bluuebunny/crossref_metadata_2025 huggingface dataset:
A Cadaveric Simulation Model to Teach Suture Placement During Sacrospinous Ligament Fixation
Authors: Evans Sarah Keenan Larkin , Myers Erinn M. , Anderson-Montoya Brittany , Vilasagar Smitha , Tarr Megan E.
Published: December 2019

Objectives

The objectives of this study were to describe a cadaveric simulation model designed to teach sacrospinous ligament fixation (SSLF) and to assess trainee performance, comfort, and skill with suture placement.

Methods

Nine invited participants from 3 institutions participated in cadaveric simulation training, consisting of a didactic lecture, technical demonstrations, and supervised execution of suture placement. Trainee self-perceived knowledge and confidence levels of SSLF were assessed before and after the educational intervention. Suture placement was assessed by expert faculty pelvic reconstructive surgeons. The number of attempts required by trainees for an anatomically safe suture placement was recorded. Participants completed a postintervention satisfaction survey.

Results

All 9 participants correctly identified where an SSLF suture should be placed on a printed image before the educational intervention, but only 33% achieved anatomically safe suture placement on the first attempt (mean ± SD, 2.88 ± 2.10 attempts). Four participants (44%) reported comfort with independently performing SSLF before the course. Of these, three (75%) required more than 1 attempt for successful suture placement. Mean ± SD distance of SSLF suture from the ischial spine was 1.90 ± 0.59 cm. All participants reported that they found the training helpful in learning the surgical steps and anatomy related to SSLF and would recommend it to others.

Conclusions

A cadaveric simulation teaching model led to improved trainee-reported confidence with the operative steps and anatomy related to SSLF. Participants’ prior knowledge of procedural steps and anatomy did not always transfer to adequate procedural skills for safe suture placement, suggesting the need for further simulation practice for fellow trainees.
Medical Nutrition Therapy for Women With Polycystic Ovary Syndrome—Does One Size Fit All? A Narrative Review on Dietary Treatment for Adolescents and Young Adults
Authors: Gordon Barbara , Agresta Eleni , Keenan Isoldi Kathy , Vargas-Rodriguez Ileana
Published: December 2019

Polycystic ovary syndrome (PCOS) affects approximately 0.76% to 3% of adolescent females. The Academy of Nutrition and Dietetics' Nutrition Care Manual includes evidence-based medical nutrition therapy guidelines for PCOS. This review compiled lifestyle considerations for adolescents and young adults (≤24 years) with PCOS and evaluated the efficacy of the Academy's guidelines for this subgroup. Findings suggest that registered dietitian nutritionists counseling adolescents and young adults with PCOS follow the Nutrition Care Manual recommendations for women of reproductive age. However, the literature also supports special considerations for assessing, counseling/educating, and monitoring adolescents and young women, which are summarized for clinicians.
Nuclear localization of Zika virus NS5 contributes to suppression of type I interferon production and response
Authors: Zhao Zikai , Tao Mengying , Han Wei , Fan Zijing , Imran Muhammad , Cao Shengbo , Ye Jing
Published: December 2019

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus, which caused an unprecedented epidemic in Latin America. Among all viral non-structural proteins in flavivirus, NS5 is the most highly conserved and has multiple crucial functions, including participating in viral RNA replication and suppressing host innate immunity. Although ZIKV NS5 prominently localizes in the nucleus during infection, its specific nuclear localization signal (NLS), and its role in viral replication and pathogenesis remain controversial. Here, we identified aa 11–90 and aa 370–406 regions that contain NLSs, which are critical for nuclear localization of ZIKV NS5. Further experiments demonstrated that nuclear localization of ZIKV NS5 predominantly participates in suppression of interferon regulatory factor 3 (IRF3)-mediated activation of type I IFN (IFN-I) transcription and inhibition of IFN-I downstream response independent of its effect on signal transducers and activators of transcription 2 (STAT2) degradation. These results suggest that subcellular localization of NS5 is important for its function on innate immune suppression, which provides new insight into ZIKV pathogenesis.
SufT is required for growth of Mycobacterium smegmatis under iron limiting conditions
Authors: Tamuhla Tsaone , Joubert Lydia , Willemse Danicke , Williams Monique J.
Published: December 2019

Iron-sulphur (FeS) clusters are versatile cofactors required for a range of biological processes within cells. Due to the reactive nature of the constituent molecules, assembly and delivery of these cofactors requires a multi-protein machinery in vivo. In prokaryotes, SufT homologues are proposed to function in the maturation and transfer of FeS clusters to apo-proteins. This study used targeted gene deletion to investigate the role of SufT in the physiology of mycobacteria, using

Mycobacterium smegmatis

as a model organism. Deletion of the sufT gene in

M. smegmatis

had no impact on growth under standard culture conditions and did not significantly alter activity of the FeS cluster dependent enzymes succinate dehydrogenase (SDH) and aconitase (ACN). Furthermore, the ΔsufT mutant was no more sensitive than the wild-type strain to the redox cycler 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), or the anti-tuberculosis drugs isoniazid, clofazimine or rifampicin. In contrast, the ΔsufT mutant displayed a growth defect under iron limiting conditions, and an increased requirement for iron during biofilm formation. This data suggests that SufT is an accessory factor in FeS cluster biogenesis in mycobacteria which is required under conditions of iron limitation.
Enhanced Cancer Subtyping via Pan-Transcriptomics Data Fusion, Monte-Carlo Consensus Clustering, and Auto Classifier Creation
Authors: Linton-Reid Kristofer , Clifford Harry , Thompson Joe Sneath
Published: December 2019

