bluuebunny/crossref_metadata_2025 huggingface dataset:
Vulnerabilities in coronavirus glycan shields despite extensive glycosylation
Authors: Watanabe Yasunori , Berndsen Zachary T. , Raghwani Jayna , Seabright Gemma E. , Allen Joel D. , McLellan Jason S. , Wilson Ian A. , Bowden Thomas A. , Ward Andrew B. , Crispin Max
Published: February 2020

Severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses (CoVs) are zoonotic pathogens with high fatality rates and pandemic potential. Vaccine development has focussed on the principal target of the neutralizing humoral immune response, the spike (S) glycoprotein, which mediates receptor recognition and membrane fusion. Coronavirus S proteins are extensively glycosylated viral fusion proteins, encoding around 69-87 N-linked glycosylation sites per trimeric spike. Using a multifaceted structural approach, we reveal a specific area of high glycan density on MERS S that results in the formation of under-processed oligomannose-type glycan clusters, which was absent on SARS and HKU1 CoVs. We provide a comparison of the global glycan density of coronavirus spikes with other viral proteins including HIV-1 envelope, Lassa virus glycoprotein complex, and influenza hemagglutinin, where glycosylation plays a known role in shielding immunogenic epitopes. Consistent with the ability of the antibody-mediated immune response to effectively target and neutralize coronaviruses, we demonstrate that the glycans of coronavirus spikes are not able to form an efficacious high-density global shield to thwart the humoral immune response. Overall, our data reveal how differential organisation of viral glycosylation across class I viral fusion proteins influence not only individual glycan compositions but also the immunological pressure across the viral protein surface.
Influenza-induced oxidative stress sensitizes lung cells to bacterial toxin-mediated necroptosis
Authors: Gonzalez-Juarbe Norberto , Riegler Ashleigh N. , Jureka Alexander S. , Gilley Ryan P. , Brand Jeffrey , Trombley John E. , Scott Ninecia R. , Dube Peter H. , Petit Chad M. , Harrod Kevin S. , Orihuela Carlos J.
Published: February 2020

Rationale

Pneumonia caused by Influenza A virus (IAV) co- and secondary bacterial infections are characterized by their severity. Previously we have shown that pore-forming toxin (PFT)-mediated necroptosis is a key driver of acute lung injury during bacterial pneumonia. Here, we evaluate the impact of IAV on PFT-induced acute lung injury during co- and secondaryStreptococcus pneumoniae(Spn) infection.

Objectives

Determine the impact of IAV infection on bacterial PFT-mediated lung epithelial cell (LEC) necroptosis. Determine the molecular basis for increased sensitivity and if inhibition of necroptosis or oxidative stress blocks IAV sensitization of LEC to PFT.

Methods

Mice and cells were challenged with IAV followed bySpn. Necroptosis was monitored by measuring cell death at fixed time points post-infection and immunofluorescent detection of necroptosis. Wildtype mice and LEC were treated with necroptosis inhibitors. Necroptosis effector molecule MLKL deficiency was tested for infection synergy. Oxidative damage to DNA and lipids as result of infection was measuredin vitroandin vivo. Necroptosis and anti-oxidant therapy efficacy to reduce disease severity was testedin vivo.

Measurements and Main Results

IAV synergistically sensitized LEC for PFT-mediated necroptosisin vitroand in murine models ofSpnco-infection and secondary infection. Pharmacological induction of oxidative stresssansvirus sensitized cells for PFT-mediated necroptosis. Necroptosis inhibition reduced disease severity during secondary bacterial infection.

Conclusions

IAV-induced oxidative stress sensitizes LEC for PFT-mediated necroptosis. This is a new molecular explanation for severe influenza-associated bacterial infections. Necroptosis inhibitors are potential therapeutic strategies to reduce IAV-primed bacterial pneumonia severity.

Here we demonstrate that Influenza A virus (IAV) infection synergistically sensitizes lung cells to bacterial pore-forming toxin (PFT)-mediated necroptosis. Moreover, this contributes to the severity of lung injury that is observed during co- and secondary infection withStreptococcus pneumoniae. IAV-induced oxidative stress was identified as a key factor contributing to cell sensitization and induction of oxidative stresssansvirus was sufficient to synergistically enhance susceptibility to PFT-mediated killing. Our results advance our understanding on the molecular basis of co- and secondary bacterial infection to influenza and identifies necroptosis inhibition and antioxidant therapy as potential intervention strategies.
Population dynamics ofDiatraeaspp. Under different climate offer conditions in Colombia
Authors: Arbeláez Julián Andrés Valencia , Giraldo Alberto Soto , Villa Gabriel Jaime Castaño , Espinosa Luis Fernando Vallejo , Gutiérrez Melba Ruth Salazar , Orozco Germán Andrés Vargas
Published: February 2020

