bluuebunny/crossref_metadata_2025 huggingface dataset:
A Survey of 25 North Carolina Health Departments/Districts on Knowledge, Attitudes, and Current Practices to Seeking Reimbursement From Third-Party Payers for Sexually Transmitted Disease Services
Authors: Kovar Cheryl L. , Carter Susan
Published: April 2017

Background

North Carolina Administrative Code 10A Chapter 41A.0204 (a) states “local health departments shall provide diagnosis, testing, treatment, follow-up, and preventive services for syphilis, gonorrhea, chlamydia, … These services shall be provided upon request and at no charge to the patient.” Although health departments/districts may bill governmental or nongovernmental insurance providers for sexually transmitted disease (STD) services, current billing practices are unknown. Because of its high STD morbidity, the eastern region of North Carolina was targeted.

Methods

Using a Qualtrics Survey developed to measure attitudes as well as knowledge and reimbursement practices, this descriptive study was performed with staff from 25 eastern North Carolina health departments/districts. Snowball sampling was used to allow for greater inclusion. Analysis of data was performed at the individual and agency level based on types of questions in the survey.

Results

For knowledge, 87% of the respondents reported being aware of the possibility of reimbursement from third-party payers/commercial insurance carriers for STD services. In regard to current billing of these services, 20 health departments/districts (80%) reported they were billing these payers. When asked about their attitude of seeking reimbursement from commercial insurance, 92% reported it was acceptable or very acceptable. But when asked if STD services should remain a free service at the health department, 55% supported and 45% did not.

Conclusions

These data provide a knowledge base for assisting health departments/districts to move forward in improving STD services as well as maximizing reimbursement from third-party payers/commercial insurance carriers when possible.
Maternal Syphilis: An Independent Risk Factor for Mother to Infant Human Immunodeficiency Virus Transmission
Authors: Kinikar Aarti , Gupte Nikhil , Bhat Jayalakshmi , Bharadwaj Renu , Kulkarni Vandana , Bhosale Ramesh , McIntire Katherine N , Mave Vidya , Suryavanshi Nishi , Patil Sandesh , Bollinger Robert , Gupta Amita
Published: April 2017

Syphilis is associated with increased human immunodeficiency virus acquisition and sexual transmission; we examined impact on human immunodeficiency virus mother-to-child transmission among mother-infant pairs enrolled in the India Six-Week Extended-Dose Nevirapine study. Maternal syphilis, diagnosed serologically using Venereal Disease Research Laboratory titer plus Treponema Pallidum Hemagglutination Assay, was associated with 2.5-fold greater risk.
HPV-negative Gastric Type Adenocarcinoma In Situ of the Cervix
Authors: Talia Karen L. , Stewart Colin J.R. , Howitt Brooke E. , Nucci Marisa R. , McCluggage W. Glenn
Published: April 2017

In recent years, a number of benign and malignant cervical glandular lesions exhibiting gastric differentiation have been described but premalignant gastric-type lesions have not been well characterized. We report a series of 9 cases of a rare form of cervical adenocarcinoma in situ (AIS) distinguished by gastric and sometimes intestinal differentiation and lack of association with human papillomavirus (HPV) infection. The lesions occurred in women aged 25 to 73 years (mean age 51 y). All cases were located at (or just proximal to) the cervical transformation zone and there was extension to the lower uterine segment in 3 cases, 2 of which also involved the endometrium. In all cases, the normal cervical glandular architecture was largely preserved but in 5 cases there was a mild degree of increased intraglandular architectural complexity. The glandular epithelium ranged from almost purely gastric in type (4 cases) to mixed gastric and intestinal (5 cases), with varying proportions of intermixed goblet cells. In contrast to the basophilic cytoplasm of normal endocervical glands, the gastric-type epithelium was typically predominantly composed of cells with eosinophilic or pale pink cytoplasm, but conspicuous foamy or clear cell cytoplasm was present in some cases. Nuclear atypia was present in all cases but was considered low-grade in 8. High-grade features such as marked nuclear pleomorphism and hyperchromasia were evident in only 1 case. Mitotic activity and apoptotic bodies were present but both were noted to be less frequent than in usual type (HPV-related) AIS. Immunohistochemically, there was usually positive staining with CK 7 (7/7 cases) and MUC 6 (7/8 cases) and some cases were positive with CK 20 (3/7), CDX2 (5/9), PAX 8 (5/9) and CEA (2/6). Estrogen receptor and progesterone receptor were usually negative, although Estrogen receptor was positive in 3 of 9 cases. p16 was negative or exhibited mosaic-type staining (nonblock staining) in all cases and there was mutation-type p53 staining in 2 of 9 cases. HPV molecular testing was negative in all 4 cases tested. We believe this unusual subtype of AIS, which we term “gastric-type AIS (gAIS),” represents a precursor to gastric-type adenocarcinoma of the cervix and suggest that gAIS and so-called “atypical lobular endocervical glandular hyperplasia” are related entities within a spectrum of premalignant gastric-type lesions for which we propose the umbrella term gAIS. The malignant potential and optimal management of gAIS are currently unknown but in one of our cases a gastric-type adenocarcinoma developed 6 years after removal of a cervical polyp which contained gAIS. The introduction of HPV vaccination will result in a relative increase in incidence of premalignant and malignant cervical glandular lesions exhibiting gastric differentiation and these will not be detected by HPV-based screening programs.
Brief Report: Prevalence of Posttreatment Controller Phenotype Is Rare in HIV-Infected Persons After Stopping Antiretroviral Therapy
Authors: Perkins Matthew J. , Bradley William P. , Lalani Tahaniyat , Agan Brian K. , Whitman Timothy J. , Ferguson Tomas M. , Okulicz Jason F. , Ganesan Anuradha
Published: April 2017

Background:

Posttreatment control of HIV infection is a rare phenomenon primarily described among those initiating treatment with antiretroviral therapy (ART) during early/acute HIV infection.

