bluuebunny/crossref_metadata_2025 huggingface dataset:
Cryptic prophages within a Streptococcus pyogenes genotype emm4 lineage
Authors: Remmington Alex , Haywood Samuel , Edgar Julia , Green Luke R. , de Silva Thushan , Turner Claire E.
Published: November 2020

The major human pathogen

Streptococcus pyogenes

shares an intimate evolutionary history with mobile genetic elements, which in many cases carry genes encoding bacterial virulence factors. During recent whole-genome sequencing of a longitudinal sample of

S. pyogenes

isolates in England, we identified a lineage within emm4 that clustered with the reference genome MEW427. Like MEW427, this lineage was characterized by substantial gene loss within all three prophage regions, compared to MGAS10750 and isolates outside of the MEW427-like lineage. Gene loss primarily affected lysogeny, replicative and regulatory modules, and to a lesser and more variable extent, structural genes. Importantly, prophage-encoded superantigen and DNase genes were retained in all isolates. In isolates where the prophage elements were complete, like MGAS10750, they could be induced experimentally, but not in MEW427-like isolates with degraded prophages. We also found gene loss within the chromosomal island SpyCIM4 of MEW427-like isolates, although surprisingly, the SpyCIM4 element could not be experimentally induced in either MGAS10750-like or MEW427-like isolates. This did not, however, appear to abolish expression of the mismatch repair operon, within which this element resides. The inclusion of further emm4 genomes in our analyses ratified our observations and revealed an international emm4 lineage characterized by prophage degradation. Intriguingly, the USA population of emm4

S. pyogenes

appeared to constitute predominantly MEW427-like isolates, whereas the UK population comprised both MEW427-like and MGAS10750-like isolates. The degraded and cryptic nature of these elements may have important phenotypic and fitness ramifications for emm4

S. pyogenes

, and the geographical distribution of this lineage raises interesting questions on the population dynamics of the genotype.
Biomarkers in the prediction of multimorbidity: scoping review
Authors: Spencer EA , Ford GA , Chan MS , Perera R , Heneghan CJ
Published: November 2020

Background

Multimorbidity presents an increasing challenge in the global ageing population. Predicting its development is necessary to help design and deliver effective healthcare.

Objective

This scoping review aimed to collate and present the body of published evidence on biomarkers and multimorbidity, identifying what work has been done and what gaps remain.

Methods

We searched the electronic databases MEDLINE, Register of Controlled Trials (Cochrane CENTRAL), CINAHL, PsycINFO, EMBASE, Scopus, Web of Science and TRIP database up until 11 August 2020 and hand-searched the reference lists of included articles.

Results

We found 34 relevant studies including 12 reporting prospective data and 22 reporting cross-sectional data. These encompassed 14 studies on serum biomarkers, 2 on molecular biomarkers, 7 on physiological biomarkers, 8 on body size biomarkers and 3 on brain function biomarkers. Most studies were undertaken in European or North American populations. There was a broadly consistent finding that obesity was associated with increased multimorbidity. Other results were more varied, reflecting the diverse range of biomarkers investigated, and lack of standardisation of multimorbidity outcome definitions.

Longitudinal studies have been set up that are maturing and further evidence can be expected over time.

Conclusion

There has been limited research on biomarkers to predict the development of multimorbidity, with minimal investigation of putative biomarkers identified in basic research. High quality research studies in this area are needed to progress the development of targeted interventions to prevent or delay the onset of multimorbidity.
Are we ready for COVID-19’s Golden Passport? Insights from a Global Physician Survey
Authors: Doraiswamy P. Murali , Chilukuri Mohan , Linares Alexandra R. , Bramstedt Katrina A.
Published: November 2020

Introduction

COVID-19 immunity passports could protect the right to free movement, but critics worry about insufficient evidence, privacy, fraud, and discrimination. We aimed to characterize the global physician community’s opinion regarding immunity passports.

Methods

Cross sectional, random stratified sample of physicians registered with Sermo, a global networking platform open to verified and licensed physicians. The survey aimed to sample 1,000 physicians divided among the USA, EU and rest of the world. The survey question on immunology asked physicians to offer their insights into whether we know enough about COVID-19 immunity and its duration to offer immunity passports at the present time.

Results

The survey was completed by 1004 physicians (67 specialties, 40 countries, 49% frontline specialties) with a mean (SD) age of 49.14 (12) years. Overall, 52% answered NO, 17% were UNCERTAIN, and 31% answered YES (P <0.05). EU physicians were more likely to sayYES but even among them it did not exceed 35% approval. US physicians (60%) were more likely to say NO.

Conclusion

Our findings suggest a current lack of support among physicians for immunity passports. It is hoped that ongoing research and vaccine trials will provide further clarity.
Current methods integrating variant functional annotation scores have limited capacity to improve the power of genome-wide association studies
Authors: Gao Jianhui , Espin-Garcia Osvaldo , Paterson Andrew D. , Sun Lei
Published: November 2020

Functional annotations have the potential to increase the power of genome-wide association studies (GWAS) by prioritizing variants according to their biological function. Focusing on variant-specific annotation meta-scores including CADD (Kircher et al., 2014) and Eigen (Ionita-laza et al., 2016), we broadly examined GWAS summary statistics of 1,132 traits from the UK Biobank (Sudlow et al., 2015) using the weighted p-value approach (Genovese et al., 2006) and stratified false discovery control (sFDR) method (Sun et al., 2006). These 1,132 traits were rated by Benjamin Neale’s lab from the Broad Institute as having medium to high confidence for their heritability estimates.