Seasonal temperature and precipitation patterns on a global scale are the main factors to identify the sharing of organisms. Accordingly, insects and plants come to adapt to combinations of various factors through natural selection, although periodic outbreaks in insect populations occur especially in areas where they have not been previously reported, is a phenomenon that is considered as a consequence of global warming. In the study, we sought estimate the distribution of the sugarcane stem borers,Diatraeaspp., under different climate scenarios. Weekly collections were carried out in four sugarcane field plots in four different towns from the Colombian department of Caldas during a consecutive year, and also from sugarcane plots from the Cauca river valley between 2010 and 2017. The influence of climatic variables on the climate in different agro-ecological zones of sugarcane crops (Saccharumsp.) was defined by using climatic data (maximum, minimum and daily temperatures; accumulated precipitation) on a daily scale between 2010 and 2017. MODESTR® was used to generate the distribution maps to estimate probability distributions subject to restrictions given by the environmental information.Diatraeaspp. is strongly influenced by the effects of climate change, considerably reducing its population niches as well as the number of individuals. The estimate of an optimal niche forDiatraeaspp. includes temperatures between 20°C and 23°C, accumulated annual rainfall between 1200 and 1500 mm, months with dry conditions, whose precipitation is below 50 mm, slopes below 0.05, crop heterogeneity with an index of 0.2 and primary production values of 1.0.

Summary statement

Diatraeaspp. is susceptible to temperature variations due to climate change, it is presumed that its adaptability could benefitDiatraeaspp. in establishing itself in new areas.
Are Antisense Proteins in Prokaryotes Functional?
Authors: Ardern Zachary , Neuhaus Klaus , Scherer Siegfried
Published: February 2020

Many prokaryotic RNAs are transcribed from loci outside of annotated protein coding genes. Across bacterial species hundreds of short open reading frames antisense to annotated genes show evidence of both transcription and translation, for instance in ribosome profiling data. Determining the functional fraction of these protein products awaits further research, including insights from studies of molecular interactions and detailed evolutionary analysis. There are multiple lines of evidence however that many of these newly discovered proteins are of use to the organism. Condition-specific phenotypes have been characterised for a few. These proteins should be added to genome annotations, and the methods for predicting them standardised. Evolutionary analysis of these typically young sequences also may provide important insights into gene evolution. This research should be prioritised for its exciting potential to uncover large numbers of novel proteins with extremely diverse potential practical uses, including applications in synthetic biology and responding to pathogens.
Deep Neural Networks Carve the Brain at its Joints
Authors: Bertolero Maxwell A. , Bassett Danielle S.
Published: February 2020

How an individual’s unique brain connectivity determines that individual’s cognition, behavior, and risk for pathology is a fundamental question in basic and clinical neuroscience. In seeking answers, many have turned to machine learning, with some noting the particular promise of deep neural networks in modelling complex non-linear functions. However, it is not clear that complex functions actually exist between brain connectivity and behavior, and thus if deep neural networks necessarily outperform simpler linear models, or if their results would be interpretable. Here we show that, across 52 subject measures of cognition and behavior, deep neural networks fit to each brain region’s connectivity outperform linear regression, particularly for the brain’s connector hubs—regions with diverse brain connectivity—whereas the two approaches perform similarly when fit to brain systems. Critically, averaging deep neural network predictions across brain regions results in the most accurate predictions, demonstrating the ability of deep neural networks to easily model the various functions that exists between regional brain connectivity and behavior, carving the brain at its joints. Finally, we shine light into the black box of deep neural networks using multislice network models. We determined that the relationship between connector hubs and behavior is best captured by modular deep neural networks. Our results demonstrate that both simple and complex relationships exist between brain connectivity and behavior, and that deep neural networks can fit both. Moreover, deep neural networks are particularly powerful when they are first fit to the various functions of a system independently and then combined. Finally, deep neural networks are interpretable when their architectures are structurally characterized using multislice network models.
Achieving symptom relief in patients with Myalgic encephalomyelitis by targeting the neuro-immune interface and inducing disease tolerance
Authors: Rodriguez Lucie S.T. , Pou Christian , Lakshmikanth Tadepally , Zhang Jingdian , Mugabo Constantin Habimana , Wang Jun , Mikes Jaromir , Olin Axel , Chen Yang , Rorbach Joanna , Juto Jan-Erik , Li Tie Qiang , Julin Per , Brodin Petter
Published: February 2020