Methods:

We examined a large, well-characterized cohort of HIV-infected Department of Defense beneficiaries for the presence of posttreatment controllers (PTCs) whom we defined as individuals with sustained viral suppression for ≥6 months after discontinuation of ART. We defined those who became viremic within 6 months of discontinuing ART as rapid viremics (RVs) and compared demographic and clinical characteristics, CD4 counts, and viral loads prior, during, and after ART discontinuation between the 2 groups.

Results:

From a cohort of 6070 patients, we identified 95 who had been treated with ART for 2 years or more who subsequently discontinued ART and had viral load assessments available after discontinuation. Four (4.2%) of these 95 met our definition of PTC. The duration of viral suppression off of ART ranged from 267 to 1058 days with 1 of the 4 restarting ART without having redeveloped a significant viremia. All 4 patients initiated ART during chronic HIV infection. Demographic and clinical characteristics of PTCs were similar to RVs.

Conclusions:

While posttreatment control has predominantly been described among individuals who initiated ART in early/acute HIV infection, we identified 4 PTCs who started ART during chronic infection suggesting that posttreatment control also occurs among such patients. The rarity of PTCs identified in our cohort is consistent with reports from previous studies.
Association of CD4+ T-cell Count, HIV-1 RNA Viral Load, and Antiretroviral Therapy With Kaposi Sarcoma Risk Among HIV-infected Persons in the United States and Canada
Authors: Dubrow Robert , Qin Li , Lin Haiqun , Hernández-Ramírez Raúl U. , Neugebauer Romain S. , Leyden Wendy , Althoff Keri N. , Achenbach Chad J. , Hessol Nancy A. , Modur Sharada P. , D'Souza Gypsyamber , Bosch Ronald J. , Grover Surbhi , Horberg Michael A. , Kitahata Mari M. , Mayor Angel M. , Novak Richard M. , Rabkin Charles S. , Sterling Timothy R. , Goedert James J. , Justice Amy C. , Engels Eric A. , Moore Richard D. , Silverberg Michael J. ,   for the North American AIDS Cohort Collaboration on Research and Design of the International Epidemiologic Databases to Evaluate AIDS
Published: April 2017

Background:

Kaposi sarcoma (KS) remains common among HIV-infected persons. To better understand KS etiology and to help target prevention efforts, we comprehensively examined a variety of CD4+ T-cell count and HIV-1 RNA viral load (VL) measures, as well as antiretroviral therapy (ART) use, to determine independent predictors of KS risk.

Setting:

North American AIDS Cohort Collaboration on Research and Design.

Methods:

We followed HIV-infected persons during 1996–2009 from 18 cohorts. We used time-updated Cox regression to model relationships between KS risk and recent, lagged, trajectory, and cumulative CD4 count or VL measures, as well as ART use. We used Akaike's information criterion and global P values to derive a final model.

Results:

In separate models, the relationship between each measure and KS risk was highly significant (P < 0.0001). Our final mutually adjusted model included recent CD4 count [hazard ratio (HR) for <50 vs. ≥500 cells/μL = 12.4; 95% confidence interval (CI): 6.5 to 23.8], recent VL (HR for ≥100,000 vs. ≤500 copies/mL = 3.8; 95% CI: 2.0 to 7.3), and cumulative (time-weighted mean) VL (HR for ≥100,000 vs. ≤500 copies/mL = 2.5; 95% CI: 1.0 to 5.9). Each P-trend was <0.0001. After adjusting for these measures, we did not detect an independent association between ART use and KS risk.

Conclusions:

Our results suggested a multifactorial etiology for KS, with early and late phases of development. The cumulative VL effect suggested that controlling HIV replication promptly after HIV diagnosis is important for KS prevention. We observed no evidence for direct anti-KS activity of ART, independent of CD4 count and VL.
Effects of Percutaneous Coronary Intervention on Viable Myocardium and Heart Function of Diabetic Patients With Chronic Total Occlusion
Authors: Zhou Guowei , Yang Wenyi , Li Liang
Published: April 2017

Objective

The aim of this study was to compare the effects of percutaneous coronary intervention (PCI) on coronary chronic total occlusion patients with (DM) or without (NDM) diabetes mellitus.

Methods

A total of 78 patients were divided into DM group and NDM group according to whether the patient has DM. The results of PCI were analyzed using quantitative coronary analysis. In addition, all the patients underwent 99mTc-MIBI (methoxyisobutylisonitrile) single-photon emission computed tomography (SPECT) and ultrasonic cardiogram in the first week and the sixth month after PCI to evaluate PCI results. During the 6-month follow-up, major adverse cardiac event (MACE) was recorded and analyzed as well.