Averaged across the 1,132 UK Biobank traits, sFDR was more robust to uninformative meta-scores, but the weighted p-value method identified more variants using CADD or Eigen, based on performance measures that included type I error control, recall, precision, and relative efficiency. Our application results were consistent with those from an extensive simulation study using three different designs, including leveraging the real genetic data combined with simulated genomic data and vice versa.

We also considered the recent FINDOR method (Kichaev et al., 2019), which leverages a set of individual 75 functional annotations into GWAS. An earlier application of FINDOR to 27 traits selected from the z7 category (SNP-heritability p-value < 1.27 × 10−12by Nealelab) detected 13%-20% additional genome-wide significant loci as compared to the standard annotation-free GWAS, which we confirmed. Moreover, across all 438 traits in the z7 category, 46,631 out 59,764 (80%) significant loci discovered are common across the three data-integration methods.

However, across all the 1,132 UK Biobank traits examined, the median [Q1,Q3] of the total numbers of new, genome-wide significant independent loci were 0 [0, 3] by FINDOR, 0 [0, 2] by weighted p-value, and 0 [0, 0] by sFDR. Notably, 162 traits (89%) in the nonsig trait category (SNP-heritability p-value > 0.05, “likely reflecting limited statistical power rather than a true lack of heritability” by Nealelab) had no new discoveries after data-integration by any of the three methods. Our findings suggest that more informative scores or new data integration methods are warranted to further improve the power of GWAS by leveraging the variant functional annotations.
Simulation and Sensitivity Analysis and Cross-Validation, Demonstrating the Utility of Genteract GxE Discovery methods
Authors: Holohan Brody , Laderman Raphael
Published: November 2020

Gene-environment interactions are at the heart of why many complex traits are not fully heritable, and why prediction of disease incidence and individual response to environmental changes based on genetics has been underwhelming in utility. Understanding these interactions is the primary limiting factor for the application of personalized medicine, but current methods are not well suited for dealing with complex traits that pose both a dimensionality and sparse data problem to unsupervised analysis methods. Genteract has developed a proprietary analytical technique that allows for detection and interpretation of GxEs regarding specific pairs of a single phenotype with a single environmental factor; these methods allow us to develop a platform that can be used to predict how individuals will respond to changes in their environment based on their genetics. To validate the methods we performed two types of testing: cross-validation against a dataset of clinical study results, and application of the methods in a simulated dataset. These tests enable a greater understanding of the methods’ utility, statistical power and predictive capabilities.
Non-targeting control for MISSION® shRNA library silences SNRPD3 leading to cell death or permanent growth arrest
Authors: Czarnek Maria , Sarad Katarzyna , Karaś Agnieszka , Kochan Jakub , Bereta Joanna
Published: November 2020

In parallel with the expansion of RNA interference techniques evidence has been accumulating that RNAi analyses may be seriously biased due to off-target effects of gene-specific shRNAs. Our work points to another possible source of misinterpretations of shRNA-based data – off-target effects of non-targeting shRNA. We found that one such control for the MISSION® library (commercialized TRC library), SHC016, is cytotoxic. Using a lentiviral vector with inducible expression of SHC016 we proved that this shRNA induces apoptosis in murine cells and, depending on p53 status, senescence or mitotic catastrophe in human tumor cell lines. We identified SNRPD3, a core spliceosomal protein, as a major SHC016 target in several cell lines and confirmed in A549 and U251 cell lines that CRISPRi-knockdown of SNRPD3 mimics the effects of SHC016 expression. Our finding disqualifies non-targeting SHC016 shRNA and adds a new premise to the discussion about the sources of uncertainty of RNAi results.
Electrophysiological signatures of acute systemic lipopolysaccharide: potential implications for delirium science
Authors: Sultan Ziyad W , Jaeckel Elizabeth R , Krause Bryan M , Grady Sean M , Murphy Caitlin A , Sanders Robert D , Banks Matthew I
Published: November 2020

Background

Novel preventive therapies are needed for postoperative delirium, which especially affects aged patients. A mouse model is presented that captures inflammation-associated cortical slow wave activity (SWA) observed in patients, allowing exploration of the mechanistic role of prostaglandin-adenosine signaling.

Methods

EEG and cortical cytokine measurements (interleukin 6 [IL-6], monocyte chemoattractant protein-1 [MCP-1]) were obtained from adult and aged mice. Behavior, SWA, and functional connectivity (alpha-band weighted phase lag index) were assayed before and after systemic administration of lipopolysaccharide (LPS) +/- piroxicam (cyclooxygenase inhibitor) or caffeine (adenosine receptor antagonist). To avoid confounds from inflammation-driven changes in movement, which alter SWA and connectivity, electrophysiological recordings were classified as occurring during quiescence or movement, and propensity score matching used to match distributions of movement magnitude between baseline and LPS.

Results

LPS produces increases in cortical cytokines and behavioral quiescence. In movement-matched data, LPS produces increases in SWA (likelihood-ratio test: χ2(4)=21.51, p=0.00057), but not connectivity (χ2(4)=6.39, p=0.17). Increases in SWA associate with IL6 (p<0.001) and MCP-1 (p=0.001) and are suppressed by piroxicam (p<0.001) and caffeine (p=0.046). Aged animals compared to adult show similar LPS-induced SWA during movement, but exaggerated cytokine response and increased SWA during quiescence.