Myalgic encephalomyelitis, ME, previously also known as chronic fatigue syndrome (CFS) is a heterogeneous, debilitating syndrome of unknown etiology responsible for long-lasting disability in millions of patients worldwide. The most well-known symptom of ME is post-exertional malaise, but many patients also experience autonomic dysregulation, cranial nerve dysfunction and signs of immune system activation. Many patients also report a sudden onset of disease following an infection. The brainstem is a suspected focal point in ME pathogenesis and patients with structural impairment to the brainstem often show ME-like symptoms. The brainstem is also where the vagus nerve originates, a critical neuro-immune interface and mediator of the inflammatory reflex which regulate systemic inflammation. Here we report the results of a randomized, placebo-controlled trial using intranasal mechanical stimulation (INMEST) targeting the vagus nuclei, and higher centers in the brain of ME-patients and induce a sustainable, ∼30% reduction in overall symptom scores after eight weeks of treatment. By performing longitudinal, systems-level monitoring of the blood immune system in these patients, we uncover chronic immune activation in ME, as well as immunological correlates of improvement that center around the IL-17 axis, gut-homing immune cells and reduced inflammation. The mechanisms of symptom relief remains to be determined, but transcriptional analyses suggest an upregulation of disease tolerance mechanisms. We wish for these results to bring some hope to patients suffering from ME and inspire researchers to help test our new hypothesis that ME is a condition caused by a failure of inducing disease tolerance upon infection and persistent immune activation.
Mapping human laryngeal motor cortex during vocalization
Authors: Eichert Nicole , Papp Daniel , Mars Rogier B. , Watkins Kate E.
Published: February 2020

The representations of the articulators involved in human speech production are organized somatotopically in primary motor cortex. The neural representation of the larynx, however, remains debated. Both a dorsal and a ventral larynx representation have been previously described. It is unknown, however, whether both representations are located in primary motor cortex. Here, we mapped the motor representations of the human larynx using fMRI and characterized the cortical microstructure underlying the activated regions. We isolated brain activity related to laryngeal activity during vocalization while controlling for breathing. We also mapped the articulators (the lips and tongue) and the hand area. We found two separate activations during vocalization – a dorsal and a ventral larynx representation. Structural and quantitative neuroimaging revealed that myelin content and cortical thickness underlying the dorsal, but not the ventral larynx representation, are similar to those of other primary motor representations. This finding confirms that the dorsal larynx representation is located in primary motor cortex and that the ventral one is not. We further speculate that the location of the ventral larynx representation is in premotor cortex, as seen in other primates. It remains unclear, however, whether and how these two representations differentially contribute to laryngeal motor control.
Metabolic defects cause hyperactive mitochondria and Parkinson’s disease-like traits
Authors: Mor Danielle E. , Sohrabi Salman , Kaletsky Rachel , Keyes Will , Kalia Vrinda , Miller Gary W. , Murphy Coleen T.
Published: February 2020

Metabolic dysfunction is a facet of many age-related neurodegenerative diseases, yet its role in disease etiology remains poorly understood1. We recently discovered a potential causal link between the branched-chain amino acid transferase,BCAT-1, and the neurodegenerative movement disorder, Parkinson’s disease (PD)2. Knockdown ofC. elegans bcat-1recapitulates PD-like features, including progressive motor deficits and neurodegeneration with age2. Using transcriptomic, metabolomic, and imaging approaches, we show thatbcat-1knockdown increases mitochondrial activity and induces oxidative damage in neurons through mTOR-independent mechanisms. We recently developed a high-throughput screening platform to identify drugs that may be repurposed for PD, and found that metformin, the leading type 2 diabetes medication, significantly improves motor function inbcat-1(RNAi)worms3. Late-in-life metformin treatment restores normal mitochondrial activity levels and protects againstbcat-1-associated neurodegeneration. Our results suggest that PD may originate as a metabolic disorder, and highlight metformin as a promising new drug candidate for PD treatment.
Network fingerprint of stimulation-induced speech impairment in essential tremor
Authors: Petry-Schmelzer Jan Niklas , Jergas Hannah , Thies Tabea , Steffen Julia K. , Reker Paul , Dafsari Haidar S. , Mücke Doris , Fink Gereon R. , Visser-Vandewalle Veerle , Dembek Till A. , Barbe Michael T.
Published: February 2020

Objective

To gain insights into structural networks associated with stimulation-induced dysarthria (SID) and to predict stimulation-induced worsening of intelligibility in essential tremor patients with bilateral thalamic deep brain stimulation (DBS).

Methods

Monopolar reviews were conducted in 14 essential tremor patients. Testing included determination of SID thresholds, intelligibility ratings and a fast syllable repetition task. Volumes of tissue activated (VTAs) were calculated to identify discriminative fibers for stimulation-induced worsening of intelligibility in a structural connectome. The resulting fiber-based atlas structure was than validated in a leave-one-out design.