Results

The first and second classes of collateral circulation between the 2 groups have significant differences (P < 0.05). Left ventricular end-diastolic volume index and left ventricular end-systolic volume index were decreased at the sixth month compared with those at the first week. Left ventricular ejection fraction was significantly increased. In both groups, the defect size significantly reduced, and percentage of radionuclide scintigraphic count significantly increased between rest and nitroglycerin interventional SPECT. After 6 months, both groups repeated nitroglycerin interventional SPECT, which showed that defect size was significantly reduced, and the percentage of radionuclide scintigraphic count was significantly increased compared with those of the first week. During the 6-month follow-up, the incidence of MACE between the 2 groups had no significant difference.

Conclusions

Percutaneous coronary intervention has beneficial effects on heart functions and MACE when performed on chronic total occlusion patients with and without DM.
Postoperative Morbidity After Radical Resection of Primary Retroperitoneal Sarcoma
Authors: MacNeill Andrea J. , Gronchi Alessandro , Miceli Rosalba , Bonvalot Sylvie , Swallow Carol J. , Hohenberger Peter , Van Coevorden Frits , Rutkowski Piotr , Callegaro Dario , Hayes Andrew J. , Honoré Charles , Fairweather Mark , Cannell Amanda , Jakob Jens , Haas Rick L. , Szacht Milena , Fiore Marco , Casali Paolo G. , Pollock Raphael E. , Barretta Francesco , Raut Chandrajit P. , Strauss Dirk C.
Published: April 2017

Objective:

To investigate the safety of radical resection for retroperitoneal sarcoma (RPS).

Background:

The surgical management of RPS frequently involves complex multivisceral resection. Improved oncologic outcomes have been demonstrated with this approach compared to marginal excision, but the safety of radical resection has not been shown in a large study population.

Methods:

The Transatlantic Retroperitoneal Sarcoma Working Group (TARPSWG) is an international collaborative of sarcoma centers. A combined experience of 1007 consecutive resections for primary RPS from January 2002 to December 2011 was studied retrospectively with respect to adverse events. A weighted organ score was devised to account for differences in surgical complexity. Univariate and multivariate logistic regression analyses were performed to investigate associations between adverse events and number and patterns of organs resected. Associations between adverse events and overall survival, local recurrence, and distant metastases were investigated.

Results:

Severe postoperative adverse events (Clavien-Dindo ≥3) occurred in 165 patients (16.4%) and 18 patients (1.8%) died within 30 days. Significant predictors of severe adverse events were age (P = 0.003), transfusion requirements (P < 0.001), and resected organ score (P = 0.042). Resections involving pancreaticoduodenectomy, major vascular resection, and splenectomy/pancreatectomy were found to entail higher operative risk (odds ratio >1.5). There was no impact of postoperative adverse events on overall survival, local recurrence, or distant metastases.

Conclusions:

A radical surgical approach to RPS is safe when carried out at a specialist sarcoma center. High-risk resections should be carefully considered on an individual basis and weighed against anticipated disease biology. There appears to be no association between surgical morbidity and long-term oncologic outcomes.
A Longitudinal Investigation of Inflammatory Markers in Colorectal Cancer Patients Perioperatively Demonstrates Benefit in Serial Remeasurement
Authors: Chan Joseph C. Y. , Diakos Connie I. , Chan David L. H. , Engel Alexander , Pavlakis Nick , Gill Anthony , Clarke Stephen J.
Published: April 2017

Objective:

To characterize the longitudinal course of the systemic inflammatory response (SIR) throughout the perioperative period. To investigate whether postoperative changes in the neutrophil-to-lymphocyte ratio (NLR) or lymphocyte-to-monocyte ratio (LMR) when compared with preoperative levels (‘conversion’) are associated with survival differences in colorectal cancer patients undergoing resection.

Background:

Recent evidence suggests that preoperative measurements of markers of the SIR including the NLR and LMR are prognostic. However, a few data exist evaluating longitudinal changes in the SIR especially in regards to their association with surgical interventions, optimal timing of assessment, and their effect on patient survival.

Methods:

Data from 6 hospitals from January 1998 to December 2012 were retrospectively collected. We examined 2280 patients with complete data. For the subgroup analysis investigating conversion, we examined 587 patients with full preoperative and postoperative data from 21 to 56 days postoperative. Patients were stratified into 4 groups for analysis of conversion in a multivariate Cox-regression model.

Results:

A longitudinal profile for the perioperative NLR and LMR was clearly characterized identifying an optimal period of remeasurement at 21 to 56 days postoperation. In multivariate analysis both NLR change group (P < 0.001) and LMR change group (P < 0.001) were independently associated with overall survival. For both biomarkers, patients with both a low preoperative and postoperative inflammatory state had the best survival. A change from the preoperative to postoperative inflammatory state was associated with a survival difference.

Conclusions:

This study characterizes the perioperative SIR profile and provides evidence for the remeasurement of SIR biomarkers postoperatively at 21 to 56 days for further prognostication.
Cured into Destitution: Catastrophic Health Expenditure Risk Among Uninsured Trauma Patients in the United States
Authors: Scott John W. , Raykar Nakul P. , Rose John A. , Tsai Thomas C. , Zogg Cheryl K. , Haider Adil H. , Salim Ali , Meara John G. , Shrime Mark G.
Published: April 2017

Objective:

To characterize the economic hardship for uninsured patients admitted for trauma using catastrophic health expenditure (CHE) risk.