Conclusions

Cytokine-SWA correlations during wakefulness are consistent with observations in patients with delirium. Absence of connectivity effects after accounting for movement changes suggests decreased connectivity in patients is a biomarker of hypoactivity. Exaggerated effects in quiescent aged animals are consistent with increased hypoactive delirium in older patients. Prostaglandin-adenosine signaling may link inflammation to neural changes and hence delirium.
Physiological role of the 3’IgH CBEs super-anchor in antibody class switching
Authors: Zhang Xuefei , Yoon Hye Suk , Chapdelaine-Williams Aimee M. , Kyritsis Nia , Alt Frederick W.
Published: November 2020

IgH class switch recombination (CSR) replaces Cμ constant region (CH) exons with one of six downstream CHS by joining transcription-targeted DSBs in the Cμ switch (S) region to DSBs in a downstream S region. Chromatin loop extrusion underlies fundamental CSR mechanisms including 3’IgH regulatory region (3’IgHRR)-mediated S region transcription, CSR center formation, and deletional CSR joining. There are ten consecutive CTCF binding elements (CBEs) downstream of the 3’IgHRR, termed the “3’IgH CBEs”. Prior studies showed that deletion of eight 3’IgH CBEs did not detectably affect CSR. Here, we report that deletion of all 3’IgH CBEs impacts, to varying degrees, germline transcription and CSR of upstream S regions, except Sγ1. Moreover, deletion of all 3’IgH CBEs rendered the 6kb region just downstream highly transcribed and caused sequences within to be aligned with Sμ, broken, and joined to form aberrant CSR rearrangements. These findings implicate the 3’IgH CBEs as a critical insulator for focusing loop extrusion-mediated 3’IgHRR transcriptional and CSR activities on upstream CH locus targets.

Significance

B lymphocytes change antibody heavy chain (IgH) isotypes by a recombination/deletion process called IgH class switch recombination (CSR). CSR involves introduction of DNA breaks into a donor switch (S) region and also into one of six downstream S regions, with joining of the breaks changing antibody isotype. A chromatin super-anchor, of unknown function, is located just downstream of the IgH locus. We show that complete deletion of this super-anchor variably decreases CSR to most S regions and creates an ectopic S region downstream of IgH locus that undergoes aberrant CSR-driven chromosomal rearrangements. Based on these and other findings, we conclude that the super-anchor downstream of IgH is a critical insulator for focusing potentially dangerous CSR rearrangements to the IgH locus.
Establishment of Multi-stage Intravenous Self-administration Paradigms in Mice
Authors: Slosky Lauren M. , Pires Andrea , Bai Yushi , Clark Nicholas , Hauser Elizabeth R. , Gross Joshua D. , Porkka Fiona , Zhou Yang , Chen Xiaoxiao , Pogorelov Vladimir M. , Toth Krisztian , Wetsel William C. , Barak Lawrence S. , Caron Marc G.
Published: November 2020

A genetically tractable animal model would provide a needed strategy to resolve the biological basis of drug addiction. Intravenous self-administration (IVSA) is the gold standard for modeling cocaine and opioid addiction in animals, but technical limitations have precluded the widespread use of IVSA in mice. Here, we describe the first IVSA paradigms for mice that capture the multi-stage nature of the disease and permit predictive modeling. Mice with long-standing indwelling jugular catheters engaged in cocaine or opioid-associated lever responding that was fixed ratio- and dose-dependent, extinguished by the withholding of drug, and reinstated by the presentation of paired cues. Machine learning revealed that vulnerability to drug seeking and relapse were predicted by a mouse’sa prioriresponse to novelty, sensitivity to drug-induced locomotion, and drug-taking behavior. Application of this behavioral and analysis approach to genetically-engineered mice will facilitate the identification of the neural circuits driving addiction susceptibility and relapse and focused therapeutic development.
Using distance on the Riemannian manifold to compare representations in brain and models
Authors: Shahbazi Mahdiyar , Shirali Ali , Aghajan Hamid , Nili Hamed
Published: November 2020

Representational similarity analysis (RSA) summarizes activity patterns for a set of experimental conditions into a matrix composed of pairwise comparisons between activity patterns. Two examples of such matrices are the condition-by-condition inner product matrix or the correlation matrix. These representational matrices reside on the manifold of positive semidefinite matrices, called the Riemannian manifold. We hypothesize that representational similarities would be more accurately quantified by considering the underlying manifold of the representational matrices. Thus, we introduce the distance on the Riemannian manifold as a metric for comparing representations. Analyzing simulated and real fMRI data and considering a wide range of metrics, we show that the Riemannian distance is least susceptible to sampling bias, results in larger intra-subject reliability, and affords searchlight mapping with high sensitivity and specificity. Furthermore, we show that the Riemannian distance can be used for measuring multi-dimensional connectivity. This measure captures both univariate and multivariate connectivity and is also more sensitive to nonlinear regional interactions compared to the state-of-the-art measures. Applying our proposed metric to neural network representations of natural images, we demonstrate that it also possesses outstanding performance in quantifying similarity in models. Taken together, our results lend credence to the proposition that RSA should consider the manifold of the representational matrices to summarize response patterns in the brain and models.
Profiling sensory neuron microenvironment after peripheral and central axon injury reveals key pathways for neural repair
Authors: Avraham Oshri , Feng Rui , Ewan Eric E. , Rustenhoven Justin , Zhao Guoyan , Cavalli Valeria
Published: November 2020

