons of the vpag and the noradrenergic neurons of the lc in aged mice ( naidoo et al . , 2009 , 2011 ) . the wake active histaminergic system originates in the tuberomammillary nucleus ( tmn ) and sends widespread projections to areas that include the cortex , thalamus and brainstem . histamine levels were found to be increased in middle aged rats when compared to young , and the level of histamine methyl transferase was decreased ( mazurkiewicz - kwilecki and prell , 1984 ) . histamine h1 , h2 and h3 receptor mrna is decreased in the aging brain ( terao et al . , 2004 ) . given these changes in the aging wake promoting neurotransmitter systems as well as wake maintaining systems , it is clear that therapies to alleviate or attenuate these changes need to be developed . , 1996 ; szymusiak et al . , 1998 ) and when lesioned results in insomnia ( lu et al . , 2000 ) . gabaergic and galaninergic inhibitory neurons from this area project to wake active histaminergic neurons ( sherin et al . , 1996 ) . interestingly , the number of activated vlpo neurons during sleep does not change in old rats ( shiromani et al . , 2000 ) although connections between these areas may become dysfunctional or degraded with age . the scn has a minor input into the vlpo , but substantial direct and indirect inputs to the dmh ( novak and nunez , 2000 ; chou et al . , 2002 ) . the dmh heavily inputs the vlpo and it would be beneficial if these pathways were examined during aging . age - associated changes in the serotonergic system affect the function of respiratory motor output during sleep . serotonergic input to the hypoglossal nucleus decreases , which is thought to lead to a decline in upper airway muscle performance ( behan and brownfield , 1999 ) . in aged rhesus monkeys , serotonin receptor 2 density reduces in the occipital and parietal cortex including the deep layers of the motor cortex ( wenk et al . , 1989 ; bigham and lidow , 1995 ) . serotonin levels also decrease in the occipital areas but do not change in the cingulate cortex in aged monkeys ( beal et al . , 1991 ) . it is likely with normal aging that changes in any neurotransmitter system affecting sleep vary across the brain .", "in many patients afflicted with neurodegenerative diseases the physical and mental consequences lead to sleep disorders ( table 1 ) . for example , sleep fragmentation can occur if the patient can not move well or insomnia may develop due to depression or feelings of helplessness . medications used to alleviate some of the motor or cognitive symptoms such as levodopa in parkinson s disease ( pd ) can also contribute to disruptions in normal sleep / wake behaviors . however , some research indicates that sleep disturbances may predict manifestation of neurodegenerative diseases ( postuma and montplaisir , 2009 ) . sleep disturbance or loss also affects metabolic and immune function ( krueger et al . , 1998 ; knutson et al . , 2007 chronic sleep loss could lead to neuronal damage resulting in altered hypothalamic pituitary adrenal axis function , cognitive deficits and memory loss . increases in the number of patients with neurodegenerative diseases may be related to or the result of a society that does not sleep . da , dopamine ; inos , inducible nitric oxide synthase . sturrock and rao ( 1985 ) , zhang et al . ( 1982 ) , foley et al . ( 2007 ) , magri et al . ( 1997 ) , touitou ( 1995 ) , whitehead et al . ( ( 1998 ) , feinberg et al . ( 1967 ) , myers and badia ( 1995 ) , brun and englund ( 1981 ) , teipel et al . ( 1985 ) , kremer et al . ( 1991 ) , arnulf et al . initially there is an increase in nighttime arousals and a decrease in sws ( vitiello et al . , 1991 ) . in the later stages , circadian disruption , severe daytime wakefulness and a reduction in rem sleep occurs , likely due to a reduction in acetylcholine ( dykierek et al . , circadian rhythm dysfunction has been proposed to be due to changes in scn and pineal functions ( wu et al . , 2007 ) . degeneration of cholinergic input from the nucleus basalis of meynert to the cortex may be responsible for some of the sleep / wake changes ( montplaisir et al . , 1995 ) . neurofibrillary tangles found in the histaminergic tmn of ad patients and amyloid- peptide ( a ) aggregation also contributes to the ad pathology . normally in the interstitial fluid a has a diurnal fluctuation with low levels during sleep and peak levels during wake . recently one study showed that prolonged wake and/or orexin administration increased levels of the a in the interstitial fluid of the brain in mice ( kang et al . administration of an orexin antagonist reduced amyloid deposits in several brain areas suggesting that manipulating sleep or the orexin system in ad patients could improve symptoms ( kang et al . , 2009 ) . although the research is sparse , melatonin , phototherapy and exercise have all had positive effects in the treatment of circadian and sleep / wake disorders of ad patients ( wu and swaab , 2007 ) . as one in three americans develops ad , there is a crucial need for more research in these therapies . rem sleep behavior disorder ( rbd ) has been associated with pd and thought to be an early manifestation ( schenck et al . , 1996 ; boeve et al . , 2003 ; postuma and montplaisir , 2009 ) . sleep attacks and excessive daytime sleepiness ( eds ) are also commonly seen in patients with pd ( factor et al . , 1990 ; diederich et al . , the degeneration begins at the brainstem and progresses rostrally , although degeneration of the dopaminergic neurons of the substantia nigra pars compacta is the main contributor to pd characteristics ( braak et al . , rbd results from pedunculopontine dysfunction and likely explains rbd manifesting previous to pd ( rye , 1997 ; boeve et al . , 2007 ) . some studies have successfully seen bright light therapy or sleep modifications reduce the symptoms of pd ( hogl et al . , 1998 ; willis and turner , 2007 ) . huntington s disease ( hd ) is a genetic disorder characterized by a polyglutamine ( cag ) repeat ( scherzinger et al . , 1999 ) . neurodegeneration is extensive throughout the brain , affecting cortical and subcortical areas but primarily affects the basal ganglia ( vonsattel et al . , 1985 ) . sleep and wake regions of the brain including the brainstem , thalamus , hypothalamus and cortex are also affected in hd ( kremer et al . , 1991 ) . the scn pacemaker is functional in mouse models of hd , so a dysfunction of the circadian circuitry is proposed to contribute to circadian abnormalities ( pallier et al . , 2007 ) . central and peripheral clock gene expression is altered as well ( morton et al . , 2005 ; maywood et al . , 2010 ) . the sleep / wake cycle is disrupted in hd patients characterized by self - reported eds , sleep fragmentation at night , and delayed sleep phase ( arnulf et al . , 2008 ; videnovic et al . , 2009 ; aziz et al . , sleep is lighter with an increase in stage 1 and a decrease in rem sleep ( arnulf et al . , 2008 ) . disruptions in the circadian and sleep / wake cycles of these patients exacerbate symptoms , increasing depression , cognitive deficits and metabolic dysfunctions ( aziz et al . , 2010 ) . it is important to note that pharmacological and behavioral manipulation of sleep and wake reduces disease progression and improves cognitive function and circadian gene expression in a mouse model of hd ( hockly et al . , 2002 ; pallier et al . , 2007 ; pallier and morton , 2009 ; maywood et al . , 2010 ) amyotrophic lateral sclerosis ( als ) is considered an age - associated neurodegenerative disease with the age of onset ranging from 40 to 70 . most als cases are sporadic and about 10% are familial . also called lou gehrig s or motor neuron disease ( boillee et al . , 2006 ) , both upper and lower motor neurons are affected . motor neurons of the motor cortex , brainstem and spinal cord gradually degenerate leading to muscle weakness , sleep disordered breathing ( sdb ) and paralysis ( kimura et al . , 1999 ) . additionally , sleep is reduced in both rem and sws stages with resulting eds ( barthlen and lange , 2000 ; lo coco et al . , 2011 ) . some patients find relief using assisted breathing such as continuous positive airway pressure ( cpap ) or bilevel positive airway pressure ( bipap ; howard et al . , 1989 ; david et al . , 1997 ; barthlen and lange , 2000 ) . amyotrophic lateral sclerosis is a complex disease of many subtypes with various genetic and environmental contributing factors . one such factor is glutamate toxicity which is decreased using the drug riluzole ( shaw and ince , 1997 ) . levels of serotonin are decreased in als patients and compensatory increases in glutamate lead to excitotoxicity . it has been suggested that motor neurons with a high density of serotonergic innervation are more susceptible to degeneration ( sandyk , 2006 ) . as melatonin has antioxidant properties and inhibits glutamate release , this reduction would further exacerbate degeneration . indeed , melatonin supplements slowed disease progression when given to a mouse model of familial als ( weishaupt et al . , 2006 ) .", "normally the scn is coupled to peripheral oscillators , although studies have shown that scn control is not necessary for sustaining oscillatory activity . if signals from central oscillators reduce in strength due to age or neurodegeneration , other cues may entrain the peripheral oscillators ( weinert , 2005 ) . unmasking mechanisms within sleep / wake systems is difficult due to the many checks and balances that ensure homeostasis . although compensation and plasticity occurs to a lesser extent in older animals , a relatively high degree is preserved ( van someren et al . , 2002 ) . this may not always be advantageous as epigenetic methylation of circadian genes has been associated with dementia ( liu et al . , 2008 ) . understanding how the aging brain can compensate and remain plastic will be beneficial to focus on more effective treatments for sleep / wake and neurodegenerative disorders .", "the co - morbidity of sleep disorders with neurodegenerative diseases suggests that changes in many of these neural areas manifests in sleep / wake and circadian dysfunction . effects on the sleep / wake and circadian systems may result from , or contribute to , the increasing pathology . some research has shown the benefit of pharmacologically or behaviorally restoring rhythms and sleep / wake for delaying pathologies ( table 1 ) . this is important to understand in a society where sleep is not considered a priority . a few points worth considering are as follows : sleep is a basic need that is made secondary to work schedules and some leisure activities for many adults . in developing children and adolescents , early school start times and late night extracurricular meetings contribute to a culture of sleep deprived , cognitively unhealthy americans . if restoring our circadian and sleep / wake cycles can ward off the deterioration of the brain , it is imperative to educate the public about the very real damage of abnormal sleep / wake cycles not only in aging individuals but at every age . the fragmented sleep / wake pattern seen in aging individuals can be due to the degeneration or dysfunction of the circadian and sleep / wake networks . uncoupling of the central and peripheral oscillators may exacerbate dysfunction via altered feedback signals or signaling pathways . it is likely that several brain regions are affected and that there are individual differences in how the sleep / wake and circadian networks degrade . additionally there may be differential plasticity and compensation in the integration of these neural systems , making the identification of applicable therapies very difficult . however , if mechanisms contributing to the normal aging process of these networks are identified , this may elucidate a general therapy for restoring sleep / wake and circadian homeostasis . it is crucial that we immediately invest our energies and resources in understanding these mechanisms as well as in the dissemination and implementation of current knowledge and therapies to the public .", "the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest ." ]

[ "it is well known that whole breast irradiation ( wbi ) after breast conserving surgeries for patients with early infiltrating breast carcinoma ( bc ) significantly reduces the likelihood of local recurrence ( lr ) . there are several evidences that lr is a predisposing factor for systemic metastasis [ 24 ] and , within this scope , radiotherapy ( rt ) is very useful for treating residual tumor cells after the surgery . the most used schedule for wbi is 50 gy delivered in 5 weeks using conventional fractionation . there is no consensus , however , regarding whether the entire breast needs to be irradiated . the accelerated partial breast irradiation ( apbi ) concept , based on confining the irradiation to the vicinity of the tumour bed , shortening the course of the treatment and allowing more convenience for patients , has contributed to changes in the rt paradigms [ 6 , 7 ] . a variety of apbi techniques , including low- or high - dose rate brachytherapy , balloon brachytherapy , localized external beam rt ( using either three - dimensional or intensity - modulated ) , and intraoperative electron or photon beam treatments , have been used with encouraging results [ 813 ] . nondedicated linear accelerators ( linacs ) capable of delivering treatment with electrons have been used for intraoperative irradiation of many other tumors [ 14 , 15 ] . these types of equipment are available in almost every rt facility and are used for daily patients ' treatments . the possibility of delivering intraoperative radiotherapy ( iort ) with electrons , without dedicated equipment , is very attractive . addressing this issue , the purpose of our paper was to assess the efficacy , toxicity , and cosmetic outcomes of iort delivered by standard linacs , during breast conserving surgeries for the treatment of early breast cancer .", "a prospective phase ii cohort study started in may 2004 at the sirio libanes hospital in sao paulo , brazil . as of july 2012 , 187 women with diagnosis of bc by percutaneous biopsy were enrolled . patients were eligible if they had unicentric invasive ductal carcinoma , with less than 3.0 cm at the largest diameter confirmed by mammography , ultrasonography , and magnetic resonance imaging ( mri ) . patients were considered ineligible if any of the following features were present : skin involvement , history of bc in the contralateral breast , or intraoperative microscopic findings of involvement of surgical margins or sentinel node ( sn ) . invasive lobular carcinoma subtype was also an exclusion criterion due to its high rate of multicentricity and multifocality . the breast conserving surgeries were performed at an operating theater located inside the radiotherapy department , contiguous to the linac suite . the quadrantectomy consisted of an en bloc resection of the parenchyma and pectoralis fascia , with at least a 2 cm macroscopic margin around the tumor . the skin over the tumor was generally removed by a circular incision , with its conservation being possible in small , deeply located tumours ( t 1.0 cm ) . after verification of clear margins by intraoperative histopathologic and cytologic exams , sn radioguided biopsy was generally performed by the unique breast incision , as previously described [ 16 , 17 ] . as for surgical aspects , the same maneuvers standardized for electron intraoperative therapy ( eliot ) by veronesi et al . once the wide local excision and the sn biopsy were performed , the glandular tissue was detached from the pectoralis major muscle , to an extension of 3 cm margin around the resected area , and the skin flaps were detached from the parenchyma at the level of the adipose lamina for 2 cm circumferentially . the surgical bed was filled with a wet compress , the wound was covered , and the patient was transferred to the rt room , where all of the materials needed for maintaining anaesthesia , including gases , were available . a three - layer disk made of lead ( down ) , aluminium ( middle ) , and silicon ( up ) was inserted underneath the gland over the muscle , to protect the normal tissue below the irradiated area and absorb the backscattered radiation . the shielding disks ( 0.5 cm thick each ) were available in three diameters ( 6 , 8 , or 10 cm ) , and the largest one fitting the space was placed . the parenchyma was approximated over the disk by separated stitches , exposing the area to the electron beam . irradiation was performed using one of two standard models of siemens linear accelerators : primus or kd2 . both machines produce electrons and are able to generate photon and electron beams with energy ranging between 6 and 21 mev . a single total dose of 21 gy prescribed at the 90% isodosis was delivered directly to the parenchyma at a rate of 300 cgy / min . the electron beam energy was chosen after measuring the gland thickness by inserting a needle perpendicularly to the parenchyma . a sterile , round collimator was connected to the gantry of the linac and gently placed into the surgical bed by appropriated mobilization of the couch and gantry ( figure 1 ) . the choice of collimator diameter was made according to each case but was usually up to 6 cm . a portal film was taken placing the film below the accelerator couch , orthogonally to the collimator , to guarantee the exact positioning of the disks . this procedure was repeated , if necessary , until the disk was considered well positioned . afterwards , the staff left the room ; the irradiation was delivered during in average 8 minutes , according to the chosen energy , under video surveillance of the vital signs of the anaesthetized patient ( figure 2 ) . the breast tissue was then reconstructed using oncoplastic techniques , preferentially outside the linac room , in the operating room [ 1921 ] . adjuvant systemic therapy was at the discretion of the physician , in accordance with current guidelines . more than half of the patients received hormone therapy alone ( 51.3% ) , 8.5% of the patients received only chemotherapy , 38.1% had both , and 1.9% patients had no adjuvant systemic therapy . follow - up was performed every 3 months in the first year and every 6 months thereafter . lr was considered as a true relapse ( tr ) , which represents regrowth of residual malignant cells in the same region of the primary tumour , or a second primary tumour ( spt ) , representing tumor growth in another quadrant , suggesting a distinct clonal origin . the presence of seromas , hematomas , fat necrosis , wound infections , and dehiscences was investigated at all time points after surgery . cosmesis evaluation was scored by the physician at least 12 months after irradiation , in accordance with the harvard criteria . briefly , the treated breast is compared with the contralateral one and the result is classified as excellent ( minimal or no difference in the size or shape ) ; good ( mild asymmetry in the size or shape ) ; fair ( obvious differences in the size and/or shape ) ; and poor ( marked change in the appearance involving more than 1/4 of the breast ) . the cumulative incidence of lr , overall survival , and bc survival were calculated using the kaplan - meier method . the spss package version 17.0 ( chicago ii ) medcalc package , 11.3.3.0 version ( mariakerke , belgium ) , was used for statistical analysis .", "of the 187 enrolled patients , 35 ( 18.7% ) were intraoperatively excluded because of sn positivity ( 18 patients ) , difficulty in obtaining clear margins ( 11 patients ) , multicentricity / multifocality ( 3 patients ) , muscle infiltration ( 1 patient ) , t3.0 cm ( 1 patient ) , and no sn identification ( 1 patient ) . the cumulative incidence of lr as the first unfavorable event was 3.2% ( 95% ci : 0.88.1 ) ( figure 3 ) . among the 5 cases of lr in the entire cohort 4 were considered to be a tr , and one had a failure in a quadrant other than the index lesion at 30-month follow - up , consistent with spt . regarding other failures , two patients ( 1.3% ) developed distant metastases , three had axillary failure ( 1.9% ) , and one patient had a contralateral tumor ( 0.6% ) . it is worth noting that among the cases with trs one had sn micrometastasis and one had lobular carcinoma , both identified only in the definitive histopathological analysis . one hundred and nine cases were followed up for at least 36 months with an estimated lr rate of 4.6% . kaplan - meier estimates of efficacy at three years were lr of 4.6% , contralateral breast tumor 0.9% , distant failure 1.8% , cancer specific survival 98.2% , and overall survival 98.0% . the cumulative incidences of first unfavorable events are outlined in table 2 , and the 3-year actuarial rates of recurrences are presented in table 3 . there were three deaths ( 1.9% ) : two related to breast cancer ( one secondary to pulmonary metastasis and another due to chemotherapy toxicity ) and one nononcologically related death . overall survival is shown in figure 4 . in the first month after surgery , 6 cases of skin erythema ( 3.9% ) , 2 wound dehiscences ( 1.3% ) , and 1 case of hematoma ( 1.9% ) were observed . evidence of late toxicity , observed after at least 6 months of follow - up , was seen in 45 patients ( 29.6% ) in a median time of 8 months ( range 824 ) . there were 21 cases ( 13.8% ) of breast fibrosis ( 13 mild and 8 severe ) and 15 cases of fat necrosis ( 9.8% ) . among these cases , there were also 3 cases of breast lymph edema and 2 cases of nipple retraction . the esthetic outcomes have shown 70.3% excellent , 14.4% good , 3.9% regular , and 3.2% of bad results . from the entire cohort , cosmetic outcomes are listed in table 4 . in figure 5 a case with an excellent esthetic result", "breast conserving surgery followed by external wbi is a well established treatment for most women with early infiltrating bc [ 24 , 25 ] . currently , more sophisticated rt techniques are available , allowing better target coverage with better normal tissue sparing . in this context , apbi is a rapidly evolving strategy , with a widespread support for its use [ 7 , 27 , 28 ] . the main biologic rationale for intraoperative partial breast irradiation is that 85% of the lr ( almost 100% of tr ) occurs in the vicinity of the tumour , next to the scar , as a consequence of the persistence of neoplastic cells that most likely possess aggressive cancer stem cell properties [ 29 , 30 ] . experimental data indicate hierarchical organisation of bc with a small number of cancer - initiating cells ( cics ) that have ability to self - renew and exhibit multilineage potential . cics , in contrast to their tumorigenic counterparts , can survive fractions of sublethal doses of rt , retaining self - renewal capacity over several generations [ 3234 ] . some properties of cics could make them a more vulnerable target to a single lethal irradiation dose , soon after the breast resection , without allowing postoperative hypoxia and time for cell repopulation . effects of iort on tumor microenvironment could improve outcomes , as it impairs local proliferation caused by surgical manipulation , inflammation , and simulation of the epithelial mesenchymal transition [ 36 , 37 ] . different rt techniques can be used with this purpose and , given that intraoperative rt with standard linacs has previously been used to treat abdominal tumors , we decided to use this form of treatment during breast conserving surgeries . the surgeries were performed at an operating theatre in the rt department , close to the linac room where the patients were transferred to receive the irradiation . this geographic characteristic by itself turned out to be a feature that helped the better feasibility of the method . however , it is still possible to transport the patient from an operative theatre far from the linac suite ( usually out of business hours ) , previously prepared to be used as an operating room . the patient transport from the operating room to the linac may be regarded as a disadvantage of the method , when compared to the treatment with a dedicated linac . but one must realize that the use of a nondedicated linac , mainly in developing countries , may represent a cost - benefit strategy . we have previously reported our outcomes with focus on technical aspects , and the highlights of the use of nondedicated machine were to explore its capability of producing higher electron beam energies rather than dedicated machines and to check the possibility of misalignment between the collimator and the shielding disks by obtaining portal films using photon beams . other advantages of iort with electrons are accurate targeting of rt and a precise definition of the tumour bed volume under direct guidance , offering very good dose homogeneity and more effectively sparing of the heart and lungs when compared to external beam rt . at the moment there are two published randomised trials focusing on single dose of rt during breast - conserving surgeries . using localized photon beams delivered by the intrabeam device ( carl zeiss meditec , oberkochen , germany ) , concluded that such approach is as efficient as conventional fractioned external beam rt for carefully selected patients . they employed two types of dedicated linear accelerators : novac 7 ( hitesys , latina , italy ) and liac ( info and tech , rome , italy ) . although they found that the rate of lr in the eliot group was within the prespecified equivalence margin of results , it was observed that this rate was significantly greater than with external radiotherapy , pointing out the necessity of defining the optimal patient selection criteria . by far , the most important benefit of iort with electrons is shortening the rt duration from the traditional 5 - 6 weeks to 58 minutes , thereby eliminating the delay in receiving rt , alleviating emotional distress , avoiding logistical difficulties in travelling to the radiation facility and ensuring 100% compliance . the rate of undertreated patientsdue to incomplete fractioned adjuvant wbi is far from ideal , especially in developing countries , being such women exposed to a higher risk of bc recurrence [ 42 , 43 ] . the key feature for the development of eliot by the italian group was the estimation of dose equivalence between the standard 60 gy divided into 30 fractions and the single dose of 21 gy . in a landmark paper , veronesi et al . presented a large phase ii study that included 1,822 cases treated with eliot using dedicated machines . after a mean follow - up period of 36.1 months , 42 women ( 2.3% ) developed a tr , 24 women ( 1.3% ) had a new primary ipsilateral tumour , and 26 women ( 1.4% ) had distant metastases as the first event . compared with conventional rt , eliot was considered a safe procedure for women with tumours measuring less than 2.5 cm , with a slightly higher lr rate . our results presented here are very similar to the results obtained by veronesi et al . the widespread use of apbi motivated the american society of therapeutic radiology and oncology to define a suitable group of patients for whom apbi is acceptable outside of clinical trials , including the following : women older than 60 years , with t1 idc , clear margins , and the absence of multicentricity , multifocality , and axillary nodes involvement . the european society for therapeutic radiology and oncology also proposed suitable conditions for apbi : age 50 years , unicentric and unifocal t1 - 2 ( 3.0 cm ) , pn0 nonlobular invasive cancer , the absence of an extensive intraductal component and lymphovascular invasion , and negative surgical margins of at least 2 mm . currently it is also known that estrogen receptor negativity is associated with increased risk of lr following apbi . this study has started before the publication of these recommendations , and part of our cases should be considered not suitable for apbi . however , some other results have pointed out that even patients who do not meet the ideal conditions may be locally treated with success [ 47 , 48 ] . anyway , since the publication of the recommendations ( 2009 ) , women under 50 years of age were no longer accepted in our study . although it might be tempting to offer iort to a large number of patients , at this time , a careful selection of suitable patients is paramount . for this reason we advocate preoperative mri which was performed in all of our patients , to better select the cases for partial breast irradiation . most likely , the traditional wbi reduces the rate of spt in the treated breast solely if they were present and occult at the time of the primary treatment . mri could potentially contribute to the more precise detection of multifocal or multicentric disease , with improvement of operative outcomes and decreased recurrence rates , although , besides the mri high diagnostic accuracy , it is always desirable to have pathological verification of the findings because of the mri high false - positive rates . the confirmation of intraoperative clear surgical margins is also mandatory , since the objective of iort is to reduce lr by treating residual malignant cells that may persist in tumour - bearing areas . with regard to efficacy moreover , complications due to local toxicity were scarce , and this form of iort led to a favorable impact on body image , as already observed by other authors . also , when oncoplastic maneuvers are required , including immediate breast reconstruction with prostheses , they are feasible and safe [ 19 , 21 ] . we consider as limitations of this study the facts that there was not a control group and that it was performed at a single institution on relatively small number of patients . in spite of these caveats , the technique was demonstrated to be feasible and was successfully implemented , with a very short learning curve . iort with electrons delivered by conventional linacs , immediately after a wide local excision , presented the expected results until now , with very good local control and cosmetic outcomes and a low toxicity rate . selected patients with early infiltrating breast carcinomas may benefit from the technique , which may represent an interesting option for developing countries ." ]

[ " heart disease , including coronary heart disease , cardiomyopathy , and heart failure , causes functional deterioration and/or failure as well as myocardial cell death . it is one of the leading causes of death in advanced countries because adult cardiomyocytes are highly differentiated and have a limited regenerative capacity ; therefore , significant loss of myocardium is mostly irreversible . cell regeneration therapy is a promising new approach for myocardial repair , [ 1 , 2 ] and in this context , there has been considerable basic research on the mechanisms of cell development into cardiomyocytes using somatic and embryonic stem ( es ) cells as well as embryonic carcinoma cells [ 36 ] . p19 embryonic carcinoma cells are one of the first among such cells to demonstrate differentiation into cardiomyocytes and have contributed extensively to the elucidation of the development mechanisms from stem cells into cardiomyocytes [ 4 , 5 , 8 ] . p19 cells are derived from a teratocarcinoma in ch3/he mice and can differentiate into all 3 germ layers . culture and differentiation of the cells is simple , and this advantage has enabled their extensive application for decades . they continuously grow in serum - supplemented media , can retain an undifferentiated cell state without a feeder cell layer unlike es cells , and their differentiation can be controlled by nontoxic reagents . primarily , cell aggregate formation in suspension culture under 0.51.0% dimethyl sulfoxide ( dmso ) followed by the reagent application in adhesion culture has been used to induce cardiomyocyte differentiation of p19 cells [ 7 , 8 , 10 ] . we examined the differentiation of p19 cells into cardiomyocytes by the above general method ; however , it resulted in large variations in the differentiation rate and low differentiation efficiencies among individual experiments , even though high differentiation efficiencies have been reported previously [ 7 , 8 , 10 ] . p19cl6 cells , clonal derivatives of p19 cells , were established by habara - ohkubo . these subline cells can be differentiated into spontaneously beating cardiomyocytes by treatment with 1% dmso in adhesion culture over a period of 10 days or weeks without cell aggregate formation in suspension culture and more efficiently as compared to the parent cells . therefore , p19cl6 cells may be more useful to examine the differentiation mechanisms of cardiomyocytes in vitro . recently , ohtsu et al . introduced a double stimulation method for cardiomyocyte differentiation from p19cl6 cells . they demonstrated that cells exposed to 10 m 5-azacytidine ( 5-aza ) for 24 hours prior to treatment with 1% dmso differentiated into cardiomyocytes more effectively than the widely used single stimulation method described above . in order to produce a large number of cardiomyocytes , we induced differentiation of p19cl6 cells into cardiomyocytes by the double stimulation method to examine the differentiation efficiency ; however , it resulted in large variations in the differentiation rates among individual experiments , as in the case of p19 cells . however , notably , the double stimulation method elicited differentiation from p19cl6 cells most efficiently among the several differentiation methods as long as it was in vitro in our laboratory . we also noticed large variations in the differentiation rates among previous reports on p19cl6 [ 1214 ] as well as p19 cells [ 8 , 10 , 15 , 16 ] and this may imply a fundamental problem of the cells in themselves and/or the differentiation methods . to overcome this difficulty , we attempted to establish a subline from p19cl6 cells , and accordingly , we could introduce a new subline of cells , that is , p19cl6-a1 cells , that could efficiently and stably differentiate into cardiomyocytes by a differentiation method based on the double stimulation method with many modifications . in the present study , we also introduce a software program , visorhythm , which can analyze the temporal variations in the beating rhythms on moving images and chart correlograms displaying the oscillated rhythms on a windows computer . because the spontaneously beating cardiomyocytes differentiated from p19cl6-a1 cells form large cell clusters , their contractions are large and strong . this feature may prove to be quite suitable for oscillated rhythm analysis based on moving images , and accordingly , we used this software to successfully display the increase in the cardiomyocyte beating rates on correlograms and dose - response curves on treatment with serially diluted cardiotonic reagents . this may indicate that p19cl6-a1 cells and the above software are useful tools for pharmacological screening tests .", "balb / c mice of either sex maintained under standard housing and feeding conditions were used . the ventricles of the hearts dissected from mice aged 8 weeks and 16.5 days post coitum ( dpc ) fetuses were examined by reverse transcription pcr ( rt - pcr ) . the cells were cultured in a tissue culture - grade dish and grown in alpha - modified eagle 's minimal essential medium ( -mem ; sigma - aldrich japan k.k . , tokyo ) supplemented with l - glutamine ( 4 mm ; gibco brl , carlsbad , ca ) or dulbecco 's modified eagle 's minimal essential medium ( dmem ; sigma - aldrich japan k.k . ) containing 510% heat - inactivated fetal bovine serum ( fbs ; jrh biosciences , lenexa , ka ) and penicillin and streptomycin ( 100 u / ml and 100 g / ml , resp . ; sigma - aldrich japan k.k . ) . several subclones were isolated from the p19cl6 cells by the common cloning ring technique . each clonal colony was expanded increasingly into larger wells and dishes and frozen until use . two types of cell colonies were derived from a single cell : one , growing horizontally to form a single cell layer on a dish and the other , growing both horizontally and vertically to form cell aggregates of multicell layers like embryoid bodies or cell aggregates in suspension culture prepared using bacterial - grade dishes [ 5 , 8 , 13 ] . both cell types were collected for the cardiac differentiation experiment ; however , the latter were considerably more efficient in differentiating into cardiomyocytes . among the several differentiation methods , the one proposed by ohtsu et al . elicited most efficient cardiomyocyte differentiation from parent p19cl6 cells as long as the experiment was in vitro in our laboratory ; however , it also led to a fundamental difficulty , that is , high variations in the differentiation efficiencies among individual experiments . hence , we modified the above method in order to adapt it to our subclonal cells as well as to the parent cells . the differentiation conditions that we primarily considered were the concentration and/or exposure duration of the differentiation reagents , the concentration of fbs in the culture medium ( -mem or dmem ) , and coating reagents . they were as follows : 5-azacytidine ( 5-aza ; sigma - aldrich japan k.k . ) : concentration : 5 , 10 , 15 , 20 , 30 , and 40 m and exposure duration : 24 , 48 , and 72 hours ; dimethyl sulfoxide ( dmso , sigma - aldrich japan k.k . ) : concentration : 0.5 , 1.0 , 1.5 , and 2.0% ; fbs in the differentiation medium : concentration : 2.5 , 5.5 , 6.5 , 7.5 , and 10% ; and the use of a culture dish coated with or without collagen ( 3.0 g / cm , type i - a ; nitta gelatin inc . , osaka ) or matrigel ( 3.0 g / cm ; bd biosciences , san jose , ca ) . accordingly , we found a subclone , designated a1 , which is the most efficiently differentiated into cardiomyocytes under the differentiation conditions below and used it for further experiments to evaluate the differentiation efficiency . we also found the optimum conditions that could be applied to the parent p19cl6 cell line , and the use of these conditions yielded almost the same results as those yielded by the method of ohtsu et al . ; the description has been provided below . the differentiation efficiencies of the subline , p19cl6-a1 , and the parent cell line , p19cl6 , were compared . the cells were plated at a density of 5.0 10 cells / well on 6-well plates or on a 35-mm tissue culture - grade dish coated with collagen ( 3.0 g / cm ) and containing the growth medium ( -mem with 6.5% fbs for p19cl6 cells or dmem with 7.5% fbs for p19cl6-a1 cells ) and were incubated in a 5% co2 atmosphere at 37c . the next day , when cells reached ~95% confluence , the medium was replaced with a growth medium containing 10 m 5-aza in order to induce differentiation . the p19cl6 and p19cl6-a1 cells were treated with 5-aza for 24 and 72 hours , respectively , and the 5-aza - containing medium was changed every 24 hours . after treatment with 5-aza , the cells were incubated in the growth medium containing 1.0% dmso for more than 16 days . the dmso - containing medium was also changed everyday in order to remove the cell debris resulting from cell death . the experimental days were numbered consecutively beginning from the day after the first day of treatment with 5-aza ( day 0 ) . p19cl6 and p19cl6-a1 cells were cultured on glass coverslips coated with collagen ( 3.0 g / cm ) . cells on day 16 were washed twice with hanks ' balanced salt solution ( hbss ; sigma - aldrich japan k.k . ) and then fixed with 4% paraformaldehyde in pbs for 30 minutes at 4c . after the fixation , cells were rinsed twice in cold pbs , treated with 0.1% triton x-100 in pbs for 20 minutes at room temperature and again rinsed twice in pbs . in order to reduce autofluorescence , cells were treated with 50 mm nh4cl in pbs for 10 minutes at room temperature . then cells were blocked in a humid chamber with pbs containing 3% normal goat serum ( ngs ; sigma - aldrich japan k.k . ) for 30 minutes and then incubated with the anticardiac -actin antibody ( sigma - aldrich japan k.k . ; 10 g / ml in pbs containing 3% ngs ) for 16 hours at 4c . after washing with pbs , cells were incubated with alexa488-conjugated goat antimouse igg ( molecular probes , inc . , eugene , or ) for 30 minutes at room temperature , cells were photographed with fluorescence microscopes ( ix71 , olympus co. , tokyo ) . the specificity of the immunofluorescence staining was verified by incubations without the primary or secondary antibodies and no specific fluorescence was observed in the control . for quantitative analysis of rates of differentiation into cardiomyocytes , we measured ratios of cardiac -actin - positive cell areas to total areas of microscopic fields using image j software ( ver . 