Background:

Medical debts are the greatest cause of bankruptcies in the United States. Injuries are often unpredictable, expensive to treat, and disproportionally affect uninsured patients. Current measures of economic hardship are insufficient and exclude those at greatest risk.

Methods:

We performed a retrospective review, using data from the 2007–2011 Nationwide Inpatient Samples of all uninsured nonelderly adults (18—64 yrs) admitted with primary diagnoses of trauma. We used US Census data to estimate annual postsubsistence income and inhospital charges for trauma-related admission. Our primary outcome measure was catastrophic health expenditure risk, defined as any charges ≥40% of annual postsubsistence income.

Results:

Our sample represented 579,683 admissions for uninsured nonelderly adults over the 5-year study period. Median estimated annual income was $40,867 (interquartile range: $21,286–$71.733). Median inpatient charges were $27,420 (interquartile range: $15,196–$49,694). Overall, 70.8% (95% posterior confidence interval: 70.7%–71.1%) of patients were at risk for CHE. The risk of CHE was similar across most demographic subgroups. The greatest risk, however, was concentrated among patients from low-income communities (77.5% among patients in the lowest community income quartile) and among patients with severe injuries (81.8% among those with ISS ≥ 16).

Conclusions:

Over 7 in 10 uninsured patients admitted for trauma are at risk of catastrophic health expenditures. This analysis is the first application of CHE to a US trauma population and will be an important measure to evaluate the effectiveness of health care and coverage strategies to improve financial risk protection.
Surgeon Age in Relation to Prognosis After Esophageal Cancer Resection
Authors: Markar Sheraz R. , Mackenzie Hugh , Lagergren Pernilla , Lagergren Jesper
Published: April 2017

Objective:

It was hypothesized that patient survival improves with increasing surgeon age up to an age where it then decreases.

Background:

Experience, physical and psychological abilities required for esophagectomy may change with increasing surgeon age.

Methods:

This population-based cohort study included all patients having undergone esophagectomy for esophageal cancer in Sweden in 1987 to 2010, with follow-up until 2016. Risk-adjusted cumulative sum (RA-CUSUM) analysis was performed to estimate the relation between surgeon age and 90-day mortality, all-cause, and disease-specific 5-year mortality. Change-points in surgeon age identified by the RA-CUSUM were then analyzed in relation to mortality using multivariable Cox regression, providing hazard ratios (HRs) with 95% confidence intervals (CIs), adjusted for age, sex, comorbidity, tumor stage, tumor histology, neoadjuvant therapy, surgeon volume, and calendar year.

Results:

Among 139 surgeons performing 1761 esophagectomies, RA-CUSUM analysis of 90-day mortality showed change-points at 43 years (downward deflection) and at 56 years (upward deflection). Both all-cause and disease-specific 5-year mortality had corresponding change-points at 52 years and 56 years. Compared with surgeon age 52 to 55 years, surgeon age ≤51 years was associated with increased 90-day mortality (HR = 1.71, 95% CI 1.01–2.90) and 5-year all-cause mortality (HR = 1.21, 95% CI 1.02–1.43), and surgeon age ≥56 years showed increased 90-day mortality (HR = 2.38, 95% CI 1.38–4.13), 5-year all-cause mortality (HR = 1.29, 95% CI 1.08–1.55), and disease-specific 5-year mortality (HR = 1.18, 95% CI 1.01–1.42).

Conclusions:

Surgeon age ≤51 and ≥56 years may increase short- and long-term mortality after esophagectomy for cancer.
Medial prefrontal cortical NMDA receptors regulate depression-like behavior and dictate limbic thalamus innervation
Authors: Miller Oliver H. , Bruns Andreas , Ammar Imen Ben , Mueggler Thomas , Hall Benjamin J.
Published: April 2017

Depression is a pervasive and debilitating neuropsychiatric disorder. A single, low dose of the NMDA receptor (NMDAR) antagonist ketamine elicits a long-lasting antidepressant response in patients with treatment-resistant major depressive disorder. Developing mechanistic understanding of how NMDAR antagonism alters synapse and circuit function is pivotal to developing translatable, circuit-based therapies for depression. Here using viral vectors, anatomical tracing, fMRI, and optogenetic-assisted circuit analysis, we assessed the role of the NMDAR subunit GluN2B in regulating cellular, synaptic, and circuit-level function and depression-related behavior. We demonstrate that post-developmental deletion of GluN2B from pyramidal neurons in medial prefrontal cortex enhances action potential output in a synaptic activity-dependent manner. GluN2B deletion dictates functional connectivity between mPFC and limbic thalamus but not ventral hippocampus and elicits antidepressant-like behavior. Our findings demonstrate that postsynaptic GluN2B exerts input-specific control of pyramidal neuron innervation, and identify a novel circuit for regulating depression-like behaviors in mice.
Decision confidence as a mapping of bayesian posterior belief
Authors: Paz Luciano , Salles Alejo , Sigman Mariano
Published: April 2017

