bluuebunny/crossref_metadata_2025 huggingface dataset:
C-terminus of the P4-ATPase ATP8A2 functions in protein folding and regulation of phospholipid flippase activity
Authors: Chalat Madhavan , Moleschi Kody , Molday Robert S.
Published: December 2016

ATP8A2 is a P4-ATPase that flips phosphatidylserine and phosphatidylethanolamine across cell membranes. This generates membrane phospholipid asymmetry, a property important in many cellular processes, including vesicle trafficking. ATP8A2 deficiency causes severe neurodegenerative diseases. We investigated the role of the C-terminus of ATP8A2 in its expression, subcellular localization, interaction with its subunit CDC50A, and function as a phosphatidylserine flippase. C-terminal deletion mutants exhibited a reduced tendency to solubilize in mild detergent and exit the endoplasmic reticulum. The solubilized protein, however, assembled with CDC50A and displayed phosphatidylserine flippase activity. Deletion of the C-terminal 33 residues resulted in reduced phosphatidylserine-dependent ATPase activity, phosphatidylserine flippase activity, and neurite extension in PC12 cells. These reduced activities were reversed with 60- and 80-residue C-terminal deletions. Unlike the yeast P4-ATPase Drs2, ATP8A2 is not regulated by phosphoinositides but undergoes phosphorylation on the serine residue within a CaMKII target motif. We propose a model in which the C-terminus of ATP8A2 consists of an autoinhibitor domain upstream of the C-terminal 33 residues and an anti-autoinhibitor domain at the extreme C-terminus. The latter blocks the inhibitory activity of the autoinhibitor domain. We conclude that the C-terminus plays an important role in the efficient folding and regulation of ATP8A2.
Harnessing molecular motors for nanoscale pulldown in live cells
Authors: Bird Jonathan E. , Barzik Melanie , Drummond Meghan C. , Sutton Daniel C. , Goodman Spencer M. , Morozko Eva L. , Cole Stacey M. , Boukhvalova Alexandra K. , Skidmore Jennifer , Syam Diana , Wilson Elizabeth A. , Fitzgerald Tracy , Rehman Atteeq U. , Martin Donna M. , Boger Erich T. , Belyantseva Inna A. , Friedman Thomas B.
Published: December 2016

Protein–protein interactions (PPIs) regulate assembly of macromolecular complexes, yet remain challenging to study within the native cytoplasm where they normally exert their biological effect. Here we miniaturize the concept of affinity pulldown, a gold-standard in vitro PPI interrogation technique, to perform nanoscale pulldowns (NanoSPDs) within living cells. NanoSPD hijacks the normal process of intracellular trafficking by myosin motors to forcibly pull fluorescently tagged protein complexes along filopodial actin filaments. Using dual-color total internal reflection fluorescence microscopy, we demonstrate complex formation by showing that bait and prey molecules are simultaneously trafficked and actively concentrated into a nanoscopic volume at the tips of filopodia. The resulting molecular traffic jams at filopodial tips amplify fluorescence intensities and allow PPIs to be interrogated using standard epifluorescence microscopy. A rigorous quantification framework and software tool are provided to statistically evaluate NanoSPD data sets. We demonstrate the capabilities of NanoSPD for a range of nuclear and cytoplasmic PPIs implicated in human deafness, in addition to dissecting these interactions using domain mapping and mutagenesis experiments. The NanoSPD methodology is extensible for use with other fluorescent molecules, in addition to proteins, and the platform can be easily scaled for high-throughput applications.
PRDM9 interactions with other proteins provide a link between recombination hotspots and the chromosomal axis in meiosis
Authors: Parvanov Emil D. , Tian Hui , Billings Timothy , Saxl Ruth L. , Spruce Catrina , Aithal Rakesh , Krejci Lumir , Paigen Kenneth , Petkov Petko M.
Published: December 2016

In mammals, meiotic recombination occurs at 1- to 2-kb genomic regions termed hotspots, whose positions and activities are determined by PRDM9, a DNA-binding histone methyltransferase. We show that the KRAB domain of PRDM9 forms complexes with additional proteins to allow hotspots to proceed into the next phase of recombination. By a combination of yeast-two hybrid assay, in vitro binding, and coimmunoprecipitation from mouse spermatocytes, we identified four proteins that directly interact with PRDM9’s KRAB domain, namely CXXC1, EWSR1, EHMT2, and CDYL. These proteins are coexpressed in spermatocytes at the early stages of meiotic prophase I, the limited period when PRDM9 is expressed. We also detected association of PRDM9-bound complexes with the meiotic cohesin REC8 and the synaptonemal complex proteins SYCP3 and SYCP1. Our results suggest a model in which PRDM9-bound hotspot DNA is brought to the chromosomal axis by the action of these proteins, ensuring the proper chromatin and spatial environment for subsequent recombination events.
Diagnosis and Treatment of Leishmaniasis: Clinical Practice Guidelines by the Infectious Diseases Society of America (IDSA) and the American Society of Tropical Medicine and Hygiene (ASTMH)
Authors: Aronson Naomi , Herwaldt Barbara L. , Libman Michael , Pearson Richard , Lopez-Velez Rogelio , Weina Peter , Carvalho Edgar M. , Ephros Moshe , Jeronimo Selma , Magill Alan
Published: December 2016

It is important to realize that leishmaniasis guidelines cannot always account for individual variation among patients. They are not intended to supplant physician judgment with respect to particular patients or special clinical situations. The IDSA and ASTMH consider adherence to these guidelines to be voluntary, with the ultimate determinations regarding their application to be made by the physician in the light of each patient's individual circumstances.
Comparative Effectiveness and Safety of Standard-, Medium-, and High-Dose Daptomycin Strategies for the Treatment of Vancomycin-Resistant Enterococcal Bacteremia Among Veterans Affairs Patients
Authors: Britt Nicholas S. , Potter Emily M. , Patel Nimish , Steed Molly E.
Published: December 2016

Background.