Sensory neurons with cell bodies in dorsal root ganglia (DRG) represent a useful model to study axon regeneration. Whereas regeneration and functional recovery occurs after peripheral nerve injury, spinal cord injury or dorsal root injury is not followed by regenerative outcomes. Regeneration of sensory axons in peripheral nerves is not entirely cell autonomous. Whether the DRG microenvironment influences the different regenerative capacities after injury to peripheral or central axons remains largely unknown. To answer this question, we performed a single cell transcriptional profiling of mouse DRG in response to peripheral (sciatic nerve crush) and central axon injuries (dorsal root crush and spinal cord injury). Each cell type responded differently to the three types of injuries. All injuries increased the proportion of a cell type that shares features of both immune cells and glial cells. A distinct subset of satellite glial cells (SGC) appeared specifically in response to peripheral nerve injury. Activation of the PPARα signaling pathway in SGC, which promotes axon regeneration after peripheral nerve injury, failed to occur after central axon injuries. Treatment with the FDA-approved PPARα agonist fenofibrate increased axon regeneration after dorsal root injury. This study provides a map of the distinct DRG microenvironment responses to peripheral and central injuries at the single cell level and highlights that manipulating non-neuronal cells could lead to avenues to promote functional recovery after CNS injuries or disease.
Mucosal vaccination with cyclic-di-nucleotide adjuvants induces effective T cell homing and IL-17 dependent protection againstM. tuberculosisinfection
Authors: Jong Robyn M. , Van Dis Erik , Nguyenla Xammy , Baltodano Alexander , Pastenkos Gabrielle , Xu Chenling , Yosef Nir , McWhirter Sarah M. , Stanley Sarah A.
Published: November 2020

The only licensed vaccine for tuberculosis,Mycobacterium bovisBacille Calmette-Guérin (BCG), is not reliably effective against adult pulmonary tuberculosis. A major hurdle to tuberculosis vaccine development is incomplete understanding of successful immunity against the causative agentMycobacterium tuberculosis. Recently, we demonstrated that a protein subunit vaccine adjuvanted with STING-activating cyclic-di-nucleotides (CDNs) robustly protects against tuberculosis infection in mice. Here we show mucosal vaccination with this vaccine induces production of T cells that home to lung parenchyma and penetrate lesions in the lung. Protection is partially dependent on IL-17, type I interferon (IFN), and IFN-γ, while the transcription factor STAT-6 is dispensable. Single cell transcriptomics reveals mucosal vaccination with a CDN vaccine increases transcriptional heterogeneity in CD4 cells, including a significant population of non-classical IFN-γ and IL-17 co-expressing Th1-Th17 cells, as well as markers of memory and activation. Th1-Th17 cells in vaccinated mice are enriched for expression of the T cell functional markersTnfsf8andIl1r1relative to more conventional Th1 cells. These data provide critical insight into the immune mediators and diversity of T cell responses that can contribute to vaccine efficacy againstM. tuberculosisinfection.
Pharmacologic manipulation of complement receptor 3 prevents dendritic spine loss and cognitive impairment after acute cranial radiation
Authors: Hinkle Joshua J. , Olschowka John A. , Williams Jacqueline P. , O’Banion M. Kerry
Published: November 2020

Cranial irradiation induces healthy tissue damage that can lead to neurocognitive complications and negatively impact patient quality of life. One type of damage associated with cognitive impairment is loss of neuronal spine density. Based on developmental and disease studies implicating microglia and complement in dendritic spine loss, we hypothesized that irradiation-mediated spine loss is microglial complement receptor 3 (CR3)-dependent, and associated with late-delayed cognitive deficits. Utilizing a model of cranial irradiation (acute, 10 Gy gamma) in C57BL/6 mice we found that male mice demonstrate irradiation-mediated spine loss and cognitive deficits whereas female mice and CR3 knockout mice do not. Moreover, pharmacological blockade of CR3 with leukadherin-1 (LA1) prevented these changes in irradiated male mice. Interestingly, CR3 KO mice showed reduced behavioral task performance suggesting that CR3 is important for normal learning and memory. Improving our understanding of irradiation-mediated mechanisms and sexual dimorphic responses is essential for the identification of novel therapeutics to reduce irradiation-induced cognitive decline and improve patient quality of life.
Identification of mutations that cooperate with defects in B cell transcription factors to initiate leukemia
Authors: Heltemes-Harris Lynn M. , Hubbard Gregory K. , La Rue Rebecca S. , Munro Sarah A. , Knudson Todd P. , Yang Rendong , Henzler Christine M. , Starr Timothy K. , Sarver Aaron L. , Kornblau Steven M. , Farrar Michael A.
Published: November 2020