We study the confidence response distributions for several two alternative forced choice tasks with different structure, and assess whether their behavioral responses are accurately accounted for as a mapping from bayesian inferred probability of having made a correct choice. We propose an extension to an existing bayesian decision making model that allows us to quantitatively compare the relative quality of different function mappings from bayesian belief onto responded confidence. We find that a simple linear rescaling from bayesian belief best fits the observed response distributions. Furthermore, the parameter values allow us to study how task structure affects differently the decision policy and confidence mapping, highlighting a dissociable effect between confidence and perceptual performance.
Decoding the functional Fes kinase signaling network topology in a lymphocyte model
Authors: Helbig Andreas O. , Kofler Michael , Gish Gerald , Sriraja Lourdes , Lorenzen Kristina , Tucholska Monika , Zhang Cunjie , Roth Frederick P. , Pawson Tony , Colwill Karen , Petsalaki Evangelia
Published: April 2017

The c-Fes protein tyrosine kinase is a proto-oncogene that can also act as a tumor suppressor. We implemented an unbiased phosphoproteomics-based analysis that establishes cognate kinase-substrate associations, and revealed that c-Fes directly phosphorylates Dok1, Ptpn18 and Sts1, facilitating recruitment of the Src inhibitory kinase Csk to these substrates. These interactions resulted in modulation of Src signaling following B-cell receptor (BCR) stimulation and subsequent alteration of the protein levels of CD19, a membrane-localized BCR co-receptor and emerging key protein affecting the development of B- and plasma cell-lymphoma. Strikingly, manipulating c-Fes expression levels drove opposing biological outcomes. Low-level exogenous c-Fes expression led to a strong increase in CD19 protein levels while high c-Fes expression abolished CD19 protein levels. Thus, we propose that a balance of c-Fes and Src signaling can regulate receptor maintenance, which may influence cellular outcome such as tumorigenesis or tumor suppression.
16S rRNA gene sequencing reveals site-specific signatures of the upper and lower airways of cystic fibrosis patients
Authors: Lucas Sarah K. , Yang Robert , Dunitz Jordan M. , Boyer Holly C. , Hunter Ryan C.
Published: April 2017

Rationale

Chronic rhinosinusitis (CRS) is an inflammatory disorder of the sinonasal mucosa associated with microbial colonization. Metastasis of sinus microbiota into the lower airways is thought have significant implications in the development of chronic respiratory disease. However, this dynamic has not been thoroughly investigated in cystic fibrosis (CF) patients, where lower airway infections are the primary driver of patient mortality. Given the high prevalence of CRS in CF patients and the proposed infection dynamic between the upper and lower airways, a better understanding of sinus-lung continuum is warranted.

Objective

To compare the microbiome of matched sinus mucus and lung sputum samples from CF subjects undergoing functional endoscopic sinus surgery (FESS) for treatment of CRS.

Methods

Mucus was isolated from the sinuses and lungs of twelve CF patients undergoing FESS. 16S ribosomal RNA gene sequencing was then performed to compare bacterial communities of the CF lung and sinus niches. Finally, functional profiling was performed to predict bacterial metagenomes from the 16S dataset, and was used to compare pathogenic bacterial phenotypes between the upper and lower airways.

Measurements and Main Results

Bacterial richness was comparable between airway sites, though sinus and lung environments differed in community evenness, with the sinuses harboring a higher prevalence of dominant microorganisms. Beta diversity metrics also revealed that samples clustered more consistently by airway niche rather than by individual. Finally, predicted metagenomes showed that anaerobic metabolism was enriched in the lung environment, while genes associated with both biofilm formation and Gram identity were not variable between sites.

Conclusions

Sinus and lung microbiomes are distinct with respect to richness and evenness, while sinus communities have a higher incidence of a dominant taxon. Additionally, ordination analyses point to sinus and lung environments as being stronger determinants of microbial community structure than the individual patient. Finally, BugBase-predicted metagenomes revealed anaerobic phenotypes to be in higher abundance in the lung relative to the sinuses. Our findings indicate that while the paranasal sinuses and lungs may still comprise a unified airway in which lower airways are seeded by sinus microbiota, these discrete airway microenvironments harbor distinct bacterial communities.
Strain-resolved microbiome sequencing reveals mobile elements that drive bacterial competition on a clinical timescale
Authors: Bishara Alex , Moss Eli L. , Tkachenko Ekaterina , Kang Joyce B. , Zlitni Soumaya , Culver Rebecca N. , Andermann Tessa M. , Weng Ziming , Wood Christina , Handy Christine , Ji Hanlee , Batzoglou Serafim , Bhatt Ami S.
Published: April 2017

Although shotgun short-read sequencing has facilitated the study of strain-level architecture within complex microbial communities, existing metagenomic approaches often cannot capture structural differences between closely related co-occurring strains. Recent methods, which employ read cloud sequencing and specialized assembly techniques, provide significantly improved genome drafts and show potential to capture these strain-level differences. Here, we apply this read cloud metagenomic approach to longitudinal stool samples from a patient undergoing hematopoietic cell transplantation. The patient’s microbiome is profoundly disrupted and is eventually dominated by Bacteroides caccae. Comparative analysis of B. caccae genomes obtained using read cloud sequencing together with metagenomic RNA sequencing allows us to predict that particular mobile element integrations result in increased antibiotic resistance, which we further support using in vitro antibiotic susceptibility testing. Thus, we find read cloud sequencing to be useful in identifying strain-level differences that underlie differential fitness.
K-mer similarity, networks of microbial genomes and taxonomic rank
Authors: Bernard Guillaume , Greenfield Paul , Ragan Mark A. , Chan Cheong Xin
Published: April 2017