Vancomycin-resistant Enterococcus bloodstream infections (VRE-BSIs) are associated with significant mortality. Daptomycin exhibits concentration-dependent activity vs VRE in vitro, yet the clinical impact of higher-dose strategies remains unclear.

Methods.

We performed a national retrospective cohort study of hospitalized Veterans Affairs patients treated with standard-dose (6 mg/kg total body weight), medium-dose (8 mg/kg total body weight), or high-dose (≥10 mg/kg total body weight) daptomycin for VRE-BSI. Patient-related, microbiological, and outcomes data were abstracted from clinical databases. The primary outcome was overall survival, evaluated by Cox regression. Secondary outcomes included 30-day mortality, time to microbiological clearance, and creatine phosphokinase (CPK) elevation.

Results.

A total of 911 patients were included (standard dose, n = 709; medium dose, n = 142; high dose, n = 60). Compared to high-dose daptomycin, both standard-dose (hazard ratio [HR], 2.68; 95% confidence interval; [CI], 1.33–3.06; P = .002) and medium-dose (HR, 2.66; 95% CI, 1.33–3.92; P = .003) daptomycin were associated with poorer survival. After adjusting for confounders, the relationship between poorer survival and standard-dose (adjusted HR [aHR], 2.58; 95% CI, 1.27–4.88; P = .004) and medium-dose (aHR, 2.52; 95% CI, 1.27–5.00; P = .008) daptomycin persisted. Thirty-day mortality was significantly lower among high-dose daptomycin–treated patients compared with other dosing strategies (risk ratio, 0.83; 95% CI, .74–.94; P = .015). Compared with standard-dose daptomycin, both medium-dose (HR, 0.78; 95% CI, .55–.90; P = .012) and high-dose daptomycin (HR, 0.70; 95% CI, .41–.84; P = .006) were associated with significantly improved microbiological clearance. No difference in the risk of CPK elevation was observed between the treatment groups (P = .504).

Conclusions.

High-dose daptomycin was associated with improved survival and microbiological clearance in VRE-BSI.
Ombitasvir/Paritaprevir/Ritonavir and Dasabuvir Combination Treatment in Patients With HIV/HCV Coinfection: Results of an Italian Compassionate Use Program
Authors: Andreoni Massimo , Teti Elisabetta , Antinori Andrea , Milazzoi Laura , Sollima Savatore , Rizzardini Giuliano , Biagio Antonio Di , Saracino Annalisa , Bruno Raffaele , Borghi Vanni , Luca Andrea De , Cattelan Annamaria , Hasson Hamid , Taliani Gloria , Monforte Antonella D’Arminio , Mastroianni Claudio Maria , Perri Giovanni Di , Bigoni Sara , Puoti Massimo , Spinetti Angiola , Gori Andrea , Boffa Nicola , Cacopardo Bruno , Giacometti Andrea , Parruti Giustino , Vullo Vincenzo , Chirianni Antonio , Pennica Alfredo , Pasquazzi Caterina , Segala Daniela , Sarmati Loredana ,   SIMIT (Società Italiana di Malattie Infettivee Tropicali)
Published: December 2016

Patients coinfected with hepatitis C virus (HCV) and human immunodeficiency virus (HIV) are at high risk of liver disease progression. We report a favorable safety profile and SVR12 rates of 96.7% among HIV/HCV coinfected patients participating in an Italian compassionate-use program of ombitasvir/paritaprevir/ritonavir + dasabuvir (OBV/PTV/r + DSV) ± ribavirin (RBV).
Delayed Diagnosis of Tuberculous Meningitis Misdiagnosed as Herpes Simplex Virus-1 Encephalitis With the FilmArray Syndromic Polymerase Chain Reaction Panel
Authors: Gomez Carlos A. , Pinsky Benjamin A. , Liu Anne , Banaei Niaz
Published: December 2016

The FilmArray meningitis/encephalitis (ME) panel is a novel syndromic, nucleic acid amplification test for diagnosis of acute meningitis and encephalitis. Emerging data on its performance are concerning for false-positive results. We present a case of tuberculous meningitis misdiagnosed as herpes simplex virus-1 encephalitis with the FilmArray ME panel. Strategies to mitigate erroneous results are discussed.
A Dynamic Transmission Model to Evaluate the Effectiveness of Infection Control Strategies
Authors: Khader Karim , Thomas Alun , Huskins W. Charles , Leecaster Molly , Zhang Yue , Greene Tom , Redd Andrew , Samore Matthew H.
Published: December 2016

Background

The advancement of knowledge about control of antibiotic resistance depends on the rigorous evaluation of alternative intervention strategies. The STAR*ICU trial examined the effects of active surveillance and expanded barrier precautions on acquisition of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) in intensive care units. We report a reanalyses of the STAR*ICU trial using a Bayesian transmission modeling framework.