Subtyping of tumor transcriptome expression profiles is a routine method used to distinguish tumor heterogeneity. Unsupervised clustering techniques are often combined with survival analysis to decipher the relationship between genes and the survival times of patients. However, the reproducibility of these subtyping based studies is poor. There are multiple reports which have conflicting subtype and gene-survival time relationship results. In this study, we introduce the issues underlying the lack of reproducibility in transcriptomic subtyping studies. This problem arises from the routine analysis of small cohorts (< 100 individuals) and use of biased traditional consensus clustering techniques. Our approach carefully combines multiple RNA-sequencing and microarray datasets, followed by subtyping via Monte-Carlo Consensus Clustering and creation of deep subtyping classifiers. This paper demonstrates an improved subtyping methodology by investigating pancreatic ductal adenocarcinoma. Importantly, our methodology identifies six biologically novel pancreatic ductal adenocarcinoma subtypes. Our approach also enables a degree of reproducibility, via our pancreatic ductal adenocarcinoma classifier PDACNet, which classical subtyping studies have failed to establish.
The association of DNA methylation with body mass index: distinguishing between predictors and biomarkers
Authors: Reed Zoe E. , Suderman Matthew J. , Relton Caroline L. , Davis Oliver S.P. , Hemani Gibran
Published: December 2019

Background

DNA methylation is associated with body mass index (BMI), but it is not clear if methylation scores are biomarkers for extant BMI, or predictive of future BMI. Here we explore the causal nature and predictive utility of DNA methylation measured in peripheral blood with BMI and cardiometabolic traits.

Methods

Analyses were conducted across the life course using the ARIES cohort of mothers (n=792) and children (n=906), for whom DNA methylation and genetic profiles and BMI at multiple time points (3 in children at birth, in childhood and in adolescence, 2 in mothers during pregnancy and in middle age) were available. Genetic and DNA methylation scores for BMI were derived using published associations between BMI and DNA methylation and genotype. Causal relationships between methylation and BMI were assessed using Mendelian randomisation and cross-lagged models.

Results

The DNA methylation scores in adult women explained 10% of extant BMI variance. However, less extant variance was explained by scores generated in the same women during pregnancy (2% BMI variance) and in older children (15-17 years; 3% BMI variance). Similarly, little extant variance was explained in younger children (at birth and at 7 years; 1% and 2%, respectively). These associations remained following adjustment for smoking exposure and education levels. The DNA methylation score was found to be a poor predictor of future BMI using linear and cross-lagged models, suggesting that DNA methylation variation does not cause later variation in BMI. However, there was some evidence to suggest that BMI is predictive of later DNA methylation. Mendelian randomisation analyses also support this direction of effect, although evidence is weak. Finally, we find that DNA methylation scores for BMI are associated with extant cardiometabolic traits independently of BMI and genetic score.

Conclusion

The age-specific nature of DNA methylation associations with BMI, lack of causal relationship, and limited predictive ability of future BMI, indicate that DNA methylation is likely influenced by BMI and might more accurately be considered a biomarker of BMI and related outcomes than a predictor. Future epigenome-wide association studies may benefit from further examining associations between early DNA methylation and later health outcomes.
Interpretations of microbial community studies are biased by the selected 16S rRNA gene amplicon sequencing pipeline
Authors: Straub Daniel , Blackwell Nia , Fuentes Adrian Langarica , Peltzer Alexander , Nahnsen Sven , Kleindienst Sara
Published: December 2019

One of the major methods to identify microbial community composition, to unravel microbial population dynamics, and to explore microbial diversity in environmental samples is DNA- or RNA-based 16S rRNA (gene) amplicon sequencing. Subsequent bioinformatics analyses are required to extract valuable information from the high-throughput sequencing approach. However, manifold bioinformatics tools complicate their choice and might cause differences in data interpretation, making the selection of the pipeline a crucial step.

Here, we compared the performance of most widely used 16S rRNA gene amplicon sequencing analysis tools (i.e. Mothur, QIIME1, QIIME2, and MEGAN) using mock datasets and environmental samples from contrasting terrestrial and freshwater sites. Our results showed that QIIME2 outcompeted all other investigated tools in sequence recovery (>10 times less false positives), taxonomic assignments (>22% better F-score) and diversity estimates (>5% better assessment), while there was still room for improvement e.g. imperfect sequence recovery (recall up to 87%) or detection of additional false sequences (precision up to 72%). Furthermore, we found that microbial diversity estimates and highest abundant taxa varied among analysis pipelines (i.e. only one in five genera was shared among all analysis tools) when analyzing environmental samples, which might skew biological conclusions.

Our findings were subsequently implemented in a high-performance computing conformant workflow following the FAIR (Findable, Accessible, Interoperable, and Re-usable) principle, allowing reproducible 16S rRNA gene amplicon sequence analysis starting from raw sequence files. Our presented workflow can be utilized for future studies, thereby facilitating the analysis of high-throughput DNA- or RNA-based 16S rRNA (gene) sequencing data substantially.