The transcription factors EBF1 and PAX5 are frequently mutated in B cell acute lymphoblastic leukemia (B-ALL). We demonstrate that Pax5+/-x Ebf1+/- compound heterozygous mice develop highly penetrant leukemia. Similar results were seen in Pax5+/-x Ikzf1+/- and Ebf1+/-x Ikzf1+/- mice for B-ALL, or in Tcf7+/-x Ikzf1+/- mice for T cell leukemia. To identify genetic defects that cooperate with Pax5 and Ebf1 compound heterozygosity to initiate leukemia, we performed a Sleeping Beauty (SB) transposon screen that identified cooperating partners including gain-of-function mutations in Stat5 (∼65%) and Jak1(∼68%), or loss-of-function mutations in Cblb (61%) and Myb (32%). These findings underscore the role of JAK/STAT5 signaling in B cell transformation and demonstrate unexpected roles for loss-of-function mutations in Cblb and Myb in leukemic transformation. RNA-Seq studies demonstrated upregulation of a PDK1>SGK3>MYC pathway; treatment of Pax5+/-x Ebf1+/- leukemia cells with PDK1 inhibitors blocked proliferation in vitro. Finally, we identified conserved transcriptional variation in a subset of genes between human leukemias and our mouse B-ALL models. Thus, compound haploinsufficiency for B cell transcription factors likely plays a critical role in transformation of human B cells and suggest that PDK1 inhibitors may be effective for treating patients with such defects.
Plume dynamics structure the spatiotemporal activity of glomerular networks in the mouse olfactory bulb
Authors: Lewis Suzanne M. , Xu Lai , Rigolli Nicola , Tariq Mohammad F. , Stern Merav , Seminara Agnese , Gire David H.
Published: November 2020

Although mice locate resources using turbulent airborne odor plumes, the stochasticity and intermittency of fluctuating plumes create challenges for interpreting odor cues in natural environments. Population activity within the olfactory bulb (OB), is thought to process this complex spatial and temporal information, but how plume dynamics impact odor representation in this early stage of the mouse olfactory system is not known. Limitations in odor detection technology have made it impossible to measure plume fluctuations while simultaneously recording from the mouse’s brain. Thus, previous studies have measured OB activity following controlled odor pulses of varying profiles or frequencies, but this approach only captures a subset of features found within olfactory plumes. Adequately sampling this feature space is difficult given a lack of knowledge regarding which features the brain extracts during exposure to natural olfactory scenes. Here we measured OB responses to naturally fluctuating odor plumes using a miniature, adapted odor sensor combined with wide-field GCaMP6f signaling from the dendrites of mitral and tufted (MT) cells imaged in olfactory glomeruli of head-fixed mice. We precisely tracked plume dynamics and imaged glomerular responses to this fluctuating input, while varying flow conditions across a range of ethologically-relevant values. We found that a consistent portion of MT activity in glomeruli follows odor concentration dynamics, and the strongest responding glomeruli are the best at following fluctuations within odor plumes. Further, the reliability and average response magnitude of glomerular populations of MT cells are affected by the flow condition in which the animal samples the plume, with the fidelity of plume following by MT cells increasing in conditions of higher flow velocity where odor dynamics result in intermittent whiffs of stronger concentration. Thus, the flow environment in which an animal encounters an odor has a large-scale impact on the temporal representation of an odor plume in the OB. Additionally, across flow conditions odor dynamics are a major driver of activity in many glomerular networks. Taken together, these data demonstrate that plume dynamics structure olfactory representations in the first stage of odor processing in the mouse olfactory system.
NMDA receptor signalling controls R-type calcium channel levels at the neuronal synapse
Authors: Glebov Oleg O.
Published: November 2020

Regulation of extracellular Ca++ influx by neuronal activity is a key mechanism underlying synaptic plasticity. At the neuronal synapse, activity-dependent Ca++ entry involves NMDA-type glutamate receptors (NMDARs) and voltage-gated calcium channels (VGCCs); the relationship between NMDARs and VGCCs, however, is poorly understood. Here, I report that neuronal activity specifically regulates synaptic levels of R-type VGCCs through synaptic NMDAR signalling and protein translation. This finding reveals a link between two key neuronal signalling pathways, suggesting a feedback mode for regulation of Ca++ signalling at the synapse.
Accelerating Drug Discovery and Repurposing by Combining Transcriptional Signature Connectivity with Docking
Authors: Thorman Alexander W. , Reigle James , Chutipongtanate Somchai , Shamsaei Behrouz , Pilarczyk Marcin , Fazel-Najafabadi Mehdi , Adamczak Rafal , Kouril Michal , Morrow Ardythe L. , Czyzyk-Krzeska Maria F. , McCullumsmith Robert , Seibel William , Nassar Nicolas , Zheng Yi , Hildeman David , Herr Andrew B. , Medvedovic Mario , Meller Jarek
Published: November 2020

The development of targeted treatment options for precision medicine is hampered by a slow and costly process of drug screening. While small molecule docking simulations are often applied in conjunction with cheminformatic methods to reduce the number of candidate molecules to be tested experimentally, the current approaches suffer from high false positive rates and are computationally expensive. Here, we present a novel in silico approach for drug discovery and repurposing, dubbed connectivity enhanced Structure Activity Relationship (ceSAR) that improves on current methods by combining docking and virtual screening approaches with pharmacogenomics and transcriptional signature connectivity analysis. ceSAR builds on the landmark LINCS library of transcriptional signatures of over 20,000 drug-like molecules and ~5,000 gene knock-downs (KDs) to connect small molecules and their potential targets. For a set of candidate molecules and specific target gene, candidate molecules are first ranked by chemical similarity to their ‘concordant’ LINCS analogs that share signature similarity with a knock-down of the target gene. An efficient method for chemical similarity search, optimized for sparse binary fingerprints of chemical moieties, is used to enable fast searches for large libraries of small molecules. A small subset of candidate compounds identified in the first step is then re-scored by combining signature connectivity with docking simulations. On a set of 20 DUD-E benchmark targets with LINCS KDs, the consensus approach reduces significantly false positive rates, improving the median precision 3-fold over docking methods at the extreme library reduction. We conclude that signature connectivity and docking provide complementary signals, offering an avenue to improve the accuracy of virtual screening while reducing run times by multiple orders of magnitude.
Identification of fungal limonene-3-hydroxylases for biotechnological menthol production
Authors: Schempp Florence M. , Strobel Ingmar , Etschmann Maria M. W. , Bierwirth Elena , Panten Johannes , Schewe Hendrik , Schrader Jens , Buchhaupt Markus
Published: November 2020