Methods

The data included admission and discharge times and surveillance test times and results. Markov chain Monte Carlo stochastic integration was used to estimate the transmission rate, importation, false negativity, and clearance separately for MRSA and VRE. The primary outcome was the intervention effect, which when less than (or greater than) zero, indicated a decreased (or increased) transmission rate attributable to the intervention.

Results

The transmission rate increased in both arms from pre- to postintervention (by 20% and 26% for MRSA and VRE). The estimated intervention effect was 0.00 (95% confidence interval [CI], −0.57 to 0.56) for MRSA and 0.05 (95% CI, −0.39 to 0.48) for VRE. Compared with MRSA, VRE had a higher transmission rate (preintervention, 0.0069 vs 0.0039; postintervention, 0.0087 vs 0.0046), higher importation probability (0.22 vs 0.17), and a lower clearance rate per colonized patient-day (0.016 vs 0.035).

Conclusions

Transmission rates in the 2 treatment arms were statistically indistinguishable from the pre- to postintervention phase, consistent with the original analysis of the STAR*ICU trial. Our statistical framework was able to disentangle transmission from importation and account for imperfect testing. Epidemiological differences between VRE and MRSA were revealed.
Using Procalcitonin to Guide Antibiotic Therapy
Authors: Rhee Chanu
Published: December 2016

Procalcitonin levels rise in response to systemic inflammation, especially of bacterial origin. Multiple randomized controlled trials have demonstrated that procalcitonin-based algorithms can safely reduce antibiotic use in 2 clinical scenarios. First, in stable, low-risk patients with respiratory infections, procalcitonin levels of <0.25 µg/L can guide the decision to withhold antibiotics or stop therapy early. Second, in critically ill patients with suspected sepsis, clinicians should not initially withhold antibiotics, but procalcitonin levels of <0.5 µg/L or levels that decrease by ≥80% from peak can guide discontinuation once patients stabilize. The recent stop antibiotics on procalcitonin guidance study (SAPS), the largest procalcitonin trial to date, demonstrated reduction in both antibiotic exposure and mortality in critically ill patients. Although procalcitonin is ready for routine use, future research should examine optimal strategies for implementation in hospitals, its real-world impact on clinical outcomes and costs, its applicability to immunocompromised patients, and the generalizability of trials to the US population.
A Proposed New Classification of Skin and Soft Tissue Infections Modeled on the Subset of Diabetic Foot Infection
Authors: Lipsky Benjamin A. , Silverman Michael H. , Joseph Warren S.
Published: December 2016

Schemes for classifying skin and soft tissue infections (SSTIs) pose limitations for clinicians and regulatory agencies. Diabetic foot infections (DFIs) are a subset of SSTIs. We developed and are proposing a classification to harmonize current schemes for SSTIs and DFIs. Existing schemes for classifying SSTIs are limited in both their usefulness to clinicians and to regulatory agencies. The guidelines on SSTI from the Infectious Diseases Society of America (IDSA) and the guidance from the US Food and Drug Administration do not adequately address many types of wound infections. However, guidelines developed by the IDSA for DFIs provide a classification scheme that has been validated and widely used. Diabetic foot infections are similar to SSTIs in pathophysiology, microbiology, and treatment and can be seen as a subset of SSTI. Thus, based on the documents noted above, and our review of the literature, we have developed a proposed classification scheme for SSTI that harmonizes well with the DFI classification. We believe this new scheme will assist clinicians in classifying most wound infections and potentially aid regulatory agencies in testing and approving new antimicrobials for these infections.
The role of nociceptive input and tissue injury on stress regulation in borderline personality disorder
Authors: Willis Franziska , Kuniss Sarah , Kleindienst Nikolaus , Naoum Janina , Reitz Sarah , Boll Sabrina , Bohus Martin , Treede Rolf-Detlef , Baumgärtner Ulf , Schmahl Christian
Published: December 2016

Approximately 60% to 90% of patients with borderline personality disorder (BPD) show nonsuicidal self-injurious behavior (NSSI) with cutting being the most frequently applied method. One of NSSI's functions is to reduce aversive tension. Previous studies have found a tension-reducing effect of painful tissue injury by an incision. It is still unclear whether this effect is based on the effect of tissue injury or the effect of pain experience, or both. The aim of this study was to determine whether tissue injury leads to a stronger stress reduction than a sole pain stimulus in patients with BPD. After stress induction, 57 BPD patients and 60 healthy controls (HCs) received either an incision or a non–tissue-injuring mechanical nociceptive stimulus (“blade”) typically perceived as painful or a non-nociceptive tactile sham stimulus (blunt end of scalpel). Participants were unaware of which procedure was applied. For stress assessment, subjective and objective parameters were measured. As immediate response to the stimulus application, we found greater stress reduction after both painful stimuli (incision and blade) in BPD patients but no difference in stress decrease between the tissue-injuring incision and the non–tissue-injuring pain stimulus (blade). Compared with HCs, incision and blade were followed by greater immediate decrease of arousal in BPD patients. Our findings confirm that among BPD patients, the nociceptive input leads to stress reduction. In contrast, the impact of tissue damage on stress reduction was relatively small. In addition, the results suggest that painful stimuli lead to a greater stress reduction in BPD patients compared with HCs.
Is There Any Association Between Use of Antidepressants and Preeclampsia or Gestational Hypertension?
Authors: Uguz Faruk
Published: December 2016

Objective

Hypertension in pregnant women is an important medical problem, which can cause morbidity and mortality in the fetus. This study reviewed the current literature examining the potential relationship between use of antidepressants during pregnancy and preeclampsia or gestational hypertension.