Results

Fibers determined as discriminative for stimulation-induced worsening of intelligibility were mainly connected to the ipsilateral precentral gyrus as well as to both cerebellar hemispheres and the ipsilateral brainstem. In the thalamic area, they ran laterally to the thalamus and postero-medially to the subthalamic nucleus, in close proximity, mainly antero-laterally, to fibers beneficial for tremor control as published by Al-Fatly et al. (2019). The overlap of the respective clinical stimulation setting’s VTAs with these fibers explained 62.4% (p<0.001) of the variance of stimulation-induced change in intelligibility in a leave-one out analysis.

Interpretation

This study demonstrates that SID in essential tremor patients is associated with both, motor cortex and cerebellar connectivity. Furthermore, the identified fiber-based atlas structure might contribute to future postoperative programming strategies to achieve optimal tremor control without speech impairment in ET patients with thalamic DBS.
Bound2Learn: A Machine Learning Approach for Classification of DNA-Bound Proteins from Single-Molecule Tracking Experiments
Authors: Kapadia Nitin , El-Hajj Ziad W. , Reyes-Lamothe Rodrigo
Published: February 2020

DNA-bound proteins are essential elements for the maintenance, regulation, and use of the genome. The time they spend bound to DNA provides useful information on their stability within protein complexes and insight into the understanding of biological processes. Single-particle tracking allows for direct visualization of protein-DNA kinetics, however, identifying whether a molecule is bound to DNA can be non-trivial. Further complications arise when tracking molecules for extended durations in processes with slow kinetics. We developed a machine learning approach, termed Bound2Learn, using output from a widely used tracking software, to robustly classify tracks in order to accurately estimate residence times. We validated our approachin silico, and in live-cell data fromEscherichia coliandSaccharomyces cerevisiae. Our method has the potential for broad utility and is applicable to other organisms.
Systematic determination of the mitochondrial proportion in human and mice tissues for single-cell RNA sequencing data quality control
Authors: Osorio Daniel , Cai James J.
Published: February 2020

Motivation

Quality control (QC) is a critical step in single-cell RNA-seq (scRNA-seq) data analysis. Low-quality cells are removed from the analysis during the QC process to avoid misinterpretation of the data. One of the important QC metrics is the mitochondrial proportion (mtDNA%), which is used as a threshold to filter out low-quality cells. Early publications in the field established a threshold of 5% and since then, it has been used as a default in several software packages for scRNA-seq data analysis and adopted as a standard in many scRNA-seq studies. However, the validity of using a uniform threshold across different species, single-cell technologies, tissues, and cell types has not been adequately assessed.

Results

We systematically analyzed 5,530,106 cells reported in 1,349 annotated datasets available in the PanglaoDB database and found that the average mtDNA% in scRNA-seq data across human tissues is significantly higher than in mouse tissues. This difference is not confounded by the platform used to generate the data. Based on this finding, we propose new reference values of the mtDNA% for 121 tissues of mice and 44 tissues of humans. In general, for mouse tissues, the 5% threshold performs well to distinguish between healthy and low-quality cells. However, for human tissues, the 5% threshold should be reconsidered as it fails to accurately discriminate between healthy and low-quality cells in 29.5% (13 of 44) tissues analyzed. We conclude that omitting the mtDNA% QC filter or adopting a suboptimal mtDNA% threshold may lead to erroneous biological interpretations of scRNA-seq data.

Availability

The code used to download datasets, perform the analyzes, and produce the figures is available athttps://github.com/dosorio/mtProportion

Contact

dcosorioh@tamu.edu

Supplementary information

Supplementary data are available atBioinformaticsonline.
Genetic loci associated with prevalent and incident myocardial infarction and coronary heart disease in the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium
Authors: Hahn Julie , Fu Yi-Ping , Brown Michael R. , Bis Joshua C. , de Vries Paul S. , Feitosa Mary F. , Yanek Lisa R. , Weiss Stefan , Giulianini Franco , Smith Albert Vernon , Guo Xiuqing , Bartz Traci M. , Becker Diane M. , Becker Lewis C. , Boerwinkle Eric , Brody Jennifer A. , Chen Yii-Der Ida , Franco Oscar H. , Grove Megan , Harris Tamara B. , Hofman Albert , Hwang Shih-Jen , Kral Brian G. , Launer Lenore J. , Markus Marcello R. P. , Rice Kenneth M. , Rich Stephen S. , Ridker Paul M. , Rivadeneira Fernando , Rotter Jerome I. , Sotoodehnia Nona , Taylor Kent D. , Uitterlinden André G. , Völker Uwe , Völzke Henry , Yao Jie , Chasman Daniel I. , Dörr Marcus , Gudnason Vilmundur , Mathias Rasika A. , Post Wendy , Psaty Bruce M. , Dehghan Abbas , O'Donnell Christopher J. , Morrison Alanna C.
Published: February 2020