More than 30,000 tons of menthol are produced every year as a flavor and fragrance compound or as medical component. So far, only extraction from plant material or chemical synthesis is possible. A sustainable alternative approach for menthol production could be a biotechnological-chemical two-step conversion, starting from (+)-limonene, which is a side product of the citrus processing industry. The first step requires a limonene-3-hydroxylase (L3H) activity that specifically catalyzes hydroxylation of limonene at carbon atom 3. Several protein engineering strategies already attempted to create limonene-3-hydroxylases from bacterial cytochrome P450 monooxygenases (CYPs or P450s), which can be efficiently expressed in bacterial hosts. However, their regiospecificity is rather low, if compared to the highly selective L3H enzymes from the biosynthetic pathway towards menthol in Mentha species. The only naturally occurring limonene-3-hydroxylase activity identified in microorganisms so far, was reported for a strain of the black yeast-like fungus Hormonema sp. in South Africa.

We have discovered further fungi that can catalyze the intended reaction and identified potential CYP-encoding genes within the genome sequence of one of the strains. Using heterologous gene expression and biotransformation experiments in yeasts, we were able to identify limonene-3-hydroxylases from Aureobasidium pullulans and Hormonema carpetanum. Further characterization of the A. pullulans enzyme demonstrated its high stereospecificity and regioselectivity, its potential for limonene-based menthol production and its additional ability to convert α-and β-pinene to verbenol and pinocarveol, respectively.

Importance

(−)-Menthol is an important flavor and fragrance compound and furthermore has medicinal uses. To realize a two-step synthesis starting from renewable (+)-limonene, a regioselective limonene-3-hydroxylase enzyme is necessary. We identified enzymes from two different fungi, which catalyze this hydroxylation reaction and represent an important module for the development of a biotechnological process for (−)-menthol production from renewable (+)-limonene.
Muscle Van Gogh-like 2 shapes the neuromuscular synapse by regulating MuSK signaling activity
Authors: Boëx Myriam , Messéant Julien , Cottin Steve , Halliez Marius , Bauché Stéphanie , Buon Céline , Sans Nathalie , Montcouquiol Mireille , Molgó Jordi , Amar Muriel , Ferry Arnaud , Lemaitre Mégane , Rouche Andrée , Langui Dominique , Baskaran Asha , Fontaine Bertrand , Strochlic Laure
Published: November 2020

The development of the neuromuscular junction (NMJ) requires dynamic trans-synaptic coordination orchestrated by secreted factors, including the morphogens of the Wnt family. Yet, how the signal of these synaptic cues is transduced, and particularly during the regulation of acetylcholine receptor (AChR) accumulation in the postsynaptic membrane remains unclear. We explored the function of Van Gogh-Like protein 2 (Vangl2), a core component of Wnt planar cell polarity signaling. We showed that the conditional genetic ablation of Vangl2 in muscle reproduces the NMJ differentiation defects in mice with constitutive Vangl2 deletion. These alterations persisted into adulthood with NMJs disassembly leading to an impairment of neurotransmission and motor function deficits. Mechanistically, we found that Vangl2 and the muscle-specific kinase MuSK acted in the same genetic pathway and that Vangl2 binds MuSK, thus controlling its signaling activity. Our results identify Vangl2 as a key player of the core complex of molecules shaping neuromuscular synapses and shed light on the molecular mechanisms underlying NMJ assembly.
Host-directed FDA-approved drugs with antiviral activity against SARS-CoV-2 identified by hierarchical in silico/in vitro screening methods
Authors: Ginex Tiziana , Garaigorta Urtzi , Ramírez David , Castro Victoria , Nozal Vanesa , Maestro Ines , García-Cárceles Javier , Campillo Nuria E. , Martinez Ana , Gastaminza Pablo , Gil Carmen
Published: November 2020

The unprecedent situation generated by the COVID-19 global emergency prompted us to actively work to fight against this pandemic by searching for repurposable agents among FDA approved drugs to shed light into immediate opportunities for the treatment of COVID-19 patients.

In the attempt to proceed toward a proper rationalization of the search for new antivirals among approved drugs, we carried out a hierarchical in silico/in vitro protocol which successfully combines virtual and biological screening to speed up the identification of host-directed therapies against COVID-19 in an effective way.