1.41 ; national institutes of health , bethesda , md ) . autofluorescence from multilayer cell regions was rather strong but -actin - specific fluorescence was easily discriminated from the autofluorescence because -actin - positive cells emerged as high - intensity cells in cell clusters ( see figure 2 ) . in this way we counted out autofluorescence regions . sixty fields were examined in each cell type and the results from six independent experiments were summarized as mean se . unpaired t - test was carried out to compare the differentiation efficiency between the two cell types . total rna was isolated from the cultured p19cl6 and p19cl6-a1 cells on days 0 , 3 , 7 , and 16 , and from the cardiac ventricles of 8-week - old adult mice and 16.5 dpc fetuses using the trizol reagent ( invitrogen japan k.k . , one microgram of total rna was transcribed into first - standard cdna using m - mlv reverse transcriptase , rnase h ( promega , madison , wi ) , and oligo ( dt ) primer according to the manufacturer 's instructions . for detection of gata4 and nkx2 - 5 , the specific transcription factors for cardiac differentiation , and of the alpha myosin heavy chain ( -mhc ) , which is indispensable for cardiomyocyte contraction , 1 l of the reaction mix ( out of the total 25 l ) as the reverse - transcribed dna template was amplified by pcr40 cycles for gata4 and nkx2 - 5 and 25 cycles for -mhc . after amplification , the pcr products were separated on 1.2% agarose gel and visualized by ethidium bromide staining . the expression levels of gata4 , nkx2 - 5 , and -mhc mrnas were compared between p19cl6 and p19cl6-a1 . they were determined from 3 independent experiments and normalized by reference to the expression levels of gapdh mrna ( 23 cycles of pcr ) . all analyses were performed on excel 2007 . the values were expressed as the mean se that was represented by ratios to the mean values of the p19cl6 cells in the basal protocol on day 16 as 1.0 according to the differentiation protocol . the following primer pairs were used : gata4 , 5-gttgtggtggtgggtttttc-3 ( forward ) and 5-tttgatgttcctgggagagg-3 ( reverse ) ; nkx2 - 5 , 5-tctccgatccatcccactttattg-3 ( forward ) and 5-ttgcgttacgcactcactttaatg-3 ( reverse ) ; -mhc , 5-tacctcatggggctgaactc-3 ( forward ) and 5-cgaacatgtggtggttgaag-3 ( reverse ) ; gapdh , 5-gacttcactcacggcaaatt-3 ( forward ) and 5-tcctcagtgtagcccaagat-3 ( reverse ) . the 35-mm dish with the differentiated cells were placed in a stage top microscope incubator ( oni - inu - f1 ; tokai hit , shizuoka , japan ) mounted on an inverted microscope ( dmirb , leica microsystems ltd . , heerbrugg , switzerland ) and incubated at 37c in a 5% co2 atmosphere . phase - contrast moving images of spontaneously beating cells were recorded through a ccd camera ( coolpix4500 ; nikon co. , tokyo ) and digitalized to mpeg2 video files ( 8 bits / channel ; 704 pixels 480 pixels / frame ; 30 frames / sec ) using an encoder ( mtu2400 fx ; canopus co. ltd . , the contraction rhythm of the spontaneously beating cells was analyzed based on the moving images using the following method based on the data analysis algorithm on the calculation of correlation coefficient as reported by yamauchi et al . . a small area of interest where brightness clearly oscillated due to the beating was selected from a moving image , and then , a reference frame was arbitrarily chosen from the moving image frames . temporal variations in the correlation coefficient were calculated between the values of mean brightness of the reference frame and those of other frames in the selected area , and accordingly , a correlogram representing the contraction rhythm of the beating cells in the area of interest was displayed by the vertical coefficient and horizontal time axis . we developed a software program , visorhythm , which calculates the temporal variations in the correlation coefficient up to 6 rectangle areas ( 150400 pixels / area ) simultaneously on a single moving image on a windows computer . the data can then be directly output into excel to chart the correlograms , and the beat - to - beat intervals are represented by the intervals between the upward and downward peaks in each correlogram in the selected areas . we examined the pharmacological reactions of spontaneously beating cardiomyocytes differentiated from the p19cl6-a1 cells to evaluate whether they are useful cellular tools for pharmacological screening tests . we used 3-isobutyl-1-methylxanthine ( ibmx ; sigma - aldrich japan k.k . ) and ouabain ( sigma - aldrich japan k.k . ) , which are well - known potent cardiotonic reagents , for this purpose . phase - contrast moving images of spontaneously beating cells were recorded before and after addition of serial dilutions of each reagent ( 10 nm , 20 nm , 70 nm , 200 nm , 700 nm , 2 m , 7 m , 20 m , and 70 m ) into the medium . the beat - to - beat intervals on exposure to certain concentrations ( 10 nm , 30 nm , 100 nm , 300 nm , 1 m , 3 m , 10 m , 30 m , and 100 m ) were obtained from the correlograms calculated on visorhythm . the median effective dose ( ed50 ) was obtained from the dose - response curve based on the beating rates on which the basal and maximum rates were represented by 0 and 100% , respectively .", "we selected the differentiation protocol suggested by ohtsu et al . for the p19cl6 cells because this method was the most efficient for cardiomyocyte differentiation from among the several differentiation methods as long as it was in vitro in our laboratory . the undifferentiated populations of the cells , however , considerably proliferated even in the differentiation medium containing 1% dmso as a differentiation inducer and 10% fbs ; hence , we reexamined the optimum concentration of fbs . we thus obtained a resultant concentration of approximately 6.5% , and accordingly , the protocol for the cells was as revised as follows . subconfluent p19cl6 cells in -mem containing 6.5% fbs were treated with 10 m 5-aza for 24 hours followed by treatment with 1% dmso for more than 16 days ( basal protocol ) . in case of the p19cl6-a1 cells , we modified the basal protocol by reexamining the conditions in each step according to the criteria of ( 1 ) rates of appearance of the beating cells per field and ( 2 ) dimensions of the beating area under a microscope . the cells showed a relatively high resistance to 5-aza , and the optimum conditions were as follows . subconfluent p19cl6-a1 cells in dmem containing 7.5% fbs were treated with 10 m 5-aza for 72 hours followed by treatment with 1% dmso for more than 14 days ( developed protocol ) . we also examined the differentiation efficiencies in culture dishes coated with or without collagen or matrigel and found the differentiation to be more effective on collagen coated dishes . the conditions for each protocol and for each cell type are summarized in table 1 . we observed the differentiation processes under an inverted microscope to compare the morphological differences between the p19cl6 and p19cl6-a1 cells . both cell types grew similarly in the beginning ( days 05 ) of the differentiation protocols . the p19cl6 and p19cl6-a1 cells reduced in number during treatment with 5-aza due to the damage by the reagent and proliferated slightly everyday thereafter during treatment with dmso . the cells started to grow vertically due to overconfluence , and multilayer cell regions appeared around day 5 . on the p19cl6 cells multilayer cell regions were generally small and appeared like scattered islands in monolayer cell regions . however , on p19cl6-a1 cells , they were connected with each other to form mesh - like structures and expanded to comprise approximately 7080% of the total area of the dishes during the procedure . this feature of the multilayer cell regions was similar to that of embryoid bodies or aggregates on culture with dmso in the nonadhesive bacteriological - grade dishes . beating cells first appeared among the p19cl6 cells differentiated by the basal protocol on day 10 and among the p19cl6-a1 cells differentiated by the developed protocol on day 11 . the clusters of beating cells increased in number and size thereafter and peaked on day 16 in both cases . the beating cell clusters from the p19cl6 cells were observed generally in the monolayer cell regions and less frequently on the boundary between the monolayer and multilayer regions ( figure 1(a ) ) . the clusters , composed of small - sized beating cells densely arranged , were generally small in the monolayer regions ( figures 1(c ) and 1(e ) ) . on the other hand , the beating cell clusters in the p19cl6-a1 cells appeared mostly in the multilayer and boundary regions ( figure 1(b ) ) . they were composed of elongated cells and formed mesh - like sheet structures ( figures 1(d ) and 1(f ) ) . they were relatively large and sometimes extended beyond the visual field under the inverted microscope through a 10 objective lens , and accordingly , the contractions were large and strong . therefore , the beating cells differentiated from the p19cl6-a1 cells on day 16 or later were suitable for contraction rhythm analyses based on moving images . in order to morphologically identify cardiomyocytes differentiated from p19cl6 and p19cl6-a1 cells clearly and to compare the differentiation efficiencies between the two , we immunocytochemically examined those cells on day 16 using anticardiac -actin antibody under immunofluorescence microscopy . cardiac -actin - positive cells from p19cl6 were various in shape ( oval , polygonal , short spindle - shaped ) and generally formed round clusters ( figures 2(a ) and 2(c ) ) . those from p19cl6-a1 were relatively large and elongated , and formed mesh - like sheet structures ( figures 2(b ) and 2(d ) ) . we measured ratios of cardiac -actin - positive cell areas to total areas of microscopic fields ; the positive cell areas were 7.0 1.1% ( mean se ) of the total areas on p19cl6 while those were 38.3 5.7% on p19cl6-a1 . the value on p19cl6-a1 was 5.5 times and significantly higher than that on p19cl6 ( p = .002 ; figure 2(e ) , table 1 ) . to compare the differentiation efficiencies between the p19cl6 and p19cl6-a1 cells , we examined the expression of the cardiac - specific transcription factors , gata4 and nkx2 - 5 , and of the cardiac - specific myosin heavy chain , -mhc , by rt - pcr on the respective cells with the respective differentiation protocols listed in table 1 on days 0 , 3 , 7 , and 16 . both gata4 and nkx2 - 5 were already expressed in both p19cl6 and p19cl6-a1 cells on day 0 , that is , in the untreated cells ( figure 3(a ) ) . the expression of the former increased from day 0 to day 16 during treatment in both groups ( figure 3(b ) ) , while that of the latter dipped on day 3 , but increased thereafter ( figure 3(c ) ) . on day 16 , when the appearance rates of the beating cells per field and the dimensions of the beating area peaked as observed under a microscope , gata4 expression did not differ significantly between the p19cl6 and the p19cl6-a1 cells ; however , nkx2 - 5 expression in the latter was significantly and 1.45 times higher than that in the former ( figure 3(c ) ) . alpha - mhc was not expressed till day 7 , although it was clearly expressed on day 16 in both groups ( figure 3(d ) ) . its expression in the p19cl6-a1 cells was significantly and 1.77 times higher than that in the p19cl6 cells ( figure 3(d ) , table 1 ) . these results indicated that p19cl6-a1 cells were more efficient than the parent in terms of cardiomyocyte differentiation . we also examined -mhc expression in the ventricles of 8-week - old adult mice and 16.5 dpc fetuses by rt - pcr to compare -mhc expression in the tissues with that in the p19cl6-a1 cells differentiated by the developed protocol on day 16 . the expression levels were higher in the adult tissue than in the other tissue , but were almost equal between the fetal tissue and the cells on day 16 ( figure 3(e ) ) . we examined the pharmacological reactions of the spontaneously beating cells differentiated from p19cl6-a1 cells by the developed protocol . for this purpose , we employed ibmx and ouabain as cardiotonic reagents , and both of them increased the beating rates . the moving images of the beating cells were recorded before and after addition of serial dilutions of each reagent . to determine the temporal variations in the beating rhythms in detail , we developed the software , visorhythm , to analyze them based on moving images and chart correlograms displaying the oscillated rhythms . figure 4(a ) is an example of a series of correlograms corresponding to the temporal variations in the contraction rhythms of the beating cells treated with 10 nm , 300 nm , and 10 m ; the beating rate was 1.30 times per second at the basal level and increased up to 2.30 times per second after the addition of 10 m ibmx . figure 4(b ) shows the dose - response curve to ibmx corresponding to the data in figure 4(a ) , and its ec50 was 950 nm . figures 4(c ) and 4(d ) illustrate another series of correlograms and a dose - response curve to ouabain , respectively . these data indicate that the p19cl6-a1 cells and visorhythm are useful tools for pharmacological screening tests .", "p19 embryonal carcinoma cells have been extensively studied for cardiac differentiation in vitro and have contributed to the elucidation of the early events in differentiation , such as the involvement of bone morphogenic proteins and their subsequent intracellular signal cascades and oxytocin and its subsequent activation of the transcription factors gata4 , nkx2 - 5 , and so on [ 5 , 7 , 8 , 10 , 19 ] . for their efficient differentiation into cardiomyocytes , prior formation of cell aggregates in suspension culture under 0.51.0% dmso followed by reagent application in adhesion culture is required ; however , molecular events occurring during aggregation and the necessity of their aggregation for differentiation have not yet been entirely understood . high cell densities at least can trigger spontaneous differentiation from p19 cells without the formation of cell aggregates and reagent treatment [ 9 , 10 ] . on the other hand , p19cl6 cells , the clonal derivative of the p19 cells , efficiently differentiate into cardiomyocytes on dmso treatment in adhesion culture without prior formation of cell aggregates , although high cell densities are required for effective differentiation . in the present study , we introduced p19cl6-a1 , a clonal derivative of p19cl6 cells , which differentiated into cardiomyocytes most efficiently among the 3 cell lines subjected to double stimulation with 5-aza and dmso in adhesion culture as reported by ohtsu et al . the p19cl6-a1 cells extensively formed multicell layers in adhesion culture under stimulation with dmso after they reached overconfluence . they were highly packed in multicell layers like the cell aggregates of p19 cells in suspension culture . p19cl6-a1-derived cardiomyocytes , which generally formed large clusters of beating cells , almost always appeared in the multilayer regions and on the boundary regions adjacent to the monolayer regions . these findings , in addition to the above differentiation characteristics in the other 2 cell types , may indicate that cell - to - cell contact all around , that is , not only from side to side but also from top to bottom , is a key event in the differentiation of p19 cells and their derivatives . because three - dimensional contact can be achieved by the formation of multicell layers , a clonal derivative differentiating more efficiently into cardiomyocytes than p19cl6-a1 may be established when the dimensions of the multilayer regions is employed as a criterion for clonal selection from p19cl6 cells . further investigations are necessary to elucidate whether multilayer formation is a crucial trigger for differentiation and to determine the molecular events occurring in the multilayer regions in this instance . we employed the double stimulation method using 5-aza and dmso as suggested by ohtsu et al . , as the basal protocol for cardiomyocyte differentiation because it elicited differentiation from the parent p19cl6 cells most efficiently among the several differentiation methods as long as it was in vitro in our laboratory . to apply it to the clonal derivative , that is , to p19cl6-a1 cells , we examined the optimum concentration and exposure time of the inducers and accordingly , the protocol for efficient differentiation was modified as summarized in table 1 . the optimum concentration of each inducer is the same as that in the basal protocol , but the exposure time of 5-aza is 3-fold longer than that for the parent cells . an almost equal and fair percentage of p19cl6 and p19cl6-a1 cells were necrotic after treatment with 10 m 5-aza for 24 and 72 hours , respectively , in the monolayer culture , and therefore , the p19cl6-a1 cells were more resistant to more prolonged exposure to the dna demethylating reagent than the p19cl6 cells . both cell types were much more resistant to the reagent than the p19 cells because the p19 cells treated with 510 m 5-aza in the monolayer culture became totally necrotic . in the present study , dmem with 7.5% fbs of the composition of the differentiation medium elicited differentiation from both p19cl6-a1 and p19cl6 cells more efficiently than -mem with 6.5% fbs and much more than -mem with 10% fbs ( data not shown ) , which has been generally used for the differentiation of p19 and p19cl6 cells [ 7 , 1115 , 2026 ] . after the 5-aza treatment , p19cl6-a1 cells proliferated slightly under dmem containing 1% dmso and 7.5% fbs and could form multicell layers in adhesion culture more extensively than p19cl6 cells under -mem containing 1% dmso and 6.5% fbs . p19cl6-a1-derived beating cells appeared mostly in the multilayer regions and formed clusters more extensively in the multilayer regions than in monolayer regions . these may indicate that more extensive multilayer formation induces the cardiomyocytes to differentiate more efficiently . however , using 10% fbs instead of 7.5% or 6.5% could not elicit more efficient differentiation of the cells probably due to stable proliferation of the undifferentiated populations in the cells even in the differentiation media containing 10% fbs . in the present study , we found that 1% dmso is optimum for the differentiation of p19cl6 and p19cl6-a1 cells . it is generally used for differentiation of p19 and p19cl6 cells [ 1115 , 20 , 2226 ] . moreover , a previous report has demonstrated that the efficiency of colony formation of p19 cells in the presence of dmso indicates the absence of toxicity at concentrations of less than 1% under -mem with 10% fbs . based on these data , we suppose that the serum concentration in combination with 1% dmso is a considerable factor for efficient differentiation . in fact , the source and lot number of fbs and calf serum used in differentiation are thought to be crucial for successful cardiac differentiation from p19 cells as well as from p19cl6 cells ( riken bioresource center , personal communication ) . in the present study , cardiac - specific transcription factors gata4 and nkx2 - 5 were expressed in untreated cells . in most previous reports p19cl6 cells untreated with inducers such as dmso and 5-aza expressed neither gata4 nor nkx2 - 5 [ 12 , 14 , 20 , 22 , 24 , 27 ] . however , there are a few reports in which gata4 and/or nkx2 - 5 expressed in p19cl6 cells before the treatment [ 23 , 26 ] . in the present study we used a medium containing 10% dmso for cell cryopreservation and cells cultured for 1 day after thawing as untreated cells ( day 0 ) for the experiments . we suppose this caused the clear expressions because the untreated cells contain cells already exposed to the dmso as a cardiac differentiation inducer probably at a high rate . there were no differences in the differentiation efficiencies under an inverted microscope between p19cl6-a1 cells with and without subcultures after thawing ( data not shown ) and the same finding was obtained on p19cl6 cells ( dr . there are 2 isoforms of cardiac mhcs , namely - and - mhc . the former is characterized by a high atpase activity and a quick contraction velocity as compared to the latter . alpha- and/or - mhc are often used as cardiac - specific markers for determining the efficiencies of cardiac differentiation of p19 and p19cl6 cells [ 8 , 11 , 13 , 14 , 20 , 25 ] . in the present study , we showed that the expression level of -mhc mrna in the p19cl6-a1 cells treated with the inducers was significantly and 1.77 times higher than that in the p19cl6 cells and almost equal to that in the heart ventricles of the mouse fetuses . we also compared the differentiation efficiency between the two cells by using cardiac -actin immunofluorescence staining and showed that the ratio of cardiac -actin - positive cell areas to total areas of microscopic fields in p19cl6-a1 cells was 38.3% and 5.5 times higher than that in p19cl6 cells . this indicates that the differentiation efficiency estimated by the immunofluorescence staining is 3.1 times higher than that by the rt - pcr . this discrepancy is likely to be caused by the multicell layer formation of p19cl6-a1 cells ; the two - dimensionally microscopic analysis may overestimate the differentiation efficiency due to the elimination of a factor in rate of formation of multicell layers and the estimation by the rt - pcr may represent the differentiation efficiency close to a real value . p19- as well as p19cl6-derived cardiomyocytes display an electrophysiological phenotype of embryonic ventricular cardiomyocytes based on the studies in which ( 1 ) they display a complete set of functional ion channels , but only limited amount of functional sodium channels , ( 2 ) the maximal diastolic potential is rather low ( approximately 40 to 60 mv ) , and ( 3 ) the shape and characteristics of the action potential of these cells resemble those of primary isolated embryonic cardiomyocytes [ 5 , 21 , 24 , 29 ] . thus , p19cl6-a1-derived cardiomyocytes may have similar properties because they are clonal derivatives of p19cl6 cells . they rhythmically and spontaneously beat and form clusters much larger than p19cl6-derived cardiomyocytes , and accordingly their contractions are larger and more obvious as observed under an inverted microscope . therefore , p19cl6-a1-derived cardiomyocytes may be suitable models for studies on the cardiac structural and functional properties during normal physiological and pathological states in vitro , particularly studies on contraction rhythm analyses based on moving images . for this purpose , that is , to figure out the temporal variations in the beating rhythms in detail , we developed the software , visorhythm , to analyze them based on moving images and chart correlograms displaying the oscillated rhythms . to illustrate the availability of p19cl6-a1-derived cardiomyocytes as physiologically functional cardiomyocytes and the software as an analytical tool for detailed analysis of beating rhythms , we employed ibmx and ouabain as potent cardiotonic reagents [ 30 , 31 ] . additionally , we found that the nonbeating cells started to contract rhythmically after the addition of these reagents ( data not shown ) . this finding is supported by a previous study in which p19-derived nonbeating cells generated action potentials upon electrical stimulation and indicates that the actual ratio of p19cl6-a1-derived cardiomyocytes is higher than that of the beating to nonbeating cells under the microscope . there are largely two different methods to analyze beating rhythms of cardiomyocytes in vitro : one is an electrophysiological method to measure electrical potential of cardiomyocytes and the other is a microscopic method to measure oscillated motions of cardiomyocytes . as for the former method , a culture plate equipped with microelectrode arrays having 64-channels are used to record electrical potentials on cultured cardiomyocytes [ 32 , 33 ] . it can analyze electrical activities of cardiomyocytes such as conduction velocities and synchronization times . as for microscopic methods , micheletto et al . introduced a free - running scanning near - field optical microscopy setup to observe live cardiomyocytes . weisensee et al . also introduced an image analysis system using the video capture board for real - time subtraction between frames of moving images to analyze motions of beating cardiomyocytes . both tools can analyze beating rates and the relative strength for inotropic force while they monitor only one object at a time . here we have introduced visorhythm to analyze temporal variations in beating rhythms of cardiomyocytes based on moving images and chart correlograms displaying the oscillated rhythms . yamauchi et al . had developed a similar software on which the temporal variations in beating rhythms could be analyzed in 1 fixed area ( 20 20 pixels ) chosen arbitrarily . in contrast , visorhythm can analyze up to 6 such areas ( 150400 pixels / area ) on each frame of one moving image simultaneously . therefore it can also analyze synchronization between cardiomyocytes . in conclusion , we used double stimulation with 5-aza and dmso in this study and showed that p19cl6-a1 cells , a new clonal derivative of p19cl6 cells , differentiated into cardiomyocytes more efficiently than the parent cells . we introduced a new software , visorhythm , that can analyze the temporal variations in the beating rhythms and can chart correlograms displaying the oscillated rhythms up to 6 areas on a single moving image simultaneously . using p19cl6-a1-derived cardiomyocytes and the software , we demonstrated that the correlograms could clearly display the enhancement of beating rates by cardiotonic reagents . these indicate that a combination of p19cl6-a1 cells and visorhythm is a useful tool that can provide invaluable assistance in inotropic drug discovery , drug screening , and toxicity testing ." ]

[ "single incision laparoscopic surgery ( sils ) for cholecystectomy procedure has been introduced as early as 1999 to achieve less pain , less scarring , and less hospitalization period . one of the major difficulties of this procedure seems to be the traction of the gallbladder in order to expose the tissues during operation without additional ports . the use of transabdominal 20 nylon sutures attached to keith needles and the use of a kirschner wire hook introduced through subcostal area are reported methods used for traction of gallbladder and better exposition of the calot triangle . however , all these techniques consist puncture of the gallbladder with sharp needles especially if it is distended and carry the risk of bile leakage and contamination afterwards . herein , we describe our technique in establishing single port access for cholecystectomy in five patients that involves the use of a 2 mm in diameter grasper , karl storz 27290f , that is generally used by urologists for percutaneous nephrolithiasis intervention .", "after induction of general anesthesia , an umbilical skin incision , 1.5 cm in length , is made vertically and a covidien sils port having 5 mm12 mm four holes is inserted to the peritoneal cavity under direct vision and pneumoperitoneum is created . thereafter , a minigrasper , karl storz 27290f , 2 mm in diameter ( figure 1 ) , was inserted thoroughly an incision , 2 mm in length , created at the transaction point of the midclavicular vertical line and umbilical horizontal line ( figure 2 ) . it is used to hold the fundus of the gallbladder gently and push the organ in an cephalad direction to visualize calot 's triangle ( figure 3 ) . this atraumatic device helped to mobilize gallbladder whenever needed during surgery without causing any wound or leakage . then , the dissection of the gallbladder was completed in a standard fashion from bottom to top and after the organ was dissected free from the liver , it was removed directly through the umbilical incision . no complications including gas leakage around the minigrasper were encountered during and after the surgery . followup was done in the first month and minimal scarring was observed in the umbilicus and the minigraspers ' small incision was almost invisible .", "single incision laparoscopic cholecystectomy ( silc ) was first described by navarra et al . in 1997 , , silc technique has been performed by many clinicians with promising results and many advantages like few complications , acceptable operation period , less scarring , less pain , and quicker recovery . there are many technical challenges in silc surgery as one of them seems to be the retraction of the fundus cephalad and traction of the hartmann 's pouch laterally to ensure safe cholecystectomy . for this purpose , some surgeons use transabdominal 20 nylon sutures attached to keith needles for fixation and assist gallbladder retraction and some other used kirschner wire hook which is introduced through subcostal area to pull gallbladder in an upright direction during the operation . yet , all these techniques consist puncture of the gallbladder with sharp needles especially if it is distended and carry the risk of bile leakage and contamination afterwards . they also have limited ability to mobilize whenever needed . to achieve gallbladder retraction and cephalad traction , this minigrasper is used not only in performing sufficient traction of the gallbladder in an upright position , but also it can allow good mobilization of the gallbladder in order to dissect calot 's triangle safely . use of this instrument has led no perforation and leakage of bile in the patients . through a very small incision , 2 mm in length , without using any port , it has been introduced directly into the abdomen under camera control and made the dissection trouble - free as the manipulation of the gallbladder could be done easily to every direction needed . no gas leakage has been noted around the instrument after direct introduction into the abdomen in all cases . the most remarking effect of using this technique was its influence on the operation time . previous reports using port systems or transabdominal sutures declare their mean operating time to be approximately at least 30 min to 70 min . our procedure even on the first patient took only 40 min to conclude without any complications . we believe learning curve would be low and it would be possible to significantly decline operation periods in following patients . in conclusion , with the help of this instrument the operation period may be lessened , less scarring is achieved and surgical procedure is done more easily and safely without any additional costs ." ]

[ "wilms tumor is the most common renal tumor in children , peaking at 23 years of age . though uniformly fatal in previous decades , the prognosis for most patients has greatly improved due to advances in chemotherapy and surgical treatment . most cases are unilateral and treated by resection at presentation , which allows for diagnostic pathology and bulk of disease removal . surgery is then followed by several weeks of adjuvant chemotherapy , with radiotherapy used mainly for cases of tumor rupture or peritoneal seeding . though primary kidney resection has long been the cornerstone of effective treatment for wilms tumors , this approach has recently been reevaluated in the context of synchronous bilateral disease . bilateral wilms tumor accounts for only 5% of cases but is associated with higher morbidity and mortality . frequently one kidney will have a much larger tumor burden , as in the present case , and surgery would consist of complete nephrectomy for one side and partial nephrectomy for the other . however , as many as 15% of stage v wilms tumor patients treated in this manner eventually develop chronic renal failure by age 20 compared with 1% of unilateral wilms tumor patients , prompting efforts to surgically spare as much normal renal tissue as possible . children 's oncology group trial aren0534calls for pre - operative neoadjuvant chemotherapy to be given in order to shrink tumors to facilitate partial nephrectomies to maximize preservation of normal renal tissue . here , we describe a young girl with bilateral wilms tumor who had marked tumor response after 12 weeks of chemotherapy . her case illustrates that tumor lysis precautions should be considered in children with a large amount of disease who undergo neoadjuvant treatment and that pathologic interpretation of the tumor is possible even after many cycles of chemotherapy .", "a normally developed 5-year - old girl presented to the emergency department with a 1 month history of worsening abdominal pain that localized to the right upper quadrant with intermittent vomiting and fevers over the week prior to presentation . on physical examination , the child was febrile to 37.6c with abdominal distension . a tender right - sided abdominal mass was found extending from the costal margin to the pelvis and beyond midline . abdominal ct imaging identified a large mass arising from the right kidney with at least one smaller lesion in the left kidney ( fig . serum wbc and electrolytes were normal , with a serum creatinine of 0.40 mg / dl and a bun of 7 mg / dl . figure 1:computed tomography of the patient 's abdominal disease at presentation ( a ) , after 6 weeks of chemotherapy per cog aren0534 ( b ) and post - surgically ( c ) . ( a ) axial abdominal ct image at presentation showing large right - sided renal mass ( 23 13 15.8 cm ; large triangle ) and small contralateral renal lesion ( small triangle ) , ( b ) axial abdominal ct after 6 weeks of chemotherapy showing marked response of the right - sided renal tumor ( 8.6 4.1 5.3 cm ) to therapy . left - sided renal renal tumor remained approximately stable in size ( 8 mm ) throughout neoadjuvant therapy . ( c ) post - surgical abdominal ct reflecting right radical nephrectomy and wedge resection of left kidney . computed tomography of the patient 's abdominal disease at presentation ( a ) , after 6 weeks of chemotherapy per cog aren0534 ( b ) and post - surgically ( c ) . ( a ) axial abdominal ct image at presentation showing large right - sided renal mass ( 23 13 15.8 cm ; large triangle ) and small contralateral renal lesion ( small triangle ) , ( b ) axial abdominal ct after 6 weeks of chemotherapy showing marked response of the right - sided renal tumor ( 8.6 4.1 5.3 cm ) to therapy . left - sided renal renal tumor remained approximately stable in size ( 8 mm ) throughout neoadjuvant therapy . ( c ) post - surgical abdominal ct reflecting right radical nephrectomy and wedge resection of left kidney . the patient was given a presumptive diagnosis of bilateral ( stage v ) wilms tumor . the family consented for her to participate in the children 's oncology group trial aren0534 which called for pre - operative chemotherapy in order to facilitate nephron - sparing surgery at the time of tumor resection . a central venous catheter was placed , and neoadjuvant chemotherapy with vincristine , dactinomycin and doxorubicin was initiated . within hours of starting her therapy , we found that lactate dehydrogenase and uric acid were both elevated ( 1134 u / l and 6.3 mg / dl , respectively ; fig . 2 ) . the patient 's hyperuricemia was felt to be consistent with tumor lysis syndrome , and allopurinol and alkalinized fluids were administered . the hyperuricemia soon resolved , and the patient maintained normal serum creatinine , potassium and phosphorus levels throughout her hospital stay . a follow - up ct scan after 6 weeks of therapy showed a marked response to chemotherapy of the right - sided lesion ( fig . we decided to administer another 6 weeks of chemotherapy to improve her candidacy for right partial nephrectomy instead of whole nephrectomy . however , despite further tumor shrinkage , total nephrectomy eventually had to be performed for the right kidney because no clear surgical plane could be identified at the time of resection ( fig . pathologic interpretation of the right- and left - sided tumors confirmed wilms tumor in each and a focus of anaplastic disease on the right ( fig . 10.8 gy of radiation to the right flank was administered because of the pathologic finding of focal anaplasia , in accordance with the aren0534 protocol . presently the patient is well , with good renal function and in remission , now roughly 6 months after her end of therapy . \n figure 2:tumor lysis metabolic abnormalities reflecting serum uric acid and creatinine levels in the context of the therapeutic timeline . \n figure 3:pathologic images from right - sided lesion after 12 weeks of neoadjuvant chemotherapy . note that the architecture of the tumor is still well preserved , confirming a diagnosis of wilms tumor . ( a ) lower - power power image ( 100 ) showing the area of anaplasia ( black triangle ) immediately adjacent to an area of scarring and fibrosis ( white triangle ) , ( b ) a region of non - anaplastic glandular wilms tumor ( 400 ) , ( c ) anaplastic focus of disease ( white triangle ; 400 ) . tumor lysis metabolic abnormalities reflecting serum uric acid and creatinine levels in the context of the therapeutic timeline . note that the architecture of the tumor is still well preserved , confirming a diagnosis of wilms tumor . ( a ) lower - power power image ( 100 ) showing the area of anaplasia ( black triangle ) immediately adjacent to an area of scarring and fibrosis ( white triangle ) , ( b ) a region of non - anaplastic glandular wilms tumor ( 400 ) , ( c ) anaplastic focus of disease ( white triangle ; 400 ) .", "wilms tumor is responsible for roughly 6% of all childhood cancers and 95% of renal tumors in the pediatric population . survivability from this malignancy now approaches 90% with proper management , including surgery , chemotherapy and radiation . historically , most wilms tumors have been resected at diagnosis , with adjuvant chemotherapy to manage micrometastatic disease . even with bilateral disease , chemotherapy would typically be administered only after the large primary tumor had been removed . however , current high - risk wilms tumor protocols , as apply to patients with bilateral disease , are designed to reduce long - term risk of renal failure by shrinking tumors with chemotherapy to enable nephron - sparing sub - total nephrectomy . clinical trials by siop ( international society of paediatric oncology ) and other consortia have found that pre - nephrectomy chemotherapy may decrease the stage of disease and risk of tumor rupture during local control ; however , this approach brings risks of tumor lysis syndrome , veno - occlusive disease and potential interference with tumor staging and pathologic diagnosis . in the present case , initial chemotherapeutic management of wilms tumor was complicated by hyperuricemia , presumably caused by tumor lysis syndrome in which metabolites are released from large numbers of cancer cells dying simultaneously in response to chemotherapy . classically , tumor lysis syndrome occurs in the setting of initial treatment of hematologic malignancies such as burkitt 's lymphoma and leukemia , presumably because of the large burden of disease and the exquisite chemosensitivity of such malignancies . laboratory findings of tumor lysis syndrome include hyperuricemia , hyperkalemia and hyperphosphatemia with secondary hypocalcemia . we reason that our patient developed hyperuricemia because of her large tumor burden , the chemosensitivity of her disease and perhaps because of the presence of tumors in her kidneys that may have physically interfered with normal glomerular filtration . many cases of tumor lysis can be managed conservatively with ample hydration , alkalization and xanthine oxidase inhibition by allopurinol . more severe or refractory cases may require administration of uric acid oxidase ( rasburicase ) , a recombinant enzyme that degrades uric acid , or hemodialysis . our case illustrates that wilms tumor , being a chemosensitive embryonal malignancy of childhood , can also be associated with tumor lysis syndrome . in the past , most patients with bilateral wilms tumors were treated up - front with surgery and therefore had much lower burdens of disease when adjuvant chemotherapy . this case illustrates that if surgery is postponed , tumor lysis precautions should be considered when chemotherapy is given to patients with large wilms tumors . this case also demonstrates that pathologic findings are preserved even after several weeks of chemotherapy ." ]