Importance

Microorganisms play an essential role in biogeochemical cycling events across the globe. Phylogenetic marker gene analysis is a widely used method to explore microbial community dynamics in space and time, to predict the ecological relevance of microbial populations, or to identify microbial key players in biogeochemical cycles. Several computational analysis methods were developed to aid 16S rRNA gene analysis but choosing the best method is not trivial. In this study, we compared popular analysis methods (i.e. Mothur, QIIME1 and 2, and MEGAN) using samples with known microbial composition (i.e. mock community samples) and environmental samples from contrasting habitats (i.e. groundwater, soil, sediment, and river water). Our findings provide guidance for choosing the currently optimal 16S rRNA gene sequencing analysis method and we implemented our recommended pipeline into a reproducible workflow, which follows highest bioinformatics standards and is open source and free to use.
PDX models reflect the proteome landscape of pediatric acute lymphoblastic leukemia but divert in select pathways
Authors: Uzozie Anuli C. , Ergin Enes K. , Rolf Nina , Tsui Janice , Lorentzian Amanda , Weng Samuel S. , Nierves Lorenz , Smith Theodore G. , Lim C. James , Maxwell Christopher A. , Reid Gregor S.D. , Lange Philipp F.
Published: December 2019

Murine xenografts of pediatric leukemia are known to accurately recapitulate genomic aberrations. How this translates to the functional capacity of the proteome is unknown. Here, we studied global protein abundance, phosphorylation, and proteolytic processing in 11 pediatric B- and T-cell acute lymphoblastic leukemia patients and 19 corresponding xenografts. Protein level differences that stratified pediatric disease subtypes at diagnostic and relapse stages were largely recapitulated in xenograft models. Patient xenografts lacked multiple human leukocyte antigens, and complement proteins, and presented incomplete response mechanisms to the host immune system which is absent in the murine model. The dominant expression of MKI67 and cell cycle proteins indicated a high proliferative capacity of xenografted cells residing in the spleen. Structural genomic changes and mutations found in patients were reflected at the protein level. The post-translational modification landscape is shaped by leukemia type and host and only to a limited degree by the patient of origin. This study portrays how genomic and host factors shape protein and post-translational modification landscapes differently, and confirms murine patient-derived xenograft as competent model system while highlighting important areas of diverging biology.
Theory of transcription bursting: Stochasticity in the transcription rates
Authors: Murugan Rajamanickam
Published: December 2019

Transcription bursting creates variation among the individuals of a given population. Bursting emerges as the consequence of turning on and off the transcription process randomly. There are at least three sub-processes involved in the bursting phenomenon with different timescale regimes viz. flipping across the on-off state channels, microscopic transcription elongation events and the mesoscopic transcription dynamics along with the mRNA recycling. We demonstrate that when the flipping dynamics is coupled with the microscopic elongation events, then the distribution of the resultant transcription rates will be over-dispersed. This in turn reflects as the transcription bursting with over-dispersed non-Poisson type distribution of mRNA numbers. We further show that there exist optimum flipping rates (αC,βC) at which the stationary state Fano factor and variance associated with the mRNA numbers attain maxima. These optimum points are connected via. Hereαis the rate of flipping from the on-state to the off-state,βis the rate of flipping from the off-state to the on-state andγris the decay rate of mRNA. Whenα=β=χwith zero rate in the off-state channel, then there exist optimum flipping rates at which the non-stationary Fano factor and variance attain maxima. Here(hereis the rate of transcription purely through the on-state elongation channel) is the optimum flipping rate at which the variance of mRNA attains a maximum andχC,κ≃ 1.72/tis the optimum flipping rate at which the Fano factor attains a maximum. Close observation of the transcription mechanism reveals that the RNA polymerase performs several rounds of stall-continue type dynamics before generating a complete mRNA. Based on this observation, we model the transcription event as a stochastic trajectory of the transcription machinery across these on-off state elongation channels. Each mRNA transcript follows different trajectory. The total time taken by a given trajectory is the first passage time (FPT). Inverse of this FPT is the resultant transcription rate associated with the particular mRNA. Therefore, the time required to generate a given mRNA transcript will be a random variable. For a stall-continue type dynamics of RNA polymerase, we show that the overall average transcription rate can be expressed aswhereis the microscopic transcription elongation rate in the on-state channel andLis the length of a complete mRNA transcript andis the stationary state probability of finding the transcription machinery in the on-state.
Cryo-EM Structure of the Fork Protection Complex Bound to CMG at a Replication Fork
Authors: Baretić Domagoj , Jenkyn-Bedford Michael , Aria Valentina , Cannone Giuseppe , Skehel Mark , Yeeles Joseph T.P.
Published: December 2019

The eukaryotic replisome, organized around the Cdc45-MCM-GINS (CMG) helicase, orchestrates chromosome replication. Multiple factors associate directly with CMG including Ctf4 and the heterotrimeric fork protection complex (Csm3/Tof1 and Mrc1), that have important roles including aiding normal replication rates and stabilizing stalled forks. How these proteins interface with CMG to execute these functions is poorly understood. Here we present 3-3.5 Å resolution cryo-EM structures comprising CMG, Ctf4, Csm3/Tof1 and Mrc1 at a replication fork. The structures provide high-resolution views of CMG:DNA interactions, revealing the mechanism of strand separation. Furthermore, they illustrate the topology of Mrc1 in the replisome and show Csm3/Tof1 ‘grips’ duplex DNA ahead of CMG via a network of interactions that are important for efficient replication fork pausing. Our work reveals how four highly conserved replisome components collaborate with CMG to facilitate replisome progression and maintain genome stability.
Two distinct HIRA-dependent pathways handle H3.3de novodeposition and recycling during transcription
Authors: Torné Júlia , Ray-Gallet Dominique , Boyarchuk Ekaterina , Garnier Mickaël , Coulon Antoine , Orsi Guillermo A. , Almouzni Geneviève
Published: December 2019