Methods

PubMed was searched for English-language reports between January 1, 1995, and December 31, 2015, by using combinations of key words pregnancy, pregnancy complications, preeclampsia, gestational hypertension, and antidepressants. Studies that reported the diagnosis of preeclampsia or gestational hypertension and use of antidepressant were included in the review.

Results

A total of 7 relevant studies that met the review criteria were examined. The studies reported that compared with nonusers adjusted relative risk of preeclampsia or gestational hypertension in antidepressant users was 1.28 to 1.53 for any antidepressant, 1.05 to 3.16 for selective serotonin reuptake inhibitors, 1.49 to 1.95 for selective serotonin-norepinephrine reuptake inhibitors, and 0.35 to 3.23 for tricyclic antidepressants. Consistently, antidepressant use during the second trimester of pregnancy was associated with increased risk of preeclampsia or gestational hypertension. However, possible contribution of severity, type, and comorbidity of underlying anxiety or depressive disorders is unclear in the current studies.

Conclusions

Although some studies have suggested a moderately increased risk in pregnancy-specific hypertensive disorders with antidepressant treatment, the current data do not allow a definitive conclusion on this topic, because the studies have many methodological limitations. In addition, the effects of untreated depression or anxiety disorders cannot be disentangled from the results.
Comparative Risk of Ventricular Arrhythmia and Sudden Cardiac Death Across Antidepressants in Patients With Depressive Disorders
Authors: Wu Chi-Shin , Tsai Yu-Ting , Hsiung Chao A. , Tsai Hui-Ju
Published: December 2016

Objective

We aimed to evaluate the risk of ventricular arrhythmia (VA) and/or sudden cardiac death (SCD) associated with antidepressant use.

Methods

A cohort study was conducted using data from Taiwan's National Health Insurance Research Database from 2001 to 2012. A total of 793,460 new antidepressant users with depressive disorders were enrolled in the study. Outcomes were defined as the first principal diagnosis of VA or SCD in the emergency department or hospital discharge records. Cox proportional hazards models with stratification of propensity score deciles were used to evaluate the relative risk of VA/SCD for antidepressants compared with selective serotonin reuptake inhibitors (SSRIs).

Results

A total of 245 VA/SCD events occurred. The incidence rate of VA/SCD among antidepressant users was 1.5 per 1000 person-years (95% confidence interval [CI], 1.3–1.7). Compared with SSRIs, the risk of VA/SCD was significantly lower for tricyclic or tetracyclic antidepressant (TCAs) (adjusted hazards ratio [aHR], 0.54; 95% CI, 0.36–0.83), but not other antidepressant classes. However, use of moderate- to high-dose TCAs carried a higher risk than low-dose TCAs (aHR, 4.37; 95% CI, 1.23–15.60). Antidepressant polypharmacy was associated with an increased risk of VA/SCD (aHR, 1.63; 95% CI, 1.07–2.49).

Conclusions

There was no difference in VA/SCD risk across antidepressant classes except that TCAs were associated with a lower risk than SSRIs. However, the observed comparative risk of TCAs might be attributable to low-dose TCA use, which is quite common in current clinical practice. It would be of importance to carry out further investigations to scrutinize the influence of antidepressants on VA/SCD.
Current Endoscopic Approaches for the Treatment of Barrett Esophagus
Authors: Brimhall Bryan , Wani Sachin
Published: December 2016

Barrett esophagus (BE) is the only identifiable premalignant condition for esophageal adenocarcinoma (EAC), a cancer associated with a poor 5-year survival rate. The stepwise pathologic progression of BE to invasive cancer provides an opportunity to halt progression and potentially decrease incidence and ultimately the morbidity and mortality related to this lethal cancer. Endoscopic eradication therapy (EET) in patients at increased risk of progression to invasive EAC (intramucosal EAC, high-grade dysplasia, and low-grade dysplasia) is a practice that is endorsed by multiple societies and has replaced esophagectomy as the standard of care for these patients. Although the effectiveness, safety, and durability of EET have been demonstrated in several studies, this review addresses the several challenges with EET that need to be considered to optimize patient outcomes. Finally, the critical role of training, competence, and quality indicators in EET are emphasized in this era of value-based health care practice.
New Standards of Care in Ischemic Stroke
Authors: Chancellor Bree K. , Ishida Koto
Published: December 2016

Background:

Ischemic arterial strokes of the ophthalmic artery and its branches and posterior cerebral artery are common causes of visual disability. Etiologies of stroke affecting the retina, optic nerve, optic radiation, and visual cortex overlap with other types of ischemic strokes. Stenosis of the internal carotid is the most common cause of central retinal artery occlusion (CRAO). One-fourth of patients with CRAO have cerebral strokes. We report recent developments in the acute treatment and secondary prevention of ischemic stroke of relevance to clinicians who encounter patients with acute vision loss.

Evidence Acquisition:

A search of Pubmed and practice guidelines over the past 5 years was performed, with a focus on significant changes in treatment and prevention of ischemic stroke.