[ "in the united states , insufficient participation in leisure time physical activity constitutes a major threat to public health . recent estimates suggest that 25% of americans do not engage in any physical activity at all . even those engaging in physical activity are usually not doing so at recommended levels . in order to promote and maintain health , the american college of sports medicine ( acsm ) recommends a minimum of 30 minutes of moderate intensity aerobic physical activity five days a week or a minimum of 20 minutes of vigorous intensity aerobic physical activity three days a week . despite these widely disseminated guidelines , the centers for disease control ( cdc ) report that americans have made no substantial progress towards achieving recommended levels of physical activity with the proportion of 1829 year olds meeting guidelines hovering around 35% and the proportion of adults 65 and older meeting guidelines at about 20% . these numbers are troubling , as aerobic exercise has been convincingly linked to the prevention of myriad negative health outcomes , including several forms of cancer . numerous studies conducted over the past two decades have explored the association between physical activity participation and cancer prevention , consistently implicating strong or probable evidence for reduced risk of colon , breast , and endometrial cancers when physical activity recommendations are followed [ 36 ] . likely mechanisms through which physical activity is believed to have an influence on cancer prevention include reduction in adiposity and changes to levels of circulating metabolic hormones and growth factors ( e.g. , estrogen , testosterone , and insulin - like growth factors ) [ 79 ] as well as influences on dna methylation [ 10 , 11 ] . in respect to prostate cancer , because physical activity activates gut motility , gastrointestinal transit time for food wastes is lessened and thus , exposure to carcinogens is attenuated . there is also research to suggest that immune function changes may mediate the relationship between physical activity and cancer development . the promising body of literature regarding the relationship between physical activity and cancer has led the national cancer institute ( nci ) to regard behavioral primary prevention of cancer ( e.g. , physical activity ) as a top priority . unfortunately , interventions designed to change behavior are typically met with only modest success , even when grounded in empirically supported theory , and behavioral adherence is reported to be a principal challenge faced by exercise promotion programs . indeed , only 50% of individuals who adopt an exercise program stay with it for more than six months [ 14 , 15 ] . researchers devoted to the goal of improved physical activity participation have suggested that one likely determinant of physical activity behavior is the way in which individuals subjectively experience exercise . in previous work , we organized genetic , physiological , subjective , and motivational factors that may contribute to the initiation and maintenance of physical activity into a conceptual transdisciplinary framework [ 16 , 17 ] . this framework has received support among both active and inactive samples , and provides the basis for the selection of phenotypes in the current study . briefly , we proposed that genetic factors influence how an individual physiologically ( e.g. , body - temperature regulation ) and subjectively ( e.g. , affective response , perceived exertion ) responds to the experience of exercise . physiological response influences how one subjectively responds to the experience of exercise ( e.g. , increased lactate during exercise may increase perceived exertion ) and these subjective responses influence motivation to exercise ( e.g. , exercise self - efficacy , exercise intentions ) . moreover , exercise behavior itself influences both how a person physiologically responds to the experience of exercise and gene expression , thereby recapitulating the framework . importantly , this framework is meant to be dynamic such that the factors selected to represent physiological response , subjective response , and/or motivation can vary depending on the goals of each individual research study . the relationship between physiological changes induced by aerobic exercise ( e.g. , regulation of body temperature , heart rate , or blood pressure during exercise ) and subjective responses to aerobic exercise ( e.g. , changes in affect during or immediately after exercise , ratings of perceived exertion or pain during an exercise bout ) is one that has a clear influence on individual differences in exercise behavior . bryan and colleagues found that physiological factors such as heart rate were related to mood response to exercise , and that mood response was a significant correlate of both motivation to exercise in the future and of current exercise behavior . additionally , subjective experiences during exercise may be influenced by interpretations of exercise - induced physiological responses . for instance , increases in lactate levels during aerobic exercise may be perceived as painful to varying degrees across individuals , and this perceived pain will in turn influence subjective exercise experiences and potentially impact motivation to engage in exercise behavior in the future . understanding potential influences on subjective response to exercise is especially important , given that affective responses to acute exercise have been found to predict long - term exercise behavior [ 19 , 20 ] . although the heritability of exercise participation in adults has been shown in twin studies to be approximately 50% ( with peak heritability of 85% , occurring at age 19 - 20 ) [ 21 , 22 ] , there is a surprising lack of research regarding the role played by genetic factors for determining physiological and affective responses to exercise . these responses may serve as promising intermediate phenotypes for the linkage of genes to broader exercise participation phenotypes . also important explained by de geus and de moor as the genetic variance causing differential responses to exercise training , given that the effects of exercise on health and fitness gains appear not to be uniform across individuals [ 24 , 25 ] . one type of gene - by - exercise interaction that is relevant to the present study is the role of exercise in reducing the phenotypic effects of some detrimental genetic variants . for example , phares et al . , showed that sedentary individuals who possess two particular polymorphisms of the adr gene have unfavorable body composition . however , these individuals experience greater loss in percent body fat after 24 weeks of aerobic training in comparison with all other genotypes . it follows that weight loss interventions for individuals with this particular genotype would likely be successful if they focused on aerobic training . thus , identifying particular genetic markers that are related to exercise behavior and physiological and affective responses to exercise may have clear implications for matching individuals to tailored exercise intervention programs . the goal of the current study is to determine whether genotypes predicted subjective physiological and affective responses to a 30-minute bout of aerobic exercise among sedentary individuals . based on the literature and on our prior analysis of the relationships among a range of exercise response phenotypes ( see for analysis and detailed information on the rationale for selection of phenotypes ) , the variables from the physiological responses to exercise domain included in our analysis were temperature , heart rate , systolic blood pressure , lactate , and norepinephrine , all measured as change scores from immediately prior to a bout of exercise to 30-minutes into the bout ( just before the end of the bout ) . genetic associations with vo2 max were also examined , as cardiovascular fitness is highly heritable [ 2730 ] , and evidence exists for a strong genetic influence on athletic performance . additionally , genetically influenced cardiovascular fitness traits play a role in determining individual experience of exercise intensity and perception of exertion during exercise . the variables selected from the subjective experience of exercise domain were affect ( i.e. , positive affect and affective valence ) , perceived pain , and rate of perceived exertion ( rpe ) , which were also change scores measured from prior to the bout to just before the end of the bout . we chose the specific genetic factors for our analyses a priori based on evidence from the literature that they were linked to processes related to physiological and subjective responses during physical activity , general health and fitness traits , or because of evidence that they moderate responses to exercise interventions . a single nucleotide polymorphism ( snp ) in the fat mass and obesity - associated protein gene ( fto ; rs9930506 ) has been associated with obesity traits such as increased bmi and weight and susceptibility to obesity . additionally , physical activity may slow down weight gain associated with the fto risk - allele . in addition , the -opioid receptor gene ( oprm1 ) may be associated with pain sensitivity such that individuals possessing the rare g allele have an increased pressure pain threshold . interestingly , this study also found gender differences in pain threshold among individuals with the g allele when heat pain was tested , such that women with this allele have lowered pain thresholds , and men exhibit higher pain thresholds . snps located within in the slit2 gene(rs1379659 ) , fam5c gene ( rs1935881 ) , kcnb2 gene ( rs10505543 ) , and rs10498091 ( an snp associated with left ventricle mass ) have all been found to be associated with echocardiography traits ( e.g. , left ventricle diastolic dimension , diameter , and systolic dimension ) in a genome - wide association study . another genome - wide analysis implicated creb1 in the prediction of submaximal exercise heart rate in response to exercise training [ 38 , 39 ] . thus , each of these snps was investigated in the current study in order to determine potential relationships with phenotypes related to physiological and affective response to an acute bout of aerobic exercise .", "participants included in the present analysis were a subset of 238 individuals from a larger intervention study ( costride ) [ 17 , 40 ] in which participants were randomly assigned to the stride exercise intervention ( costride ) or a health - and - wellness contact control condition ( hw ) . participants were men and women ( ages 1845 ) who reported less than 90 minutes on average of at least moderate - intensity physical activity per week for the past three months . individuals were excluded if they smoked cigarettes , were on a restricted diet , were taking psychotropic medications , were receiving treatment for any psychiatric disorder , were diabetic , had a history of cardiovascular or respiratory disease , had the flu or illness within the last month , or were pregnant ( if female ) . all participants were required to be willing to be randomized to an intervention condition , to give informed consent , to be able to engage in moderate - intensity physical activity , to have a body mass index ( bmi ) between 18 and 37.5 , and to have a regular menstrual cycle ( if female ) . all participants were recruited from the denver - metro area and the university of colorado boulder community . the data reported herein are from assessments conducted prior to randomization , and the analysis and questions addressed are unique to this investigation . as described in detail below , we used the illumina human 1 m duov3 dna analysis beadchip to genotype the dna samples . the bead chips accommodate 4 samples each , and we ran a total of 50 bead chips . thus , this experiment allowed for the genotyping of 200 individuals total . due to limitations in funding , we were unable to genotype the remaining 38 individuals in the sample . thus , individuals with the most complete baseline data ( baseline dna sample , self - report questionnaire assessments , vo2 max fitness assessment , and submaximal exercise session ) were selected to be genotyped out of the full sample . statistical tests revealed no significant differences on demographic variables between participants who were included in genotyping procedures and those who were not included in genotyping procedures ( details available from the first author ) . this reduced sample ( n = 200 ) was comprised primarily of females ( n = 160 ) and most participants identified as white ( n = 137 ) , followed by hispanic / latino ( n = 22 ) , asian american ( n = 22 ) , african american ( n = 9 ) , native american ( n = 5 ) , and mixed ethnicity ( n = 5 ) , the average age of participants at baseline was 28.68 ( sd= 7.86 ) years old and mean body mass index ( bmi ; weight in kg / height in m ) was 25.18 ( sd = 4.72 ) . on average participants reported an average of 28.14 minutes of voluntary physical activity in the past week ( sd= 50.95 ) , and reached an average vo2 max peak of 34.06 ml / kg / min ( sd= 8.11 ) . prior to randomization to intervention condition and after giving informed consent , participants completed three sessions : ( 1 ) an orientation ( baseline ) session in which self - report questionnaire assessments were completed , ( 2 ) a vo2 max cardiovascular fitness assessment , and ( 3 ) a submaximal exercise session . prior to exercise sessions , each participant was instructed to eat a meal comprised of both carbohydrates and protein and to consume at least 300 calories two hours before coming into the lab ( e.g. , if a participant is scheduled to come into the lab at 12:00 p.m. a researcher instructed him / her to eat the 300 calorie meal at 10:00 a.m. and no later ) . participants were also instructed to drink at least 17 oz . of water two hours prior to coming into the lab . participants were instructed not to exercise on their own prior to the laboratory session , and not to consume alcohol during the 24 hours prior to testing . further details regarding recruitment , selection of measures , and study procedures are available elsewhere . consistent with established procedures , maximal oxygen capacity ( vo2 max ) was assessed during a balke protocol ( a graded , incremental exercise test ) on a motorized treadmill . vo2 max was assessed with online computer - assisted open - circuit spirometry using the medgraphics cardi02/cp system . prior to the fitness test , saliva samples ( 5 ml ) were collected for dna extraction and measurements of height and weight were taken for calculation of bmi . approximately one week after the fitness test , participants completed a standardized , short 30-minute bout of physical activity on the treadmill at 65% of their previously established vo2 max , calculated during the fitness test ( vo2 max test session ) . prior to beginning activity , an intravenous catheter was inserted by a nurse to collect blood samples during the bout . intensity was maintained by measuring oxygen uptake and expired co2 for two to three minutes at the beginning of exercise and at 10 and 20 minutes during exercise . \n lactate concentration and catecholamine levels ( epinephrine and norepinephrine ) were collected via blood samples immediately before activity began ( 11.5 ml ) , and 10 ( 5.5 ml ) and 30 ( 11.5 ml ) minutes into activity . tympanic temperature was measured by taking an average of 2 - 3 temperature readings at each measurement . readings of temperature , blood pressure , and heart rate were taken before activity , at 10 minutes , 20 minutes , and 30 minutes ( directly before completion ) during activity . subjective experiences during exercise were assessed at six points during the submaximal session : five minutes prior to activity , immediately before activity began , and 10 , 20 , and 30 minutes into activity ( directly before completion of the session ) . the present study focuses only on change scores created from subtracting the values obtained immediately before the exercise bout began from the values obtained 30 minutes into the bout . for the time points that occurred 10 , 20 , and 30 minutes into the exercise bout , participants were assessed while they were exercising the bout was not interrupted to make these assessments . while participants continued their session on the treadmill , a research assistant held up cards with the questionnaire items displayed on them . participants indicated the number that they felt reflected their current subjective states , and their responses were manually recorded . physiological measures were obtained at these time points using the iv catheter that was inserted prior to the bout . positive affect was assessed using 3 items from the 12-item physical activity affect scale ( paas ) . participants rated their current state for each item using a 5-point scale ( 0 = do not feel to 4 = feel very strongly ) . the adjectives assessed by the 3-item positive affect subscale were enthusiastic , energetic , and upbeat ( = .81 ) . affective valence during exercise was assessed using the 11-point single - item feeling scale ( fs ) , which ranges from 5 = very bad to + 5 = very good . perceived exertion was assessed using the 15-point single - item rating of perceived exertion ( rpe ) that ranges from 6 to 20 ( 6 = no exertion at all , 20 = maximal exertion ) . perceived pain was assessed using a single - item 12-point borg category ratio-10 scale ( cr10 ) ( 0 = no pain at all , 10 = extremely intense pain ) . samples were genotyped on the illumina infinitum assay platform using the human 1 m duov3 dna analysis beadchip ( illumina , inc . , san diego , ca , usa ) following the manufacturer 's protocol at the university of colorado , boulder . in this assay , the dna is then resuspended in hybridization buffer and applied to the bead chip array for an overnight incubation . the amplified and fragmented dna samples anneal to locus - specific 50-mers ( covalently linked to one of over 1,000,000 bead types ) during the hybridization step . the samples then undergo single - base extension and staining , followed by more washing . the arrays are allowed to dry , and then scanned using the illumina iscan system . genotype calls were made using illumina 's genomestudio software in conjunction with the genome studio genotyping module . additionally , we excluded snps with a minor allele frequency ( maf ) of < 10% and snps that showed significant deviation from hardy - weinberg equilibrium ( p < 1 10 ) . following these quality control checks , although we used a genome - wide approach to genotyping , we only tested a total of 14 snps which were selected for analysis in this study based upon their potential association with aerobic exercise response phenotypes suggested by prior studies ( see table 2 for hardy - weinberg p values and minor allele frequencies for each snp tested ) . our search was conducted primarily using pubmed , and was focused on snps that were directly associated with specific phenotypes of interest and to traits that may be associated with those phenotypes . analyses were run using the snp and variation suite for genetic analysis ( svs ) ( version 7.5.6 , golden helix inc . , bozeman , mt).the 14 snps selected for inclusion based on our search were tested for associations with the phenotypes using a correlational trend test assuming additive effects of allele dosages for each snp ( i.e. , homozygous for the minor allele = 0 ; heterozygous = 1 ; homozygous for the major allele = 2 ) .", "these analyses focused on correlations between particular snps suggested by the relevant literature and exercise response phenotypes drawn from our previous work and the existant exercise literature . due to the fact that both exercise phenotypes and candidate snps were selected a priori based on the literature as well as our transdisciplinary framework , critical alpha for all tests was maintained at the .05 level for all analyses . additionally , given that the aim of this study was to examine changes in physiological and subjective responses to exercise over the course of the 30-minute exercise bout , it was not necessary to compare subjects cross - sectionally at the baseline or 30-minute time points . rather , all phenotype values were determined using a change score created by subtracting each subject 's baseline values from the values obtained by that subject 30 minutes after the exercise bout began . in order to determine whether allele frequencies for all snps examined in this study were significantly different across racial / ethnic groups , additionally , major and minor alleles , as well as minor allele frequencies ( mafs ) for caucasians , african americans , asians , and hispanic / latino participants are reported in table 3 . significant differences in genotype across racial / ethnic groups were found for three snps , rs1935881 ( 10,200 ) = 19.25 , p = .037 , rs1799971 ( 10,200 ) = 38.13 , p < .001 , and rs8044769 ( 10,200 ) = 21.54 , p = .018 . for rs1935881 , the maf is lower among asians . for rs1799971 , an maf of 0 was found in african americans . for rs8044769 , results of associations between snps and exercise response phenotypes presented below are uncorrected ( no pca correction applied ) . a total of 10 different phenotypes were examined for association with genetic variants in this study . given that many of these phenotypes may have common underlying physiological bases , we tested for associations between these phenotypes . in the following results , all phenotypes tested and reported ( except for vo2 max ) refer to a change score created by subtracting preexercise values from the values obtained 30 minutes into the exercise bout . vo2 max was significantly correlated with change in lactate ( r = .177 , < .05 ) , heart - rate ( r = .434 , p < .01 ) , systolic blood pressure ( r = .193 , p < .01 ) , and rate of perceived exertion ( r = .157 , p lactate change was correlated with temperature change ( r = .214 , p < .01 ) , heart rate change ( r = .429 , p < .01 ) , systolic blood pressure change ( r = .208 , p < .01 ) , change in affective valence ( as measured by the feeling scale ) ( r = .173 , p < .05 ) , and pain change ( r = .215 , p < .05 ) . norepinephrine change was significantly correlated with positive affect change ( r = .174 , p < .05 ) and pain change ( r = .165 , p < .05 ) . temperature change was significantly correlated with heart - rate change ( r = .172 , p < .05 ) and affective valence change ( r = .149 , p < .05 ) . heart rate change was significantly correlated with systolic blood pressure change ( r = .185 , p < .05 ) and rate of perceived exertion change ( r = .231 , p < .05 ) . rate of perceived exertion change was significantly correlated with affective valence change ( r = .163 , p < .05 ) and pain change ( r = .316 , p < .01 ) . finally , positive affect change was significantly correlated with affective valence ( r = .455 , p < .05 ) . the creb1 snps rs2360969 and rs2253206 were associated with temperature change during exercise ( rs2360969 , r = .17 , p = .02 ; rs2253206 , r = .17 , p = .02 ) indicating that for rs2360969 , individuals with the t allele had greater changes in temperature over the course of the exercise , and for rs2253206 , individuals with the a allele had greater changes in temperature during exercise . these same snps were also significantly associated with vo2 max ( rs2253206 , r = .17 , p = .01 ; rs2360969 , r = .14 p = .049 ) , such that for rs2253206 , individuals with the g allele had higher vo2 max , and for rs2360969 , individuals with the c allele had higher vo2 max . the oprm1 snp rs1799971 was significantly associated with lactate change during exercise ( r = .17 , p = .02 ) , norepinephrine change during exercise ( r = .16 , p = .03 ) , and change in rpe during exercise ( r = .14 , p = .048 ) , indicating that individuals with the rare g allele had greater changes in lactate , norepinephrine , and rate of perceived exertion change over the course of exercise . the fto snp rs8044769 was related to change in positive affect during exercise ( r = .16 , p = .03 ) , and individuals with the c allele had greater change in positive affect over the course of the exercise . the fto snp rs3751812 was associated with positive affect change during exercise ( r = .14 , p = .04 ) , such that individuals with the t allele experienced greater changes in positive affect . the fto snp rs9941349 was significantly related to change in systolic blood pressure during exercise ( r = .15 , p = .04 ) , and individuals with the t allele experienced greater increases in systolic blood pressure during exercise . the fto snp rs7201850 was significantly related to change in systolic blood pressure during exercise ( r = .17 , p = .027 ) , with individuals possessing the t allele experiencing greater increases in systolic blood pressure over the course of the exercise bout . the slit2 snp rs1379659 was associated with norepinephrine change during exercise ( r = .18 , p = .01 ) , with individuals with the g allele experiencing greater changes in norepinephrine during exercise . finally , the fam5c snp rs1935881 was associated with change in norepinephrine during exercise ( r = .16 , p = .03 ) . individuals with the g allele had greater changes in norepinephrine over the course of the exercise bout ( all associations initially reported in the manuscript changed only slightly after applying the pca correction . pca corrected p - values for genotype - phenotype associations are as follows : rs1799971 and norepinephrine change ( p = .104 ) , rs1799971 and rpe change , ( p = .038 ) , rs8044769 and positive affect change ( p = .038 ) , rs3751812 and positive affect ( p = .036 ) , rs1935881 and norepinephrine change ( p = .059 ) , rs1379659 and norepinephrine change ( p = .010 ) , rs9941349 and systolic blood pressure change ( p = .053 ) , rs7201850 and systolic blood pressure change ( p = .04998 ) , rs2360969 and temperature change ( p = .031 ) , rs2253206 and temperature change ( p = .066 ) , rs1799971 and lactate change ( p = .015 ) , rs2253206 and vo2 max ( p = .035 ) , and rs2360969 and vo2 max ( p = .032 ) ) . due to the fact that several of the variants that were associated with a particular phenotype were in the same gene , it is likely that these snps are in high - linkage disequilibrium with one another . these snp sets within single genes are rs3751812 and rs8044769 in fto , both significantly associated with positive affect change , rs2253206 and rs2360969 in creb1 , both significantly associated with temperature change as well as vo2 max , and rs7201850 and rs9941349 , both in fto , both significantly associated with systolic blood pressure change . to examine whether these snps were in ld , we ran correlations on each set of 2 snps in the same gene that were associated with the same phenotype . the correlation between rs2360969 and rs2253206 was .805 ( p < .01 ) , the correlation between rs3751812 and rs8044769 was .676 ( p < .01 ) , and the correlation between rs7201850 and rs9941349 was .938 ( p < .01 ) . in order to further explore the direction of the relationship of genotype on exercise response , we graphed the adjusted means for each genotype of three snps which demonstrated particularly robust relationships with exercise response phenotypes . we graphed the relationship between rs2360969 and temperature 30 minutes into the exercise bout , between rs1799971 and rpe 30 minutes into the exercise bout , and between rs8044769 and positive affect score 30 minutes into the exercise bout . as can be seen in figure 1 , individuals with the tt genotype of rs2360969 showed the highest temperature after 30 minutes of aerobic exercise , controlling for baseline temperature . in figure 2 , we show that individuals with the ag / gg genotypes on rs1799971 show greater rpe after 30 minutes of aerobic exercise than individuals with the aa genotype , controlling for baseline rpe . in figure 3 , we show that individuals with the cc genotype in rs8044769 show the highest ratings of positive affect after 30 minutes of aerobic exercise , controlling for baseline positive affect .", "the present study replicated prior findings suggesting that snps in the creb1 , fto , oprm1 , slit2 , and fam5c genes are all related to phenotypes encompassing various responses to exercise . our study tested conceptually relevant phenotypes that to date had not been explored in this way inanyother exercise research . given that the physiological response to aerobic exercise involves a complex interplay of metabolic , cardiovascular , musculoskeletal , ventilator , and hormonal functions , these genes and snps are likely to explain only a small portion of the variability in individual differences in response to aerobic exercise . subjective responses to exercise may be yet more complex , involving sociocultural factors , effects of previous exercise experiences , and anticipated consequences / rewards of exercise . additionally , our findings suggest that individuals performing equivalent bouts of aerobic exercise may have vastly different subjective perceptions of this exercise ( overall experiences which can range from negative to positive ) , and that these perceptions may be influenced by genotype . giving sedentary individuals information about their propensity to respond to exercise in a particular way could provide useful insight , allowing these individuals to temper their expectations of what aerobic exercise should feel like for them or allowing intervention designers to incorporate external reinforcement contingencies ( e.g. , social interaction ) for individuals who are less likely to experience intrinsic rewards from exercise . despite several inherent limitations , the present study 's findings linking genetic variants to exercise responses among sedentary individuals presents promising initial evidence associating genes and exercise behavior . however , it is unlikely that variation at a single genetic locus could fully explain variation in physiological and subjective responses to exercise more possibly , there are many genetic variables influencing this phenotype , each of which contributes only bya small fraction of the observed variation . when combined into a genetic composite , these loci would likely correlate more strongly with phenotypic response . so , although the correlations between genotype and exercise response found in this study are not large , they represent a necessary first step in forming genetic composite scores that are likely to be more highly correlated and significantly predictive of exercise responses . in summary , linking snps to specific physiological and psychological mechanisms that contribute to exercise response will assist in informing individually tailored exercise programs , as well as deepen our understanding of the relationship between genetics , physiology , and psychology underlying health behaviors associated with cancer prevention . our study showed that for rs3751812 , the presence of a t allele increased change in positive affect during exercise . this finding is somewhat at odds with previous work suggesting that tt individuals have higher bmi on average . rs3751812 was found to have a strong association with bmi in african - derived populations , with the tt genotype predicting increased bmi . however , the relationship between bmi and positive affective response to exercise is unclear because the hassanein study did not include information about exercise behavior of participants . it is possible that rs3751812 individuals are predisposed to have higher bmi , but if they engage in aerobic exercise , they are likely to have a more positive affective response . this is one example of how knowledge about the effect of a particular genotype could be used to prescribe tailored interventions for overweight individuals with the tt genotype , exercise could be recommended as a more effective weight loss tool , given that these individuals have a more positive affective response to exercise . we also found that for rs8044769 , the tt individuals had greater changes in positive affect during exercise . in a hispanic american sample , rs8044769 was found to be weakly associated with waist - to - hip ratio , and the c allele showed an association with variation in bmi . prior research suggests that the c allele of rs8044769 is associated with greater variation in bmi . this snp seems to be related to body fat mass , predisposition to obesity , and response to aerobic exercise yet the nature of this relationship requires further exploration . creb1 is a key component of long - term cardiac memory formation ( specific t - wave patterns on an electrocardiogram ) , as well as long - term memory formation in the brain [ 54 , 55 ] . our results indicate that for the creb1 snp rs2253206 , individuals with the a allele ( ag genotypes , and to a greater extent aa genotypes ) have a greater change in temperature during exercise . if greater temperature change while exercising translates into a more unpleasant subjective exercise experience , then our findings suggest that the aa individuals ( and to a lesser extent ag individuals ) may have less pleasant subjective experiences of exercise than gg individuals . rs2253206 was shown to be strongly associated with heart rate ( hr ) change in response to a 20-week endurance training program , with gg and ag genotypes and showing 57% and 20% better change in hr than the aa participants . our results make sense in the context of the rankinen findings , as the aa individuals may have more unpleasant exercise experiences due to increased temperature , which could influence their ability to exercise effectively ( and thus decrease the heart rate improvements they can obtain from an exercise intervention ) . we also found that this snp was related to vo2 max ( an indicator of cardiovascular fitness ) , such that the gg individuals had greater vo2 max than ag individuals , who had greater vo2 max than aa individuals . these results also coincide with our findings and the findings from previous research , as gg individuals may be more fit to begin with , and also more capable of gaining increased fitness through training , due to the fact that they experience exercise as less painful . additionally , we found that for rs2360969 , tt individuals experienced greater change in temperature than did ct and cc individuals . rs2360969 has also been shown to be related to heart rate response endurance training [ 38 , 39 ] , however , these studies did not state direction of effect for this snp . in our analysis , rs1799971 ( the a118 g polymorphism ) was related to rpe , as well as to lactate change during exercise and norepinephrine change during exercise . for all three of these phenotypes , individuals with the rare g allele showed greater change during the exercise bout . previous research on this snp has found that individuals with the g allele ( genotypes of either ag or gg ) demonstrated higher pressure pain thresholds than individuals with the aa genotype . this study also found that when heat pain was tested , a sex by genotype interaction emerged , such that the g allele was associated with lower pain ratings among men but higher pain ratings among women . the a118 g variant has greater binding affinity for -endorphin ( an exogenous opioid that activates the mu opioid receptor ) , which is one possible mechanism by which this snp could influence pain sensitivity . the relationship between rs1799971 and subjective responses to pain may extend to the pain and exertion experienced during aerobic exercise . given that our sample was 79.5% female , our findings of greater lactate , norepinephrine and rpe change over the course of exercise for the gg / ag group is in the same direction as the findings for females in the fillingim study . these results lend further support to the idea that individuals ( and perhaps particularly women ) with the ag / gg genotype have lowered pain threshold , and the present study suggests increases in lactate and norepinephrine as possible physiological explanations , at least in the context of aerobic exercise - induced pain . prior studies have shown that rs1379659 in fam5c is associated with echocardiographic traits , and specifically left ventricular systolic dimension . the results of our study suggest that it is also associated with change in norepinephrine in response to exercise . to date , research has not examined the relationship between fam5c and aerobic exercise response . given the connection between this gene and cardiac function , examining the potential relationship between fam5c and aerobic exercise would provide a logical next step for research in this area . previous research has demonstrated an association between the slit2 snp rs1935881 and echocardiographic traits , specifically left ventricular diastolic dimension . the results of this study suggest that it is also related to norepinephrine change during exercise . further research is needed to elucidate more specific relationships between slit2 and response to aerobic exercise . the genes discussed above represent potential candidates for further explanation in terms of their relationship to exercise response phenotypes . more than a decade 's worth of research on the psychophysiological responses associated with exercise has demonstrated that the subjective experience of exercise , how sensations are remembered , anticipated , and interpreted , is closely tied to subsequent exercise behavior [ 14 , 19 , 20 , 47 , 57 ] . a better understanding of the genetic basis for subjective responses to aerobic exercise may have the potential to lead to more effective and sophisticated intervention designs . eventually , these advances in the basic science of exercise response could lead to the implementation of interventions tailored on the level of individual genetic variants . primary prevention of cancer through behavioral intervention is now a top priority of the nci . this approach is intuitive given that approximately 30% of total cancer deaths are related to energy imbalance ( e.g. , excessive adiposity ) [ 58 , 59 ] . physical inactivity is not the only contributing factor to energy imbalance , but it is a major contributing factor as trends clearly show that the least physically active regions of the country are also the most obese . the hopeful perspective on behavioral intervention for physical activity is that even small increases in the total amount of participation accumulated per week stands to lead to meaningful differences in cancer risk . for example , found evidence for a 38% reduction in risk for breast cancer with every additional 60 minutes of physical activity engaged in per week . the link between physical activity participation and reduced risk for cancer , especially of the colon , breast , and endometrium is convincing , but also dependent upon good adherence [ 36 , 61 ] . for this reason , it is imperative that researchers continue to search for ways to improve the likelihood of adherence to behavioral interventions . one way to achieve this goal may be through increasing the amount of focus that is placed on subjective response phenotypes and their underlying genetic variants . developing a better understanding of the link between genes , exercise - relevant physiological mechanisms , and the resulting exercise - response phenotypes is a first step towards tailoring individualized exercise programs that would likely increase adherence and lead to improved health outcomes and decreased rates of cancer and other diseases . as with all research that involves genetic analyses , we can not rule out the possibility that other genetic factors , including rare or common snps , insertions , deletions , or copy number variants , could play a role in determining the physiological responses to exercise that were measured in this study . the phenotypes investigated in this study are likely to be polygenic traits , such that numerous genes and snps other than those examined in the present study may all contribute to these exercise response phenotypes . in contrast , the extent of the pleiotropic effects of the genes and snps investigated in this study are unknown . thus , it is possible that the polymorphisms that influence exercise response may also be more strongly associated with other , possibly unrelated phenotypes that led to our findings . another limitation of note is the present study 's lack of power to detect moderation effects of demographic variables . it is possible that variables such as age or ethnicity could moderate the associations between genetic variation and response to exercise . additionally , the results of this investigation are based on one single , standardized , bout of moderate - intensity exercise . for this reason , our results can not be generalized to subjective exercise experiences that occur under less regulated circumstances ( i.e. , when type of activity , intensity , and duration are individually determined ) . despite this limitation , there are many examples from the literature in which subjective responses to exercise are measured and analyzed based on a single bout of standardized exercise ( e.g. , [ 19 , 20 , 6265 ] ) and therefore , our procedures and analyses are in concert with the approach previously established by the field . importantly , the purpose of the present investigation was to understand how genetic variants are associated with particular subjective responses to exercise when the parameters of the exercise experience are standardized across all individuals . in the present study , this level of standardization was achieved by having all participants perform the same activity ( treadmill walking ) , for the same duration ( 30 minutes ) , at the same intensity ( 65% of each individual participant 's previously established vo2 max ) . further , efforts were made to standardize variables external to the exercise bout as well ( i.e. , instructions detailing recommended calorie and water consumption prior to the bout described in section 2 ) . it remains to be seen whether the snps and genes reported in this study to be related to exercise response phenotypes would show an association to these same phenotypes in other studies examining different types , duration , and intensity of exercise sessions . however , as noted , the size of the associations changed negligibly after a pca correction , suggesting that the population substructure did not play a major role . overall , replication is needed in order to confirm findings from the present study , and to better understand the functional significance of these genes and snps in relation to physiological and subjective responses to aerobic exercise .", "the purpose of this paper was to explore the genetic underpinnings of individual physiological and subjective responses to aerobic exercise . one strength of this study was its focus on a sedentary population , a group that has been rarely tested in terms of associations between genetics and exercise phenotypes . the relationship between particular genetic variants and responses to exercise has important implications for the prevention of cancer via increasing exercise behavior in sedentary populations . future studies designed to test genetic influences on a wide range of exercise response phenotypes would help to advance this goal , potentially leading to a panel of markers important for characterizing the physiological and subjective response to exercise . moreover , giving feedback to sedentary individuals regarding the genetic basis for their strengths and weaknesses in fitness / exercise / sports activities could be a potentially useful motivational tool for increasing exercise behavior [ 13 , 66 ] . in sum , expanding our understanding of the association between genetics and exercise response phenotypes has a myriad of implications for helping to increase exercise behavior in sedentary individuals , an outcome which is crucially important for the reduction of morbidity and mortality associated with cancer ." ]