The packaging of DNA into nucleosomes represents a challenge for transcription. Nucleosome disruption and histone eviction enables RNA Polymerase II progression through DNA, a process that compromises chromatin integrity and the maintenance of epigenetic information. Here, we used the imaging SNAP-tag system to distinguish new and old histones and monitor chromatin re-assembly coupled to transcription in cells. First, we uncovered a loss of both old variants H3.1 and H3.3 that depends on transcriptional activity, with a major effect on H3.3. Focusing on transcriptionally active domains, we revealed a local enrichment in H3.3 with dynamics involving both new H3.3 incorporation and old H3.3 retention. Mechanistically, we demonstrate that the HIRA chaperone is critical to handle both new and old H3.3, and showed that this implicates different pathways. The de novo H3.3 deposition depends strictly on HIRA trimerization as well as its partner UBN1 while ASF1 interaction with HIRA can be bypassed. In contrast, the recycling of H3.3 requires HIRA but proceeds independently of UBN1 or HIRA trimerization and shows an absolute dependency on ASF1-HIRA interaction. Therefore, we propose a model where HIRA can coordinate these distinct pathways for old H3.3 recycling and new H3.3 deposition during transcription to fine-tune chromatin states.
Inclusion of Oxford Nanopore long reads improves all microbial and phage metagenome-assembled genomes from a complex aquifer system
Authors: Overholt Will A. , Hölzer Martin , Geesink Patricia , Diezel Celia , Marz Manja , Küsel Kirsten
Published: December 2019

Assembling microbial and phage genomes from metagenomes is a powerful and appealing method to understand structure-function relationships in complex environments. In order to compare the recovery of genomes from microorganisms and their phages from groundwater, we generated shotgun metagenomes with Illumina sequencing accompanied by long reads derived from the Oxford Nanopore sequencing platform. Assembly and metagenome-assembled genome (MAG) metrics for both microbes and viruses were determined from Illumina-only assemblies and a hybrid assembly approach. Strikingly, the hybrid approach more than doubled the number of mid to high-quality MAGs (> 50% completion, < 10% redundancy), generated nearly four-fold more phage genomes, and improved all associated genome metrics relative to the Illumina only method. The hybrid assemblies yielded MAGs that were on average 7.8% more complete, with 133 fewer contigs and a 14 kbp greater N50. Furthermore, the longer contigs from the hybrid approach generated microbial MAGs that had a higher proportion of rRNA genes. We demonstrate this usefulness by linking microbial MAGs containing 16S rRNA genes with extensive amplicon dataset. This work provides quantitative data to inform a cost-benefit analysis on the decision to supplement shotgun metagenomic projects with long reads towards the goal of recovering genomes from environmentally abundant groups.
LYS3encodes a prolamin-box-binding transcription factor that controls embryo growth in barley and wheat
Authors: Orman-Ligeza Beata , Borrill Philippa , Chia Tansy , Chirico Marcella , Doležel Jaroslav , Drea Sinead , Karafiátová Miroslava , Schatlowski Nicole , Solomon Charles U. , Steuernagel Burkhard , Wulff Brande B. H. , Uauy Cristobal , Trafford Kay
Published: December 2019

Mutations at theLYS3locus in barley have multiple effects on grain development, including an increase in embryo size and a decrease in endosperm starch content. The gene underlyingLYS3was identified by genetic mapping and mutations in this gene were identified in all four barleylys3alleles.LYS3encodes a transcription factor called Prolamin Binding Factor (PBF). Its role in controlling embryo size was confirmed using wheat TILLING mutants. To understand howPBFcontrols embryo development, we studied its spatial and temporal patterns of expression in developing grains. ThePBFgene is expressed in both the endosperm and the embryos, but the timing of expression in these organs differs.PBFexpression in wild-type embryos precedes the onset of embryo enlargement inlys3mutants, suggesting thatPBFsuppresses embryo growth. We predicted the down-stream target genes ofPBFin wheat and found them to be involved in a wide range of biological processes, including organ development and starch metabolism. Our work suggests thatPBFmay influence embryo size and endosperm starch synthesis via separate gene control networks.

HIGHLIGHTS

LYS3encodes a transcription factor called Prolamin Binding Factor (PBF) that is expressed in grains only.

Wheat and barleyLYS3/PBFmutants have enlarged embryos suggesting that this gene suppresses embryo growth.