Results:

Recent randomized controlled trials provide Level I evidence for the use of endovascular therapy with current stent retriever devices for patients with large vessel anterior circulation occlusions within 6 hours of presentation. Number needed to treat to achieve one additional patient with an independent functional outcome was in the range of 3–7, and benefit was additive to that of intravenous tissue plasminogen activator alone. Paroxysmal atrial fibrillation (AF) is a major cause of cryptogenic stroke with incidence expected to rise with the aging population. Since 2014, prolonged 30-day cardiac monitoring has been recommended as a part of transient ischemic attack and stroke workup in patients with cryptogenic stroke. Even longer term monitoring of 6 months to 1 year with external and implantable loop recorders improves rates of diagnosing AF. First available in 2010, the novel anticoagulants—dabigatran, apixaban, rivaroxaban, and edoxaban—have been compared with warfarin in the prevention of stroke in patients with nonvalvular AF. Apixaban demonstrated superiority in safety and efficacy, with the novel anticoagulants as a group having favorable risk–benefit profile at higher dosages compared with standard warfarin therapy.

Conclusions:

Endovascular therapy is now standard of care for eligible patients with anterior large vessel occlusions. Prolonged cardiac monitoring is recommended for patients with cryptogenic stroke. The novel anticoagulants are an alternative to warfarin in patients with AF.
Serum extracellular vesicle depletion processes affect release and infectivity of HIV-1 in culture
Authors: Liao Zhaohao , Muth Dillon C. , Eitan Erez , Travers Meghan , Lehrmann Elin , Witwer Kenneth W.
Published: December 2016

Extracellular vesicles (EVs, including exosomes and microvesicles) are involved in intercellular communication in health and disease and affect processes including immune and antiviral responses. Ultracentrifuged serum is depleted of EVs and, when used in culture media, reduces growth and viability of some cell types. In this study, we examined the effects of serum EV depletion processes on HIV-1 replication in primary cells and cell lines, including two HIV-1 latency models. Increased HIV-1 production was observed in certain EV-depleted conditions, along with cell morphology changes and decreased cell viability. Add-back of ultracentrifuge pellets rescued baseline HIV-1 production. Primary cells appeared to be less sensitive to EV depletion. ACH-2 and U1 latency models produced more HIV-1 under EV-depleted conditions, while virus produced under processed serum conditions was more infectious. Finally, changes in cellular metabolism and gene expression were associated with EV-depleted culture. In conclusion, the EV environment of HIV-1 infected cells has a substantial effect on virus production and infectivity. EV-dependence of cell cultures should be examined carefully along with other experimental variables. However, EVs may not be the only particles depleted by ultracentrifugation or other processes. Effects of EVs may be accompanied by or confused with those of closely associated or physically similar particles.
The detection of covariation of mRNA levels of large sets of genes across multiple human populations
Authors: Quan Yu , Xie Chao , Williams Rohan B. H. , Little Peter F. R
Published: December 2016

In this study, we analyse RNA-Seq data from panels of human lymphoblastoid cell lines (LCLs) to identify covariation in the mRNA levels of large numbers of genes. Such large scale covariation may have biological origin or be due to technical variation in analysis (generally referred to as batch effects). We show that batch effects cannot explain this covariation by demonstrating reproducibility across different human populations and across different methods of analysis. This view is also supported by enrichment of single and combinations of transcription factors (TFs) binding to cognate promoter regions, enrichment of genes shown to be sensitive to the knockdown of individual TFs, enrichment of functional pathways, and finally enrichment of protein-protein interactions in proteins encoded by groups of covarying genes. The properties of the groups of covarying genes are therefore most readily explained by the influence of cumulative variations in the effectors of gene expression that act intranson cognate genes. We suggest that covariation has functional outcomes by showing that covariation of 83 genes involved in the spliceosome pathway accounts for 8–16% of the variation in the alternative splicing patterns of genes expressed in human LCLs.
Cannabis use and risk of schizophrenia: a Mendelian randomization study
Authors: Vaucher Julien , Keating Brendan J. , Lasserre Aurélie M. , Gan Wei , Lyall Donald M. , Ward Joey , Smith Daniel J. , Pell Jill P. , Sattar Naveed , Paré Guillaume , Holmes Michael V.
Published: December 2016

Cannabis use is observationally associated with an increased risk of schizophrenia, however whether the relationship is causal is not known. To determine the nature of the association between cannabis use on risk of schizophrenia using Mendelian randomization (MR) analysis, we used ten genetic variants previously identified to associate with cannabis use in 32,330 individuals. Genetic variants were used in a MR analyses of the association of genetically determined cannabis on risk of schizophrenia in 34,241 cases and 45,604 controls from predominantly European descent. Estimates from MR were compared to a metaanalysis of observational studies reporting effect estimates for ever use of cannabis and risk of schizophrenia or related disorders. Genetically determined use of cannabis was associated with increased risk of schizophrenia (OR of schizophrenia for users vs. non-users of cannabis: 1.37; 95%CI, 1.09 to 1.67; P-value=0.007). The corresponding estimate from observational analysis was 1.50 (95% CI, 1.10 to 2.00; P-value for heterogeneity = 0.88). The genetic instrument did not show evidence of pleiotropy on MR-Egger (Egger test, P-value=0.292) nor on multivariable MR accounting for tobacco exposure (OR of schizophrenia for users vs. nonusers of cannabis, adjusted for ever vs. never smoker: 1.41; 95% CI, 1.09-1.83). Furthermore, the causal estimate remained robust to sensitivity analyses. These findings strongly support a causal association between genetically determined use of cannabis and risk of schizophrenia. Such robust evidence may inform public health message about the risks of cannabis use, especially regarding its potential mental health consequences.
Review: Population Structure in Genetic Studies: Confounding Factors and Mixed Models
Authors: Martin Lana S. , Eskin Eleazar
Published: December 2016