Alignment-free (AF) methods have recently been adopted to infer phylogenetic trees. However, the evolutionary relationships among microbes, impacted by common phenomena such as lateral genetic transfer and rearrangement, cannot be adequately captured in a strictly tree-like structure. Bacterial and archaeal genomes consist of highly conserved regions, e.g. ribosomal RNA genes (commonly used as phylogenetic markers), more-variable regions and extrachromosomal elements, i.e. plasmids (that contain genes critical under a selective condition e.g. antibiotic resistance). The impact of these elements on genome-scale inference of microbial phylogeny remains little known. Here, using an AF approach, we inferred phylogenomic networks of microbial life based on 2785 completely sequenced bacterial and archaeal genomes, and systematically assessed the impact of ribosomal RNA genes and plasmid sequences in this network. Our results indicate that k-mer similarity can correlate with taxonomic rank of microbes. Using a relational database approach, we linked the implicatedk-mers to annotated genomic regions (thus functions), and defined core functions in specific phyletic groups and genera. We found that, in most phyla, highly conserved functions are often related to Amino acid metabolism and transport, and Energy production and conversion. Our findings indicate that AF phylogenomics can be used to infer reticulate relationships in a scalable manner and provide new perspective into microbial biology and evolution.
Integrated design, execution, and analysis of arrayed and pooled CRISPR genome editing experiments
Authors: Canver Matthew C. , Haeussler Maximilian , Bauer Daniel E. , Orkin Stuart H. , Sanjana Neville E. , Shalem Ophir , Yuan Guo-Cheng , Zhang Feng , Concordet Jean-Paul , Pinello Luca
Published: April 2017

CRISPR genome editing experiments offer enormous potential for the evaluation of genomic loci using arrayed single guide RNAs (sgRNAs) or pooled sgRNA libraries. Numerous computational tools are available to help design sgRNAs with optimal on-target efficiency and minimal off-target potential. In addition, computational tools have been developed to analyze deep sequencing data resulting from genome editing experiments. However, these tools are typically developed in isolation and oftentimes not readily translatable into laboratory-based experiments. Here we present a protocol that describes in detail both the computational and benchtop implementation of an arrayed and/or pooled CRISPR genome editing experiment. This protocol provides instructions for sgRNA design with CRISPOR, experimental implementation, and analysis of the resulting high-throughput sequencing data with CRISPResso. This protocol allows for design and execution of arrayed and pooled CRISPR experiments in 4-5 weeks by non-experts as well as computational data analysis in 1-2 days that can be performed by both computational and non-computational biologists alike.
Enterococcus faecalismodulates immune activation and slows healing during wound infection
Authors: Long Chong Kelvin Kian , Tay Wei Hong , Janela Baptiste , Adeline Yong Mei Hui , Liew Tze Horng , Madden Leigh , Keogh Damien , Sebastian Barkham Timothy Mark , Ginhoux Florent , Becker David Laurence , Kline Kimberly A.
Published: April 2017

Enterococcus faecalisis one of most frequently isolated bacterial species in wounds yet little is known about its pathogenic mechanisms in this setting. Here, we used a mouse wound excisional model to characterize the infection dynamics ofE. faecalisand show that infected wounds result in two different states depending on the initial inoculum. Low dose inocula were associated with short term, low titer colonization whereas high dose inocula were associated with acute bacterial replication and long term persistence. High dose infection and persistence were also associated with immune cell infiltration, despite suppression of some inflammatory cytokines and delayed wound healing. During high dose infection, the multiple peptide resistance factor (MprF) which is involved in resisting immune clearance, contributes toE. faecalisfitness. These results comprehensively describe a mouse model for investigatingE. faecaliswound infection determinants, and suggest that both immune modulation and resistance contribute to persistent, non-healing wounds.
Linking dendroecology and association genetics: Stress responses archived in tree rings associate with SNP genotypes inAbies alba (Mill.)
Authors: Heer Katrin , Behringer David , Piermattei Alma , Bässler Claus , Fady Bruno , Jehl Hans , Liepelt Sascha , Lorch Sven , Piotti Andrea , Vendramin Giovanni Guiseppe , Weller Max , Ziegenhagen Birgit , Büntgen Ulf , Opgenoorth Lars
Published: April 2017

Genetic association studies in forest tress would greatly benefit from information on tree response to environmental stressors over time. Dendroecology can close this gap by providing such time series measurements. Here, we jointly analyzed dendroecological and genetic data to explore the genetic basis of resistance, recovery and resilience to episodic stress in silver fir.

We used individual level tree-ring data to characterize the growth patterns of surviving silver fir (Abies alba) during the forest dieback in the 1970s and 1980s in Central Europe and associated them with SNPs in candidate genes.

Most trees at our study sites in the Bavarian Forest experienced severe growth decline from 1974 until the mid-1980s, which peaked during the drought year of 1976. Using the machine learning algorithm random forest, we identified 15 candidate genes that were associated with the variance in resistance, resilience and recovery among trees in this period.