The down-stream target genes ofPBFin wheat are predicted to be involved in a wide range of biological processes including organ development and starch metabolism.
Revealing the impact of recurrent and rare structural variants in multiple myeloma
Authors: Rustad Even H , Yellapantula Venkata D , Glodzik Dominik , Maclachlan Kylee H , Diamond Benjamin , Boyle Eileen M , Ashby Cody , Blaney Patrick , Gundem Gunes , Hultcrantz Malin , Leongamornlert Daniel , Angelopoulos Nicos , Auclair Daniel , Zhang Yanming , Dogan Ahmet , Bolli Niccolò , Papaemmanuil Elli , Anderson Kenneth C. , Moreau Philippe , Avet-Loiseau Herve , Munshi Nikhil , Keats Jonathan , Campbell Peter J , Morgan Gareth J , Landgren Ola , Maura Francesco
Published: December 2019

The landscape of structural variants (SVs) in multiple myeloma remains poorly understood. Here, we performed comprehensive classification and analysis of SVs in multiple myeloma, interrogating a large cohort of 762 patients with whole genome and RNA sequencing. We identified 100 SV hotspots involving 31 new candidate driver genes, including drug targets BCMA (TNFRSF17) andSLAMF7. Complex SVs, including chromothripsis and templated insertions, were present in 61 % of patients and frequently resulted in the simultaneous acquisition of multiple drivers. After accounting for all recurrent events, 63 % of SVs remained unexplained. Intriguingly, these rare SVs were associated with up to 7-fold enrichment for outlier gene expression, indicating that many rare driver SVs remain unrecognized and are likely important in the biology of individual tumors.
Relationships between community composition, productivity and invasion resistance in semi-natural bacterial microcosms
Authors: Jones Matt Lloyd , Rivett Damian W. , Pascual-García Alberto , Bell Thomas
Published: December 2019

Experiments with artificial communities have suggested that invasion resistance in microbial communities is often a side-effect of community members’ contribution towards overall community productivity (broadly defined as cumulative cell density and/or growth rate). However, few experiments have investigated this in natural microbial communities. We conducted experimental invasions of two bacterial species (Pseudomonas fluorescensandPseudomonas putida) into laboratory microcosms inoculated with 680 different mixtures of bacteria derived from naturally-occurring microbial communities collected in the field. Using 16S amplicon sequencing to characterise microcosm starting composition, and high-throughput assays of community phenotypes including productivity and invader survival, we determined that productivity is a key predictor of invasion resistance in natural microbial communities, substantially mediating the effect of composition on invasion resistance. The results suggest that similar general principles govern invasion in artificial and natural communities, and that factors affecting resident community productivity should be a focal point for future microbial invasion experiments.
Robust detection of oncometabolic aberrations by1H-13C heteronuclear single quantum correlation in live cells and intact tumorsex-vivo
Authors: Barekatain Yasaman , Yan Victoria C , Arthur Kenisha , Ackroyd Jeffrey J. , Khadka Sunada , De Groot John , Huse Jason T. , Muller Florian L.
Published: December 2019

Extensive efforts have been made to use non-invasive1H magnetic resonance (MR) spectroscopy to quantify metabolites that are diagnostic of specific disease states. Within the realm of precision oncology, these efforts have largely centered on quantifying 2-hydroxyglutarate (2-HG) in tumors harboring isocitrate dehydrogenase 1 (IDH1) mutations. As many metabolites have similar chemical shifts, the resulting1H spectra of intact biological material are highly convoluted, limiting the application of1H MR to high abundance metabolites. Hydrogen-Carbon Heteronuclear single quantum correlation1H-13C HSQC is routinely employed in organic synthesis to resolve complex spectra but has received limited attention for biological studies. Here, we show that1H-13C HSQC offers a dramatic improvement in sensitivity compared to one-dimensional (1D)13C NMR and dramatic signal deconvolution compared to 1D1H spectra in an intact biological setting. Using a standard NMR spectroscope without specialized signal enhancements features such as magic angle spinning, metabolite extractions or13C-isotopic enrichment, we obtain well-resolved 2D1H-13C HSQC spectra in live cancer cells, inex-vivofreshly dissected xenografted tumors and resected primary tumors. We demonstrate that this method can readily identify tumors with specific genetic-driven oncometabolite alterations such asIDHmutations with elevation of 2-HG as well asPGD-homozygously deleted tumors with elevation of gluconate. These data support the potential of1H-13C HSQC as a non-invasive diagnostic tool for metabolic precision oncology.
Ca2+ions promote fusion of Middle East Respiratory Syndrome coronavirus with host cells and increase infectivity
Authors: Straus Marco R. , Tang Tiffany , Lai Alex L. , Flegel Annkatrin , Bidon Miya , Freed Jack H. , Daniel Susan , Whittaker Gary R.
Published: December 2019