[ "dental implants are now a reliable solution for the functional and esthetic rehabilitation of partially and completely edentulous patients ; this has been demonstrated by long - term clinical trials , with survival rates of greater than 95% [ 13 ] . in order to achieve long - term survival , osseointegration of the dental implant needs to occur ; that is , a direct connection must be established between the bone and the implant surface , without the interposition of fibrous tissue ; once established , this close bond must be maintained over time , resulting in a clinically asymptomatic fixation of the implant under functional load . osseointegration is a complex phenomenon and depends on many factors ; some are related to the implant ( material , macroscopic design , and implant surface ) , others to the surgical - prosthetic protocol ( surgical technique , loading conditions , and time ) , and others to the patient ( quantity / quality of bone at the receiving site and the host response ) [ 4 , 5 ] . although survival rates of dental implants are now high , there still remains a seemingly unavoidable number of failures : either cases in which correctly placed implants do not integrate with the bone or cases of peri - implant tissue infection [ 6 , 7 ] . to be specific , failure to osseointegrate and peri - implantitis are the most frequent causes of early implant failure [ 3 , 6 , 7 ] . such events occur during the early stages of healing ( within 2 - 3 months of implantation ) and therefore before the implant is functionally loaded with the prosthetic restoration ; these failures are unevenly distributed within the general population and tend to occur in some subjects in particular . in these individuals early failures occur even when optimal materials are used , surgical protocols are strictly followed , and the quantity / quality of bone at the recipient site is sufficient [ 68 ] . all these observations would suggest the existence of specific patient - related risk factors ; this prompts an investigation into the regulatory mechanisms controlling bone metabolism , bone remodelling , and bone turnover [ 9 , 10 ] . it is a fat - soluble vitamin which promotes the absorption of calcium in the intestine and regulates calcium and phosphate homoeostasis in the tissues and it is a fundamental element in the mineralization of bones and teeth [ 1113 ] . it also acts as a hormone and is vital for the health of the blood vessels and the brain [ 14 , 15 ] . it has been demonstrated that vitamin d plays a crucial role in the health of the cardiovascular tract , the immune system , and the respiratory tract [ 18 , 19 ] . vitamin d in an inactive form ( cholecalciferol or vitamin d3 ) is ingested or produced in the skin on exposure to sunlight [ 11 , 12 ] . this inactive form undergoes double hydroxylation in the liver and the kidneys and is thereby transformed into its active form , known as either calcitriol or 1,25-dihydroxyvitamin d3 [ 11 , 12 ] . this active form exerts its action on various tissues by binding to the vitamin d receptors and regulating the transcription of specific target genes [ 1223 ] . serum levels of vitamin d in the 25(oh)d form are the most accurate way of determining vitamin d status : a subject with < 10 ng / ml is considered to be vitamin d deficient ; one with 1030 ng / ml is considered to have low levels of vitamin d. the optimal blood level of vitamin d is a value > 30 ng / ml [ 12 , 13 ] . vitamin d deficiency is high in the general population : in italy , for example , it is estimated that about 80% of people can be deficient , particularly in the northern regions where exposure to the sun is lower . this deficiency increases with age and encompasses the majority of the elderly population of italy who are not taking vitamin d supplements . until a few years ago , the guidelines estimated that the daily intake of vitamin d required to maintain adequate blood levels was 200 iu ( 5 mcg ) in adults aged between 19 and 50 , 400 iu ( 10 mcg ) in adults aged between 51 and 69 , and at least 600 iu ( 15 mcg ) in those over 70 [ 12 , 13 ] . these guidelines have now been revised upwards and it is currently believed that the amount of vitamin d which should be taken daily is 2000 iu ( 50 mcg ) and up to 4000 ( 100 mcg ) in the case of , for example , pregnant women [ 12 , 13 ] . there is now substantial literature on the negative effects of low levels of vitamin d , especially in severely compromised patients : vitamin d deficiency seems to be associated with increased mortality , cardiovascular events , and reduced functioning of the immune and musculoskeletal systems [ 1519 , 2123 ] . on the other hand , normalizing levels of vitamin d can lead to substantial benefits for critically ill patients , with effects on the muscles , the respiratory system , the heart , and the immune system [ 18 , 21 , 23 ] . despite the importance of vitamin d and its effects on bone metabolism [ 11 , 12 ] few studies have , to date , investigated the effects of its depletion on the osseointegration of dental implants [ 9 , 2435 ] : almost all these studies have been done on animal models [ 2432 ] and very few on humans [ 3335 ] . the purpose of this retrospective study was therefore to investigate any possible correlation between low blood levels of vitamin d and early implant failure ( failure occurring in the four months prior to the full restoration of the implant , because of a lack of osseointegration or because of infection ) .", "all patients who had been treated with morse - taper connection dental implants ( leone implant system , florence , italy ) [ 3 , 8 ] inserted to support fixed prosthetic restorations in one single dental centre ( gravedona , como , italy ) , in the period between january 2003 and december 2015 , were evaluated for possible enrollment into this retrospective study . patients were enrolled into the study if they were over 18 years of age , had good oral and general health , and had not undergone bone regenerative therapy prior to implant placement . the exclusion criteria were incomplete medical records , the presence of specific systemic diseases ( uncontrolled diabetes mellitus , immunodeficient states , and bleeding disorders ) , and the abuse of alcohol and drugs ; patients undergoing radiotherapy and chemotherapy and those who were pregnant were also excluded . all the data used for the study were obtained from the medical records of the patients enrolled . the patient data was evaluated ; this included gender ( male or female ) , age at time of surgery , history of chronic periodontal disease , smoking habits , and serum vitamin d levels . vitamin d levels were taken from blood tests , which had been requested two weeks prior to surgery . the medical records also contained a range of information as regards the implant or implants , that is , their site ( maxilla or mandible ) , location ( incisor , canine , premolar , and molar ) , the length and diameter of the implant , the type of prosthetic restoration , and the loading conditions . these included their cause ( lack of osseointegration in the absence of infection , infection of the peri - implant tissues or peri - implantitis , or implant failure due to progressive bone loss caused by to prosthetic overload ) . it also included their classification : early failure , occurring in the early healing period , that is , the four months after implant placement , prior to the placement of restoration and loading , or late failure , occurring after loading . there were also details of any possible biological complications ( peri - implant mucositis and peri - implantitis ) and/or prosthetic complications ( mechanical and/or technical ) . all implants were inserted under the same strict protocol by the same specialist ( c. m. ) who had 25 years ' experience in implant dentistry , in the period between january 2003 and december 2015 . the implants were inserted after raising a full thickness mucoperiosteal flap ; the implant site preparation and implant placement were performed in compliance with modern surgical protocols and in accordance with the manufacturer 's instructions . after placement , cover screw was positioned and the implants were submerged . immediately after positioning , patients were prescribed antibiotic coverage with 2 g of amoxicillin ( or 600 mg of clindamycin in patients allergic to penicillins ) for 6 days . patients were given detailed instructions on oral hygiene and were prescribed chlorhexidine 0.12% mouth rinse twice a day for 6 days . the implants were left to heal submerged for a total period of 4 months , to allow undisturbed healing and achieve osseointegration . after 4 months of undisturbed healing , the patient was recalled for the implant to be uncovered . two weeks later , the sutures were removed and an impression was taken for the manufacture of the temporary restoration . the temporary restoration was maintained in situ for 3 months , in order to monitor the response of the implant , as well as the peri - implant tissues , to masticatory load ; at the end of this period , the temporary restoration was replaced with the final restoration . the final restorations were metal porcelain , cemented with a zinc oxide - eugenol cement . a periapical radiograph was taken to check on the sealing of the restoration . all patients were included in a follow - up protocol with an annual check - up at one of the scheduled professional oral hygiene sessions . early implant failure , occurring within 4 months after implant placement and therefore prior to placement of the prosthetic restoration and the functional load of the implant , was the primary outcome studied . early implant failures were divided into two different categories : ( a ) early failures due to lack of osseointegration and subsequent implant mobility , in the absence of clinical signs of infection ; ( b ) early failures due to infection of the bone tissue around the implant , with inflammation ( peri - implantitis ) of peri - implant tissues and the presence of fistula , pain , swelling , pus and/or exudate , pocket depth > 6 mm with bleeding , and marginal bone resorption > 2.5 mm . all the data retrieved from the individual medical records were recorded on a generic spreadsheet ( excel , microsoft office , redmond , ma , usa ) which was used for the descriptive , qualitative , and quantitative analyses . the mean , standard deviation , median , and confidence intervals were calculated for the quantitative variables ( e.g. , patients ' age and vitamin d levels in serum ) . a patient - based technique was used to calculate implant survival . in this analysis , the event was implant failure : thus in patients receiving more than one implant , the occurrence of even a single implant failure led to the patient being classified as a failure . the influence of different variables on implant survival was taken into consideration : gender ( male or female ) , age at time of surgery ( three age groups were examined : < 40 , 4060 years , and > 60 years ) , smoking habits ( regardless of the actual number of cigarettes smoked ) , a history of chronic periodontitis , and serum levels of vitamin d. in the analysis of serum levels of vitamin d , three classes of patients were considered : severely deficient patients ( serum vitamin d < 10 ng / ml ) , patients with low levels ( serum vitamin d between 10 and 30 ng / ml ) , and patients with adequate levels ( serum vitamin d > 30 ng / ml ) . the influence of each of these variables on implant survival was calculated using the chi square test . the overall implant survival , the survival within the different groups , and the analysis of the influence of the different variables on survival were all made using dedicated statistical analysis software ( spss 17.0 , spss inc . ,", "of the 915 patients originally evaluated for enrollment in this study , 93 presented with conditions corresponding to the exclusion criteria and were therefore excluded from the assessment . by contrast , 822 patients ( mean age 57.3 14.2 years ; median age 58 ; range 1890 ; and 95% ci , 56.358.2 ) , receiving 1625 implants , did not have any of the conditions contained in the exclusion criteria and were therefore enrolled into this retrospective study . the distribution of patients by groups , with relative incidence of failures , was reported in table 1 . in total , 27 early failures were recorded ( 19 due to failure of osseointegration and 8 due to peri - implant tissue infection ) , with an overall incidence of 3.2% . no differences were observed in the incidence of early failures between males and females ( p = 0.97 ) nor according to age at time of surgery ( p = 0.98 ) . although the percentage of early failures in smokers was slightly higher than that detected in nonsmokers , there was no statistically significant difference ( p = 0.56 ) between these two groups of patients . the same was true for patients with a history of periodontal disease ; they displayed a slightly higher incidence of early failures than patients who had not been affected by periodontitis , but this difference was not significant ( p = 0.73 ) . the average serum level of vitamin d in the general population was 29.9 ng / ml ( 12.1 ; median 29 ; range 573 ; and 95% ci , 29.130.7 ) . in patients in whom early implant failure occurred , the average serum level of vitamin d was 25.5 ( 13.2 ; median 24 ; range 855 ; and 95% ci , 20.630.4 ) . statistical analysis reported a rather low incidence of early failures ( 2.2% ) in patients with blood vitamin d levels > 30 ng / ml . the incidence of early failure was almost double in patients with insufficient serum levels of vitamin d ( 1030 ng / ml ) and became even higher ( 9.0% ) in patients with serious vitamin d deficiency . although the statistical analysis revealed a trend toward an increased incidence of failure in patients with severe vitamin d deficiency , the analysis did not reveal a statistically significant difference ( p = 0.15 ) in the incidence of early implant failure in these three groups of patients . similar results ( p = 0.14 ) were obtained comparing the incidence of failures in the group of severely deficient patients ( 2/22 : 9.0% ) with the incidence of failures in all other patients ( 25/800 : 3.1% ) . finally , the statistical analysis did not reveal a significant difference ( p = 0.13 ) when comparing the incidence of failures in the group of patients with serum vitamin d levels > 30 ng / ml ( 9/394 : 2.2% ) with the incidence of failures in all other patients ( 18/428 : 4.2% ) .", "a relatively small number of experimental studies has attempted to investigate the effects of vitamin d on the osseointegration of dental implants [ 2432 ] . the majority of these studies would appear to indicate a positive effect of vitamin d on osseointegration , but it is not yet entirely clear whether supplementation would promote the healing of peri - implant bone tissue clinically [ 24 , 3335 ] . a recent review of the literature on animal studies has shown that vitamin d supplementation can stimulate new bone formation and increase the contact between the bone and the surface of titanium implants . specifically , kelly et al . demonstrated that vitamin d deficiency could significantly compromise the establishment of osseointegration of ti6al4v implants in rats . in an experimental study on ovariectomized rats , the authors demonstrated that vitamin d deficiency could impair the formation of peri - implant bone ; the normalization of blood levels via supplementation of vitamin d stimulated new bone formation . similar results were reported by zhou et al . , who found an increase in osseointegration in osteoporotic rats given vitamin d supplements , and wu et al . , who demonstrated an increase in the percentage of contact between bone and implant in diabetic rats given vitamin d supplements . finally , liu et al . reported that the administration of vitamin d could increase the fixation of dental implants in mice suffering from chronic kidney disease . a further possibility for study , in order to understand the effects of the administration of vitamin d on bone healing of the peri - implant tissues , is that of coating the implant surface with vitamin d [ 3032 ] . evaluated the effect of the topical application of vitamin d to the surface of implants inserted in postextraction sockets in dogs , with histological and histomorphometric analyses of tissues removed at 12 weeks . topical application of vitamin d increased the percentage of bone to implant contact of 10% . similarly promising results were reported by cho et al . in a histological and histomorphometric study on rabbits , where the coating of anodized implant surfaces with a solution of poly(d , l - lactide - co - glycolide ) plga and 1,25-dihydroxyvitamin d3 ( 1,25-(oh)2d3 ) stimulated the apposition of new bone on fixtures . finally , in a further experimental work in rabbits , implants with a surface coated in 1,25-(oh)2d3 have shown an improved tendency to osseointegrate compared to noncoated implants ; however , this difference was not statistically significant . unfortunately , very few clinical studies have so far investigated the effects of vitamin d deficiency on osseointegration and on bone regeneration in dentistry [ 3335 ] . this is probably due to the fact that there are many factors which can determine the success or failure of dental implants ; the attention of clinicians has been mostly focused on drawing up surgical and prosthetic protocols and identifying new materials and implant surfaces to improve osseointegration , rather than on the analysis of patient - related risk factors [ 69 ] . in a recent clinical work , alvim - pereira et al . found no relationship between polymorphism of the vitamin d receptor and implant failure . in a randomized , investigated the effects of supplementation with a combination of vitamin d3 ( 5000 iu ) and calcium ( 600 mg ) on the formation of new bone following maxillary sinus lift . ten patients were assigned to the test group and given vitamin d and calcium ; ten other patients were assigned to the control group and received only calcium . six to eight months after surgery for bone regeneration , bone samples were taken for histological analysis during implant placement . although supplementation with vitamin d3 would have increased the serum levels of vitamin d with potentially positive effects on bone remodelling at the cellular level , no statistically significant difference was demonstrated between the two groups at the histological level . the results of our study would appear to suggest that a severe deficiency of vitamin d in the blood might be related to an increase in the incidence of early implant failure . in fact , the incidence of early implant failure was rather low ( 2.2% ) in patients with normalized levels of vitamin d in the blood ( > 30 ng / ml ) , rose to almost double ( 3.9% ) in patients with insufficient serum levels ( 1030 ng / ml ) , and were rather high ( 9.0% ) in patients characterized by severe deficiency states . however , despite the tendency to an increased incidence of early failure in patients characterized by deficiency states , the differences between the three groups of patients were not statistically significant ( p = 0.15 ) . our study also confirms that the serum values of vitamin d in the local population are rather low : we found that the proportion of patients with insufficient levels was 49.4% and that the percentage with a severe deficiency was 2.7% . this is not surprising , as most of the patients treated came from northern italy and southern switzerland , regions where exposure to sunlight is somewhat reduced for long periods of the year . in the light of this , the administration of vitamin d in the weeks prior to placement of a dental implant could be useful , particularly in patients with severe deficiency states ; in these patients , vitamin d supplementation should be maintained for the whole life , in order to guarantee a good remodelling of the bone around the implant . this study has the distinction of being one of the first clinical studies carried out on a large number of patients to investigate the possibility of an association between low blood levels of vitamin d and the incidence of early failure in implantology . by restricting the analysis to early failures , occurring in the first period of healing and therefore prior to placement of the prosthetic restoration , we were able to focus our research and avoid a range of factors ( linked to the restoration itself and the prosthetic load ) which could have confused the issue in the study . it is , in fact , well known that implant survival and thus osseointegration depend on a large number of factors ( related to the surgical and prosthetic protocol , the materials used , and lastly the patient ) [ 4 , 5 ] and it can be difficult to identify which of them might be determining the success or failure of the treatment . in order to avoid this and to limit the confounding factors , the same materials were used for all the patients in this study ( the same implant system for all the patients ) [ 1 , 3 , 8 ] . in addition , the same surgical protocol was used , involving submerged healing in the absence of prosthetic loading . thus the only possible confounding factors were the different quantity and quality of bone at the implant receiving sites and the patients ' responses : these are unavoidable factors . however , some of those categories of patients most at risk of implant failure ( patients undergoing bone regeneration to create the conditions for the positioning of the implant fixtures or those with particular medical conditions which might increase the risk of treatment failure ) were excluded in the present study . it is a retrospective work , in which the number of patients having a severe deficiency of vitamin d in the blood was low ( only 22 ) ; thus the presence of even just one less failure in this group would have led to quite different results . it is possible that some residual confounding may have biased the association between vitamin d and implant failures that we observed . for instance , this study did not investigate the influence of other patient - related factors ( e.g. , the bone quality ) which can affect implant survival in the period immediately following implant placement . in addition , if subjects with low levels of vitamin d were also likely to receive more than 1 implant , their risk of being classified as failures may increase . however , no patient in this study experienced more than 1 failure , and the probability of implant failure was not higher ( 1.5% versus 2.1% ) in presence of another implant . therefore , randomized , controlled clinical trials are needed to confirm the presence of an association between low serum levels of vitamin d and an increase in the incidence of early failure in implantology . it would be appropriate to assess whether supplementation of vitamin d in the weeks before the operation could lead to a reduction in early failures , whether due to lack of osseointegration or implant infection . further scientific studies with an appropriate design and a more rigorous statistical analysis will therefore be required in order to thoroughly investigate this issue .", "until now , very few studies , and those mainly on animal models , have involved assessing the influence of blood levels of vitamin d levels on the osseointegration of dental implants . although most of these studies have shown that the administration of vitamin d can improve the healing of the peri - implant bone tissue , it is not yet clear whether vitamin d supplements can promote the osseointegration of dental implants . our retrospective clinical study aimed to investigate if there is a link between low levels of vitamin d in the blood and an increased risk of early implant failures . although the incidence of early implant failures was higher in patients with low serum levels of vitamin d , our study failed in proving an effective link between low levels of vitamin d in the blood and an increased risk of early implant failure . further higher level studies ( prospective controlled trials or , even better , randomized controlled clinical trials ) with a more rigorous statistical analysis are therefore needed to investigate this issue . if an association between low serum levels of vitamin d and higher risk of early implant failure could be demonstrated , the clinician could give a set dose of vitamin d in the weeks before surgery , in order to normalize serum levels and obtain a positive effect on the healing process ." ]

[ "root canal treatment is an effective , less invasive and ideal treatment modality for pulpally involved tooth and salvaging it from extraction . cleaning , shaping and three dimensional obturation of root canal system are essential steps in root canal treatment . the bacteria impervious seal that is essential for success is provided by the root canal sealer and obturation material . introduced mineral trioxide aggregate ( mta ) , a tricalcium silicate cement as a perforation repair material . being biocompatible and bioactive it gained popularity for pulp capping , pulpotomy , apexification and as root end filling material . recently , it is finding application as a root canal sealer and as obturation material . when used as a root canal sealer , mta has the ability to regenerate periodontal ligament and form cementum in the root canal space and accessory canals , thus closing the leeway spaces that can result in the treatment failure . more recently , sealers based on mta have been introduced and found to have good sealing ability and higher push - out bond strengths . in addition , sealers based on mta demonstrated apatite - like deposits in contact with physiological solutions and a biocompatibility similar to mta . a sealer of good working consistency could be developed by mixing mta with adequate quantities of water - soluble polymer . epoxy resin sealers like ah plus was found to bond better to the core obturation material and root dentine . they have the advantages such as less shrinkage , high radio opacity , low solubility , better periapical repair and biocompatibility . studies have evaluated the success of root canal treatment , the incidence of pain and healing ability and many other aspects of the root canal sealers both in vivo and in vitro . evaluation of post - operative pain with visual analogue scale ( vas ) , periapical healing with periapical index ( pai ) are widely used methods and morphometric evaluation with vixwin pro digital image analysis software is a new , reliable and accurate technique . zinc oxide eugenol is an efficient and proven root canal sealer while epoxy resin has equivalent properties to compete with it . this study was done to compare the clinical and radiological outcome of mta or epoxy resin as a root canal sealers compared to zinc oxide eugenol sealer using gutta - percha as the obturating material in teeth with periapical radiolucency .", "this study was conducted in patients in an age group of 18 - 50 years with teeth indicated for root canal treatment . teeth with calcified canals , retreatment cases , pregnant patients , systemic diseases and lactating mothers were excluded . the 45 teeth indicated for root canal treatment were allotted into three groups with 15 teeth in each group . the cases were assigned into any one of the following groups : \n group 1 : zinc oxide eugenol ( tubliseal ) as root canal sealer ( n = 15)group 2 : epoxy resin ( ah plus ) as root canal sealer ( n = 15)group 3 : mta ( proroot mta ) as root canal sealer ( n = 15 ) . \n group 1 : zinc oxide eugenol ( tubliseal ) as root canal sealer ( n = 15 ) group 2 : epoxy resin ( ah plus ) as root canal sealer ( n = 15 ) group 3 : mta ( proroot mta ) as root canal sealer ( n = 15 ) . caries was excavated and if necessary pre - endodontic management was done with composite resin . access cavity was prepared with access preparation kit ( dentsply maillefer , ballaigues , switzerland ) . after irrigation with 2.5% sodium hypochlorite ( naocl ) ( prime dental products , thane , india ) , a k - file of appropriate size was introduced in the root canal and working length was verified with propex ii apex locator ( dentsply maillefer , ballaigues , switzerland ) . this was confirmed by taking a radiograph using parallel cone technique with the help of a film positioning device ( endoray ii , densply rinn . cleaning and shaping was done with k - files ( sybron endo , orange , ca ) and protaper rotary system ( dentsply maillefer , ballaigues , switzerland ) for all the teeth . 2.5% naocl , ethylenediaminetetraacetic acid ( anabond stedman , kanchipuram , india ) and normal saline ( baxter , alathur , india ) were used as irrigants . after cleaning and shaping , the canals were dried and medicated with calcium hydroxide ( endo cal , m dent , bkk , thailand ) mixed with normal saline . after 1 week , patients were recalled and the intracanal medicament was removed and evaluated . once the patient was free of pain , discomfort and canals were dry , the teeth were obturated according to their groups . for group 1 : zinc oxide eugenol ( tubliseal , kerr / sybron , romulus , mi ) was used as root canal sealer . the apical extent of the master cone was confirmed with radiograph and the canals were dried . the root canal was coated with the sealer using lentulospirals ( densply canada , woodbridge , canada ) in a slow speed handpiece ( nsk , tochigi , japan ) . obturation was performed with gutta - percha cones and sealer by lateral compaction technique . for group 2 : epoxy resin ( ah plus , dentsply detrey , konstanz , germany ) was used as a sealer . mta ( proroot mta , densply tulsa , johnson city ) was used as root canal sealer in group 3 . to improve the handling properties of mta and to get a sealer like consistency , the powder was mixed with propylene glycol in a mixing pad . mta sealer was coated in canal walls using lentulo spirals in a slow speed handpiece and obturated as in groups 1 and 2 . permanent restorations were done with composite resin ( filtek z 350 , 3 m espe ) and full coverage restoration if necessary after obturation . follow - up evaluations were done after an interval of 1 week and after 6 months . before commencing the evaluation for pain , every patient was explained about the usage of vas using the following criteria : immediately after obturation and placement of coronal seal every patient was asked to mark the pain intensity using a 10 cm vas . all subjects were recalled after 1 week of post - obturation for evaluation of pain and clinically examined . immediate post - obturation radiograph ( base line data ) was evaluated for pai score and recorded in the evaluation sheet using the criteria described by orstavik . at the 6 month follow - up visit , again a radiograph was taken using parallel cone technique and the pai score was assessed [ figure 1 ] . sample radiographs : ( a ) pre - operative , ( b ) immediate post - operative and ( c ) after 6 months for the groups respectively immediately after obturation , a digital radiograph was taken ( base line data ) with gendex rvg unit for morphometric analysis ( gendex dental systems , hatfiled , pa , usa ) . the image was then subjected to morphometric area measurement with the area measurement tool in the vixwin pro digital image analysis software ( version 1.3 , gendex dental systems , hatfiled , pa , usa ) by outlining the radiolucency . the area measured was recorded in the evaluation sheet . at the 6 month , again the same procedure was repeated and the area was measured using the same criteria as mentioned before [ figure 2 ] . area measurement by outlining the radiolucency using vixwin pro digital image analysis software wilcoxon 's signed rank test was used for comparison of pain and pai score within the groups at different intervals . kruskall - wallis analysis of variance test followed by mann - whitney u test was used for comparison of pain and pai scores between groups at different intervals . wilcoxon 's signed rank test was used for the comparison of area among the three groups at base line and at 6 months .", "before commencing the evaluation for pain , every patient was explained about the usage of vas using the following criteria : immediately after obturation and placement of coronal seal every patient was asked to mark the pain intensity using a 10 cm vas . all subjects were recalled after 1 week of post - obturation for evaluation of pain and clinically examined .", "immediate post - obturation radiograph ( base line data ) was evaluated for pai score and recorded in the evaluation sheet using the criteria described by orstavik . at the 6 month follow - up visit , again a radiograph was taken using parallel cone technique and the pai score was assessed [ figure 1 ] . sample radiographs : ( a ) pre - operative , ( b ) immediate post - operative and ( c ) after 6 months for the groups respectively", "immediately after obturation , a digital radiograph was taken ( base line data ) with gendex rvg unit for morphometric analysis ( gendex dental systems , hatfiled , pa , usa ) . the image was then subjected to morphometric area measurement with the area measurement tool in the vixwin pro digital image analysis software ( version 1.3 , gendex dental systems , hatfiled , pa , usa ) by outlining the radiolucency . the area measured was recorded in the evaluation sheet . at the 6 month , again the same procedure was repeated and the area was measured using the same criteria as mentioned before [ figure 2 ] .", "wilcoxon 's signed rank test was used for comparison of pain and pai score within the groups at different intervals . kruskall - wallis analysis of variance test followed by mann - whitney u test was used for comparison of pain and pai scores between groups at different intervals . wilcoxon 's signed rank test was used for the comparison of area among the three groups at base line and at 6 months .", "pain comparison was performed intergroup and intragroup at three specific time periods . the standard deviation ( sd ) of vas score immediately after obturation for the groups are 8.46 ( 11.26 ) , 10.46 ( 14.86 ) and 7.63 ( 8.46 ) respectively . group 2 was found to have more vas score ( p = 0.19 ) than other two groups but was statistically not significant ( p = 0.19 ) . when the pain was compared within the group at 1 week and at 6 months , there was no difference . vas score after 6 months were 1.63 , 1.06 and 0.33 respectively and there was no statistically significant difference [ tables 1 and 2 ] . comparison of pain between groups measured by vas at baseline , 1 week and at 6 months comparison of pain within group measured by vas at baseline , 1 week and at 6 months there was a highly significant ( hs ) difference in the pai score was found on comparing the base line and at 6 months . however , no difference among the groups was found at any interval [ table 3 ] . comparison of periapical index score within groups at baseline and at 6 months the sd of area measured immediately after obturation for the groups were 8.14 ( 8.40 ) , 9.57 ( 9.20 ) and 4.32 ( 5.20 ) respectively and area after 6 months were 5.4 ( 5.7 ) , 6.80 ( 8.11 ) and 4.14 ( 3.93 ) respectively . there was a hs difference in the area measurement comparing the base line and at 6 months , but no difference among the groups at any interval [ table 4 ] .", "pain comparison was performed intergroup and intragroup at three specific time periods . the standard deviation ( sd ) of vas score immediately after obturation for the groups are 8.46 ( 11.26 ) , 10.46 ( 14.86 ) and 7.63 ( 8.46 ) respectively . group 2 was found to have more vas score ( p = 0.19 ) than other two groups but was statistically not significant ( p = 0.19 ) . when the pain was compared within the group at 1 week and at 6 months , there was no difference . vas score after 6 months were 1.63 , 1.06 and 0.33 respectively and there was no statistically significant difference [ tables 1 and 2 ] . comparison of pain between groups measured by vas at baseline , 1 week and at 6 months comparison of pain within group measured by vas at baseline , 1 week and at 6 months", "there was a highly significant ( hs ) difference in the pai score was found on comparing the base line and at 6 months . however , no difference among the groups was found at any interval [ table 3 ] .", "the sd of area measured immediately after obturation for the groups were 8.14 ( 8.40 ) , 9.57 ( 9.20 ) and 4.32 ( 5.20 ) respectively and area after 6 months were 5.4 ( 5.7 ) , 6.80 ( 8.11 ) and 4.14 ( 3.93 ) respectively . there was a hs difference in the area measurement comparing the base line and at 6 months , but no difference among the groups at any interval [ table 4 ] .", "invasion of microorganisms into the pulp is responsible for the pathogenesis and necrosis of the vital tissue . elimination of infection from the root canal system followed by its maintenance was found to induce healing . root canal sealers along with obturation material will provide a bacteria proof seal of the root canal system , preventing the leeway space and communications between the intracanal and extracanal environments . the present study was designed to compare the clinical and radiological outcome of three different root canal sealers . no difference in the age , sex , tooth type , tooth distribution , root morphology , pai score and area measurement at baseline suggest that the groups were equally distributed [ table 5 ] . baseline values of vas score , pai scores and area measurement zinc oxide eugenol was used as a positive control in this study . this is because it has a history of long - term clinical usage with definitive success rate . it has good handling properties , sealing ability , minimal tissue toxicity , less water solubility , antimicrobial property periapical repair and good radio opacity . ah plus is an epoxy resin based sealer that is widely used because of its compatibility with resin based materials used for obturation and post - endodontic restorations . because of better flow and long setting time , ah plus sealer can penetrates deeper into the surface micro irregularities as well inside the lateral root canals . these properties lead to greater intertwining of the sealer with dentin structure , which , together with the cohesion among the cement molecules provides greater adhesiveness and resistance to dislodgment . mta mixed with propylene glycol was used as a root canal sealer in this study . mta sets hard and its setting time is long enough ( 165 5 min ) to be used as a sealer . calcium sulfate sealers based on mta has demonstrated apatite - like deposits in contact with physiological solutions and a biocompatibility similar to mta . pain was evaluated at 3 time periods - immediately after obturation , 1 week after obturation and 6 months after obturation . on comparison of pain between groups , there was no statistically significant difference in pain experienced by the subjects in any of the time intervals evaluated . clinical difference in pain intensity was observed by patients with epoxy resin sealer experiencing more pain , but was not statistically significant . the results of this study are in accordance with results obtained by alacam in his study . he found no statistical significant difference between the presence and type of post - operative discomfort and the type of sealer utilized . comparison with in the group at different interval has provided a hs difference immediately after the procedure and after 1 week or after 6 months . according to a meta - analysis by nixdorf et al . , the incidence of persistent tooth pain after endodontic therapy is 5.3% . the radiological outcome of root canal treatment using three different sealers was evaluated with pai . when compared with in the group , there is a highly statistically significant difference in the pai score at baseline and after 6 months . this dictates that healing of the periapical lesions may take longer time period than 6 months . but , according to a study by jean camps , there was a noticeable change in the periapical lesion after comparing the radiographs taken immediately after treatment , after 3 months and 6 months of endodontic therapy . however , there was no increase in pai score in any of the groups . this implies that all the three sealers used were highly efficient in inducing healing of the periapical tissues . morphometric area measurement was done using vixwin pro digital image analyzing software ( gendex dental systems ) . the periapical lesions were circumscribed by drawing the outline and the software provided the measured area in cm . there was no difference in the baseline data . when compared at different time periods within the same group there was a decrease in the area measured in all the groups after a period of 6 months . there are no previous clinical studies evaluating area measurement using vixwin pro digital image analysis software as criteria for success of endodontic therapy . so , comparison of results with previous studies in the literature is not possible , but ideally it can be judged that , pai records the periapical status of a tooth based on previous reference radiographs , which is more subjective and differs between the examiners . area measurement makes it more objective , better comparison between pre - operative and follow - up evaluation with less inter examiner bias . in this study , mta was mixed with propylene glycol to get a sealer consistency . however in the future studies , an ideal powder liquid ratio with the vehicle should be established . though radiographically detectable difference in periapical lesions can be appreciated in 6 months as used in this study , further longer period evaluation should be carried out in the future . furthermore , the removal of material from the root canal system , if retreatment is indicated should also be evaluated . being a biocompatible material and having the property to induce the deposition of cementum and periapical repair , mta can prove to be a better material than other sealers . the scope of this study was to evaluate the effectiveness of mta as a root canal sealer considering its radiological and clinical success rate , usage and feasibility . further long - term clinical trials with more criteria should be conducted to conclude the superiority of mta among other root canal sealers .", "among the three root canal sealers used in this study , no sealer was proven to be superior . mta has performed equally well when compared with the zinc oxide eugenol and ah plus . further long - term clinical trials with more criteria should be conducted to conclude the superiority among these materials ." ]

[ "osteoarthritis ( oa ) is characterized by cartilage breakdown , synovial fibrosis , and osteophyte formation . the main clinical symptom of oa is chronic joint pain , which is typically treated using analgesic drugs , such as nonsteroidal anti - inflammatory drugs and corticosteroids [ 1 , 2 ] . however , due to potential adverse effects [ 3 , 4 ] and variable efficacy of these drugs for providing symptomatic pain relief , more effective analgesics are needed to improve oa treatment and patient care . in addition , because only a few effective disease - modifying drugs are available for oa [ 5 , 6 ] , a better understanding of the mechanisms that drive oa pain is required to guide drug development . nerve growth factor ( ngf ) is a widely known pain mediator and plays a critical role in the modulation of oa pain [ 811 ] . the neutralization of ngf with tanezumab , an anti - ngf monoclonal antibody , has robust analgesic effects on oa pain [ 8 , 10 , 11 ] . due to these effects , the regulation of ngf has been investigated in several in vitro and in vivo studies [ 12 , 13 ] . evidence from these studies suggests that the activity of ngf is mediated by inflammatory cytokines . for example , in an experimental arthritic mouse model , il-1 , but not tnf- , increased ngf levels in knee joints . however , the mechanism regulating ngf expression in synovial tissue ( st ) remains unclear . st contains macrophage- and fibroblast - like cells in the lining layer [ 1416 ] . the treatment of cultured synovial oa fibroblasts with il-1 and tnf- induces the production of ngf . more recent studies have shown that macrophages produce a number of inflammatory cytokines , including il-1 , il-6 , and tnf- , which contribute to oa progression and associated joint pain [ 1721 ] . despite these findings , the regulation of ngf expression in synovial macrophages is not fully understood . str / ort mice are a well - characterized oa model that spontaneously develop oa with a progression resembling that of humans [ 2224 ] . synovial hyperplasia is also observed in str / ort mice , and we previously showed that the cd11b+ macrophage population in st str / ort mice is higher than that found in c57bl/6j mice [ 17 , 18 ] . in addition , we reported that compared to synovial fibroblasts ( cd11b ) , synovial macrophages produce high levels of il-1 and tnf- in str / ort mice . here , we characterized the expression profiles of several inflammatory cytokines and ngf in the st of str / ort mice . in addition , the regulation of ngf expression by inflammatory cytokines in synovial macrophages and fibroblasts was also examined .", "nine - month - old male str / ort ( average body weight , 40.6 4.1 g ) and c57bl/6j ( control ; average body weight , 32.8 1.2 g ) mice ( charles river laboratories , inc . , specific pathogen - free colonies of each mouse line were maintained at nippon charles river laboratories ( kanagawa , japan ) in a semibarrier system with a controlled environment ( temperature : 23 2c ; humidity : 55% 10% ; lighting : 12 h light / dark cycle ) . all experimental protocols were approved by the kitasato university school of medicine animal care committee . c57bl/6j and str / ort mice were sacrificed by the intramuscular injection of a mixture of medetomidine , midazolam , and butorphanol tartrate . after removing skin with a scalpel , st was harvested , and total rna was then extracted from the harvested tissue using trizol ( invitrogen , carlsbad , ca , usa ) , according to the manufacturer 's instructions . the extracted total rna was used as a template for first - strand cdna synthesis using superscript iii rt ( invitrogen ) in pcr reaction mixtures consisting of 2 l cdna , specific primer set ( 0.2 m final concentration ) , and 12.5 l sybr premix ex taq ( takara , kyoto , japan ) in a final volume of 25 l . the primers for il6 and ngf were designed using primer blast software and were synthesized by hokkaido system science co. , ltd . the other primers used in this study were designed based on previously published primer sequences . the sequences of the pcr primer pairs used in this study are listed in table 1 . quantitative pcr was performed using a cfx-96 real - time pcr detection system ( bio - rad , hercules , ca , usa ) . the pcr cycles consisted of an initial denaturation step at 95c for 1 min , followed by 40 cycles of 95c for 5 s and 60c for 30 s. mrna expression was normalized to the levels of glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) mrna . the gene expression levels in the st of str / ort mice were compared with those in the st of c57bl/6j mice ( str / ort / c57bl6j ) . in addition , the gene expression levels in cd11b+ cells were compared with those in cd11b cells ( cd11b+/cd11b ) . st samples were collected from both knees of five str / ort mice , and mononuclear cells were then isolated from the collected tissue by digestion with type i collagenase for 2 h at 37c . the obtained mononuclear cells were suspended in 500 l phosphate - buffered saline ( pbs ) containing biotinylated anti - cd11b antibody . following a 30 min incubation at 4c , the cells were washed with pbs , mixed with streptavidin - labelled magnetic particles ( bd imag streptavidin particles plus - dm ; bd biosciences , tokyo , japan ) , and incubated for 30 min on ice in an imag separation system ( bd biosciences ) . warmed ( 37c ) -minimum essential medium ( mem ) was added to the cell suspension to collect unbound ( cd11b - negative ) cells , and an additional 3 ml -mem was added to collect cd11b - positive cells after removing the tub from the magnetic support . the cd11b - positive and cd11b - negative cells were collected by centrifugation at 300 g for 10 min , and the obtained cells were cultured in -mem in six - well plates . tnf- , il-1 , il-6 , and ngf expression in the cells was analyzed by reverse transcription - polymerase chain reaction ( rt pcr ) . st - derived mononuclear cells , including synovial macrophages and fibroblasts , which were isolated as described above , were cultured in -mem in six - well plates . after a 1-week incubation at 37c in a 5% co2 incubator , synovial fibroblasts were incubated with either mouse recombinant tnf- ( 25 ng / ml ) , il-1 ( 50 ng / ml ) , or il-6 ( 100 ng / ml ) ( biolegend , san diego , ca , usa ) for 24 h. cells that were not treated with any cytokines were used as controls . to examine the effect of tnf- on synovial macrophages and fibroblasts , cd11b - positive and cd11b - negative cells were collected from st , as described above , and were then cultured in -mem in six - well plates . after a 1-week incubation at 37c in a 5% co2 incubator , cd11b - positive and negative cells were incubated with mouse recombinant tnf- ( 25 ng / ml ) or il-1 ( 50 ng / ml ) for 24 h. cells that were not treated with tnf- were used as controls . the treated and control cells were harvested for total rna isolation , as described above , and ngf expression was analyzed by rt pcr . ten l3 and l4 drg samples were harvested from the 9-month - old c57bl/6j and str / ort mice and were then immersed in a buffered paraformaldehyde fixative at 4c overnight . the samples were further incubated in pbs containing 20% sucrose for 24 h at 4c . after freezing in liquid nitrogen , each specimen was sectioned at 10 m thickness on a cryostat ( leica microsystems , cm3050s , wetzlar , germany ) and was then treated for 90 min at room temperature with a blocking solution of pbs containing 0.05% tween-20 and 1% skim milk . the sectioned drg specimens were stained with rabbit antibodies against calcitonin gene - related peptide ( cgrp ; 1 : 1000 ; immunostar , hudson , wi , usa ) and transient receptor potential vanilloid 1 ( trpv1 ; 1 : 500 ; calbiochem , san diego , ca , usa ) by incubation for 18 h at 4c . the drg sections were then incubated with alexa 488-conjugated goat anti - rabbit igg ( for cgrp immunoreactivity , 1 : 1000 ; molecular probes ) and alexa 488-conjugated goat anti - rabbit igg ( for trpv1 immunoreactivity , 1 : 1000 ; molecular probes ) . the immunostained sections were visualized using a fluorescence microscope ( axiovert 200 , zeiss , jena , germany ) in a treatment - blinded manner . the numbers of cgrp- and trpv - positive cells were counted , and the proportion of these cells to the total number of nucleated cells in drg was calculated for each drg sample . differences between c57bl/6j and str / ort mice were examined using the t - test . all statistical analyses were performed with spss software ( version 11.0 ; spss , inc . , chicago , il , usa ) . a p value of < 0.05 was considered statistically significant .", "real - time pcr analysis of the genes encoding tnf- , il-1 , il-6 , and ngf showed that the expression levels of these genes were significantly elevated in the st of str / ort mice compared to those in control c57bl/6j mice ( figures 1(a)1(d ) ) . activated macrophages reportedly produce higher levels of inflammatory cytokines , including tnf- , il-1 , and il-6 , than tissue - resident macrophages [ 18 , 26 , 27 ] . to determine whether macrophages in st also produce inflammatory cytokines , expression of the genes encoding tnf- , il-1 , and il-6 in cd11b - positive cells isolated from the st of str / ort mice was examined by real - time pcr . tnf- , il-1 , and il-6 gene expression in cd11b - positive cell fractions was higher than that in cd11b - negative cell fractions ( figures 2(a)2(c ) ) . in contrast , no differences in the gene expression of ngf were detected between the two examined cell fractions ( figure 2(d ) ) . real - time pcr analysis revealed that the gene expression of ngf increased significantly in isolated synovial cells in the presence of exogenously added tnf- and il-1 compared to untreated control cells ( figure 3 ) . in contrast , the gene expression of ngf was not affected in il-6-treated synovial cells ( figure 3 ) . ngf expression was also significantly increased in the presence of exogenously added tnf- in isolated populations of synovial fibroblasts and macrophages compared to untreated control cells ( figure 4(a ) ) . ngf expression was also significantly increased in the presence of exogenously added il-1 in isolated populations of synovial fibroblasts , but not in synovial macrophages ( figure 4(b ) ) . to investigate pain - related sensory innervation by ngf , the proportion of trpv1- and cgrp - positive cells in the drg of str / ort mice was investigated by immunohistochemistry . the number of trpv1- ( figures 5(a ) , 5(b ) , and 5(e ) ) and cgrp - positive cells ( figures 5(b ) , 5(d ) , and 5(f ) ) in the drg of str / ort mice was significantly higher compared to that found in the drg of c57bl/6j mice .", "in the present study investigating the mechanisms underlying the regulation of ngf and development of oa pain , higher expression of the genes encoding tnf- , il-1 , il-6 , and ngf was observed in the st of an oa str / ort mouse model compared to that in c57bl/6j mice . in addition , ngf expression was specifically detected in the cd11b - positive and cd11b - negative cell fractions isolated from st , whereas higher tnf- , il-1 , and il-6 gene expression was observed in the cd11b - positive cell fractions . notably , the treatment of cultured synovial cells with tnf- and il-1 stimulated ngf expression and tnf- also stimulated ngf expression in cd11b - positive and cd11b - negative cell fractions . the number of trpv1- and cgrp - positive cells in the drg of str / ort mice was significantly higher compared to that found in the drg of c57bl/6j mice . taken together , these findings suggest that tnf- regulates ngf expression in both synovial fibroblasts and macrophages and that il-1 regulates ngf expression in synovial fibroblasts and that these factors may contribute to the development of pain in oa patients by innervation of the peripheral nervous system . ngf is upregulated in human synovial fibroblasts , suggesting that this factor plays an important role in oa pathology [ 12 , 13 , 28 ] . ngf elevated inflammatory knee joint and tnf- and il-1 stimulate synovial fibroblasts to produce ngf . reported that ngf expression is upregulated in the knee joint of a carrageenan - treated inflammatory arthritis model and is further stimulated by the intraarticular injection of tnf- and il-1 . further , tnf- and il-1 treatment of cultured synovial fibroblasts derived from a human oa patient also increased ngf production . however , because these studies consisted of analyses of whole knee joints or cultured synovial fibroblasts exogenously stimulated with tnf- and il-1 , the cell populations responsible for regulating ngf and producing tnf- and il-1 in st were not conclusively determined . here , elevated expression of the inflammatory cytokines tnf- , il-1 , and il-6 was observed in the st of str / ort mice , and tnf- and il-1 were increased in synovial macrophages isolated from this oa model . in addition , ngf expression was stimulated by tnf- and il-1 in the synovial fibroblast fraction . taken together , these findings suggest that tnf- and il-1 stimulate ngf expression in synovial fibroblasts . a recent immunohistochemical analysis revealed that ngf not only is localized to fibroblasts , but is also produced by a specific population of macrophages in human st . however , the factor regulating ngf in macrophages was not determined . in the present study , isolated synovial macrophages and fibroblasts from str / ort mice expressed ngf at similar levels to each other . ngf expression in the macrophage fraction was also stimulated by tnf-. taken together , these findings indicate that tnf- stimulates ngf expression in both synovial fibroblasts and macrophages . ngf plays a key role in the generation of acute and chronic pain and peripheral sensitization [ 3033 ] . recent evidence suggests that the trpv1 ion channel and cgrp are involved in the development of ngf - induced persistent mechanical and thermal hypersensitivity in rats [ 30 , 31 ] . ngf acutely modulates trpv1 activity [ 32 , 33 ] and also stimulates cgrp expression in drg neurons in vitro and in vivo . consistent with this speculation , inhibition of ngf by treatment with tanezumab was recently shown to have high analgesic efficacy in oa knees compared with placebo . in the present study , cgrp- and trpv1-positive cells , which are localized in drg neurons in monoiodoacetate- ( mia- ) treated oa animals [ 35 , 36 ] , were increased in the drg of str / ort compared to c57bl/6j mice , suggesting that the elevation of synovial ngf in str / ort mice contributes to peripheral sensitization . however , several studies have shown that human oa chondrocytes also produce ngf by inflammatory cytokines [ 37 , 38 ] . further investigations are needed to clarify the contribution of chondrocytes derived ngf on peripheral sensitization . first , the mechanism underlying the development of oa pain that was proposed based on the present results was based on cross - sectional analysis and therefore no data on the effects of the observed changes on the progression of oa are available . second , although ngf was found to be elevated in the st of oa mice , it remains to be determined if the elevation of synovial ngf levels contributes to peripheral sensitization . finally , present study did not confirm that the protein levels of these cytokines correlated with the expression levels . in conclusion , the present results support the hypothesis that tnf- regulates ngf expression in synovial fibroblasts and macrophages and il-1 regulates ngf expression in synovial fibroblasts in oa mice . tnf- and il-1 may therefore regulate ngf signaling in oa joints and be suitable therapeutic targets for treating oa pain ." ]

[ "\n hepatitis b virus ( hbv ) is a major health problem , with approximately 400 million chronically infected people worldwide , and 1560% of the normal population in many african countries may be positive for one or more of the serological markers of hepatitis b virus infection . these chronically infected patients not only are at an increased stage of developing liver cirrhosis and hepatocellular carcinoma , but also serve as a potential reservoir of infection . the major structural protein of virus envelope , hepatitis b surface antigen ( hbsag ) , is universally considered as a diagnostic marker of hbv infection . the absence of hbsag in the serum and the presence of antibodies to core antigen ( anti - hbc ) usually indicate resolved infection . occult hbv infection ( obi ) usually has a serological evidence of previous hbv infection that has been described in a few cases . hiv coinfection has been reported to modify the natural history of hbv with potential consequences on morbidity and mortality . data on obi in art untreated hiv patients is limited from a vast number of african countries like nigeria , where prevalence of hbv monoinfection , mode of transmission , viral genotype , and mutational pattern varies considerably in different parts of the country . no previous study from nigeria on prevalence on obi among any groups has been carried out . this is particularly important as exposure to hbv is common among hiv - infected cases because of shared routes of transmission . notably , there is considerable variation in prevalence of hiv / hbv coinfection according to geographic regions and exposure risk . successful implementation of art leads to immune reconstitution that can potentially result in immune mediated liver injury in the setting of hbv coinfection . some studies have reported an association between obi and elevated transaminase ; therefore identification of obi is of importance .", "of the 1,200 hiv - infected patients enrolled in the haart clinic of the specialist hospital , ikole , ekiti state , nigeria , from october , 2012 , to april , 2013 , we identified 980 hbsag negative patients ( art - nave subjects ) . among them , 188 were selected for the study by a simple random method . informed consent was obtained from the patients , and the institutional committee approved the study protocol . all samples were tested for hbsag , anti - hbs , anti - hbc , anti - hcv , and anti - hiv using elisa ( drg diagnostics , marburg , germany ) . all anti - hbc positive samples were retested for hbsag as well as for anti - hbc , and only repeat positive samples were included in the study . dna was extracted from all the serum samples using qiaamp dna blood mini kit ( qiagen gmbh , hilden , germany ) following the manufacturers ' instructions . briefly , samples ( 200 l ) were incubated with protease and lysis buffer . after incubation , there were two washing steps , and the nucleic acids were eluted in a volume of 50 l of elution buffer . the presence of hbv dna was examined in all samples using a routine diagnostic pcr . primer pairs were designed from the highly conserved overlapping regions of the s and p genes of the hbv genome . a nested pcr was performed : outer primer pairs were hbpr134 ( sense ) 5-tgctgctatgcctcatcttc-3 and hbpr135 ( antisense ) 5-cagagacaaaagaaaattgg-3 and the inner primer pairs were hbpr75 ( sense ) 5-caaggttatgttgcccgtttgtcc-3 and hbpr94 ( antisense ) 5-ggtataaagggactcacgatg-3. pcr amplifications were carried out in 25 l reaction volumes with 5 ng of genomic dna , 10x pcr buffer ( 20 mm tris - hcl ph 8.4 , 50 mm kcl ; qiagen ) , 2 mm of dntps , 50 ng of each primer , and 1 u ampli taq gold dna polymerase ( applied biosystems ) on a ptc 200 cycler ( peltier thermal cycler watertown , massachusetts , usa ) . thermal cycling parameters were initial denaturation at 94c for 2 min , followed by 35 cycles of 30 sec at 94c denaturation , 30 sec at 52c annealing temperature , and 45 sec at 72c extension , followed by a final extension of 5 min at 72c . thermal cycling parameters remained the same as in the first pcr round except for the number of cycles that is increased to 40 cycles of amplification . each pcr product ( 5 l ) was analysed by electrophoresis in 2% agarose gels . a positive control ( hbv plasmid dna ) and a negative control of the master mix only were integrated to each run to validate the pcr products that yielded a 340 bp fragment .", "quantification of hbv dna was performed with quantitative real - time pcr using a previously described procedure in a geneamp 7300 sequence analyzer ( applied biosystems , perkin - elmer , foster city , ca ) . hbv - plasmid dna was used to generate a standard curve following a serial 10-fold dilution .", "mean age and all the numerical data were analysed using student 's t - test . the chi - square test and fisher 's exact test were used to compare categorical data . for the purpose of our study ,", "the demographic , biochemical , and virological parameters of the study group are summarized in table 1 . the mean age was 35 ( range : 367 ) years . the majority ( 45% ) had multiple sexual partners and 25% of the subjects had a history of concomitant alcohol use . overall , 29/96 ( 29.2% ) of patients were reactive for anti - hbc , an indication of prior exposure to hbv dna , and majority 6/8 ( 75% ) of the patients were female ( table 2 ) . thus , in the total study population , 21/188 ( 11.2% ) of patients were identified as obi and 62.5% of the obi patients had cd4 count less than 200 cells / mm . averagely the hbv viral load was < 50 copies / ml in the obi samples examined by quantitative pcr . serum levels of ast and alt were higher among patients with obi in comparison to anti - hbc positive hbv dna negative individuals , but the difference failed to reach standard significance ( p = 0.13 and p = 0.07 ) , respectively . the comparison of different demographic , biochemical , and virological factors between hbv dna positive and negative cases was illustrated in table 3 . the distribution of the study participants as per the 1993 revised classification system for hiv infection and expanded surveillance case definition for aids among adolescents and adults was as shown in table 4 . ", "the present study represents a comprehensive cross - sectional analysis of prevalence of obi in an art nave hiv positive cohort comprising various risk groups . most previous studies looking at the clinical effects of obi in hiv include a large number of patients on anti - hbv drugs as a component of art . this study describes the risk factors associated with obi , frequently of anti - hbc positivity and its possible values as a serological marker for identifying hiv - infected patients who benefit from hbv dna assay . we found the prevalence of occult hbv to be 11.2% among a random selected group of hiv - infected patients . the prevalence of obi in hiv positive individuals varies worldwide between 0 and 90% , depending on the geographic regions , risk factors , and the exposure involved . in the present study , the prevalence of anti - hbc ( 29.2% ; 28 of 96 ) and obi ( 28.6% ; 8 of 28 ) among the art nave hiv positive cohort was higher compared to previous report on blood donors from studies done in areas of india , areas which reported 21.3% obi among the hbsag negative anti - hbe positive donors . within nigeria , hbv and hcv coinfection among hcv - infected patients most of the previous studies on hbv / hiv co - infection are aimed at detecting hbv prevalence in the hiv population are based on hbsag positivity ( prevalence 9.9 to 11% ) , but reports on obi are scarce . a previous study on intravenous drug users in northeastern india detected a prevalence of 15.9% . among the obi cases , the rate of anti - hbs was lower ( 2/8 ; 25% ) , which may be due to the fact that hiv - infected patients are prone to lose anti - hbs immunity at a higher frequency than the general population . previous reports suggested that the lower hbv replication was associated with milder hepatic damage . among the subjects with obi , elevated alt or ast however , our study is cross - sectional ; therefore evaluation of long term clinical significance of obi should be better addressed by follow - up studies . the low level of viral load obtained in this study buttress the findings in another study that showed that showed that almost all obi cases are infected with replication incompetent hbv , revealing a strong suppression of overall replication activity and gene expression , thereby resulting in a significant reduced viral load . hiv patients are screened for concomitant chronic hepatitis b using hbsag elisa , and it is not considered cost - effective to perform hbv dna testing for all hiv patients in our resource - poor setting . our study tried to identify possible clinical and serological markers which could guide dna testing in these patients . obi is reported to be common among hcv infection , but we found its prevalence to be low among our study group . however , anti - hcv was not tested among the other hiv positive samples attending the specialist hospital , ikole , ekiti state , nigeria . furthermore , none of the risk factors were found to be statistically significant markers of obi and can not be used as an independent marker for identifying patients who should benefit from hbv dna estimation . however , as one third of the anti - hbc positive negative patients were positive for hbv dna ( 8 of 28 ) , it is recommended that hiv positive patients with hbsag negative / anti - hbc positive patterns should be tested for the presence of hbv dna irrespective of their anti - hbs status . nevirapine is commonly included in the first line art regimens at most treatment centers in nigeria . our study identified only 21 subjects with obi , and all the samples were collected from a single center indicating that results might differ in setting with significant different demographic characteristics .", "a main implication of the presently viable data is therefore further emphasizing the need for efficient hbv vaccination programs . overall , the present study highlights the need for screening hbv before the initiation of any haart containing anti - hbv regimens in hbv / hiv coinfected patients . it necessitates the use of nat for effective laboratory diagnosis of occult hbv infections in hiv positive patients , especially in developing countries where these assays are not widely available ." ]

[ "epilepsy is generally deemed intractable or refractory to treatment if the use of two or more appropriately chosen antiepileptic drugs ( aeds ) fails to adequately control seizures . the aeds in question must be at the maximum tolerated doses , at a sufficient therapeutic level and not be discontinued due to side effects . a less commonly used definition of intractability requires monthly seizures for a period of 18 months.1 despite optimal medical management with appropriate aeds , 20%30% of adults and 10%40% of children diagnosed with epilepsy continue to suffer from seizures.28 furthermore , after the failure in efficacy of two aeds , the chance of achieving seizure freedom by introducing subsequent drug regimens has been shown to be less than 10%.2,812 intractable epilepsy is a significant problem for the quality of life ( qol ) of the individual patients . persistent seizures heavily impact on the patients behavior , cognitive ability and psychosocial well - being . it has been shown that this condition can lead to poor academic achievements , increased probability of unemployment , low self - esteem , anxiety and depression , social isolation and ultimately to decreased quality of life.13,14 in addition , the unremitting seizures can pose a significant financial burden , with patients suffering from poor seizure control reported to represent approximately 25% of epilepsy cases but accounting for greater than 85% of epilepsy associated costs , mainly due to repeated hospital admissions and extensive investigations.15 further to the financial burden , patients with refractory seizures have higher mortality rate than patients whose seizures are well - controlled , who have mortality rates comparable to the general population.16 this is likely to be due to differences in the underlying etiology rather than the effect of seizures per se . patients with refractory seizures have higher rates of structural abnormalities or inborn errors of metabolism ( iem ) . in addition , sudden unexpected death in epilepsy ( sudep ) is forty times more common in patients who continue to suffer from seizures compared to patients who are seizure free.17 the number of new aeds has significantly increased in the last 20 years , effectively doubling the number of anticonvulsants available for physicians to prescribe . whilst the efficacy of new molecules in controlling seizures has not dramatically increased , safety and tolerability profiles are significantly better than for the older aeds . many of the new drugs have novel mechanisms of action and exhibit fewer drug - interactions . when considering the most appropriate choice of treatment this is of particular importance in children with refractory epilepsy , a large proportion of whom present specific age - dependent epilepsy syndromes . accurate identification of the epilepsy syndrome is the foundation for the choice of an appropriate treatment and increases the likelihood of seizure remission.18 in some instances , identifying etiologies can radically alter the potential treatment options , for example metabolic treatments such as the ketogenic diet for epilepsies resulting from iem.19 surgery may be the most appropriate treatment option in some cases , especially where strong evidence exists for its efficacy , such as for the anterior temporal lobectomy in drug - resistant temporal lobe epilepsy.20 however , the most appropriate timing for surgery remains debated . when surgery is not a suitable treatment option , neurostimulation therapies such as vagus nerve stimulation ( vns ) may be considered . this review will outline the evidence for the use of different treatment options for patients with intractable seizures . whilst the focus will be on the pharmacological options , we will also review the extent and quality of evidence supporting the efficacy of surgical , metabolic , neurostimulatory and herbal interventions in the context of the associated side - effects , safety and patient preferences .", "before considering any changes to pharmacological treatment in a patient with refractory seizures , confirmation that the events are epileptic in origin is essential . it is estimated that up to 30% of patients seen in specialist epilepsy clinics present with non - epileptic attacks alone or in combination with epileptic seizures.21 in these patients , the prescription of aeds is often inappropriate and can lead to significant side effects , in addition to the financial burden to the health services . in many patients with refractory seizures , video - electroencephalography ( video - eeg ) monitoring is essential to characterize the events and reach diagnostic conclusions with a high level of confidence . a better understanding of the underlying etiology can inform treatment decisions and lead to optimal seizure control over a shorter time period . this is also the case for patients who suffer from a specific epilepsy syndrome , for whom the importance of correct classification at an early stage should also be emphasized . the correct identification and classification of seizures and epilepsy types allows evidence - based choice of syndrome - specific or etiology - specific treatment intervention , as is the case for valproate treatment as a first line agent in juvenile myoclonic epilepsy.18 this stepwise approach to correct diagnosis and subsequent treatments options is outlined in figure 1 . patients with refractory seizures have , by definition , tried at least two appropriate aeds and their seizures have failed to be adequately controlled . at this point the physician faces the decision to either add a further aed to the current treatment or to pursue sequential monotherapy . if sequential monotherapy is chosen , the drugs that are failing to control seizures are slowly tapered off and replaced with different aeds which can potentially improve seizure control . sequential monotherapy was the main treatment paradigm from the 1970s through to the 1990s , when sodium channel blocking aeds were the only treatment option . combining drugs with the same mechanism of action lead to a slightly better seizure control , but at the expense of a marked increase in adverse drug interactions and pharmacodynamic amplification of side effects.22 this practice changed in the 1990s , when new aeds were introduced with novel mechanisms of action . these drugs exhibited fewer pharmacokinetic interactions , exhibited fewer side effects when combined and raised the possibility of synergistic drug combinations . clinical practice over the past 20 years has been characterized by rational polytherapy : numerous drug combinations have been considered and a wealth of evidence on which combinations are most effective and where drug interactions are most likely to occur has accumulated.23 when contemplating polypharmacy for a patient with epilepsy , it is important to consider the concept of total drug load in addition to the number of drugs being prescribed . although general agreement on the definition has not been reached , drug load is usually defined as the total amount of drug exposure for a treatment regimen , and has been quantified as the prescribed daily dose of aed(s ) divided by the defined daily dose for the aed(s ) as outlined by the world health organization . drug load has been demonstrated to correlate with the number of adverse