Background Genome-wide association studies have identified multiple genomic loci associated with coronary artery disease, but most are common variants in non-coding regions that provide limited information on causal genes and etiology of the disease. To better understand etiological pathways that might lead to discovery of new treatments or prevention strategies, we focused our investigation on low-frequency and rare sequence variations primarily residing in coding regions of the genome while also exploring associations with common variants. Methods and Results Using samples of individuals of European ancestry from ten cohorts within the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium, both cross-sectional and prospective analyses were conducted to examine associations between genetic variants and myocardial infarction (MI), coronary heart disease (CHD), and all-cause mortality following these events. Single variant and gene-based analyses were performed separately in each cohort and then meta-analyzed for each outcome. A low-frequency intronic variant (rs988583) in PLCL1 was significantly associated with prevalent MI (OR=1.80, 95% confidence interval: 1.43, 2.27; P =7.12 × 10 -7 ). Three common variants, rs9349379 in PHACTR1 , and rs1333048 and rs4977574 in the 9p21 region, were significantly associated with prevalent CHD. Four common variants (rs4977574, rs10757278, rs1333049, and rs1333048) within the 9p21 locus were significantly associated with incident MI. We conducted gene-based burden tests for genes with a cumulative minor allele count (cMAC) 5 and variants with minor allele frequency (MAF) < 5%. TMPRSS5 and LDLRAD1 were significantly associated with prevalent MI and CHD, respectively, and RC3H2 and ANGPTL4 were significantly associated with incident MI and CHD, respectively. No loci were significantly associated with all-cause mortality following a MI or CHD event. Conclusion This study confirmed previously reported loci influencing heart disease risk, and one single variant and three genes associated with MI and CHD were newly identified and warrant future investigation.
Molecular Characterization of invasiveEnterobacteriaceaefrom Pediatric Patients in Central and Northwestern Nigeria
Authors: Duru Carissa , Olanipekun Grace M , Odili Vivian , Kocmich Nicholas J , Rezac Amy , Ajose Theresa O , Medugu Nubwa , Ebruke Bernard , Esimone Charles , Obaro Stephen
Published: February 2020

Background

Bacteremia is a leading cause of death in developing countries but etiologic evaluation is infrequent and empiric antibiotics are not evidence-based. Very little is known about the types of extended-spectrum β-lactamases (ESBL) in pediatric bacteremia patients in Nigeria. We evaluated the patterns of ESBL resistance in children enrolled into surveillance for community acquired bacteremic syndromes across health facilities in Central and Northwestern Nigeria.

Method

Blood culture from suspected cases of sepsis from children age less than 5 years were processed using automated Bactec®incubator System from Sept 2008-Dec 2016.Enterobacteriaceawere identified to the species level using Analytical Profile Index (API20E®) identification strip and antibiotic susceptibility profile was determined by the disc diffusion method. The multidrug resistant strains were then screened and confirmed for extended spectrum beta lactamase (ESBL) production by the combination disc method as recommended by Clinical and Laboratory Standard Institute (CLSI). Real time PCR was used to elucidate the genes responsible for ESBL production characterize the resistance genes

Result

Of 21,000 children screened from Sept 2008-Dec 2016, 2,625(12.5%) were culture-positive. A total of 413Enterobacteriaceaeavailable for analysis were screened for ESBL. ESBL production was detected in 160/413(38.7%), comprisingKlebsiella pneumoniae105/160(65.6%),Enterobacter cloacae21/160(13.1%),Escherichia coli22/160(13.8%),Serratiaspecies 4/160(2.5%),Pantoeaspecies 7/160(4.4%) andCitrobacterspecies 1/160(0.6%). Of the 160 ESBL-producing isolates, high resistance rates were observed among ESBL-positive isolates for Ceftriaxone (92.3%), Aztreonam (96.8%), Cefpodoxime (96.25%), Cefotaxime (98.75%) and sulphamethoxazole-trimethoprim (90%), while 87.5 %, 90.63%, and 91.87% of the isolates were susceptible to Imipenem, Amikacin and Meropenem respectively. Frequently detected resistance genes wereblaTEM 83.75%) (134/160), and,blaCTX-M 83.12% (133/160) followed byblaSHV genes 66.25% (106/160). Co-existence ofblaCTX-M,blaTEM andblaSHV was seen in 94/160 (58.8%),blaCTX-M andblaTEM in 118/160 (73.8%),blaTEM andblaSHV in 97/160 (60.6%) andblaCTX-M andblaSHV in 100/160 (62.5%) of isolates tested.