Middle East respiratory syndrome coronavirus (MERS-CoV) is a major emerging zoonotic infectious disease. Since its first outbreak in 2012, the virus has repeatedly transmitted from camels to humans with 2,468 confirmed cases, causing 851 deaths. To date, there are no efficacious drugs and vaccines against MERS-CoV, increasing its potential to cause a public health emergency. A critical step in the life cycle of MERS-CoV is the fusion with the host cell with its spike (S) protein as main determinant of viral entry. Proteolytic cleavage of S exposes its fusion peptide (FP), which initiates membrane fusion. Previous studies on the related severe acute respiratory syndrome coronavirus (SARS-CoV) FP have shown that calcium (Ca2+) plays an important role for fusogenic activity via a Ca2+binding pocket with conserved glutamic acid (E) and aspartic acid (D) residues. SARS-CoV and MERS-CoV FP share a high sequence homology and here, we investigated whether Ca2+is required for MERS-CoV fusion by substituting E and D residues in the MERS-CoV FP with neutrally charged alanines. Upon verifying mutant cell surface expression and proteolytic cleavage, we tested the mutants ability to mediate infection of pseudo-particles (PPs) on host cells without and with Ca2+. Our results demonstrate that intracellular Ca2+enhances MERS-CoV WT PPs infection by approximately two-fold and that E891 is a crucial residue for Ca2+interaction. Electron spin resonance revealed that this enhancement could be attributed to Ca2+increasing MERS-CoV FP fusion-relevant membrane ordering. Intriguingly, isothermal calorimetry titration showed that MERS-CoV FP binds one Ca2+, as opposed to SARS-CoV FP which binds two. Our data suggests that there are significant differences in FP-Ca2+interactions of MERS-CoV and SARS-CoV FP despite their high sequence similarity and that the number of Ca2+ions interacting with the FP has implications on the fusion dynamics of the virus.
Integrase-RNA interactions underscore the critical role of integrase in HIV-1 virion morphogenesis
Authors: Elliott Jennifer , Eschbach Jenna E. , Koneru Pratibha C. , Li Wen , Chavez Maritza Puray , Townsend Dana , Lawson Dana , Engelman Alan N. , Kvaratskhelia Mamuka , Kutluay Sebla B.
Published: December 2019

A large number of HIV-1 integrase (IN) alterations, referred to as class II substitutions, exhibit pleotropic effects during virus replication. However, the underlying mechanism for the class II phenotype is not known. Here we demonstrate that all tested class II IN substitutions compromised IN-RNA binding in virions by one of three distinct mechanisms: i) markedly reducing IN levels thus precluding formation of IN complexes with viral RNA; ii) adversely affecting functional IN multimerization and consequently impairing IN binding to viral RNA; iii) directly compromising IN-RNA interactions without substantially affecting IN levels or functional IN multimerization. Inhibition of IN-RNA interactions resulted in mislocalization of the viral ribonucleoprotein complexes outside the capsid lattice, which led to premature degradation of the viral genome and IN in target cells. Collectively, our studies uncover causal mechanisms for the class II phenotype and highlight an essential role of IN-RNA interactions for accurate virion maturation.
Testing Hormone-specific Antibody Probes for Presumptive Detection and Separation of Contributor Cell Populations in Trace DNA Mixtures
Authors: Miller Jennifer M. , Brocato Emily R. , Yadavalli Vamsi K. , Greenspoon Susan A. , Ehrhardt Christopher J.
Published: December 2019

“Touch” or trace DNA evidence represent a significant proportion of samples analyzed by forensic science laboratories. Because these samples frequently contain multiple contributors and are often challenging to analyze due to low DNA concentrations and frequent degradation, front end techniques to simplify the mixture prior to DNA profiling could significantly impact case processing and enhance success rates. The goal of this study was to investigate whether targeting hormone molecules within the cell with antibody probes could be used to selectively label and then physically isolate contributor cell populations in trace biological samples. The separation of male and female cells into distinct fractions could reduce the complexity of the mixture prior to DNA profiling. To accomplish this, we first tested the specificity of fluorescently labelled anti-testosterone and anti-dihydrotestosterone antibody probes to epidermal cells from both male and female individuals. Results show that male and female cell populations can be effectively labeled using anti-testosterone and anti-dihydrotestosterone antibody probes and that distinct differences in binding efficiency and resulting median fluorescence of cell populations were observed between several individuals. These differences were then used to design sorting criteria for physically isolating each cell population in two-person epidermal cell mixtures using fluorescence activated cell sorting (FACS). DNA profiling of separated fractions in combination with probabilistic modeling demonstrated that some cell mixtures could be enriched for one contributor in separated cell fractions and yielded statistically more discriminating profiles compared to those generated from the original mixtures. Other mixtures tested showed less evidence of effective cell separation possibly due to a number of factors including imbalance of contributor DNA ratio, intra-sex variation of antibody binding efficiency, and contributions of extracellular or cell-free DNA in the mixture sample. Screening and separation of trace DNA samples with this approach may be presumptive and ultimately constrained by specific parameters of the original mixture, however, antibody binding optimization may mitigate some of these influences.
Phase separation-mediated formation of condensed GIT/PIX enzyme complex module for compartmentalized signaling
Authors: Zhu Jinwei , Zhou Qingqing , Xia Yitian , Lin Lin , Li Jianchao , Peng Mengjuan , Zhang Rongguang , Zhang Mingjie
Published: December 2019

Cells compartmentalize enzymes for broad physiological functions such as efficient metabolic reactions and spatiotemporally controlled signaling. A given enzyme or enzyme complex can participate in multiple cellular processes in response to different signal inputs by forming different cellular compartments. Here, we demonstrate that association of GIT1 and β-Pix, a pair of GTPase regulatory enzymes involved in diverse cellular processes, leads to autonomous condensation of the complex via phase separation without additional scaffolding molecules. The atomic structure of the GIT/PIX complex reveals the molecular basis governing the phase separation-mediated condensation of the GIT1/β-Pix complex. Importantly, the GIT1/β-Pix condensates can function as a versatile modular membrane-less organelle- like structure for distinct cellular compartmentalization by binding to upstream proteins such as Paxillin in focal adhesions, Shank3 in neuronal synapses, and Scribble in cellular junctions. Thus, phase separation-mediated formation of condensed enzyme complexes provides a powerful way of dynamically concentrating limited amounts of cooperating enzymes to specific cellular compartments for optimal signaling.
Widespread diversity in the transcriptomes of functionally divergent limb tendons
Authors: Disser Nathaniel P , Ghahramani Gregory C , Swanson Jacob B , Wada Susumu , Chao Max L , Rodeo Scott A , Oliver David J , Mendias Christopher L
Published: December 2019