To this end a multi-target virtual screening approach focused on host-based targets related to viral entry followed by the experimental evaluation of the antiviral activity of selected compounds has been carried out. As a result, five different potentially repurposable drugs interfering with viral entry, cepharantine, clofazimine, metergoline, imatinib and efloxate, have been identified.
Cellular Synchronisation through Unidirectional and Phase-Gated Signalling
Authors: Roth Gregory , Misailidis Georgios , Tsiairis Charisios D.
Published: November 2020

Multiple natural and artificial oscillator systems achieve synchronisation when oscillators are coupled. The coupling mechanism, essentially the communication between oscillators, is often assumed to be continuous and bidirectional. However, the cells of the presomitic mesoderm synchronise their gene expression oscillations through Notch signalling, which is intermittent and directed from a ligand-presenting to a receptor-presenting cell. Motivated by this mode of communication we present a phase-gated and unidirectional coupling mechanism. We identify conditions under which it can successfully bring two or more oscillators to cycle in-phase. In the presomitic mesoderm we observed the oscillatory dynamics of two synchronizing cell populations and record one population halting its pace while the other keeps undisturbed, as would be predicted from our model. For the same system another important prediction, convergence to a specific range of phases upon synchronisation is also confirmed. Thus, the proposed mechanism accurately describes the coordinated oscillations of the presomitic mesoderm cells and provides an alternative framework for deciphering synchronisation.
Forever Young: Structural Stability of Telomeric Guanine-Quadruplexes in Presence of Oxidative DNA Lesions
Authors: Miclot Tom , Corbier Camille , Terenzi Alessio , Hognon Cécilia , Grandemange Stéphanie , Barone Giampaolo , Monari Antonio
Published: November 2020

Human telomeric DNA (h-Telo), in G-quadruplex (G4) conformation, is characterized by a remarkable structural stability that confers it the capacity to resist to oxidative stress producing one or even clustered 8-oxoguanine lesions. We present a combined experimental/computational investigation, by using circular dichroism in aqueous solutions, cellular immunofluorescence assays and molecular dynamics (MD) simulations, that identifies the crucial role of the stability of G4s to oxidative lesions, related also to their biological role as inhibitors of telomerase, an enzyme overexpressed in most cancers associated to oxidative stress.
Kinome-wide RNAi screen uncovers role of Ballchen in maintenance of gene activation by trithorax group in Drosophila
Authors: Khan Muhammad Haider Farooq , Akhtar Jawad , Umer Zain , Shaheen Najma , Shaukat Ammad , Mithani Aziz , Anwar Saima , Tariq Muhammad
Published: November 2020

Polycomb group (PcG) and trithorax group (trxG) proteins are evolutionary conserved factors that contribute to cell fate determination and maintenance of cellular identities during development of multicellular organisms. The PcG behaves as repressors to maintain heritable patterns of gene silencing and trxG act as anti-silencing factors by maintaining activation of cell type specific genes. Genetic and molecular analysis has revealed extensive details about how different PcG and trxG complexes antagonize each other to maintain cell fates, however the cellular signaling components that contribute to maintenance of gene expression by PcG/trxG remain elusive. Here, we report an ex vivo kinome-wide RNAi screen in Drosophila aimed to identify cell signaling genes that facilitate trxG to counteract PcG mediated repression. From the list of trxG candidates, Ballchen (BALL), a histone kinase, known to phosphorylate histone H2A at threonine 119 (H2AT119p), was characterized as a trxG regulator. The ball mutant exhibit strong genetic interaction with Polycomb (Pc) and trithorax (trx) mutants and loss of BALL also affects expressions of trxG target genes in ball mutant embryos. BALL co-localizes with Trithorax on chromatin and depletion of BALL results in increased H2AK118 ubiquitination, a histone mark central to PcG mediated gene silencing. Moreover, analysis of genome-wide binding profile of BALL shows an overlap with 85% known binding sites of TRX across the genome. Both BALL and TRX are highly enriched at actively transcribed genes, which also correlate with presence of H3K4me3 and H3K27ac. We propose that BALL mediated signal positively contributes to the maintenance of gene activation by trxG by counteracting the repressive effect of PcG.
Linking meta-omics to the kinetics of denitrification intermediates reveals pH-dependent causes of N2O emissions and nitrite accumulation in soil
Authors: Frostegård Åsa , Vick Silas HW , Lim Natalie YN , Bakken Lars R , Shapleigh James P
Published: November 2020

Denitrifier community phenotypes often result in transient accumulation of denitrification (NO3−→NO2−→NO→N2O→N2) intermediates. Consequently, anoxic spells drive NO-, N2O- and possibly HONO-emissions to the atmosphere, affecting both climate and tropospheric chemistry. Soil pH is a key controller of intermediate levels, and while there is a clear negative correlation between pH and emission of N2O, NO2− concentrations instead increase with pH. These divergent trends are probably a combination of direct effects of pH on the expression/activity of denitrification enzymes, and an indirect effect via altered community composition. This was studied by analyzing metagenomics/transcriptomics and phenomics of two soil denitrifier communities, one of pH 3.8 (Soil3.8) and the other 6.8 (Soil6.8). Soil3.8 had severely delayed N2O reduction despite early transcription of nosZ, encoding N2O reductase, by diverse denitrifiers, and of several nosZ accessory genes. This lends support to a post-transcriptional, pH-dependent mechanism acting on the NosZ apo-protein or on enzymes involved in its maturation. Metagenome/metatranscriptome reads of nosZ were almost exclusively clade I in Soil3.8 while clade II dominated in Soil6.8. Reads of genes and transcripts for NO2−-reductase were dominated by nirK over nirS in both soils, while qPCR-based determinations showed the opposite, demonstrating that standard primer pairs only capture a fraction of the nirK community. The -omics results suggested that low NO2− concentrations in acidic soils, often ascribed to abiotic degradation, are primarily due to enzymatic activity. The NO reductase gene qnor was strongly expressed in Soil3.8, suggesting an important role in controlling NO. Production of HONO, for which some studies claim higher, others lower, emissions from NO2− accumulating soil, was estimated to be ten times higher from Soil3.8 than from Soil6.8. The study extends our understanding of denitrification-driven gas emissions and the diversity of bacteria involved and demonstrates that gene and transcript quantifications cannot always reliably predict community phenotypes.
The intraflagellar transport protein IFT20 recruits ATG16L1 to early endosomes to promote autophagosome formation in T cells
Authors: Finetti Francesca , Cassioli Chiara , Cianfanelli Valentina , Zevolini Fabrizia , Onnis Anna , Gesualdo Monica , Brunetti Jlenia , Cecconi Francesco , Baldari Cosima T.
Published: November 2020