events better than the number of aeds being prescribed.24 it has been argued that for the use of multiple aeds to be rational , the combination of two drugs must provide supra - additive therapeutic efficacy , whereby the combined efficacy of the two drugs is greater than when the drugs are given alone . alternatively , combining aeds may be more advantageous than prescribing monotherapy at standard doses if they demonstrate simple additive efficacy but less than additive side effects . however , pharmacokinetic drug interactions often contraindicate specific drug combinations . some of the traditional aeds , including phenobarbital , phenytoin and carbamazepine are metabolized in the liver and are potent inducers of the cytochrome p450 ( cyp450 ) system . these drugs act by blocking voltage - dependent sodium channels preventing the repetitive and uncontrolled neuronal discharges seen in epilepsy . phenobarbital , in addition , enhances gaba mediated inhibition , which further acts to prevent the repetitive neuronal firing.25 in the developed world , phenobarbital is now prescribed rarely due to its sedative side effect profile . however due to its relatively low cost it is regularly prescribed in developing countries . all of the drugs described above induce hepatic enzymes and decrease the serum concentration of additional aeds that may be prescribed . as a consequence of this effect , each drug may impede the other , by preventing effective serum concentrations being reached.22 it is therefore advised that the combination of an enzyme - inducing aed with another aed which is metabolized in the liver requires particular caution . interactions with other medications should also be considered when prescribing hepatic enzyme inducing drugs , particularly in patients taking exogenous contraceptive hormones , immunosuppressant therapy , a number of corticosteroids and some antineoplastic chemotherapeutic agents , all of which can be the subject of enzyme - induction . physicians should also be aware of the additional adverse effects that enzyme - inducing drugs can produce , including secondary hyperparathyroidism , reduced serum estosterone concentrations , menstrual irregularities and elevated cholesterol levels , which may impact on a patient s cardiovascular disease risk . consequently , it has been suggested that the older , enzyme - inducing aeds should not be considered as first - line treatment options for patients with refractory seizures.26 among older aeds , valproate is characterized by a number of mechanisms of action , including sodium channel blockade and potentiation of gaba mediated inhibition . it is also metabolized by the liver , but has no enzyme - inducer properties . the newer aeds , including gabapentin , levetiracetam , pregabalin , vigabatrin , lamotrigine , oxcarbazepine , topiramate and zonisamide , are characterized by a more favorable tolerability profile , as their hepatic enzyme induction properties are minimal or absent . many of these drugs have novel or unknown mechanisms of action and demonstrate similar levels of efficacy and fewer side effects , less adverse pharmacokinetic drug interactions and are tolerated better by patients.25 when compared to carbamazepine in double - blind randomized control trials for treatment of partial onset seizures , gabapentin , lamotrigine and oxcarbazepine were found to have an similar efficacy but better levels of tolerability.2729 until recently , direct comparison studies in treatment - refractory epilepsy populations have been sparse . a recent systematic review and meta - analysis of studies on patients with refractory partial epilepsy suggested that topiramate and levetiracetam have a greater efficacy in controlling seizure frequency than the other new aeds.30 the same study found that gabapentin is likely to be less effective than other new aeds . in terms of tolerability , oxcarbazepine and topiramate were associated with significantly more withdrawals and therefore more poorly tolerated than the other new aeds . gabapentin and levetiracetam , on the other hand , were associated with significantly fewer withdrawals and were therefore better tolerated . it should be noted that these conclusions were based on indirect comparisons and should be treated with caution . although the newer aeds exhibit fewer side effects than traditional aeds , the potential for significant cognitive and behavioral side effects should be considered when prescribing . drugs that have a mechanism of action involving the potentiation of gaba mediated inhibition such as vigabatrin , tiagabine , topiramate and gabapentin have been associated with sedation and negative effects on mood . some patients who are prescribed these aeds are at risk of developing depression as a side effect , especially patients with a past psychiatric history.31 topiramate has been associated with unfavorable cognitive effects , including impaired concentration , psychomotor slowing , language regression , comprehension problems and memory deficits.32 other new aeds which act by inhibiting the glutamatergic excitatory neurotransmission have been associated with an taking advantage of these properties , lamotrigine may be considered in patients with concomitant depression , apathy and hypersomnia.31 in addition , psychotic symptoms have been reported as adverse events following aed administration , especially with topiramate and levetiracetam , although the extent that the medication plays in producing these symptoms remains uncertain.33 it is recommended that baseline mood and cognitive ability is taken into account when deciding which aed to prescribe in refractory patients , in order to minimize any impact that prescribing may have on mood and cognition . in addition to the above evidence , clinical studies on polytherapy are informative when considering using a combination of aeds . one study on 47 patients with cognitive impairment showed that seizures were controlled more effectively by a combination of phenobarbital and phenytoin or phenobarbital and carbamazepine , than by phenytoin and carbamazepine combination.34 this suggested that a combination of two sodium - channel blocking aeds does not improve seizure control significantly . other studies indicate that a combination of carbamazepine and valproate or carbamazepine and vigabatrin is more effective in seizure control than a combination of carbamazepine and phenytoin , although these studies were not controlled for drug concentration.35,36 the aed combination for which there is the most convincing evidence is lamotrigine and valproate . a large study aiming to assess the efficacy of lamotrigine monotherapy also evaluated the effect on seizure control whilst taking lamotrigine as add - on to the first aed ( carbamazepine , phenytoin or valproate ) . this study showed that patients who were taking lamotrigine and valproate experienced an 83% reduction in seizures , whilst those taking the lamotrigine and carbamazepine or lamotrigine and phenytoin combinations experienced only a 43% and 34% reduction in seizures , respectively.37 these findings should be interpreted with some caution , however , as different doses of lamotrigine were used in each combination .", "for resective surgery to be a viable option , the patient with treatment - refractory epilepsy should ideally have a single epileptogenic focus in a non - eloquent cortical region ( ie , not involved in key language , memory or motor processes ) . it should be noted that exceptional cases do exist , where larger resections such as hemispherectomies are acceptable if the seizures are severe and the benefit gained from such surgery can be justified.38 major surgery such as corpus callosotomy can be considered as last treatment option in palliative care for patients with intractable seizures . this invasive intervention is usually aimed at preventing secondary generalization in patients who are regularly experiencing loss of consciousness and a high frequency of seizure - related injuries . most epileptologists consider surgical management as second line therapy for patients with intractable seizures.39 this is especially true for patients with drug - resistant temporal lobe epilepsy ( tle ) , for whom temporal lobectomy can represent an effective treatment option . a randomized control trial found that 58% of patients randomly allocated to surgery for temporal lobe resection did not present with any seizures which impaired consciousness after one year , compared to 8% in the treatment arm receiving the optimum medication regimen ( p < 0.001).20 a multi - center study which followed 339 patients with epilepsy for 2 years post - operatively showed that temporal lobe resection is the most effective type of resective surgery in this patient population : 68% of the patients who received temporal lobectomies experienced seizure remission for 2 years , compared to 50% of the patients who underwent extratemporal resections.40 another non - randomized controlled study which followed up patients with refractory seizures , assigned either to surgical treatment or continued drug therapy , found that 44.6% of 242 patients with pharmacoresistant tle who received surgery were seizure - free for 12 months , compared to 4.3% of those who received aeds only ( p < 0.001).41 it should be emphasized , however , that not all patients with tle have the same probability of achieving seizure freedom through temporal lobe resection . a meta - analysis of 83 studies including 7,343 patients undergoing epilepsy surgery showed that the proportion of patients experiencing seizure freedom for at least 5 years post - operatively was highest in those who underwent temporal lobe resection ( 66% ) , even though a substantial proportion of patients undergoing occipital , parietal or frontal lobe resections demonstrated post - operative seizure freedom ( 46% , 46% and 27% respectively).42 it should be noted that post - operative seizure freedom for the first few years following surgery might not persist long - term . isolated relapses can still occur many years after the procedure , with a probability of seizure relapse of 4% per year for the 5 years following surgery.43 overall , patients with structural abnormalities apparent on pre - operative imaging studies , such as hippocampal sclerosis or foreign tissue lesions are more likely to experience post - operative seizure freedom compared to patients with no obvious lesion.4446 when considering surgery for refractory epilepsy , there are a number of risks that need to be taken into account alongside the potential benefits . the surgery itself can cause a substantial period of disability , with patients typically having to take 48 weeks off work or school.47 the most common medical complications are deep vein thrombosis , infection and transient endocrine abnormalities . psychiatric complications are reported post - resection in 25% of the patients , including transient dysphoria , depression and occasionally mania.48 although less commonly reported , permanent neurological and neuropsychological complications can result from resective surgery . these differ depending on the site of resection , and tend to be more common and disabling if the site is close to areas of the cortex involved in key cognitive functions . furthermore , the procedure has the possibility to create new lesions which may become epileptogenic and trigger subsequent seizures.47 on the other hand , patients are often withdrawn from aeds following surgery , and therefore relieved from many of the adverse effects of these medications . surgical procedures are generally considered after the failure of two appropriate drug regimens , although the number of unsuccessful trials with aeds , alone or in combination , before considering surgery remains a matter of debate.47,49 when contemplating surgery , it is important to assess the likelihood of seizure remission using further drug regimens compared to surgical resection , whilst considering the risks associated with such procedures . although current evidence suggests that temporal lobe surgery is the most effective treatment option after the failure of two aeds , the same can not be said for other surgical procedures , such as frontal lobe resection , whereby the overall benefit from surgery may not be greater than trying alternative drug regimens . in these cases , the risks for each treatment option should be weighed up on a case - by - case basis until more definitive data is available to guide therapeutic decisions . patients with intractable epilepsy who are not suitable for epilepsy surgery or those who continue to experience seizures post - operatively may benefit from neurostimulation . this treatment option for patients with intractable epilepsy involves electrical stimulation of the nervous system using surgically implanted devices , or non - invasive stimulation as is the case for repetitive transcranial magnetic stimulation ( rtms ) . vagal nerve stimulation ( vns ) therapy is the only neurostimulation method to have received approval by the usa food and drug administration , whilst both vns and deep brain stimulation ( dbs ) are approved for use in patients with refractory epilepsy in the uk . in vns therapy , intermittent electrical stimulation is delivered to the left vagus nerve , which has ascending fibers with widespread connections to the limbic , autonomic and reticular brain regions . the reduced thalamic blood flow that is observed in vns has been proposed to underlie the mechanism for seizure reduction.50 in addition to a number of retrospective studies , efficacy for this treatment option has been demonstrated in two randomized , placebo - controlled , double - blind trials , which reported a median seizure reduction of 24.5%28.0% in the group receiving high level vns compared to just 6.1%15.0% in patients receiving low level vns ( p = 0.01 and p = 0.04 , respectively).51,52 the risks associated with implantation of the vns device are relatively low , with a 3%5% chance of infection.5355 vocal cord dysfunction , throat discomfort , change in voice quality and sleep apnea can present post - operatively , and such consequences should be discussed with the patient when considering this treatment . an additional factor that should be considered when planning vns therapy is that patients are usually unable to undergo magnetic resonance imaging ( mri ) after implantation , since the changing magnetic fields may induce electrical currents in the device.56 dbs involves electrical stimulation of specific subcortical nuclei , which have widespread neural connections . the anterior nucleus of the thalamus is often the target of dbs due to its widespread projections to the limbic system . in a randomized controlled trial of 109 patients with refractory epilepsy who had dbs electrodes implanted in the anterior nucleus of the thalamus there was a 29% greater reduction in seizures for 54 patients who had the stimulator switched on compared to 55 patients who had their stimulation turned off after a blinded period of 3 months ( p = 0.002).57 infection , hemorrhage and stimulation - induced seizures were amongst the most frequently reported complications . in the same study , impaired memory and higher levels of depression were observed in the group receiving the active treatment . dbs has been recently licensed in the uk , and nice guidelines suggest that this method should be used in highly selected cases , due to the lack of efficacy data and the associated risks.58 the centromedian nucleus is also a potential location for dbs , since it is part of the reticulo - thalamo - cortical system which is involved in mediating cortical excitability and wakefulness . one double - blind crossover trial reported a 30% reduction in generalized tonic - tonic seizures when the device was turned on compared to when it was off , although this change was not significant , possibly due to the small sample size of 7 patients.59 further evidence for centromedian nucleus stimulation efficacy can be found in a longitudinal study which reported good efficacy for generalized tonic - clonic seizures , atypical absences and tonic seizures , however caution should be taken in interpreting these findings because of the lack of control groups.60 unlike other methods of neurostimulation , responsive neurostimulation ( rns ) does not deliver electrical stimulation at specific frequencies throughout the day . instead , the rns device is composed of a combined recorder and stimulator device , which detects clinically relevant epileptiform discharges and delivers an appropriate electrical stimuli in response . a recent multicenter double - blind randomized controlled trial in adults with intractable partial seizures demonstrated a reduction in seizure frequency of 37.9% in the treatment arm compared to 17.3% in the control group ( p = 0.012).61 the fourth and only non - invasive neurostimulation technique , low frequency rtms , is thought to suppress cortical excitability either via inducing long term depression or alternatively by enhancing gabaergic inhibition.62 although evidence for its efficacy is limited , a number of small - scale studies suggest antiepileptic properties ( see fregni and pascual - leone for a comprehensive review).63 over the past century , the efficacy of dietary therapies for intractable seizures has increasingly been recognized . currently there are four main types of metabolic therapy in use for the reduction of seizures . three of these have a high fat content and low carbohydrate content the classic ketogenic diet , the medium chain triglyceride ( mct ) diet and the modified atkins diet ( mad)and the fourth controls the carbohydrate quality , the low glycemic index treatment ( lgit ) . these treatments are usually considered in patients with intractable epilepsy who have failed to respond to several aeds and are not suitable candidates for surgery . the classic ketogenic diet is a high fat and low carbohydrate diet that uses long chain fatty acids ( lcfas ) as its main source of fat . typically the patient must consume 34 grams of fat for every 1 gram of carbohydrate plus protein . as a result , between 86% and 90% of the calories from this diet are provided from lipids.64 the mct diet provides more ketones per calorie consumed than in the classic ketogenic diet , which uses mainly lcfas . in mct , more carbohydrates and proteins can be consumed for every gram of fat . the mad recommends increased consumption of foods high in fat whilst restricting the amount of carbohydrates . unlike the classic ketogenic and mct diets , there is no limit on the amount of protein consumed or the total number of calories per day.65 the typical mad uses a ratio of 1 gram of fat for every gram of carbohydrate plus protein , and consequently is less restrictive than the other two high fat diets . the lgit restricts carbohydrate intake to food items with a glycemic index of less than 50 at 4060 grams per day , in order to prevent large fluctuations in blood glucose concentrations , which are thought to exacerbate seizures.66 there is a large body of evidence demonstrating the efficacy of the metabolism - based therapies for refractory seizures , with the majority focusing on the classic ketogenic diet . two meta - analyses of observational studies showed that 15.6%15.8% of patients on the diet can achieve seizure freedom whilst 33.0%55.8% of the patients can have a > 50% reduction in number of seizures.67 one of the only randomized controlled trials to investigate the efficacy of the classic ketogenic diet followed children with intractable or focal - onset epilepsy who were randomized to a ketogenic diet or continuation of a normal diet for a 3-month period . of the patients who received the ketogenic diet , 38% had a seizure reduction of > 50% . this was significantly greater than the control group , where only 6% had > 50% seizure reduction ( p < 0.001).68 the results of a follow - up study comparing the effectiveness of the lcfa versus the mct diet suggest there is no increased benefit using either of the two diets for seizure reduction.69 despite the lack of prospective randomized control trials , the mad has also demonstrated substantial efficacy in controlling seizures . of the patients who have been studied on the mad , 45% have demonstrated 50%90% seizure reduction and 28% have > 90% seizure reduction . similar results have been reported in two retrospective studies of the lgit , with 38%73% of patients achieving > 50% seizure reduction.70,71 the best candidates for the mad tend to be adults and adolescents , for whom compliance on the lcfa and mct diets is an issue.64 this is because the food consumed on the mad is similar to that being consumed by peers . the same can be said about the lgit , which in addition can be considered for patients who find the other 3 high - fat diets unpalatable . there is a body of evidence suggesting that metabolism - based therapy can be considered earlier in the treatment plan for patients with specific epilepsy syndromes . one notable example is west syndrome ( infantile spasms ) , where the ketogenic diet is successfully used as a first line agent.72 the same diet is also thought to be especially effective in unspecified symptomatic generalized epilepsy , multifocal epilepsy , juvenile absence epilepsy , continuous spike and slow wave of sleep , myoclonic - astatic epilepsy ( mae or doose syndrome ) , severe myoclonic epilepsy of infancy ( dravet syndrome ) , and seizures in patients with tuberous sclerosis complex and landau - kleffner syndrome.68,73,74 ketogenic diets are also recommended for patients suffering from glucose transporter defects and pyruvate dehydrogenase deficiency , who are unable to utilize glucose for brain metabolism.75,76 the most common side effects of dietary treatments are constipation , acidosis , temporary hypercholesterolemia , kidney stones and hunger.64 growth restriction has also been observed in children.77 the ketogenic diet is contraindicated in patients who are unable to use ketone bodies as an energy source or those who require high levels of glucose . such conditions include pyruvate decarboxylase deficiency , primary carnitine deficiency , fatty acid oxidation abnormalities , the porphyrias and some mitochondrial disorders.78 generally , as with aeds , it is suggested that after adhering to dietary therapy for a period of 2 years , the treatment should slowly be tapered off . many patients who become seizure free whilst adhering to the diets remain seizure free off the diet . this has been demonstrated in one study , which reported the risk of seizures recurring when stopping the diet at only 20%.79 it has been hypothesized that the dietary therapies have a long - term , disease modifying effects , although the observed phenomenon could just be explained by the natural history of the seizure disorder.64 in addition to showing high levels of efficacy , similar to many aeds , the dietary interventions are generally quite tolerable , can have associated positive effects such as weight loss,80 and demonstrate effectiveness in a relatively short time period . whilst aeds may take several weeks to be titrated up to a therapeutic dose and vns may take months or years to show an optimal effect,81 the effects of a ketogenic diet can be seen within a few days . furthermore , the diets are cost effective compared to many aeds.64 in practice , a median of 5 aeds are prescribed before considering a metabolic treatment such as the ketogenic diet.82 furthermore , 60% of child neurologists only use a ketogenic diet as a last resort treatment.83 it is therefore suggested that the metabolic treatments should be considered earlier in the course of treatment , especially in patients with refractory seizures who are not suitable for or willing to undergo resective surgery . herbal treatments are generally viewed as alternative and complementary forms of medicine by both patients and physicians . there is , however , a small body of evidence demonstrating the effectiveness of some herbs at controlling seizures . out of all the herbal treatments , cannabinoids have the largest body of evidence supporting their use as anticonvulsants . one randomized , double - blind controlled trial reported 50% of patients with secondary generalized epilepsy receiving cannabidiol becoming virtually seizure - free for the duration of the study . however , only 15 patients were included and to our knowledge no follow - up study has been performed.84 the use of cannabis for epilepsy has been legalized in canada and licensed in 14 states within the united states , although it is prohibited in most european countries.85huperzine a. has been shown to be effective in animal models of epilepsy , and a clinical trial to assess its effectiveness has been planned.86,87 there is also laboratory data supporting the efficacy of kava ( piper methysticum ) and mistletoe ( viscum sp ) , but no clinical evidence exists to support their use . a number of other herbal treatments that are commonly used to treat seizures have either been shown to have no effect on the severity of seizures , or in fact demonstrate proconvulsant properties and exacerbate the condition . in addition , much evidence is anecdotal , or when clinical studies have been performed , these often display poor methodology and do not have sufficient levels of statistical power to make firm conclusions from . further , good quality evidence in the form of controlled trials is required to justify the use of herbal treatments in a clinical setting ( see pearl et al for a comprehensive review).88", "for resective surgery to be a viable option , the patient with treatment - refractory epilepsy should ideally have a single epileptogenic focus in a non - eloquent cortical region ( ie , not involved in key language , memory or motor processes ) . it should be noted that exceptional cases do exist , where larger resections such as hemispherectomies are acceptable if the seizures are severe and the benefit gained from such surgery can be justified.38 major surgery such as corpus callosotomy can be considered as last treatment option in palliative care for patients with intractable seizures . this invasive intervention is usually aimed at preventing secondary generalization in patients who are regularly experiencing loss of consciousness and a high frequency of seizure - related injuries . most epileptologists consider surgical management as second line therapy for patients with intractable seizures.39 this is especially true for patients with drug - resistant temporal lobe epilepsy ( tle ) , for whom temporal lobectomy can represent an effective treatment option . a randomized control trial found that 58% of patients randomly allocated to surgery for temporal lobe resection did not present with any seizures which impaired consciousness after one year , compared to 8% in the treatment arm receiving the optimum medication regimen ( p < 0.001).20 a multi - center study which followed 339 patients with epilepsy for 2 years post - operatively showed that temporal lobe resection is the most effective type of resective surgery in this patient population : 68% of the patients who received temporal lobectomies experienced seizure remission for 2 years , compared to 50% of the patients who underwent extratemporal resections.40 another non - randomized controlled study which followed up patients with refractory seizures , assigned either to surgical treatment or continued drug therapy , found that 44.6% of 242 patients with pharmacoresistant tle who received surgery were seizure - free for 12 months , compared to 4.3% of those who received aeds only ( p < 0.001).41 it should be emphasized , however , that not all patients with tle have the same probability of achieving seizure freedom through temporal lobe resection . a meta - analysis of 83 studies including 7,343 patients undergoing epilepsy surgery showed that the proportion of patients experiencing seizure freedom for at least 5 years post - operatively was highest in those who underwent temporal lobe resection ( 66% ) , even though a substantial proportion of patients undergoing occipital , parietal or frontal lobe resections demonstrated post - operative seizure freedom ( 46% , 46% and 27% respectively).42 it should be noted that post - operative seizure freedom for the first few years following surgery might not persist long - term . isolated relapses can still occur many years after the procedure , with a probability of seizure relapse of 4% per year for the 5 years following surgery.43 overall , patients with structural abnormalities apparent on pre - operative imaging studies , such as hippocampal sclerosis or foreign tissue lesions are more likely to experience post - operative seizure freedom compared to patients with no obvious lesion.4446 when considering surgery for refractory epilepsy , there are a number of risks that need to be taken into account alongside the potential benefits . the surgery itself can cause a substantial period of disability , with patients typically having to take 48 weeks off work or school.47 the most common medical complications are deep vein thrombosis , infection and transient endocrine abnormalities . psychiatric complications are reported post - resection in 25% of the patients , including transient dysphoria , depression and occasionally mania.48 although less commonly reported , permanent neurological and neuropsychological complications can result from resective surgery . these differ depending on the site of resection , and tend to be more common and disabling if the site is close to areas of the cortex involved in key cognitive functions . furthermore , the procedure has the possibility to create new lesions which may become epileptogenic and trigger subsequent seizures.47 on the other hand , patients are often withdrawn from aeds following surgery , and therefore relieved from many of the adverse effects of these medications . surgical procedures are generally considered after the failure of two appropriate drug regimens , although the number of unsuccessful trials with aeds , alone or in combination , before considering surgery remains a matter of debate.47,49 when contemplating surgery , it is important to assess the likelihood of seizure remission using further drug regimens compared to surgical resection , whilst considering the risks associated with such procedures . although current evidence suggests that temporal lobe surgery is the most effective treatment option after the failure of two aeds , the same can not be said for other surgical procedures , such as frontal lobe resection , whereby the overall benefit from surgery may not be greater than trying alternative drug regimens . in these cases , the risks for each treatment option should be weighed up on a case - by - case basis until more definitive data is available to guide therapeutic decisions .", "patients with intractable epilepsy who are not suitable for epilepsy surgery or those who continue to experience seizures post - operatively may benefit from neurostimulation . this treatment option for patients with intractable epilepsy involves electrical stimulation of the nervous system using surgically implanted devices , or non - invasive stimulation as is the case for repetitive transcranial magnetic stimulation ( rtms ) . vagal nerve stimulation ( vns ) therapy is the only neurostimulation method to have received approval by the usa food and drug administration , whilst both vns and deep brain stimulation ( dbs ) are approved for use in patients with refractory epilepsy in the uk . in vns therapy , intermittent electrical stimulation is delivered to the left vagus nerve , which has ascending fibers with widespread connections to the limbic , autonomic and reticular brain regions . the reduced thalamic blood flow that is observed in vns has been proposed to underlie the mechanism for seizure reduction.50 in addition to a number of retrospective studies , efficacy for this treatment option has been demonstrated in two randomized , placebo - controlled , double - blind trials , which reported a median seizure reduction of 24.5%28.0% in the group receiving high level vns compared to just 6.1%15.0% in patients receiving low level vns ( p = 0.01 and p = 0.04 , respectively).51,52 the risks associated with implantation of the vns device are relatively low , with a 3%5% chance of infection.5355 vocal cord dysfunction , throat discomfort , change in voice quality and sleep apnea can present post - operatively , and such consequences should be discussed with the patient when considering this treatment . an additional factor that should be considered when planning vns therapy is that patients are usually unable to undergo magnetic resonance imaging ( mri ) after implantation , since the changing magnetic fields may induce electrical currents in the device.56 dbs involves electrical stimulation of specific subcortical nuclei , which have widespread neural connections . the anterior nucleus of the thalamus is often the target of dbs due to its widespread projections to the limbic system . in a randomized controlled trial of 109 patients with refractory epilepsy who had dbs electrodes implanted in the anterior nucleus of the thalamus there was a 29% greater reduction in seizures for 54 patients who had the stimulator switched on compared to 55 patients who had their stimulation turned off after a blinded period of 3 months ( p = 0.002).57 infection , hemorrhage and stimulation - induced seizures were amongst the most frequently reported complications . in the same study , impaired memory and higher levels of depression were observed in the group receiving the active treatment . dbs has been recently licensed in the uk , and nice guidelines suggest that this method should be used in highly selected cases , due to the lack of efficacy data and the associated risks.58 the centromedian nucleus is also a potential location for dbs , since it is part of the reticulo - thalamo - cortical system which is involved in mediating cortical excitability and wakefulness . one double - blind crossover trial reported a 30% reduction in generalized tonic - tonic seizures when the device was turned on compared to when it was off , although this change was not significant , possibly due to the small sample size of 7 patients.59 further evidence for centromedian nucleus stimulation efficacy can be found in a longitudinal study which reported good efficacy for generalized tonic - clonic seizures , atypical absences and tonic seizures , however caution should be taken in interpreting these findings because of the lack of control groups.60 unlike other methods of neurostimulation , responsive neurostimulation ( rns ) does not deliver electrical stimulation at specific frequencies throughout the day . instead , the rns device is composed of a combined recorder and stimulator device , which detects clinically relevant epileptiform discharges and delivers an appropriate electrical stimuli in response . a recent multicenter double - blind randomized controlled trial in adults with intractable partial seizures demonstrated a reduction in seizure frequency of 37.9% in the treatment arm compared to 17.3% in the control group ( p = 0.012).61 the fourth and only non - invasive neurostimulation technique , low frequency rtms , is thought to suppress cortical excitability either via inducing long term depression or alternatively by enhancing gabaergic inhibition.62 although evidence for its efficacy is limited , a number of small - scale studies suggest antiepileptic properties ( see fregni and pascual - leone for a comprehensive review).63", "over the past century , the efficacy of dietary therapies for intractable seizures has increasingly been recognized . currently there are four main types of metabolic therapy in use for the reduction of seizures . three of these have a high fat content and low carbohydrate content the classic ketogenic diet , the medium chain triglyceride ( mct ) diet and the modified atkins diet ( mad)and the fourth controls the carbohydrate quality , the low glycemic index treatment ( lgit ) . these treatments are usually considered in patients with intractable epilepsy who have failed to respond to several aeds and are not suitable candidates for surgery . the classic ketogenic diet is a high fat and low carbohydrate diet that uses long chain fatty acids ( lcfas ) as its main source of fat . typically the patient must consume 34 grams of fat for every 1 gram of carbohydrate plus protein . as a result , between 86% and 90% of the calories from this diet are provided from lipids.64 the mct diet provides more ketones per calorie consumed than in the classic ketogenic diet , which uses mainly lcfas . in mct the mad recommends increased consumption of foods high in fat whilst restricting the amount of carbohydrates . unlike the classic ketogenic and mct diets , there is no limit on the amount of protein consumed or the total number of calories per day.65 the typical mad uses a ratio of 1 gram of fat for every gram of carbohydrate plus protein , and consequently is less restrictive than the other two high fat diets . the lgit restricts carbohydrate intake to food items with a glycemic index of less than 50 at 4060 grams per day , in order to prevent large fluctuations in blood glucose concentrations , which are thought to exacerbate seizures.66 there is a large body of evidence demonstrating the efficacy of the metabolism - based therapies for refractory seizures , with the majority focusing on the classic ketogenic diet . two meta - analyses of observational studies showed that 15.6%15.8% of patients on the diet can achieve seizure freedom whilst 33.0%55.8% of the patients can have a > 50% reduction in number of seizures.67 one of the only randomized controlled trials to investigate the efficacy of the classic ketogenic diet followed children with intractable or focal - onset epilepsy who were randomized to a ketogenic diet or continuation of a normal diet for a 3-month period . of the patients who received the ketogenic diet , 38% had a seizure reduction of > 50% . this was significantly greater than the control group , where only 6% had > 50% seizure reduction ( p < 0.001).68 the results of a follow - up study comparing the effectiveness of the lcfa versus the mct diet suggest there is no increased benefit using either of the two diets for seizure reduction.69 despite the lack of prospective randomized control trials , the mad has also demonstrated substantial efficacy in controlling seizures . of the patients who have been studied on the mad , 45% have demonstrated 50%90% seizure reduction and 28% have > 90% seizure reduction . similar results have been reported in two retrospective studies of the lgit , with 38%73% of patients achieving > 50% seizure reduction.70,71 the best candidates for the mad tend to be adults and adolescents , for whom compliance on the lcfa and mct diets is an issue.64 this is because the food consumed on the mad is similar to that being consumed by peers . the same can be said about the lgit , which in addition can be considered for patients who find the other 3 high - fat diets unpalatable . there is a body of evidence suggesting that metabolism - based therapy can be considered earlier in the treatment plan for patients with specific epilepsy syndromes . one notable example is west syndrome ( infantile spasms ) , where the ketogenic diet is successfully used as a first line agent.72 the same diet is also thought to be especially effective in unspecified symptomatic generalized epilepsy , multifocal epilepsy , juvenile absence epilepsy , continuous spike and slow wave of sleep , myoclonic - astatic epilepsy ( mae or doose syndrome ) , severe myoclonic epilepsy of infancy ( dravet syndrome ) , and seizures in patients with tuberous sclerosis complex and landau - kleffner syndrome.68,73,74 ketogenic diets are also recommended for patients suffering from glucose transporter defects and pyruvate dehydrogenase deficiency , who are unable to utilize glucose for brain metabolism.75,76 the most common side effects of dietary treatments are constipation , acidosis , temporary hypercholesterolemia , kidney stones and hunger.64 growth restriction has also been observed in children.77 the ketogenic diet is contraindicated in patients who are unable to use ketone bodies as an energy source or those who require high levels of glucose . such conditions include pyruvate decarboxylase deficiency , primary carnitine deficiency , fatty acid oxidation abnormalities , the porphyrias and some mitochondrial disorders.78 generally , as with aeds , it is suggested that after adhering to dietary therapy for a period of 2 years , the treatment should slowly be tapered off . many patients who become seizure free whilst adhering to the diets remain seizure free off the diet . this has been demonstrated in one study , which reported the risk of seizures recurring when stopping the diet at only 20%.79 it has been hypothesized that the dietary therapies have a long - term , disease modifying effects , although the observed phenomenon could just be explained by the natural history of the seizure disorder.64 in addition to showing high levels of efficacy , similar to many aeds , the dietary interventions are generally quite tolerable , can have associated positive effects such as weight loss,80 and demonstrate effectiveness in a relatively short time period . whilst aeds may take several weeks to be titrated up to a therapeutic dose and vns may take months or years to show an optimal effect,81 the effects of a ketogenic diet can be seen within a few days . furthermore , the diets are cost effective compared to many aeds.64 in practice , a median of 5 aeds are prescribed before considering a metabolic treatment such as the ketogenic diet.82 furthermore , 60% of child neurologists only use a ketogenic diet as a last resort treatment.83 it is therefore suggested that the metabolic treatments should be considered earlier in the course of treatment , especially in patients with refractory seizures who are not suitable for or willing to undergo resective surgery .", "herbal treatments are generally viewed as alternative and complementary forms of medicine by both patients and physicians . there is , however , a small body of evidence demonstrating the effectiveness of some herbs at controlling seizures . out of all the herbal treatments , cannabinoids have the largest body of evidence supporting their use as anticonvulsants . one randomized , double - blind controlled trial reported 50% of patients with secondary generalized epilepsy receiving cannabidiol becoming virtually seizure - free for the duration of the study . however , only 15 patients were included and to our knowledge no follow - up study has been performed.84 the use of cannabis for epilepsy has been legalized in canada and licensed in 14 states within the united states , although it is prohibited in most european countries.85huperzine a. has been shown to be effective in animal models of epilepsy , and a clinical trial to assess its effectiveness has been planned.86,87 there is also laboratory data supporting the efficacy of kava ( piper methysticum ) and mistletoe ( viscum sp ) , but no clinical evidence exists to support their use . a number of other herbal treatments that are commonly used to treat seizures have either been shown to have no effect on the severity of seizures , or in fact demonstrate proconvulsant properties and exacerbate the condition . in addition , much evidence is anecdotal , or when clinical studies have been performed , these often display poor methodology and do not have sufficient levels of statistical power to make firm conclusions from . further , good quality evidence in the form of controlled trials is required to justify the use of herbal treatments in a clinical setting ( see pearl et al for a comprehensive review).88", "patients with treatment refractory seizures continue to represent a large proportion of patients diagnosed with epilepsy . the fact that recurrent seizures and associated side effects from aeds significantly impact on patient s qol means that treatment decisions should be made in light of the best evidence for improved seizure control , enhanced qol and the greatest tolerability profile . confirmation that the events are epileptogenic in origin and early identification of an underlying etiology or specific epilepsy syndrome is essential to guide appropriate treatment for which syndrome or etiology specific efficacy exists . this may be in the form of monotherapy , polytherapy or alternative treatment options including surgery , neurostimulation , metabolic treatments and herbal remedies . despite the fact that decisions to alter a treatment regimen may be influenced by local availability and funding , physicians should balance the evidence for efficacy in seizure reduction with the associated risks , side effects and patient preference ." ]