A genome-wide association study (GWAS) seeks to identify genetic variants that contribute to the development and progression of a specific disease. Over the past 10 years, new approaches using mixed models have emerged to mitigate the deleterious effects of population structure and relatedness in association studies. However, developing GWAS techniques to effectively test for association while correcting for population structure is a computational and statistical challenge. Using laboratory mouse strains as an example, our review characterizes the problem of population structure in association studies and describes how it can cause false positive associations. We then motivate mixed models in the context of unmodeled factors.
Ras/ERK-signalling promotes tRNA synthesis and growth via the RNA polymerase III repressor Maf1 inDrosophila
Authors: Sriskanthadevan-Pirahas Shrivani , Deshpande Rujuta , Lee Byoungchun , Grewal Savraj S.
Published: December 2016

The small G-protein Ras is a conserved regulator of cell and tissue growth. These effects of Ras are mediated largely through activation of a canonical RAF-MEK-ERK kinase cascade. An important challenge is to identify how this Ras/ERK pathway alters cellular metabolism to drive growth. Here we report on stimulation of RNA polymerase III (Pol III)-mediated tRNA synthesis as a growth effector of Ras/ERK signalling inDrosophila. We find that activation of Ras/ERK signalling promotes tRNA synthesis both in vivo and in culturedDrosophilaS2 cells. We also show that Pol III function is required for Ras/ERK signalling to drive proliferation in both epithelial and stem cells inDrosophilatissues. We find that the transcription factor Myc is required but not sufficient for Ras-mediated stimulation of tRNA synthesis. Instead we show that the main way that Ras promotes Pol III function and tRNA synthesis is by inhibiting the nuclear localization and function of the Pol III repressor Maf1. We propose that inhibition of Maf1 and stimulation of tRNA synthesis is one way by which Ras signalling enhances protein synthesis to promote cell and tissue growth.
Revising mtDNA haplotypes of the ancient Hungarian conquerors with next generation sequencing
Authors: Neparáczki Endre , Kocsy Klaudia , Tóth Gábor Endre , Maróti Zoltán , Kalmár Tibor , Bihari Péter , Nagy István , Pálfi György , Molnár Erika , Raskó István , Török Tibor
Published: December 2016

As part of the effort to create a high resolution representative sequence database of the medieval Hungarian conquerors we have resequenced the entire mtDNA genome of 24 published ancient samples with Next Generation Sequencing, whose haplotypes had been previously determined with traditional PCR based methods. We show that PCR based methods are prone to erroneous haplotype or haplogroup determination due to ambiguous sequence reads, and many of the resequenced samples had been classified inaccurately. The SNaPshot method applied with published ancient DNA authenticity criteria is the most straightforward and cheapest PCR based approach for testing a large number of coding region SNP-s, which greatly facilitates correct haplogroup determination.
An Estradiol-Inducible Promoter Enables Fast, Graduated Control of Gene Expression in Fission Yeast
Authors: Ohira Makoto J. , Hendrickson David G. , Scott McIsaac R. , Rhind Nicholas
Published: December 2016

The fission yeastSchizosaccharomyces pombelacks a diverse toolkit of inducible promoters for experimental manipulation. Available inducible promoters suffer from slow induction kinetics, limited control of expression levels and/or a requirement for defined growth medium. In particular, noS. pombeinducible promoter systems exhibit a linear dose response, which would allow expression to be tuned to specific levels. We have adapted a fast, orthogonal promoter system with a large dynamic range and a linear dose response, based on β-estradiol-regulated function of the human estrogen receptor, for use inS. pombe. We show that this promoter system, termed Z3EV, turns on quickly, can reach a maximal induction of 20 fold, and exhibits a linear dose response over its entire induction range, with few off target effects. We demonstrate the utility of this system by regulating the mitotic inhibitor Wee1 to create a strain in which cell size is regulated by β-estradiol concentration. This promoter system will be of great utility for experimentally regulating gene expression in fission yeast.
A synthesis of over 9,000 mass spectrometry experiments reveals the core set of human protein complexes
Authors: Drew Kevin , Lee Chanjae , Huizar Ryan L. , Tu Fan , Borgeson Blake , McWhite Claire D. , Ma Yun , Wallingford John B. , Marcotte Edward M.
Published: December 2016

Macromolecular protein complexes carry out many of the essential functions of cells, and many genetic diseases arise from disrupting the functions of such complexes. Currently there is great interest in defining the complete set of human protein complexes, but recent published maps lack comprehensive coverage. Here, through the synthesis of over 9,000 published mass spectrometry experiments, we present hu.MAP, the most comprehensive and accurate human protein complex map to date, containing >4,600 total complexes, >7,700 proteins and >56,000 unique interactions, including thousands of confident protein interactions not identified by the original publications. hu.MAP accurately recapitulates known complexes withheld from the learning procedure, which was optimized with the aid of a new quantitative metric (k-cliques) for comparing sets of sets. The vast majority of complexes in our map are significantly enriched with literature annotations and the map overall shows improved coverage of many disease-associated proteins, as we describe in detail for ciliopathies. Using hu.MAP, we predicted and experimentally validated candidate ciliopathy disease genesin vivoin a model vertebrate, discovering CCDC138, WDR90, and KIAA1328 to be new cilia basal body/centriolar satellite proteins, and identifying ANKRD55 as a novel member of the intraflagellar transport machinery. By offering significant improvements to the accuracy and coverage of human protein complexes, hu.MAP (http://proteincomplexes.org) serves as a valuable resource for better understanding the core cellular functions of human proteins and helping to determine mechanistic foundations of human disease.
Complete fold annotation of the human proteome using a novel structural feature space
Authors: Middleton Sarah A. , Illuminati Joseph , Kim Junhyong
Published: December 2016