Lymphocyte homeostasis, activation and differentiation crucially rely on basal autophagy. The fine-tuning of this process depends on autophagy-related (ATG) proteins and their interaction with the trafficking machinery that orchestrates the membrane rearrangements leading to autophagosome biogenesis. The underlying mechanisms are as yet not fully understood. The intraflagellar transport (IFT) system, known for its role in cargo transport along the axonemal microtubules of the primary cilium, has emerged as a regulator of autophagy in ciliated cells. Growing evidence indicates that ciliogenesis proteins participate in cilia-independent processes, including autophagy, in the non-ciliated T cell. Here we investigate the mechanism by which IFT20, an integral component of the IFT system, regulates basal T cell autophagy. We show that IFT20 interacts with the core autophagy protein ATG16L1 through its CC domain, which is also essential for its pro-autophagic activity. We demonstrate that IFT20 is required for the association of ATG16L1 with the Golgi complex and early endosomes, both of which have been identified as membrane sources for phagophore elongation. This involves the ability of IFT20 to interact with proteins that are resident at these subcellular localizations, namely the golgin GMAP210 at the Golgi apparatus and Rab5 at early endosomes. GMAP210 depletion, while leading to a dispersion of ATG16L1 from the Golgi, did not affect basal autophagy. Conversely, IFT20 was found to recruit ATG16L1 to early endosomes tagged for autophagosome formation by the BECLIN 1/VPS34/Rab5 complex, which resulted in the local accumulation of LC3. Hence IFT20 participates in autophagosome biogenesis under basal conditions by regulating the localization of ATG16L1 at early endosomes to promote autophagosome biogenesis. These data identify IFT20 as a new regulator of an early step of basal autophagy in T cells.
The cerebellum is involved in internal and external speech error monitoring
Authors: Runnqvist Elin , Chanoine Valérie , Strijkers Kristof , Patamadilok Chotiga , Bonnard Mireille , Nazarian Bruno , Sein Julien , Anton Jean-Luc , Belin Pascal , Alario F.- Xavier
Published: November 2020

An fMRI study examined how speakers inspect their own speech for errors. In a word production task, we observed enhanced involvement of the right posterior cerebellum for trials that were correct, but on which participants were more likely to make a word-as compared to a non-word error. Furthermore, comparing errors to correctly produced utterances, we observed increased activation of the same cerebellar region, in addition to temporal and medial frontal regions. Within the framework associating the cerebellum to forward modelling of upcoming actions, this indicates that forward models of verbal actions contain information about word representations used for error monitoring even before articulation (internal monitoring). Additional resources relying on speech perception and conflict monitoring are deployed during articulation to detect overt errors (external monitoring). In summary, speech monitoring seems to recruit a network of brain regions serving domain general purposes, even for abstract levels of processing.
Leveraging both individual-level genetic data and GWAS summary statistics increases polygenic prediction
Authors: Albiñana Clara , Grove Jakob , McGrath John J. , Agerbo Esben , Wray Naomi R. , Werge Thomas , Børglum Anders D. , Mortensen Preben Bo , Privé Florian , Vilhjálmsson Bjarni J.
Published: November 2020

The accuracy of polygenic risk scores (PRSs) to predict complex diseases increases with the training sample size. PRSs are generally derived based on summary statistics from large meta-analyses of multiple genome-wide association studies (GWAS). However, it is now common for researchers to have access to large individual-level data as well, such as the UK biobank data. To the best of our knowledge, it has not yet been explored how to best combine both types of data (summary statistics and individual-level data) to optimize polygenic prediction. The most widely used approach to combine data is the meta-analysis of GWAS summary statistics (Meta-GWAS), but we show that it does not always provide the most accurate PRS. Through simulations and using twelve real case-control and quantitative traits from both iPSYCH and UK Biobank along with external GWAS summary statistics, we compare Meta-GWAS with two alternative data-combining approaches, stacked clumping and thresholding (SCT) and Meta-PRS. We find that, when large individual-level data is available, the linear combination of PRSs (Meta-PRS) is both a simple alternative to Meta-GWAS and often more accurate.
Crystallization of a nonclassical Kazal-type Carcinoscorpius rotundicauda serine protease inhibitor, CrSPI-1, complexed with subtilisin. Corrigendum
Authors: Shenoy Rajesh T. , Thangamani Saravanan , Ho Bow , Sivaraman J. , Ding Jeak Ling
Published: November 2020

The name of the first author in the article by Tulisas et al. [(2009), Acta Cryst. F65, 533–535] is corrected.