[ "in august of 2005 , hurricane katrina devastated the new orleans area with high wind , heavy rainfall , and a storm surge of about 7 m which caused the collapse of the levee system surrounding the city . approximately 80% of the city was flooded to varying depths for many weeks before the us army corps of engineers was able to implement temporary levee repairs and install emergency pumping capacity . in the aftermath of the flood event , the infrastructures of the city along with residences and commercial buildings were grossly contaminated with sediments deposited by the floodwaters and subsequently by microbial overgrowth supported by the residual moisture , high humidity , and elevated temperatures in the area . after floodwaters had receded , various surveys were conducted for measurement of indicators of microbial contamination in air , dust , and damaged building materials , including total and culturable mold spores , fungal fragments , mycotoxins , 13--d - glucan , and bacterial endotoxin . generally , observed levels of microbial contaminants in these surveys were elevated , often extremely so , and were relatable to the depth and duration of flooding , and indoor levels were typically higher than those in the surrounding outdoor environment [ 15 ] . subsequent to the posthurricane flooding event , there has been extensive rebuilding in the new orleans area . residents who personally performed repairs of their properties as well as various skilled and unskilled laborers working in the construction and building maintenance trades were at risk for inhalation exposures to dust containing microbial and other agents during demolition , removal , and repair of flood - damaged and contaminated infrastructure and building materials [ 68 ] . exposures to microbial contaminants in agriculture , waste management , and in water - damaged and moldy buildings have been linked to various upper and lower respiratory illnesses and adverse effects including rhinitis , hayfever , toxic pneumonitis ( tp ) , hypersensitivity pneumonitis ( hp ) , and respiratory infections including pneumonia and exacerbation or initiation of asthma [ 912 ] . the potential for respiratory illness arising from inhalation exposure to bioaerosols and microbial contaminants during restoration activities in the post - hurricane katrina environment was of particular concern . as a part of a 5-year longitudinal study investigating the risk of respiratory illness associated with work in and around flood - damaged structures in post - hurricane katrina new orleans , baseline findings from initial cross - sectional survey are reported .", "the study cohort consisted of 791 adults residing or working in the greater new orleans metropolitan area . study participants were recruited from several sources : ( 1 ) employees of three large institutions in the city of new orleans , two of which are academic and the third is a branch of local government ( n = 488 total ) . all three institutions experienced heavy flood damage to their buildings and facilities and utilized their regular staff as well as contract labor to perform restoration work . recruiting from the academic institutions focused primarily on workers from departments normally engaged in maintenance , custodial , and facilities services . support personnel ( clerical , managerial , etc . ) from the targeted departments were included in the recruitment . ( 2 ) members of a local union hall for the skilled and unskilled building trades ( n = 63 ) . ( 3 ) private building contractors and self - employed tradesmen ( n = 95 ) . ( 4 ) other residents of the new orleans area ( n = 145 ) , many of whom performed restoration work on their own properties . overall , 54% of the study cohort reported a skilled or unskilled trade as their primary occupation , including carpentry ( n = 50 ) , electrician ( n = 27 ) , plumbing ( n = 12 ) , paint / drywall ( n = 21 ) , hvac ( n = 12 ) , groundskeeping ( n = 20 ) , general construction ( n = 102 ) , general maintenance ( n = 36 ) , operating / building engineering ( n = 18 ) , and being mechanic / machinist ( n = 15 ) . an additional 15% of study participants worked in custodial or janitorial services ( n = 115 ) . testing was conducted in a mobile laboratory van outfitted with spirometry and interview work stations and ancillary equipment . the mobile laboratory van was moved to the work locations or union hall of the study participants for the duration of their respective testing period , generally 2 to 3 weeks , and to the parking lots of several large building supply stores , in order to allow private contractors and self - employed construction tradesmen to participate . spirometry testing procedures and equipment have been previously described and comply with both the original and updated american thoracic society spirometric test criteria . spirograms were collected with a sensormedics model 1022 dry rolling seal spirometer interfaced to a laptop computer running omi spirometry software version 5.05.9 ( occupational marketing , inc . , all spirometric testing was conducted by the same individual who is a member of the research staff and is a certified pulmonary function technician ; in addition , all spirometric test results were quality assured and interpreted by senior study investigators . predicted lung function parameters and lower limit of normal ( lln ) lung function values for forced expiratory volume in one second ( fev1 ) , forced vital capacity ( fvc ) , and fev1/fvc ratio were computed from predictive equations developed by hankinson et al . . separate predictive equations were used for caucasians , african americans , and latinos . predicted values for study participants of asian heritage were calculated using the equations for caucasians . in addition to race , the predicted values were based on age , gender , and height . the lln values were calculated by subtracting 1.645 see from the predicted values , where see was the standard error of the estimate and 1.645 is the 95th percentile of a standard normal distribution . those participants with chronic obstructive pulmonary disease ( copd ) were identified according to the gold criteria , that is , fev1/fvc % predicted less than 70% and fev1% predicted less than 80% ; however , only prebronchodilator lung function values were available and thus may not have adequately differentiated asthma ( with reversible obstruction ) from copd . ever asthma on questionnaire were therefore excluded from the analyses of copd prevalence as a function of exposure . a demographic , medical , smoking , and occupational questionnaire it was based on a modified version of the standardized questionnaire reported by burrows et al . , which accounts for a variety of putative and established risk factors and potential confounders for the development of airways disease including asthma , allergic disease , historical confounding exposures , serious childhood respiratory illness , cigarette smoking history , environmental tobacco smoke , and age , gender , and race . additional questions were designed to capture the development of specific symptoms after hurricane katrina that might be associated with living and working in the post - katrina environment . these included post - hurricane katrina onset of asthma , sinus symptoms , pneumonia , and transient fever and cough absent infection , with the latter used as an indicator of possible hypersensitive ( hp ) or toxic ( tp ) reaction . asthma was defined dichotomously and required a positive response to both of the following questions : have you ever had asthma or attacks of shortness of breath with wheezing in the chest when not having a cold ? followed by the response to the question how old were you when your asthma started ? was used in conjunction with the participant 's date of birth to determine whether asthma onset was after september 30 , 2005 ( post - katrina new onset asthma ) . dyspnea was also defined dichotomously and required a positive response to the question do you have shortness of breath when hurrying on level ground ? the interview also included queries on pre- and post - katrina work and occupation , and detailed information was gathered on time spent after hurricane katrina performing five specific types of hurricane / flood remediation work : demolition and ripout , trash removal , landscape restoration , sewer line repair , and mold remediation . participation in any of these work activities , herein identified as restoration work , was assumed to result in occupational or vocational exposure to flood - related contaminants . participants were asked to report the number of hours spent in each of the five restoration work activities , for each year since the hurricane up to the point of interview , and the type and relative frequency of any respiratory protective equipment that may have been used during the work . restoration of personal property was included in the total time spent in restoration work along with any from the subject 's regular employment . the study protocol was approved by the authors ' institutional review board and all study participants provided a written informed consent . for the current hayfever compared to current trouble with pollen , grass , or fur , analysis indicated that the parallel question significantly enhanced the positive response rate for these symptoms ( 22% claiming current hayfever versus 39% claiming sensitivity to pollen , grass , or animal fur ; p < 0.0001 ) . similar but nonsignificant results were observed for ever and current asthma versus attacks of dyspnea and for chronic bronchitis versus copd . the unadjusted prevalence rate ratios for each symptom or condition for those doing any restoration work versus those not doing any restoration work were calculated within smoking categories based on 2 2 contingency tables . the prevalence rate ratio was defined as prr = p1/p2 , where p1 = a / n1 and p2 = c / n2 represent the sample proportion of exposed ( n1 ) and unexposed ( n2 ) individuals with disease . if a and b represent the number of exposed subjects who do and do not have disease , respectively , and c and d represent the number of unexposed subjects who do and do not have disease , respectively , the asymptotic 95% confidence interval for prevalence rate ratio is calculated using the following standard logarithmic transformation : ln(prr)1.96b / an1+d / cn2 . exponential transformations on the confidence limits of this log transformed interval provided the asymptotic 95% confidence intervals for prevalence rate ratio . multiple logistic regression analyses were used to compute adjusted prevalence odds ratios for each symptom or condition per 100 hours of restoration work as well as to compute asymptotic 95% confidence intervals for prevalence odds ratios . due to significant interactions between gender and total hours of restoration work , logistic regression analyses were performed separately by gender and adjusted for age ( because of a significant correlation with prevalence of pneumonia ) and smoking categories . all interactions between age , smoking category , use of respiratory protective equipment ( ever versus never ) , and total hours of restoration work were considered and were not significant . multiple linear regression related % p fev1 , fvc , and fev1/fvc to restoration work hours , use of respiratory protection , gender , asthma classification , and smoking category . all possible interactions were considered and were not significant , and no significant exposure associations were detected .", "the majority of the study cohort was african american and male ( table 1 ) . current smokers comprised 28% of the cohort , whereas 18% were ex - smokers and 54% had never smoked . 3.7% of the study cohort reported having pneumonia after hurricane katrina and almost half reported newly developing sinus symptoms . among those reporting never having had asthma prior to hurricane katrina ( n = 539 ) , about 4.5% reported new onset asthma . episodes of transient fever and cough occurring after hurricane katrina were reported by about 29% of the study cohort . multiple episodes were also common in this reporting group : the median number of episodes was 3 , and 10% of the group reported having 12 or more such occurrences . overall , lung function parameters were somewhat depressed in the cohort ( tables 1 and 4 ) and correlated with cigarette smoking and presence of current asthma symptoms . percent predicted ( % p ) fev1 averaged 93.4% ( sd : 16.0 ) for current smokers , while ex- and never smokers had a mean level of 96.0% ( sd : 15.4 ) , p = 0.037 by t - test . the proportions of the cohort falling below lln for fev1 and fvc were also somewhat elevated ( 5% being the expected proportion based on the definition of lln ) , particularly for the current and ex - smokers , as expected . participants reporting current asthma symptoms had a mean % p fev1 of 89.2% ( sd : 21.8 ) and % p fvc of 91.8% ( sd : 16.8 ) , while asthmatics without current symptoms had a mean % p fev1 of 95.8% ( sd : 18.0 ) and % p fvc of 96.7% ( sd : 15.2 ) . participants who never had asthma had mean % p fev1 of 96.1% ( sd : 13.6 ) and % p fvc of 96.4% ( sd : 13.6 ) . almost 75% of the study participants reported having performed some restoration work activity after hurricane katrina ( n = 587 ) , and details on the actual time spent in these activities were self - reported by 474 or 81% of this group ( table 2 ) . demolition / ripout was the most commonly reported restoration work activity , followed by landscape restoration and trash / debris removal . the distributions of time spent in these activities were highly skewed because many study subjects worked as much as 16 hours per day , seven days per week , for extended durations after hurricane katrina . the majority of the study subjects also reported time spent in more than one type of restoration work activity . for the total combined hours spent in any of the specific restoration work activities , the mean and median values reported by 474 subjects with complete data were 1646 and 620 hours , respectively . among those who reported performing restoration work , 80.1% reported some use of respiratory protective equipment ( i.e. , filtering facepiece or air - purifying cartridge respirator ) . 202 study subjects reported no time spent in restoration work . the prevalence rates for post - hurricane katrina episodes of transient fever and cough and new onset sinusitis were significantly elevated for those reporting any restoration work ( prr : 1.7 and 1.3 , resp . ; table 3 ) . the prevalence rate ratios were statistically significant for ex- and never smokers but not current smokers . the prevalence rate ratios for post - hurricane katrina new onset asthma were elevated for the overall cohort ( prr = 2.2 ) and especially for ex- and never smokers ( prr = 2.7 ) but were not statistically significant ( p = 0.09 ) ; the overall lack of significance may have been due in part to the low incidence ( 29 cases ) and the reduced size of the base population ; that is , only those who never had asthma prior to hurricane katrina . statistically significant elevations in prevalence rate ratios for those having done any restoration work were not observed for pneumonia , dyspnea , copd , and being below lln for any of the lung function parameters . \n figure 1 illustrates the prevalence of respiratory symptoms and conditions with quartiles of reported time spent in post - hurricane katrina restoration work activities ( proportions for new onset asthma and pneumonia are multiplied by 10 in the figure for purposes of scaling ) . the unadjusted proportions for several post - hurricane katrina symptoms and conditions , including transient fever and cough , dyspnea , new onset sinus symptoms , new onset asthma , and pneumonia , show trends of increasing prevalence with time in restoration work . statistically significant increases in prevalence odds ratio with restoration work time were observed only for transient fever and cough , for new onset sinus symptoms , and for dyspnea . when analyzed by logistic regression , the prevalence of fever and cough was statistically significantly associated with restoration work time , but only for men , with a prevalence odds ratio of 1.016 per 100 hours . likewise , for new onset sinus symptoms in men , the prevalence odds ratio was 1.042 per 100 hours . in contrast , only women exhibited a statistically significant association between the prevalence of dyspnea and restoration work time , the odds ratio being 1.031 per 100 hours . the restoration work time - gender interactions observed in the logistic regression analysis of the prevalence odds ratios for these symptoms may be confounded with job type since women in the study tended to be in the custodial / janitorial occupations , whereas men were more likely to be in the building , construction , or maintenance trades . the exposures associated with restoration work done within these two broad categories of occupation are likely to be qualitatively and quantitatively different . the prevalence odds ratios for post - katrina new onset asthma and for pneumonia with restoration work time were elevated but were not statistically significant by logistic regression analysis . for new onset asthma the crude , unadjusted proportions in each of the restoration work time quartiles ( figure 1 ) were all higher than for participants with no restoration work time ( 1.99.1% versus 1.7% ) . for pneumonia , the unadjusted prevalence among those with no restoration work was 3.0% whereas subjects in each of the first three quartiles of restoration work time exhibited higher prevalence rates ( 3.25.1% ) ; however , those in the highest quartile of restoration work time had a pneumonia prevalence rate of only 2.5% . the group mean values for % p fev1 , fvc , and fev1/fvc ratios generally were depressed but were within 5% of the normal except for smokers performing restoration work . current smokers who did restoration work showed lower overall predicted lung function compared to smokers who did not ; however , multiple linear regression analysis yielded no statistically significant correlations of any of the lung function parameters with restoration work time after adjustment for smoking , gender , asthma status , and use of respiratory protective equipment .", "the results of this study suggest moderate adverse impact on respiratory health from time spent in post - hurricane katrina flood restoration activities . published reports and public health surveillance systems generally did not show increases in emergency room visits or hospitalizations resulting from exposures in the post - hurricane katrina environment , although there have been a few reports of increased risk for respiratory effects . in a survey of 525 new orleans firefighters , 79% had contact with floodwaters following hurricane katrina , and 38% reported new onset respiratory symptoms including sinus congestion , throat irritation , and cough . the prevalence rate ratio for those who had contact with floodwater versus those who did not was 1.9 and was statistically significant . first responders would have had significant exposures to flood sediments and associated contaminants , in addition to microbial agents . inhalation exposure to aerosolized sediment collected in the aftermath of hurricane katrina was also shown to elicit significant pulmonary inflammation , increased airways resistance , and airway hyperreactivity in a mouse model . there were widespread anecdotal reports of persistent nonproductive cough , often with sore throat and rhinorrhea , in the population residing in new orleans in the fall of 2005 . an investigation of this phenomenon by the louisiana department of health and hospitals concluded that visits to medical facilities for respiratory complaints in the population of new orleans were not related to exposure to dust or molds at the residence or at work . it is likely that katrina cough was an irritant phenomenon resulting from a dry fall season with high levels of airborne particulate matter , coinciding with the start of the regular allergy and flu seasons [ 21 , 24 ] . the prevalence of episodes of fever and cough in the present study population is clearly elevated for those who have done restoration work . however , given the overall strong correlation with restoration work time , the common reports of multiple and distinct episodes of fever and cough , and the inclusion of the febrile component in the symptom complex , tp is likely to be underlying many of these reports and appears to be a common adverse effect of restoration work exposures in the post - hurricane katrina environment . unlike hypersensitivity pneumonitis , it is uncertain whether toxic pneumonitis and inhalation fevers result in significant lasting decrements in lung function , and functional parameters are expected to return to baseline upon recovery from an episode [ 25 , 26 ] . this study did not identify any restoration work - related decrements in functional parameters , nor in the prevalence of being below lln . the world health organization , in its report on guidelines on indoor air quality related to damp indoor spaces and mold , concluded that the evidence is inadequate to identify an association between damp indoor environments or the presence of mold with risk of alterations in lung function . however , in a recent study of 6,443 individuals in the european community respiratory health survey , lung function measurements across 9 years showed statistically significant excess declines in fev1 of 2.25 ml / year and an additional 7.43 ml / year for women who reported dampness in the home and visible damp spots in the bedroom , respectively . annual excess declines of such small magnitude are difficult to detect over a short time period and are unlikely to result in a detectable group difference in function , measured cross - sectionally , after only a few years in the post - hurricane katrina environment , as in this baseline study . however , our study population is being evaluated annually over the course of a 5-year period , and currently undetectable decrements in lung function may reach the level of significance when measured directly over this extended period of time . it is generally accepted that exposure to flood - related microbial contaminants can exacerbate existing asthma , and there is a mounting evidence that such exposures increase the risk of development of new asthma [ 11 , 12 ] . in a recent extensive review and meta - analysis of the literature from 1980 to 2010 , overall odds ratios of 1.49 ( c.i . : 1.281.72 ) and 1.68 ( c.i . : 1.481.90 ) were found for the associations of asthma and wheezing , respectively , in children living in homes with visible mold . in this study , there was an observable elevation in prevalence of new onset asthma after hurricane katrina which increased with increasing quartiles of restoration work time but was not statistically significant . the lack of significance may be due in part to a self - selection process occurring in the cohort , with some study subjects who developed new onset asthma in the wake of hurricane katrina avoiding or terminating further restoration work exposure because of personal health concerns . furthermore , the reported restoration work time is at the time of the interview , not at the time of development of new onset asthma , which always preceded the interview . some of the study subjects may have continued to engage in and increase their time in restoration work to varying extent after developing asthma , but there is no information on the magnitude of this confounding nor its effect on the analysis . this study has several additional limitations : as noted , health outcomes were assessed cross - sectionally , and there was a significant time element over which the baseline information was collected for the entire study population . those subjects evaluated later in the study period could therefore have opportunity for greater amounts of time spent in restoration work with concomitant increase in risk for development of respiratory effects . as the longitudinal component of the study moves to completion , additional reports of development of respiratory symptoms and conditions are coming to light , and the ability to detect ongoing small excess decrements in lung function will also increase . exposure to flood - related contaminants was assumed to be related to reported time spent performing restoration work . however , there is no information as to a particular individual 's exposure intensity nor can it be assumed that all exposures associated with restoration work activities were qualitatively or quantitatively similar . finally , the reliance on self - reporting of respiratory symptoms and conditions could have led to misclassification due to recall bias .", "this study provides further evidence that workers performing restoration work on flood - damaged structures are at risk of respiratory health impacts from exposure to microbial - contaminated dust and debris . moderate adverse respiratory health effects including toxic pneumonitis and sinusitis were commonly reported in the study cohort , and the prevalence of new onset asthma among restoration workers was noticeably elevated . while it is unclear from this cross - sectional analysis whether restoration work exposures have adversely affected pulmonary function in the population , the functional parameters overall are depressed in the cohort . ongoing longitudinal health surveillance of this study cohort , along with a quantitative exposure assessment , will examine whether there is an increased risk for long - term or irreversible effects on respiratory health and how the risks relate to the nature and magnitude of the exposures occurring during posthurricane flood restoration work ." ]

[ "\n road traffic injuries are responsible for the death of 1.23 million people around the world annually ( 1 ) . in terms of disability adjusted life year ( daly ( ( 1 ) . road traffic injuries ranked ninth in 1999 , and are expected to rise to the third place by 2020 ( 2 ) . these events account for 1 - 2% of gross national product ( gdp ) of different countries ( 3 ) . in iran , cost of road traffic injuries is 2.19% of gdp that is significantly higher than the global average ( 4 ) . \n \n more than 20% of deaths caused by traffic crashes belong to pedestrians . according to the estimates , about 273000 pedestrians around the world lost their lives due to traffic crashes in 2010 ( 5 ) . in 2013 , 22% of all fatalities as a result of road traffic crashes in the european union(eu ) were fatalities of pedestrians ( 6 ) . studies have shown that most traffic crashes related to pedestrians , particularly in high - income countries , occur in interurban streets and roads ( 7 ) . for example , in the european union countries , 70% of these crashes occur in cities , and this figure is 76% in the u.s . pedestrians are associated with the highest contribution of deaths caused by traffic crashes in the world s densely populated cities . for instance , in mumbai and delhi , pedestrians accounted for 78% and 53% of traffic fatalities , respectively ( 9 ) . according to the statistics of tehran traffic police , more than 6,000 traffic crashes causing injury and more than 100 traffic crashes causing death annually occur in tehran . in fact , more than 40% of deaths from traffic crashes in tehran occur for pedestrians ( 11 ) . \n \n given the importance of this issue , in the recent years , many studies have been conducted in different parts of the world , especially in densely populated cities , on the role of environmental and demographic factors in frequency , spatial distribution , and severity of traffic crashes related to pedestrians in urban areas , using different methods of spatial analysis and a variety of statistical models ( 12 - 21 ) . \n \n in the recent years , due to the increase in automobile production , traffic load has dramatically increased in urban and suburban streets and roads of iran , while these transport infrastructures have not been developed qualitatively and quantitatively commensurate with car production . since a major proportion of traffic crashes in iran occur due to environmental factors , especially the quality of roads and streets and as the capacity of streets and roads , studying the urban and interurban roads and streets , particularly in densely populated cities with heavy traffic such as tehran , in terms of the environmental factors involved in traffic crashes can be helpful to more accurately identify the environmental factors of such crashes and their contribution . the results of such studies can be used in better planning for reduction of this type of risk factors . hence , this study aimed to determine the high - risk areas and spatially analyze the traffic crashes causing death to pedestrians in tehran in 2013 and 2014 . tehran is the largest city and the capital of iran , with an area of more than 612 square kilometers ( 22 ) . tehran is divided into 22 districts , 370 parishes , and 560 traffic zones , with more than 535 km of highway and 445 arterial streets ( 24 ) . \n", "statistical analyses were performed by descriptive and inferential statistics in spss and arc - gis . analytic surveys were conducted in the following steps : first , the exact location of traffic crashes causing death to pedestrians was extracted by reviewing the files of crashes in the database of tehran traffic police . second , according to the universal transverse mercator ( utm ) , geographical coordinates of the traffic crashes locations with the accuracy of one to three meters were recorded and saved in a computer . the utm system divides the earth into 60 zones , each 6 of longitude in width and uses a secant transverse mercator projection in each zone . the point of origin of each utm zone is the intersection of the equator and the zone 's central meridian . \n \n third , using arc - gis , different layers of geographic information were put on each other and mapping was extracted . finally , global moran s index with euclidean distance method was used to study and analyze the distribution pattern of traffic crashes related to pedestrians in terms of being either cluster or scattered . moran s i index is the most common index used for measuring the spatial autocorrelation to determine how close are the objects compared to other close objects . \n \n a strong autocorrelation occurs when the values of a variable that are spatially close to each other are correlated . in other words , if the spatial objects or the values of variables related to them are randomly distributed in space , there must apparently be no relationship between them . moran s index studies the distribution pattern of these spatial objects by considering the values of the studied variable in terms of being cluster or scattered . if the value of moran 's index is equal to + 1 or near + 1 , it implies a complete cluster pattern or existence of autocorrelation . if this value is zero , it means that the pattern is random or multipolar accumulation . \n \n finally , if moran 's index is equal to -1 or near -1 , it shows that the crashes follow a scattered pattern . hot spots analysis : for this analysis , global getis - ord g index with euclidean distance method was calculated for each feature . z - score of this index shows that to what extent the studies feature has been distributed cluster - like , which may be statistically significant . in this study , this index was used to display the areas with high , low or moderate frequency of traffic crashes associated with pedestrians . getis - ord g index values were interpreted based on comparing the observed values and the expected values . if the observed values of getis - ord g index in an area are more than the expected values , that area is considered among the hot spots , and if the observed values are less than the expected values , that area is considered among the cold spots . \n", "\n one hundred ninety - eight incidents were studied ; 92 of which , ( 46.4% ) occurred in april 2013 to march 2014 and other 106 cases ( 63.6% ) occurred in april 2014 to march 2015 . the highest and the lowest frequency of crashes belonged to january ( 26 cases ) and june ( 10 cases ) . figure 1 demonstrates the trend of the traffic crashes causing death to pedestrians in 2013 - 2015 in tehran . \n distribution of fatal crashes associated with pedestrians in tehran by different months : 2013 - 2015 \n overall , 158 cases ( 79.8% ) of crashes have occurred in tehran highways . azadegan highway , which stretches from northwest to southeast of tehran , had the highest frequency of crashes ( 25 cases ) . during the study period , fatal road traffic injuries that caused the death of pedestrians occurred most frequently in traffic zones located in the west , east and south of tehran . figure 2 depicts the distribution of studied crashes in tehran and their position to highways , and urban to interurban bus terminals . \n mapping of fatal crashes associated with pedestrians in tehran and their position relative to highways and urban and interurban passenger terminals : 2013 - 2015 \n among the 22 districts of tehran , districts located in the south , east , and northwest of tehran , as well as districts located in the center of the city , have shown the highest and the lowest frequency of traffic crashes , respectively . figure 3 displays the distribution of studied crashes in the 22 districts of tehran in 2013 and 2014 . \n mapping of fatal crashes associated with pedestrians in the 22 districts of tehran in 2013 - 2015 \n result revealed that the density of events in the areas with dominant transportation , industrial and military land use was higher than the areas with dominant residential land use ( fig . 