Conclusion

Our results indicate a high prevalence of ESBL resistance to commonly used antibiotics inEnterobacteriaceaeisolates from bloodstream infections in children in this study. Careful choice of antibiotic treatment options and further studies to evaluate transmission dynamics of resistance genes could help in the reduction of ESBL resistance in these settings.
New Tricks for an old molecule: Preserved antibacterial activity of ribosomal protein S15 during evolution
Authors: Qu Baozhen , Ma Zengyu , Yao Lan , Gao Zhan , Zhang Shicui
Published: February 2020

Previous studies show that some ribosomal proteins possess antimicrobial peptide (AMP) activity. However, information as such remains rather fragmentary and limited. Here we demonstrated for the first time that amphioxus RPS15, BjRPS15, was a previously uncharacterized AMP, which was not only capable of identifying Gram-negative and -positive bacteria via interaction with LPS and LTA but also capable of killing the bacteria. We also showed that both the sequence and 3D structure of RPS15 and its prokaryotic homologs were highly conserved, suggesting its antibacterial activity is universal across widely separated taxa. Actually this was supported by the facts that the residues positioned at 45-67 formed the core region for the antimicrobial activity of BjRPS15, and its prokaryotic counterparts, includingNitrospiraeRPS1933-55,AquificaeRPS1933-55andP. syringaeRPS1950-72,similarly displayed antibacterial activities. BjRPS15 functioned by both interaction with bacterial membrane via LPS and LTA and membrane depolarization as well as induction of intracellular ROS. Moreover, we showed that RPS15 existed extracellularly in amphioxus, shrimp, zebrafish and mice, hinting it may play a critical role in systematic immunity in different animals. In addition, we found that neither BjRPS15 nor its truncated form BjRPS1545-67were toxic to mammalian cells, making them promising lead molecules for the design of novel peptide antibiotics against bacteria. Collectively, these indicate that RPS15 is a new member of AMP with ancient origin and high conservation throughout evolution.

Author summary

Ribosomal protein, a component of ribonucleoprotein particles, is traditionally known involved in protein synthesis in a cell. Here we demonstrated for the first time that amphioxus ribosomal protein 15 was a novel antibacterial protein, capable of recognizing Gram-negative and -positive bacteria as well as killing them. It killed the bacteria by a combined mode of action of disrupting bacterial membrane integrity and inducing radical oxygen species production. We also showed that both eukaryotic ribosomal protein 15 and its prokaryotic counterpart ribosomal protein 19 possessed antibacterial activity, indicating that the antibacterial property is universal for this family of molecules. Moreover, we found that ribosomal protein 15 was present in the circulation system of various animals including shrimp, amphioxus, zebrafish and mice, suggesting it may physiologically play a key role in systematic immunity. Altogether, our study provides a new angle for understanding the biological function of ribosomal proteins.
Autophagy-associated Production of Antimicrobial Peptides hBD1 and LL37 Exhibits Anti-Bacillus Calmette-Guérin Effects in Lung Epithelial Cells
Authors: Wang Rui-ning , Liu Hong-lin , Chen Yao-xin , Wen Qian , Zhou Xin-ying , Wang Jin-li , Yang Jia-hui , Li Yan-fen , Han Zhen-yu , Ma Li
Published: February 2020

Antimicrobial peptides (AMPs) constitute important groups of bactericidal polypeptides against various microorganisms that exhibit their anti-bacteria activity through cleavage of precursor peptides into the active form of 50–100 amino acids in length. Various AMP cleavage mechanisms have been reported in different cell types; however, those inMycobacterium tuberculosis(MTB)-infected lung epithelial cells remain unknown. In the present study, we found that MTB-infected lung epithelial cells expressed high level of the AMPs hBD1 and LL37 to kill intracellular MTB as the first-line immune barrier against MTB infection. Notably, their production in the lung epithelial cells was closely related to the function of autophagosomes and lysosomes. Experimental induction of autophagy in lung epithelial cells could enhance the expression of active hBD1 and LL37 at the post-transcriptional level, whereas silencing of these two active AMPs could decrease the bactericidal effect of autophagy. These findings indicated that cleavage of peptide precursors to form active AMPs might constitute a previously unrecognized antibacterial mechanism of autophagy.

Author summary

LM and RW conceived and designed the experiments; RW performed the experiments and analyzed the data; QW analyzed the data and contributed reagents/materials/analysis tools; HL performed the experiments; XZ, JY, YL and ZH analyzed the data. LM and RW drafted the manuscript.
Junctional and cytoplasmatic contributions in wound healing
Authors: Mosaffa Payman , Tetley Robert J. , Rodríguez-Ferran Antonio , Mao Yanlan , Muñoz José J.
Published: February 2020

Wound healing is characterised by the re-epitheliation of a tissue through the activation of contractile forces concentrated mainly at the wound edge. While the formation of an actin purse string has been identified as one of the main mechanisms, far less is known about the effects of the viscoelastic properties of the surrounding cells, and the different contribution of the junctional and cytoplasmic contractilities.