Tendon is a functionally important connective tissue that transmits force between skeletal muscle and bone. Previous studies have evaluated the architectural designs and mechanical properties of different tendons throughout the body. However, less is known about the underlying transcriptional differences between tendons which may dictate their designs and properties. Therefore, our objective was to develop a comprehensive atlas of the transcriptome of limb tendons in adult mice and rats using systems biology techniques. We selected the Achilles, forepaw digit flexor, patellar, and supraspinatus tendons due to their divergent functions and high rates of injury and tendinopathies in patients. Using RNA sequencing data, we generated the Comparative Tendon Transcriptional Database (CTTDb) that identified substantial diversity in the transcriptomes of tendons both within and across species. Approximately 30% of transcripts were differentially regulated between tendons of a given species, and nearly 60% of the transcripts present in anatomically similar tendons were different between species. Many of the genes that differed between tendons and across species are important in tissue specification and limb morphogenesis, tendon cell biology and tenogenesis, growth factor signaling, and production and maintenance of the extracellular matrix. This study indicates that tendon is a surprisingly heterogenous tissue with substantial genetic variation based on anatomical location and species.

Key Points

Tendon is a hypocellular, matrix-rich tissue that has been excluded from comparative transcriptional atlases. These atlases have provided important knowledge about biological heterogeneity between tissues, and our manuscript addresses this important gap.

We performed measures on four of the most studied tendons, the Achilles, forepaw flexor, patellar, and supraspinatus tendons of both mice and rats. These tendons are functionally distinct and are also among the most commonly injured, and therefore of important translational interest.

Approximately one-third of the transcriptome was differentially regulated between Achilles, forepaw flexor, patellar, and supraspinatus tendons within either mice or rats. Nearly two thirds of the transcripts that are expressed in anatomically similar tendons were different between mice and rats.

The overall findings from this study identified that although tendons across the body share a common anatomical definition based on their physical location between skeletal muscle and bone, tendon is a surprisingly genetically heterogeneous tissue.
Polygenic Risk for Skin Autoimmunity Impacts Immune Checkpoint Blockade in Bladder Cancer
Authors: Khan Zia , Di Nucci Flavia , Kwan Antonia , Hammer Christian , Mariathasan Sanjeev , Rouilly Vincent , Carroll Jonathan , Fontes Magnus , Acosta Sergio Ley , Guardino Ellie , Chen-Harris Haiyin , Bhangale Tushar , Mellman Ira , Rosenberg Jonathan , Powles Thomas , Hunkapiller Julie , Chandler G. Scott , Albert Matthew L.
Published: December 2019

PD-1 and PD-L1 act to restrict T-cell responses in cancer and contribute to self-tolerance. Consistent with this role, PD-1 checkpoint inhibitors have been associated with immune-related adverse events (irAEs), immune toxicities thought to be autoimmune in origin. Analyses of dermatological irAEs have identified an association with improved overall survival (OS) following anti-PD-(L)1 therapy, but the factors that contribute to this relationship are poorly understood. We collected germline whole genome sequencing data from IMvigor211, a recent phase 3 randomized controlled trial comparing atezolizumab (anti-PD-L1) monotherapy to chemotherapy in bladder cancer. We found that high vitiligo, high psoriasis, and low atopic dermatitis polygenic risk scores (PRSs) were associated with longer OS under anti-PD-L1 monotherapy as compared to chemotherapy, reflecting the Th17 polarization of these diseases. PRSs were not correlated with tumor mutation burden, PD-L1 immunohistochemistry, nor T-effector gene signatures. Shared genetic factors impact risk for dermatological autoimmunity and anti-PD-L1 monotherapy in bladder cancer.
Finding a suitable library size to call variants in RNA-seq
Authors: Quaglieri Anna , Flensburg Christoffer , Speed Terence P , Majewski Ian J
Published: December 2019

Background

RNA-Seq allows the study of both gene expression changes and transcribed mutations, providing a highly effective way to gain insight into cancer biology. When planning the sequencing of a large cohort of samples, library size is a fundamental factor affecting both the overall cost and the quality of the results. While several studies analyse the effect that library size has on differential expression analyses, sensitivity analysis for variant detection has received far less attention.

Results

We simulated shallower sequencing depths by downsampling 45 AML samples that are part of the Leucegene project, which were originally sequenced at high depth. We compared the sensitivity of six methods of recovering validated mutations on the same samples. The methods compared are a combination of three popular callers (MuTect, VarScan, and VarDict) and two filtering strategies. We observed an incremental loss in sensitivity when simulating libraries of 80M, 50M, 40M, 30M and 20M fragments, with the largest loss detected with less than 30M fragments (below 90%). The sensitivity in recovering indels varied markedly between callers, with VarDict showing the highest sensitivity (60%). Single nucleotide variant sensitivity is relatively consistent across methods, apart from MuTect, whose default filters need adjustment when using RNA-Seq. We also analysed 136 RNA-Seq samples from the TCGA-LAML cohort, assessing the change in sensitivity between the initial libraries (average 59M fragments) and after downsampling to 40M fragments. When considering single nucleotide variants in recurrently mutated myeloid genes we found a comparable performance, with a 3% average loss in sensitivity using 40M fragments.