With our study we show that the unique possibility of phenotypic time series archived in tree-rings are a powerful resource in genetic association studies. We call for a closer collaboration of dendroceologists and forest geneticists to focus on integrating individual tree level signals in genetic association studies in long lived trees.
Windows of opportunity for Ebola virus infection treatment and vaccination
Authors: Nguyen Van Kinh , Hernandez-Vargas Esteban A.
Published: April 2017

Ebola virus (EBOV) infection causes a high death toll, killing a high proportion of EBOV infected patients within 7 days. Comprehensive data on EBOV infection are very fragmented, hampering efforts in developing therapeutics and vaccines against EBOV. Under this circumstance, mathematical models become valuable resources to explore potential controlling strategies. In this paper, we employed experimental data of EBOV-infected nonhuman primates (NHPs) to construct a mathematical framework for determining windows of opportunity for treatment and vaccination. Considering a prophylactic vaccine based on recombinant vesicular stomatitis virus expressing the EBOV glycoprotein (VSV-EBOV), we found that the time window can be subject-specific, but vaccination could be protective if a subject is vaccinated during a period from one week to four months before infection. For the case of a therapeutic vaccine based on monoclonal antibodies (mAbs), a single dose might resolve the invasive EBOV replication even it was administrated as late as four days after infection. Our mathematical models can be used as building blocks for developing therapeutic and vaccine modalities as well as for evaluating public health intervention strategies in outbreaks. Future laboratory experiments will help to validate and refine the estimates of the windows of opportunity proposed here.
Non-parametric estimation of population size changes from the site frequency spectrum
Authors: Waltoft Berit Lindum , Hobolth Asger
Published: April 2017

The variability in population size is a key quantity for understanding the evolutionary history of a species. We present a new method, CubSFS, for estimating the changes in population size of a panmictic population from the site frequency spectrum. First, we provide a straightforward proof for the expression of the expected site frequency spectrum depending only on the population size. Our derivation is based on an eigenvalue decomposition of the instantaneous coalescent rate matrix. Second, we solve the inverse problem of determining the variability in population size from an observed SFS. Our solution is based on a cubic spline for the population size. The cubic spline is determined by minimizing the weighted average of two terms, namely (i) the goodness of fit to the SFS, and (ii) a penalty term based on the smoothness of the changes. The weight is determined by cross-validation. The new method is validated on simulated demographic histories and applied on data from nine different human populations.
A cassava drought inducible CC-type glutaredoxin,MeGRX232, negatively regulates drought tolerance inArabidopsisby inhibition of ABA-dependent stomatal closure
Authors: Ruan Meng-Bin , Yang Yi-Ling , Guo Xin , Wang Xue , Wang Bin , Yu Xiao-Ling , Zhang Peng , Peng Ming
Published: April 2017

CC-type glutaredoxins (GRXs) are a land plant-specific GRX subgroup that evolved from CGFS GRXs, and participate in organ development and stress responses through the regulation of transcription factors. Here, genome-wide analysis identified 18 CC-type GRXs in the cassava genome, of which six (MeGRX058, 232, 360, 496, 785, and892) were induced by drought and ABA stress in cassava leaves. Furthermore, we found that overexpression ofMeGRX232results in drought hypersensitivity in soil-grown plants, with a higher water loss rate, but with increased tolerance of mannitol and ABA inArabidopsison the sealed agar plates. The ABA induced stomatal closure is impaired inMeGRX232-OE Arabidopsis. Further analysis reveals that the overexpression ofMeGRX232leads to more ROS accumulation in guard cells. MeGRX232 can interact with TGA5 fromArabidopsisand MeTGA074 from cassavain vitroandin vivo. The results of microarray assays show thatMeGRX232-OEaffected the expression of a set of drought and oxidative stress related genes. Taken together, we demonstrated that CC-type GRXs involved in ABA signal transduction and play roles in response to drought through regulating stomatal closure.

Novelty statement:

We found that drought and ABA stress induced the transcription of CC-type glutaredoxins (GRXs) in cassava leaves. Ectopic expression of one of them,MeGRX232inArabidopsisaffected the sensitivity to abscisic acid (ABA) and mannitol, and caused drought hypersensitivity by impairment of ABA-dependent stomatal closure.
Perturbation of PTEN-PI3K/AKT Signalling Impaired Autophagy Modulation in Dystrophin-Deficient Myoblasts
Authors: Dain Yazid Muhammad , Smith Janet
Published: April 2017

Alteration of single protein regulation has given a massive implication in Muscular Dystrophy pathogenesis. Herein, we investigated the contribution of defected dystrophin that has impaired PI3K/Akt signalling and subsequently reduced autophagy in dystrophin-deficient myoblasts. In this study, dfd13 (dystrophin-deficient) and C2C12 (non-dystrophic) myoblasts were cultured in low mitogen condition for 10 days to induce differentiation. Analyses of protein expression has been done by using immunoblot technique, immunofluorescence and flow cytometry. In our myoblasts differentiation system, the dfd13 myoblasts did not achieved terminal differentiation as fewer myotube formation and fast-myosin heavy chain expression almost not detected. Immunoblot analysis showed that PTEN expression is profoundly increased in dfd13 myoblasts throughout the differentiation day. As a result, the PI3K activity is decreased and has caused serine/threonine kinase Akt inactivation. Both residues; Thr308 and Ser473, on Akt were found not phosphorylated. The mTOR activation by Ser2448 phosphorylation was decreased indicates an impairment for raptor and rictor binding. Unable to form complexes; mTORC1 target protein, p70S6K1 activation was found reduced at the same time explained un-phosphorylated-Akt at Ser473 by rictor-mTORC2. As one of Akt downstream protein, transcription factor FoxO3 regulation was found impaired as it was highly expressed and highly mainly localised in the nucleus in dfd13 towards the end of the differentiation day. This occurrence has caused higher activation of autophagy related genes; Beclin1, Atg5, Atg7, in dfd13 myoblasts. Autophagosome formation was increased as LC3B-I/II showed accumulation upon differentiation. However, ratio of LC3B lipidation and autophagic flux were shown decreased which exhibited dystrophic features. As a conclusion, destabilisation of plasma membrane owing to dystrophin mutation has caused the alteration of plasma membrane protein regulation particularly PTEN-PI3K, thus impaired autophagy modulation that critical for myoblasts development.
RNA helicase, DDX27 regulates skeletal muscle growth and regeneration by modulation of translational processes
Authors: Bennett Alexis H , O’Donohue Marie-Francoise , Gundry Stacey R. , Chan Aye T. , Widrick Jeffery , Draper Isabelle , Chakraborty Anirban , Zhou Yi , Zon Leonard I. , Gleizes Pierre-Emmanuel , Beggs Alan H. , Gupta Vandana A
Published: April 2017