In this paper we simulate the wound healing process, resorting to a hybrid vertex model that includes cell boundary and cytoplasmatic contractilities explicitly, together with a differentiated viscoelastic rheology based on an adaptive rest-length. From experimental measurements of the recoil and closure phases of wounds in theDrosophilawing disc epithelium, we fit tissue viscoelastic properties. We then analyse in terms of closure rate and energy requirements the contributions of junctional and cytoplasmatic contractilities.

Our results suggest that reduction of junctional stiffness rather than cytoplasmatic stiffness has a more pronounced effect on shortening closure times, and that intercalation rate has a minor effect on the stored energy, but contributes significantly to shortening the healing process, mostly in the later stages.

Author summary

We simulate the wound healing process of epithelia in the absence of substrate. By analysing the recoil process we are able to fit the viscoelastic properties of the monolayer, and study the influence of contractility at junctions and at the interior polymer network. We numerically simulate the whole wound opening and closure process, and inspect which mechanism has a more pronounced effect in terms of energy barrier and wound closure rate. We conclude that while junctional stiffness seems to be more effective than bulk stiffness at speeding up the closure, the increase of intercalation process is the mechanism with the lowest energy cost.
Redundant Trojan horse and endothelial-circulatory mechanisms for host-mediated spread ofCandida albicansyeast
Authors: Scherer Allison K. , Blair Bailey A. , Park Jieun , Seman Brittany G. , Kelley Joshua B. , Wheeler Robert T.
Published: February 2020

The host innate immune system has developed elegant processes for the detection and clearance of invasive fungal pathogens. These strategies may also aid in the spread of pathogensin vivo, although technical limitations have previously hindered our ability to view the host innate immune and endothelial cells to probe their roles in spreading disease. Here, we have leveraged zebrafish larvae as a model to view the interactions of these host processes with the fungal pathogenCandida albicans in vivo. We examined three potential host-mediated mechanisms of fungal spread: movement inside phagocytes in a “Trojan Horse” mechanism, inflammation-assisted spread, and endothelial barrier passage. Utilizing both chemical and genetic tools, we systematically tested the loss of neutrophils and macrophages and the loss of blood flow on yeast cell spread. Both neutrophils and macrophages respond to yeast-locked and wild typeC. albicansin our model and time-lapse imaging revealed that macrophages can support yeast spread in a “Trojan Horse” mechanism. Surprisingly, loss of immune cells or inflammation does not alter dissemination dynamics. On the other hand, when blood flow is blocked, yeast can cross into blood vessels but they are limited in how far they travel. Blockade of both phagocytes and circulation reduces rates of dissemination and significantly limits fungal spread distance from the infection site. Together, this data suggests a redundant two-step process whereby (1) yeast cross the endothelium inside phagocytes or via direct uptake, and then (2) they utilize blood flow or phagocytes to travel to distant sites.

Author summary

AlthoughCandida albicansis the most common cause of fungal bloodstream infection, we know little about how it spreads from one tissue to another. Host processes often inadvertently assist pathogens that can hijack immune cells or induce endothelial endocytosis. Here we have used transparent zebrafish to visualize how specific host cells and processes contribute to yeast spreadin vivo.We find thatCandidais a sophisticated pathogen that uses redundant strategies to disseminate in the host: it can either use host immune cells or cross into the circulatory system and use blood flow. These data suggest that both of these mechanisms must be targeted to limitCandidadissemination during infection.
Loss of Corneal Raver2 Expression in Aniridia Associated Keratopathy
Authors: Das Subrata K. , Carroll Lara , Holt Derick , Zhang Xiaohui , Uehara Hironori , Archer Bonnie , Ambati Jayakrishna , Ambati Balamurali K.
Published: February 2020

Purpose

The aim of this study was to evaluate downstream factors of Pax6 associated with the aniridic corneal phenotype. We characterize the ocular expression ofRaver2, a novel heterogeneous ribonucleoprotein (hnRNP).

Methods

Genome wide microarrays of corneal RNA from non-aniridic mice (C57BL/6J and MRL/MpJ) and corneas from aniridia model, Pax6+/-(SEY) mice were performed.Raver2expression profiles were validated using qPCR and western blot. Raver2 immunohistochemistry on corneas of Pax6+/-mice and aniridic humans were compared to control specimens, and insitu-hybridization was used to analyze developmental expression ofRaver2.