Recognition of protein structural fold is the starting point for many structure prediction tools and protein function inference. Fold prediction is computationally demanding and recognizing novel folds is difficult such that the majority of proteins have not been annotated for fold classification. Here we describe a new machine learning approach using a novel feature space that can be used for accurate recognition of all 1,221 currently known folds and inference of unknown novel folds. We show that our method achieves better than 96% accuracy even when many folds have only one training example. We demonstrate the utility of this method by predicting the folds of 34,330 human protein domains and showing that these predictions can yield useful insights into biological function, including the prediction of functional motifs relevant to human diseases. Our method can be applied tode novofold prediction of entire proteomes and identify candidate novel fold families.
Systematic morphological profiling of human gene and allele function reveals Hippo-NF-κB pathway connectivity
Authors: Rohban Mohammad H , Singh Shantanu , Wu Xiaoyun , Berthet Julia B , Bray Mark-Anthony , Shrestha Yashaswi , Varelas Xaralabos , Boehm Jesse S , Carpenter Anne E
Published: December 2016

We hypothesized that human genes and disease-associated alleles might be systematically functionally annotated using morphological profiling of cDNA constructs, via a microscopy-based Cell Painting assay. Indeed, 50% of the 220 tested genes yielded detectable morphological profiles, which grouped into biologically meaningful gene clusters consistent with known functional annotation (e.g., the RAS-RAF-MEK-ERK cascade). We used novel subpopulation-based visualization methods to interpret the morphological changes for specific clusters. This unbiased morphologic map of gene function revealed TRAF2/C-REL negative regulation of YAP 1/WWTR1-responsive pathways. We confirmed this discovery of functional connectivity between theNF-κBpathway and Hippo pathway effectors at the transcriptional level, thereby expanding knowledge of these two signaling pathways that critically regulate tumor initiation and progression. We make the images and raw data publicly available, providing an initial morphological map of major biological pathways for future study.
Paired CRISPR/Cas9 guide-RNAs enable high-throughput deletion scanning (ScanDel) of a Mendelian disease locus for functionally critical non-coding elements
Authors: Gasperini Molly , Findlay Gregory M. , McKenna Aaron , Milbank Jennifer H. , Lee Choli , Zhang Melissa D. , Cusanovich Darren A. , Shendure Jay
Published: December 2016

The extent to which distal non-coding mutations contribute to Mendelian disease remains a major unknown in human genetics. Given that a gene’s in vivo function can be appropriately modeled in vitro, CRISPR/Cas9 genome editing enables the large-scale perturbation of distal non-coding regions to identify functional elements in their native context. However, early attempts at such screens have relied on one individual guide RNA (gRNA) per cell, resulting in sparse mutagenesis with minimal redundancy across regions of interest. To address this, we developed a system that uses pairs of gRNAs to program thousands of kilobase-scale deletions that scan across a targeted region in a tiling fashion (“ScanDel”). As a proof-of-concept, we applied ScanDel to program 4,342 overlapping 1- and 2- kilobase (Kb) deletions that tile a 206 Kb region centered onHPRT1, the gene underlying Lesch-Nyhan syndrome, with median 27-fold redundancy per base. Programmed deletions were functionally assayed by selecting for loss ofHPRT1function with 6-thioguanine.HPRT1exons served as positive controls, and all were successfully identified as functionally critical by the screen. Remarkably,HPRT1function appeared robust to deletion of any intergenic or deeply intronic non-coding region across the 206 Kb locus, indicating that proximal regulatory sequences are sufficient for its expression. A sparser mutagenesis screen of the same 206 Kb with individual gRNAs also failed to identify critical distal regulatory elements. Although our screen did find programmed deletions and individual gRNAs with putative functional consequences that targeted exon-proximal non-coding sequences (e.g. the promoter), long-read sequencing revealed that this signal was driven almost entirely by rare, unexpected deletions that extended into exonic sequence. These targeted validation experiments defined a small region surrounding the transcriptional start site as the only non-coding sequence essential toHPRT1function. Overall, our results suggest that distal regulatory elements are not critical forHPRT1expression, and underscore the necessity of comprehensive edited-locus genotyping for validating the results of CRISPR screens. The application of ScanDel to additional loci will enable more insight into the extent to which the disruption of distal non-coding elements contributes to Mendelian diseases. In addition, dense, redundant, large-scale deletion scanning with gRNA pairs will facilitate a deeper understanding of endogenous gene regulation in the human genome.
Detection and characterization of low and high genome coverage regions using an efficient running median and a double threshold approach
Authors: Desvillechabrol Dimitri , Bouchier Christiane , Kennedy Sean , Cokelaer Thomas
Published: December 2016

Motivation

Next Generation Sequencing (NGS) provides researchers with powerful tools to investigate both prokaryotic and eukaryotic genetics. An accurate assessment of reads mapped to a specific genome consists of inspecting thegenome coverageas number of reads mapped to a specific genome location. Most current methods use the average of the genome coverage (sequencing depth) to summarize the overall coverage. This metric quickly assess the sequencing quality but ignores valuable biological information like the presence of repetitive regions or deleted genes. The detection of such information may be challenging due to a wide spectrum of heterogeneous coverage regions, a mixture of underlying models or the presence of a non-constant trend along the genome. Using robust statistics to systematically identify genomic regions with unusual coverage is needed to characterize these regions more precisely.