Conclusions

Between 30M and 40M fragments are needed to recover 90%-95% of the initial variants on recurrently mutated myeloid genes. To extend this result to another cancer type, an exploration of the characteristics of its mutations and gene expression patterns is suggested.
Anin situatlas of mitochondrial DNA in mammalian tissues reveals high content in stem/progenitor cells
Authors: Chen Jiayu , Zheng Qizhi , Peiffer Lauren B. , Hicks Jessica L. , Haffner Michael C. , Rosenberg Avi Z. , Levi Moshe , Wang Xiaoxin X. , Ozbek Busra , Yegnasubramanian Srinivasan , De Marzo Angelo M.
Published: December 2019

Mitochondria regulate ATP production, metabolism and cell death. Alterations in mitochondrial DNA (mtDNA) sequence and copy number are implicated in aging and organ dysfunction in diverse inherited and sporadic diseases. Since most measurements of mtDNA use homogenates of complex tissues, little is known about cell type-specific mtDNA copy number heterogeneity in normal physiology, aging and disease. Thus, the precise cell types whose loss of mitochondrial activity and altered mtDNA copy number that result in organ dysfunction in aging and disease have often not been clarified. Here, we validated anin situhybridization approach to generate a single cell resolution atlas of mtDNA content in mammalian tissues. In hierarchically organized self-renewing tissues, higher levels of mtDNA were observed in stem/proliferative compartments compared to differentiated compartments. Striking zonal patterns of mtDNA levels in the liver reflected the known oxygen tension gradient. In the kidney, proximal and distal tubules had markedly higher mtDNA levels compared to cells within glomeruli and collecting duct epithelial cells. Decreased mtDNA levels were visualized in renal tubules as a function of aging, which was prevented by calorie restriction. We provide a novel approach for quantifying species- and cell type-specific mtDNA copy number and dynamics in any normal or diseased tissue and can be used for monitoring the effects of interventions in animal and human studies.
Correlation ofp53and DNA repair gene mutation patterns in human malignancies indicates different tumour suppression mechanisms of p53
Authors: Xue Xuemin , Dong Lin , Xue Liyan , Lu Yong-jie
Published: December 2019

P53 suppresses tumorigenesis through multiple cellular functions/mechanisms. Recently, Janic A, et al. reported that DNA repair pathways are critical mediators of p53-dependent tumor suppression. We showed, by mining cBioPortal data of a range of human cancers, that the tendency of ‘mutual exclusivity’ of mutations inp53and DNA repair genes only exist in very limited human cancer types. In the majority of human cancers,p53mutations are equally distributed between DNA repair gene mutation positive and negative cases and in a number of human cancers, p53 and DNA repair gene mutations have a tendency of co-occurrence. These different correlation patterns ofp53and DNA repair gene mutations in human malignancies may reflect different critical molecular/cellular pathways activated by p53 in different organs or cell types to suppress tumorigenesis.
Physiological Adaptability and Parametric Versatility in a Simple Genetic Circuit
Authors: Chure Griffin , Kaczmarek Zofii A. , Phillips Rob
Published: December 2019

The intimate relationship between the environment and cellular growth rate has remained a major topic of inquiry in bacterial physiology for over a century. Now, as it becomes possible to understand how the growth rate dictates the wholesale reorganization of the intracellular molecular composition, we can interrogate the biophysical principles underlying this adaptive response. Regulation of gene expression drives this adaptation, with changes in growth rate tied to the activation or repression of genes covering enormous swaths of the genome. Here, we dissect how physiological perturbations alter the expression of a circuit which has been extensively characterized in a single physiological state. Given a complete thermodynamic model, we map changes in physiology directly to the biophysical parameters which define the expression. Controlling the growth rate via modulating the available carbon source or growth temperature, we measure the level of gene expression from a LacI-regulated promoter where the LacI copy number is directly measured in each condition, permitting parameter-free prediction of the expression level. The transcriptional output of this circuit is remarkably robust, with expression of the repressor being largely insensitive to the growth rate. The predicted gene expression quantitatively captures the observations under different carbon conditions, indicating that the bio-physical parameters are indifferent to the physiology. Interestingly, temperature controls the expression level in ways that are inconsistent with the prediction, revealing temperature-dependent effects that challenge current models. This work exposes the strengths and weaknesses of thermodynamic models in fluctuating environments, posing novel challenges and utility in studying physiological adaptation.

Significance

Cells adapt to changing environmental conditions by repressing or activating gene expression from enormous fractions of their genome, drastically changing the molecular composition of the cell. This requires the concerted adaptation of transcription factors to the environmental signals, leading to binding or releasing of their cognate sequences. Here, we dissect a well characterized genetic circuit in a number of physiological states, make predictions of the response, and measure how the copy number of a regulator and its gene target are affected. We find the parameters defining the regulators behavior are remarkably robust to changes in the nutrient availability, but are susceptible to temperature changes. We quantitatively explore these two effects and discuss how they challenge current models of transcriptional regulation.