Gene expression in a tissue-specific context depends on the combined efforts of epigenetic, transcriptional and post-transcriptional processes that lead to the production of specific proteins that are important determinants of cellular identity. Ribosomes are a central component of the protein biosynthesis machinery in cells; however, their regulatory roles in the translational control of gene expression in skeletal muscle remain to be defined. In a genetic screen to identify critical regulators of myogenesis, we identified a DEAD-Box RNA helicase, DDX27, that is required for skeletal muscle growth and regeneration. We demonstrate that DDX27 regulates ribosomal RNA (rRNA) maturation, and thereby the ribosome biogenesis and the translation of specific transcripts during myogenesis. These findings provide insight into the translational regulation of gene expression in myogenesis and suggest novel functions for ribosomes in regulating gene expression in skeletal muscles.

AUTHOR SUMMARY

Inherited skeletal muscle diseases are the most common form of genetic disorders with primary abnormalities in the structure and function of skeletal muscle resulting in the impaired locomotion in affected patients. A major hindrance to the development of effective therapies is a lack of understanding of biological processes that promote skeletal muscle growth. By performing a forward genetic screen in zebrafish we have identified mutation in a RNA helicase that leads to perturbations of ribosomal biogenesis pathway and impairs skeletal muscle growth and regeneration. Therefore, our studies have identified novel ribosome-based disease processes that may be therapeutic modulated to restore muscle function in skeletal muscle diseases.
Natural variation in a single amino acid underlies cellular responses to topoisomerase II poisons
Authors: Zdraljevic Stefan , Strand Christine , Seidel Hannah S. , Cook Daniel E. , Doench John G. , Andersen Erik C.
Published: April 2017

Many medications, including chemotherapeutics, are differentially effective from one patient to the next. Understanding the causes of these population-wide differences is a critical step towards the development of personalized treatments and improvements to existing medications. Here, we investigate natural differences in sensitivity to anti-neoplastic drugs that target topoisomerase II, using the model organismCaenorhabditis elegans. We show that wild isolates ofC. elegansvary in their sensitivity to these drugs, and we use an unbiased statistical and molecular genetics approach to demonstrate that this variation is explained by a methionine-to-glutamine substitution in topoisomerase II (TOP-2). The presence of a non-polar methionine at this residue increases hydrophobic interactions between TOP-2 and the poison etoposide, as compared to a polar glutamine. We hypothesize that this stabilizing interaction results in increased genomic instability in strains that contain a methionine residue. The residue affected by this substitution is conserved from yeast to humans and is one of the few differences between the two human topoisomerase II isoforms (methionine in hTOPIIα and glutamine in hTOPIIβ). We go on to show that this substitution influences binding and cytotoxicity of etoposide and two additional topoisomerase II poisons in human cell lines. These results explain why hTOPIIα and hTOPIIβ are differentially affected by various poisons and demonstrate the utility ofC. elegansin understanding the genetics of drug responses.
Hypothesis: Hypoxia inducesde novosynthesis of NeuGc gangliosides in humans through CMAH domain substitute
Authors: Bousquet Paula A. , Sandvik Joe Alexander , Edin Nina F. Jeppesen , Krengel Ute
Published: April 2017

Immunotherapy is a growing field in cancer research. A privileged tumor-associated antigen that has received much attention isN-glycolyl (NeuGc) GM3. This ganglioside is present in several types of cancer, but is almost undetectable in human healthy tissues. However, its non-hydroxylated variant, NeuAc GM3, is abundant in all mammals. Due to a deletion in the human gene encoding the key enzyme for synthesis of NeuGc, humans, in contrast to other mammals, cannot synthesize NeuGc GM3. Therefore the presence of this ganglioside in human cancer cells represents an enigma. It has been shown that hypoxic conditions trigger the expression of NeuGc gangliosides, which not only serve as attractive targets for cancer therapy, but also as diagnostic and prognostic tumor marker. Here, we confirm hypoxia-induced expression of the NeuGc GM3 ganglioside also in HeLa cells and reveal several candidate proteins, in particular GM3 synthase and subunit B of respiratory complex II (SDHB), that may be involved in the generation of NeuGc GM3 by SILAC-based proteome analysis. These findings have the potential to significantly advance our understanding of how this enigmatic tumor-associated antigen is produced in humans, and also suggest a possible mechanism of action of anti-tumor antibodies that recognize hypoxia markers, such as 14F7.