Results

We implemented an efficient running median algorithm to estimate the genome coverage trend. The distribution of the normalized genome coverage is then estimated using a Gaussian mixture model. Az-score statistics is then assigned to each base position and used to separate the central distribution from the regions of interest (ROI) (i.e., under and over-covered regions). Finally, a double threshold mechanism is used to cluster the genomic ROIs. HTML reports provide a summary with interactive visual representations of the genomic ROIs.

Availability

An implementation of the genome coverage characterization is available within theSequanaproject. The standalone application is calledsequana_coverage. The source code is available onGitHub(http://github.com/sequana/sequana), and documentation onReadTheDocs(http://sequana.readtheodcs.org). An example of HTML report is provided onhttp://sequana.github.io.

Contact

dimitri.desvillechabrol@pasteur.fr,thomas.cokelaer@pasteur.fr
New single-molecule imaging of the eisosome BAR domain protein Pil1p reveals filament-like dynamics
Authors: Lacy Michael M. , Baddeley David , Berro Julien
Published: December 2016

Molecular assemblies can have highly heterogeneous dynamics within the cell, but the limitations of conventional fluorescence microscopy can mask nanometer-scale features. We have developed a novel, broadly applicable, fluorescent labeling and imaging protocol, called Single-molecule Recovery After Photobleaching (SRAP), which allowed us to reveal the heterogeneous dynamics of the eisosome, a multi-protein structure on the cytoplasmic face of the plasma membrane in fungi. By fluorescently labeling only a small fraction of cellular Pil1p, the core eisosome BAR domain protein in fission yeast, we visualized whole eisosomes and, after photobleaching, recorded the binding of individual Pil1p molecules with ~20 nm precision. Further analysis of these dynamic structures and comparison to computer simulations allowed us to show that Pil1p exchange is spatially heterogeneous, supporting a new model of the eisosome as a dynamic filament.

Abbreviations used

BAR

Bin/Amphiphysin/Rvs domain

FRAP

Fluorescence Recovery After Photobleaching

mEGFP

monomeric Enhanced Green Fluorescent Protein

SiR647

silicon-rhodamine 647

TIRF

Total Internal Reflection Fluorescence
Resolving the Functional Significance ofBRCA1RING Domain Missense Substitutions
Authors: Paquette Andrew , Tao Kayoko , Clark Kathleen A , Stark Alex W , Rosenthal Judith , Snow Angela K , Bell Russell , Thompson Bryony A , Unger Joshua , Milash Brett A , Pappas Lisa , Gertz Jason , Varley Katherine E , Thomas Alun , Boucher Ken , Foulkes William D , Goldgar David E , Tavtigian Sean V
Published: December 2016

Part 1

Development and calibration of suitably accurate functional assays forBRCA1RING domain and BRCT domain missense substitutions could dramatically accelerate clinical classification of rare missense substitutions observed in that gene. Leveraging data from 68,000 full sequence tests ofBRCA1andBRCA2, plus data from the limited number of already classifiedBRCA1RING domain missense substitutions, we used logistic regression and related techniques to evaluate threeBRCA1RING domain assays. These were recently described high throughput yeast 2-hybrid and E3 ubiquitin ligase assays, plus a newly developed mammalian 2-hybrid assay. While there were concerns about the accuracy of the yeast 2-hybrid assay and the indirect nature of the ubiquitin ligase assay, the mammalian 2-hybrid assay had excellent correlation with existing missense substitution classifications. After calibration, this assay contributed to classification of one newly reportedBRCA1missense substitution. In principal, the mammalian 2-hybrid assay could be converted to a high-throughput format that would likely retain suitable accuracy.

Part 2

How does one achieve clinically applicable classification of the vast majority of all possible sequence variants in disease susceptibility genes? BRCA1 is a high-risk susceptibility gene for breast and ovarian cancer. Pathogenic protein truncating variants are scattered across the open reading frame, but all known missense substitutions that are pathogenic because of missense dysfunction are located in either the amino-terminal RING domain or the carboxy-terminal BRCT domain. Heterodimerization of the BRCA1 and BARD1 RING domains is a molecularly defined obligate activity. Hence, we tested every BRCA1 RING domain missense substitution that can be created by a single nucleotide change for heterodimerization with BARD1 in a Mammalian 2-hybrid (M2H) assay. Downstream of the M2H laboratory assay, we addressed three additional challenges: assay calibration, validation thereof, and integration of the calibrated results with other available data such as computational evidence and patient/population observational data to achieve clinically applicable classification. Overall, we found that about 20% of BRCA1 RING domain missense substitutions are pathogenic. Using a Bayesian point system for data integration and variant classification, we achieved clinical classification of about 89% of observed missense substitutions. Moreover, among missense substitutions not present in the human observational data used here, we find an additional 47 with concordant computational and functional assay evidence in favor of pathogenicity; these are particularly likely to be classified as Likely Pathogenic once human observational data become available.