4 ) . during the study period , fatal road traffic injuries that caused the death of pedestrians occurred most frequently in traffic zones located in the west , east and south of tehran . \n mapping of fatal crashes associated with pedestrians in tehran and their position relative to land use in 2013 - 2015 \n figure 5 depicts the distribution pattern of traffic crashes causing death to pedestrians in traffic zones . moran s i index value showed that the studied crashes had a cluster - like distribution ( p<0.001 ) . \n distribution pattern of fatal crashes associated with pedestrians in tehran by traffic areas in 2013 - 2015 \n the hot spots in tehran in terms of traffic crashes causing death to pedestrians included the western , southern , northern , partly eastern suburbs , and cold spots were mainly located in the central areas of tehran ( fig . getis - ord general g index showed that the distribution of hot and cold spots of the studied crashes was statistically significant ( p<0.001 ) . \n low and high - risk traffic areas in terms of frequency of fatal crashes associated with pedestrians in tehran in 2013 - 2015", "\n the results of this study revealed that occurrence of the traffic crashes causing death among pedestrians in tehran during different months of 2013 and 2014 does not follow a specific and fixed trend . for instance , the highest and the lowest frequencies of studied crashes in 2013 have been recorded in january - february and october , respectively , while the highest and the lowest frequencies belonged to may and december , respectively . however , it can be generally concluded that the number of crashes in this period during the summer months ( especially june and august ) is fewer than the winter months ( especially january and march ) . since some studies have shown that the frequency of traffic crashes associated with pedestrians increases with increased pedestrian volume ( 10,25,26 ) , these seasonal differences can be attributed to changes in pedestrian volume . since some studies such as those conducted by cloutier , cottrill , green , miranda - moreno , mcarthur et.al found that the number of schools or students is associated with the frequency of traffic crashes associated with pedestrians in urban areas ( 17,26 - 29 ) , school holidays and reduced traffic of students in june and august can be one of the reasons for the reduced frequency of such crashes during these months . therefore , better organization of students traffic and promotion of the safety of the streets near schools can be considered as one of the strategies for reducing the deaths from traffic crashes related to pedestrians in tehran . \n \n this study showed that about 80% of the studied crashes have occurred in highways and freeways . highways located at the main western and southern entrances and exits of tehran such azadegan highway ( from northwest to southeast of tehran ) and karaj special road , which connects karaj to 36 km west of tehran had the highest share of such crashes . these results are consistent with the findings of studies conducted by wier et.al in san francisco and mueler et al . in washington that showed that frequency of traffic crashes associated with pedestrians since traffic load in the western and southern highways of tehran is higher than other areas and there is an intense congestion of different means of transport in some of these areas , one reason for the high frequency of fatal crashes in these areas would be the high traffic load and congestion of different means of transportation . therefore , the reasons of this problem need to be identified by conducting more studies . in addition , the necessary measures should be taken to reduce the rate of traffic crashes through organizing the passage of vehicles and pedestrians and separating the passage of light and heavy means of transportation in the western and southern highways of tehran . \n \n distribution of studied crashes in terms of proximity to or remoteness from the west ( azadi ) and south ( khazaneh ) terminals showed that the density of traffic crash was relatively high at the end of the roads leading to azadi passenger terminal such as tehran - karaj freeway , karaj special road , afsarieh bridge , and the eastern part of azadegan highway . thus , one of the reasons for the high frequency of traffic crashes causing death to pedestrians in south and west of tehran is the location of azadi and khazaneh passenger terminals in these areas . on the other hand , distribution of the studied crashes in terms of proximity to or remoteness from urban passenger terminals does not follow a specific pattern and the frequency of traffic crashes associated with pedestrians is very low near these passenger terminals , especially those far from interurban passenger terminals . therefore , it can be concluded that the streets and roads around the urban passenger terminals are of low risk in terms of traffic crashes associated with pedestrians . \n \n comparison of the 22 districts of tehran in terms of the frequency of fatal crashes involving pedestrians shows that the highest frequency of this type of crashes has occurred in district 2 , which accounts for 13.6% of the total cases , while this district covers only 8% of the total area of tehran . given that most crashes in district 2 have happened on highways , the relative high length of highways could be mentioned as one of the reasons for the higher frequency of traffic crashes associated with pedestrians in district 2 of tehran . highways in this district , with a length of about 61 km , account for about 11% of all highways of tehran . \n \n results of this study revealed that high - risk traffic areas in terms of crashes causing death to pedestrians during the studied years are mostly located in the west and south of tehran . high value of moran s i index indicated the distribution of crashes by the 560 traffic areas of tehran followed a cluster - like pattern . in other words , the location of occurrence of these crashes in tehran did not follow a random distribution . in addition , findings demonstrated that the hot spots of the studied crashes were mostly located in the west and south of tehran , while the cold spots were mainly focused in the central parts of the city . since getis - ord general g index showed that the distribution of hot and cold spots of the studied crashes is statistically significant , it can be stated that some certain factors were involved in non - random distribution traffic crashes causing death to pedestrians in tehran and high frequency of these crashes in high - risk areas . due to the environmental and structural differences between the high - risk and low risk areas , which are mainly located in the center of tehran , environmental factors similar studies in other parts of the world , including the studies conducted by slaughter et.al in new york , anderson in london , taquechel et.al in atlanta , wang and kockelman in texas , siddiqui et.al in florida and kuhlmann et.al in colorado , have also shown that environmental factors are highly involved in non - random distribution of traffic crashes related to pedestrians ( 12,14,16,31 - 33 ) . \n \n it seems that one of the factors determining the distribution of the studied crashes in tehran , which is the higher frequency of crashes in the suburban areas than the central part of the city , is the difference between drivers and pedestrians in terms of traffic culture . the drivers and pedestrians in central parts of the city have higher education levels and respect traffic regulations more than those in the suburban areas . higher traffic load on the outskirts of the city compared to the downtown was another reason for this issue . enforcement of regulation of traffic limits and alternative passage of vehicles with even or odd license plates in the central parts of tehran causes the central parts of the city to have a lighter traffic load than the margins of tehran . \n \n this was one of the few studies in iran that has used the files drawn by police experts for conducting an applied research to determine the high - risk areas in a metropolis . using these files , we recorded the geographical coordinates of the location of crashes and we prepared a map to determine the distribution pattern of traffic crashes causing death to pedestrians and their position relative to other spatial objects . \n \n this study was conducted using only the data of crashes registered by tehran traffic police . comparison of the statistics provided by the police and forensic medicine organization showed that the actual number of fatal crashes in tehran is more than those mentioned in the database of tehran traffic police and the information related to some crashes causing death does not exist in this database . therefore , it is not possible to acquire information on their location of occurrence through the police files . \n \n in such studies , it is better that the relevancy of the death of the injured by traffic crashes to these crashes be defined based on the time of death after crash . this time in different countries varies between 6 days to one month , and in iran , this time has been defined to be one month . however , due to the lack of consistency between the data provided by tehran traffic police and forensic medicine organization , relevancy of the death of the injured by traffic crashes to these crashes in this study was determined based on the data extracted from data based on tehran traffic police . \n", "\n various districts of tehran , excluding central districts , are considered high - risk areas . therefore , the majority of traffic crashes causing death to pedestrians have occurred on highways located at the main entrance ; that is , districts located in south , east and northwest of tehran . significant cluster - like distribution of crashes by the 560 traffic zones of tehran and existence of hot spots in terms of the studied crashes in the western and southern parts of tehran indicates the decisive role of environmental factors in occurrence of traffic crashes causing death to pedestrians . moreover , it is necessary to organize the pedestrians traffic in high - risk areas . \n", "\n this study was part of a thesis conducted in shahid beheshti university of medical sciences . we would like to sincerely thank all who helped us in this study , especially gen . hosseini , the commander of tehran traffic police , personnel of crashes department of tehran traffic police , particularly lt . soroush , headquarters of public health faculty and members of the epidemiology department of shahid beheshti university of medical sciences ." ]

[ "since its first application in 1980 , extracorporeal shock wave lithotripsy ( swl ) has become the preferred treatment method in many ureteric and kidney stone diseases . advances in stone treatment in the endoscopic age , such as the ability to perform retrograde intrarenal surgery more frequently and almost independently of the size of the stone and percutaneous nephrolithotripsy ( pnl ) gaining less invasive features defined as mini and micro , can be listed as developments that have hindered the preference of swl . the wide use of swl is due to its higher efficacy in selected cases while its low morbidity rates is one of the most important advantages of this method , making it the first treatment choice in many cases today , despite the other treatment alternatives that are available . however , there are conditions that limit the use of the method and affect its success . among the factors that affect the success and results of swl are ; the type of lithotripter ; stone - related factors such as the size , structure , number , and localization ; the anatomy and the functioning of the kidney ; and patient specific structural features [ 3 , 4 , 5 ] . al ansari et al . investigated the prognostic factors affecting the success of swl in 427 patients and they demonstrated that in cases with renal stones larger than 30 mm , the stone 's size , localization , number , radiologic renal features and congenital renal anomalies were significant factors , while ureteral stent use , age , gender , and the nature of the stone ( de novo or recurrent ) had no effect . . reached the same conclusions in their study of 2954 cases with renal stones that were smaller than 30 mm . in addition , factors such as the presence of additional interventions pre- and post - swl , complications , and costs can also affect the efficacy of the treatment [ 2 , 3 , 4 , 7 ] . the routine use of ureteral stent prior to swl is not recommended in renal stone cases despite the lack of any defined criteria in the guidelines [ 2 , 8 ] . while the use of ureteral stents can reduce post - swl complications such as obstruction and renal colic , it does not prevent steinstrasse formation and infectious complications , and does not increase stone - free rates [ 8 , 9 , 10 ] . patient discomfort , pain in bladder , and issues related to urination that are associated with ureteral stent use can often be experienced . according to kirkali et al . , pre - swl ureteral stent use should be preferred only in solitary kidney patients . the goal of our retrospective study of over 1361 patients was to compare the stone - free rates , steinstrasse formation , treatment efficacy and complications between patients with renal pelvic stones with and without pre - swl stent and to contribute to medical literature based on real life experiences .", "this is a retrospective study conducted by scanning the medical data of 1378 patients treated with swl for renal pelvic stones at our clinic between 1995 and 2011 . seventeen patients who had percutaneous nephrostomy tube placement prior to swl were excluded from the study . the median age of the 1361 patients included in the study was 40 ( 1 - 85 ) years . all patients had routine renal function tests , urinalysis and urine culture , coagulation tests , kidney - ureter - bladder x - ray ( kub ) , intravenous pyelography ( ivp ) , and ultrasonography ( usg ) before swl . an abdominal contrast - free computerized tomography ( ct ) patients with urinary system infections were treated with antibiotics according to their culture results prior to swl . uncontrolled infections , coagulation dysfunctions , ureteropelvic junction obstruction , and pregnancy were considered contraindications for swl . the swl procedure was carried out via siemens lithostar lithotripter ( siemens medizinische technik , erlangen , germany ) . the size of the stone was calculated in squared centimeters , by multiplying the widest width and length observed in kub . when multiple stones were observed , the sum of their sizes was calculated . in order to avoid statistical bias in this study , the patients were separated into 3 groups based on the size of the stone : 1 cm ( group 1 ) , 1.12 cm ( group 2 ) , and > 2 cm ( group 3 ) . table 1 displays the patient characteristics , prior interventions for stones on the same side , stone characteristics , and treatment features of the 1361 patients . specialized in swl and the energy and shock wave count for each patient were determined by the same physician . prior to the procedure , an ureteral stent ( percuflex plus 4.8 f x 26 cm , boston scientific , quincy , ma , usa ) was placed in solitary kidney patients , patients with renal ectasia of grade 2 , and patients with obstruction symptoms lasting a long period of time ( > 1 month ) . swl was performed on all patients in the supine position , under fluoroscopic control and as an outpatient procedure . fourteen patients had the procedure under anesthesia where 0.10.2 mg / kg midazolam and 0.5 mg alfentanil were used for analgesic sedation . the treatment was initiated with 13 kilovolt ( kv ) and was increased with 0.3 kv increments up to the highest level that the patient could tolerate . the procedure was ended when full fragmentation of the stones was observed in the fluoroscopic control . the procedure was considered unsuccessful in cases where fragmentation was not achieved at the end of the 3 session and/or in patients who wanted to try another treatment . patients were given hydration , analgesic and antispasmolytic treatments during the sessions and the first post - treatment week . patients were evaluated with kub and usg at the end of the first week after the procedure and at 3-month follow - ups . ct was not performed on any patients who did not display significant symptoms or if hydronephrosis was not detected in their usg . size of the stone(s ) , auxiliary procedures ( with or without stent use ) , radiologic evaluations at 3-month follow - up , and complications were evaluated retrospectively . statistical analyses were performed using chi - square , fisher 's exact and mann - whitney u tests . using the efficacy coefficient equation ( eq ) : stone free % / ( 100% + re - treatment + auxiliary procedures % ) x 100 . \n characteristics of patients , stones and treatments", "the grouping of 1361 patients according to the renal pelvis stone size was as follows : 514 patients in group 1 , 530 in group 2 , and 317 in group 3 . there were 178 patients ( 13% ) who had stent placement prior to the procedure according to the aforementioned criteria . the number of patients in groups 1 , 2 and 3 who had pre - swl stent placement was 30 ( 6% ) , 44 ( 8% ) , and 104 ( 33% ) , respectively . patient and stone characteristics by groups and stent placement are presented in table 2 , while treatment features by groups are shown in table 3 . the average number of sessions was found to be significantly higher among the patients with stent placement in all groups ( group 1 : p = 0.022 ; group 2 and 3 : p = 0.000 ) , while the proportion of stone - free patients was similar across patients with and without stent placement in group 1 ( 86.4% and 73.3% ; p = 0.06 ) , a significant difference was observed in group 2 ( without stent 80.2% vs. with stent 56.8% ; p= 0.000 ) and group 3 ( without stent 75.1% vs. with stent 64.4% ; p= 0.047 ) . treatment eq for patients without and with stent placement , and for all patients within the groups were 64% , 46% , and 63.7% , respectively , for group 1 ; 52% , 30% , and 49% , respectively , for group 2 ; and 43% , 33% , and 40% , respectively , for group 3 . the distribution of patients with and without stent placement according to steinstrasse formation was 6.7% and 5.8% ( p = 0.692 ) in group 1 , 11.4% and 15.8% ( p = 0.431 ) in group 2 , and 26% and 22.5% ( p = 0.500 ) in group 3 , respectively . according to these findings , no significant difference was observed between the patients with and without stent placement within the groups in terms of steinstrasse formation . gender distribution and side of the stone localization yielded similar results across groups , as well . while the rates of solitary kidney patients were significantly different across patients with and without stent placement in groups 1 and 2 ( p = 0.000 ) , no such significant difference was observed in group 3 ( p = 0.209 ) . none of the major complications such as stent migration , infection , pyelonephritis or stent breakages were observed in any of the patients with stent placement in this study . of the 178 patients with stent placement , 68 ( 38% ) complained of frequent urination and pain in the bladder and kidney area that was associated with the stent but these issues were resolved via symptomatic therapies . \n comparison of characteristics of patients and stones according to the groups treatment characteristics according to groups", "ureteral stents are mostly used to enable continuation of drainage in the presence of complications such as stone , tumor or obstruction between the kidney and the bladder . the pieces of the stones broken down still have a risk of causing obstruction in the ureter following swl and this condition is associated with the size of the stone . use of ureteral stent in swl is generally not recommended and there are various studies on the matter . although ureteral stent is useful to prevent complications such as obstruction and renal colic following swl , it does not protect from steinstrasse formation or infectious complications and does not increase the proportion of stone - free patients [ 8 , 9 , 10 ] . in our study , the stone - free rate following swl was found to be higher for renal pelvis stones > 1 cm in patients without stent placement , although no difference was observed regarding steinstrasse formation . as for the demographic characteristic of our patient series , the female / male ratio , side ratio , stone load , and the number of swl sessions were in line with the literature findings . libby et al . demonstrated that ureteral stents reduced morbidity in case of stones of sizes > 2.5 cm . on the other hand , reported that the swl morbidity with or without stent placement is similar to that of pnl in patients with stones of > 2 cm . groeneveld recommended ureteral stent placement in case of stones that are greater than 3 cm , if swl with prior debulking or swl alone will be performed . however , the benefits of ureteral stent use prior to swl are still disputable [ 17 , 18 ] . low et al . , with their 179-patient retrospective series , determined that there was no difference between the stone - free rates of patients with or without stent placement in their 1-month and 3-month evaluations . sulaiman et al . reported that ureteral stents did not make a difference in steinstrasse formation in cases of stones that greater than 2 cm . preminger et al . also failed to detect a difference between patients with and without stent placement in terms of their stone - free rates , independent of the stone load and shock strength . reported that stent use did not cause a significant difference in the stone - free rates of large kidney stones or reduce post - swl morbidity and that their use in routine care is not necessary . however , in hollowel 's inquiry of urologic specialists in the united states , it was determined that most of the urologists declared that they use stents in case of stones that are greater than 2 cm in size although there was no objective data about their usage . we believe that the urologists desire to stay on the safe side in terms of colic - type pain or obstructions following swl for relatively larger stones play a role in the decision . in sfoungaristas 's study investigating stent use in ureter stones that are 4 - 10 mm in size , stent use was reported to reduce stone - free rates and that it negatively affected the post - swl quality of life . in pettenali et al . 's retrospective study , stent use was reported to reduce swl success in cases of proximal ureteral stones that are larger than 8 mm . . 's retrospective analysis showed that , in cases of kidney stones larger than 30 mm , steinstrasse formation was more common among patients with stent placement , and that the sole removal of the stent would be sufficient for these patients ; and in cases of stones that are between 2030 mm , they reported no difference between patients with and without stent placement in terms of steinstrasse formation . they do not recommend stent use in the case of stones smaller than 20 mm in size . mustafa et al . also reported that the placement of a ureteral stent for the purpose of improving stone free rates or enhancing the passage of fragments during swl is unnecessary in renal stones with diameters less than 2.5 cm . though medical literature specifies that stents generally do not affect swl results or at least do not have a negative effect on stone - free rates , we state in our series that ureteral stents were observed to have a negative effect on the stone - free rates among stones greater than 1 cm in size . according to this finding , while stone - free rates do not vary across groups with or without stent placement in case of stones up to 1 cm , stent use was observed to have a negative impact on the stone - free rates in groups 2 and 3 . again , the eq values were higher among patients without stent placement in all groups . this is contradictory with the predisposition of stent use with increase in stone size and in fact shows that ureteral stents may reduce the efficacy of swl as stone size increases . in all 3 groups of our series , we believe that the difficulty the stent creates in focusing on the stone and the additional sessions performed for the stones traveling down the ureter plays a significant role in these findings . furthermore , in such cases stents can also block the particles that could migrate downwards under normal conditions . in terms of steinstrasse formation , no significant difference was observed between the patients with and without stent placement in any of the 3 groups and ureteral stents were observed to be unable to prevent steinstrasse formation . literature reports migration , stent breakage , encrustation , infection , pyelonephritis , and stone formation as stent - related complications [ 26 , 27 ] . 's evaluation of 290 cases with stent placement that had uretero - renoscopy and swl , the 12-week rates for encrustation was 76.3% , migration was 3.7% , and breaking of stent was 0.3% . joshi et al . also reported that 60% of the patients had stent- related symptoms of overactive bladder such as increased urination frequency and urge incontinence . while we did not observe complications such as migration , encrustation , breaking or pyelonephritis in our series , about 38% of the patients had symptomatic ( frequency , urgency , hematuria , dysuria , and in some patients a colic - like pain ) complaints . we believe that urinary reflux related to stent may play a role especially if the patient with a full bladder experiences colicky pain after urination . these complaints were generally controlled via symptomatic treatments and none of the patients required stent removal due to these symptoms . we believe that informing the patients about the possible symptoms that can be experienced following stent placement will allow them to tolerate the ureteral stents better . the retrospective nature , lack of randomization in terms of ureteral stent use prior to swl and that only 13% of patients had stent placement may be considered the limitations of this study . on the other hand , these limitations are avoided by the sole use of specialists with 25 years of swl experience ( n.t . ) in the performance of all procedures , which contributed significantly to our study with respect to the standardization provided in ureteral stent preference and adjustment of the number and strength of shock waves . we believe that our patient series with cases of various stone loads has a lot to contribute to the medical literature on this matter . the usage of kub and usg for monitoring the treatment results , but not ct could also be argued . kub and usg are preferred in the aim to avoid the risk of high radiation levels and financial burden on the health system especially in symptom - free patients . 's study on this topic with 2759 patients between the ages of 1876 years reported that usg , compared to ct , does not increase complications , pain score , admission rates to the emergency department , or hospitalization but does reduce exposure to radiation .", "in our study , the stone - free rate following swl was found to be higher for renal pelvis stones > 1 cm in patients without stent placement , although no difference was observed regarding steinstrasse formation . our results showed that ureteral stents were observed to have a negative effect on the stone - free rates among stones greater than 1 cm in size . the results of this study support the idea of limiting the application of stents during swl and prevent possible complications as well as reduce the financial cost of treatment . considering that ureteral stents reduce stone - free rates , we believe that they should be preferred in special cases such as solitary kidney patients or those with long - term obstruction .", "" ]

[ "noncommunicable diseases , including type 2 diabetes mellitus ( t2 dm ) , are considered the leading cause of death in the world and , in brazil , constitute the main cause of diseases . t2 dm assumes an important role in this context given that it is considered a worldwide epidemic disease ; in 2011 it appeared among the ten leading causes of death in the world and it is prospected that number of cases will continue to increase . the ones most frequently being discussed are the increase in population life expectancy , changes in lifestyle ( including unbalanced diet and physical inactivity ) , and obesity . diabetes can be defined as a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion , insulin action , or both . the underlining cause of t2 dm can be attributed to a combination of resistance to the action of the hormone , increased production of insulin in a compensatory manner , and inadequate secretory response . on the other hand , according to a study conducted by kaprio et al . , the heredity has an input around 47% in susceptibility to t2 dm ; thus , genetic factors play an important role in the development of the pathology . some facts corroborate the importance of heritability in t2 dm : the greater concordance between monozygotic twins than among dizygotic twins and a wide variation in the prevalence of t2 dm in epidemiological studies with different ethnic groups as well as positive results in numerous other genetic studies . in this regard , it should be noted that even as more than 30 genes associated with t2 dm have been identified , the contribution of each individual gene in the disease susceptibility is very small . additionally , most of these genes identified are related to dysfunction of pancreatic cells . several reports suggest that the characteristics of each individual , including genetic polymorphisms , may confer differences in the occurrence of diabetes and other complex diseases such as cancer and heart disease . withal , in most cases , a positive association between polymorphisms of these genes and t2 dm are not reproducible in the analysis of a different population . two polymorphisms in the promoter region of the tnfa gene have been described , one present at position -308 . in this regard , swaroop et al . demonstrated an association between the adipocytokine tnf- and development of insulin resistance . this systematic review was conducted to provide a comprehensive assessment of the association between the polymorphism in the gene tnfa -308g / a and the presence of t2 dm .", "the literature search was made through the virtual health library ( vhl ) which uses as bibliographic databases the publications found in sources such as lilacs , medline , and other information bases such as open educational resources , websites , and scientific events . any publications with case - control studies , on january 21 , 2016 , that show the relationship between polymorphisms of tnfa gene -308g / a and susceptibility to type 2 diabetes mellitus ( t2 dm ) were surveyed . the search terms used were as follows : diabetes and tnf or tumor necrosis factor , and polymorphism . the filters are selected in accordance with the main subjects of the following terms : type 2 diabetes mellitus , tumor necrosis factor alpha , single nucleotide polymorphism , and genetic polymorphism . the filter base on the type of study included solely the term case - control study . inclusion criteria were as follows : ( 1 ) the study must have been case - control ; ( 2 ) the study should have assessed type 2 diabetes mellitus and -308g / a polymorphism of tnfa gene , even if associated with other comorbidities ; and ( 3 ) the study should have provided sufficient data , including the number or frequency of alleles and genotypes . studies were excluded if ( 1 ) there were commentaries , case reports , or non - case - control or meta - analyses studies ; ( 2 ) they did not report sufficient data ; ( 3 ) they presented data from other types of diabetes or did not specify what kind patients had ; and ( 4 ) they showed polymorphism in another coding region of the tnfa gene or ( 5 ) if the sample was composed of only dm patients . data from eligible studies were extracted independently in accordance with the inclusion and exclusion criteria . for each study the following characteristics were collected : the authors , the study title , the year of publication , where it was published , the sample size , the tnfa -308g / a genotypes with their corresponding simple frequencies and percentages for the control and the case group , and the study 's p value and odds ratio , when calculated .", "the literature search found articles published starting from 2000 , given that the year the complete mapping of the human genome was made available . advances in molecular biology technology since this project have been quick and , thus , increasing the number of genetic studies published for the association of certain genetic polymorphisms with various pathologies . in the case of literature search for articles with case studies that related polymorphism position -308 of the tnfa gene with diabetes , the result presented forty - three publications between the years 2003 and 2015 , ten of which met the criteria inclusion . thirteen studies presented results for other types of diabetes ; eleven related to type 1 diabetes and two to gestational diabetes . two others did not specify what type of diabetes had patients in the case group . two of these studies did not show enough genetic data for analysis . in relation to tnfa gene , seven studies presented polymorphisms in region other than the -308 position and three publications had single nucleotide polymorphism in a gene that was not tnfa . other studies were excluded due to different methodological designs in their inclusion criteria : two meta - analysis , one being observational and the other prospective and , relative to the sample . furthermore , there was a publication that presented data of diabetic patient 's polymorphisms in both the control and the case group . the number and homogeneity of the included studies presented were too limited to allow a meta - analysis . relate the polymorphism in gene studied with t2 dm and coronary heart disease , while another two ( buraczynska et al . , dabhi and mistry ) relate association frequencies in diabetic patients with nephropathy or renal failure . finally , five studies reported data that directly relate polymorphism in question with t2 dm ( shiau et al . , bouhaha et al . , guzmn - flores et al . one of which also relates it with obese t2 dm patients ( bouhaha et al . ) . the general characteristics of the studies included in this review are summarized in table 1 . in three studies conducted in spain , hungary , and morocco significant associations between the tnfa -308g / a polymorphism and the risk of t2 dm were identified [ 11 , 17 , 20 ] : two of them in patients with other comorbidities such as coronary heart disease and atherosclerosis . in the present study , therefore , we infer that although there was no evidence of heterogeneity in the association between polymorphisms in the tnfa -308g / a gene and increased risk for development of t2 dm , the presence of positive results may mean that few studies have not been enough to clarify this relationship , and besides there is no documented evidence in various ethnic groups . due to contradictory results , this systematic review is aimed at providing a comprehensive assessment of the association between tnfa -308g / a and phenotype involving type 2 diabetes .", "the concern regarding the quantification and qualification of the genetic impact on phenotypic characteristics started after the first complete human sequence after genomic era . as a consequence , health research delved into the genetic information as a way to find answers to complex disease questions . this type of research has shown high prevalence and increasing prospects in a number of cases , such as diabetes . for this purpose , various portions of the human genome are now analyzed in search for genetic variations among individuals that could explain these diseases . studies seeking interactions between these variations , in particular , have been very promising . in the case of diabetes , a syndrome whose etiology is complicated and involves several risk factors for its development and progression , some studies on single nucleotide polymorphisms were suggested as causing the phenotypic characteristics and responsible for the increased risk of developing pathology [ 2527 ] . to study the risk factors for a disease is to study the possibility of a certain event happening . within the epidemiological concepts , the term is used to predict the likelihood of healthy individuals , exposed to certain factors , developing a given disease . this does not necessarily mean that it would occur ; nevertheless the presence of these factors makes the individual more vulnerable to manifest the disease . a risk factor x may be the trigger of various diseases , and several risk factors can cooperate for the genesis of a common disease . environmental factors such as eating habits [ 2931 ] and physical inactivity [ 3234 ] , in addition to the concomitant presence of diverse mutations in many genes , need to be considered so that the contribution of each of them is understood . linkage research studies genetic markers and specific polymorphisms in a family group that has the disease . this , thus , allows the researchers a way to better study the regions of a chromosome that is affected . after the connection is made to the chromosome , one can test for association of polymorphisms for identifying a genotype specific to a given disease in groups of people who have the targeted disease [ 34 , 35 ] . several association studies have discussed the importance of clarifying the relationship between genetic polymorphisms and the development of type 2 diabetes . the search for candidate genes was initially focused on possible genes encoding proteins directly involved in the pathophysiology of t2 dm : related encoders , for example , production , secretion or activity of insulin , or development of the pancreas [ 3638 ] . gradually , however , emerging studies suggested that several other factors could contribute to the development of t2 dm , as the expression of proinflammatory cytokines and other molecules that act in the inflammatory process and that appear to play a critical role in the development of chronic complications of dm . several studies have shown the influence of various polymorphisms in proinflammatory cytokines genes as a risk of development of obesity , diabetes , and metabolic syndrome . one of these cytokines , tumor necrosis factor ( tnf- ) , has been investigated since diabetics have elevated levels of it circulating . tnf- has also been implicated as an insulin resistance - causing factor associated with the pathogenesis of t2 dm . tnf- is a proinflammatory cytokine that acts in the regulation of cell proliferation , differentiation , and apoptosis . among the various polymorphisms describing the tnfa gene , the -308g > a variant located in the promoter region excels by affecting the expression of its gene . the presence of the a allele at nucleotide -308 increases transcription of tnfa gene approximately twofold , therefore increasing the production of this cytokine . under this circumstance , it is expected that the polymorphic genotype may be associated with an increased frequency of diabetes and , for the proper association , of case - control studies can be used . in this review , several studies with the frequency of genotypes in control and case group were observed for a better understanding of the population genetic profile and a possible association between polymorphism of tnfa gene and t2 dm in groups with different ethnicities . the influence of genetic polymorphisms on certain diseases may be found in a population and yet not in another ; this may impact the frequencies and distribution of a given polymorphism in distinct population . the difference in influence may be due to racial variantion as well as other factors . . in addition , some studies have included a proportionally small sample size in relation to the country 's population and most of them define the case of groups in t2 dm patients who have concomitant comorbidities . case - control studies were selected in this review and were made with the people of spain , taiwan , poland , india , tunisia , mexico , hungary , and morocco ; nonetheless a positive association was only found in the spanish , hungarian , and moroccan population . sefri et al . in their meta - analysis , which considers 21 case - control studies published until august 2013 , argue that no significant associations were found between the polymorphism in the studied region of the tnfa gene and risk of developing t2 dm . their finding was consistent with a previous meta - analysis , which included data from 18 association studies . in these studies , publications with different ethnic groups were included ; among them were africans , asians , caucasians , and other populations . so , when comparing the frequencies of polymorphic genotypes ( ga and aa ) in the groups of case - control study ( described in table 1 ) , it is possible to observe that these genotypes are presented more frequently in the group that has the disease . the exceptions to this were the studies in tunisia and india ; the most frequent genotype in type 2 diabetic patients was the gg . in all other countries this evidence supports the assumption that polymorphisms in the tnfa gene and its association with other aspects , both genetic and environmental , may represent an important risk factor for type 2 diabetes mellitus .", "risk factors not modifiable or irreversible in nature , such as genetic profiles , refer to the individual characteristics . even though these factors may not be changed , the identification of their presence in individuals and families may enable health professionals to advise change in the lifestyle of these patients , avoiding early manifestation of t2 dm ." ]