Results

Microarray analysis of corneal RNA identifiedRaver2as the gene with the strongest differential expression profile across non-aniridic and aniridic mouse strains. Consistent with mouse data, we found thatRaver2is expressed at high levels in healthy human corneal epithelium, and is downregulated in the corneas of human aniridia patients.

Conclusions

These findings suggest thatRaver2has an evolutionarily conserved developmental role downstream ofPax6in corneal development and maintenance.
A non-canonical Hippo pathway regulates spindle disassembly and cytokinesis during meiosis inSaccharomyces cerevisiae
Authors: Paulissen Scott M. , Hunt Cindy A. , Slubowski Christian J. , Yu Yao , Truong Dang , Mucelli Xheni , Nguyen Hung T. , Newman-Toledo Shayla , Neiman Aaron M. , Huang Linda S.
Published: February 2020

Meiosis in the budding yeastSaccharomyces cerevisiaeis used to create haploid yeast spores from a diploid mother cell. During meiosis II, cytokinesis occurs by closure of the prospore membrane, a membrane that initiates at the spindle pole body and grows to surround each of the haploid meiotic products. Timely prospore membrane closure requiresSPS1, which encodes a STE20-family GCKIII kinase. To identify genes that may activateSPS1, we utilized a histone phosphorylation defect ofsps1mutants to screen for genes with a similar phenotype and found thatcdc15shared this phenotype.CDC15encodes a Hippo-like kinase that is part of the mitotic exit network. We find that Sps1 complexes with Cdc15, that Sps1 phosphorylation requires Cdc15, and thatCDC15is also required for timely prospore membrane closure. We also find thatSPS1, likeCDC15, is required for meiosis II spindle disassembly and sustained anaphase II release of Cdc14 in meiosis. However, the NDR-kinase complex encoded byDBF2/DBF20 MOB1which functions downstream ofCDC15in mitotic cells, does not appear to play a role in spindle disassembly, timely prospore membrane closure, or sustained anaphase II Cdc14 release. Taken together, our results suggest that the mitotic exit network is rewired for exit from meiosis II, such thatSPS1replaces the NDR-kinase complex downstream ofCDC15.
Drosophilalarval epidermal cells only exhibit epidermal aging when they persist to the adult stage
Authors: Wang Yan , Burra Sirisha , Galko Michael J.
Published: February 2020

Holometabolous insects undergo a complete transformation of the body plan from the larval to the adult stage. InDrosophila, this transformation includes replacement of larval epidermal cells (LECs) by adult epidermal cells (AECs). AECs inDrosophilaundergo a rapid and stereotyped aging program where they lose both cell membranes and nuclei. Whether LEC’s are capable of undergoing aging in a manner similar to AECs remains unknown. Here, we address this question in two ways. First, we looked for hallmarks of epidermal aging in larvae that have a greatly extended third instar and/or carry mutations that would cause premature epidermal aging at the adult stage. Such larvae, irrespective of genotype, did not show any of the signs of epidermal aging observed in the adult. Second, we developed a procedure to effect a heterochronic persistence of LECs into the adult epidermal sheet. LECs embedded within the adult epidermal sheet undergo clear signs of epidermal aging; they form multinucleate cells with each other and with the surrounding AECs on the same schedule as the AECs themselves. Our data reveals that epidermal aging in holometabolousDrosophilais a stage-specific phenomenon and that the capacity of LECs to respond to aging signals does exist.

Summary Statement

We show thatDrosophilalarval epidermal cells do not age at the larval stage. They do, however, exhibit signs of aging if they persist into the adult.
Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform
Authors: Nhu Thao Tran Thi , Labroussaa Fabien , Ebert Nadine , V’kovski Philip , Stalder Hanspeter , Portmann Jasmine , Kelly Jenna , Steiner Silvio , Holwerda Melle , Kratzel Annika , Gultom Mitra , Laloli Laura , Hüsser Linda , Wider Manon , Pfaender Stephanie , Hirt Dagny , Cippà Valentina , Crespo-Pomar Silvia , Schröder Simon , Muth Doreen , Niemeyer Daniela , Müller Marcel A. , Drosten Christian , Dijkman Ronald , Jores Joerg , Thiel Volker
Published: February 2020

Reverse genetics has been an indispensable tool revolutionising our insights into viral pathogenesis and vaccine development. Large RNA virus genomes, such as from Coronaviruses, are cumbersome to clone and to manipulate inE. colihosts due to size and occasional instability1-3. Therefore, an alternative rapid and robust reverse genetics platform for RNA viruses would benefit the research community. Here we show the full functionality of a yeast-based synthetic genomics platform for the genetic reconstruction of diverse RNA viruses, including members of theCoronaviridae, FlaviviridaeandParamyxoviridaefamilies. Viral subgenomic fragments were generated using viral isolates, cloned viral DNA, clinical samples, or synthetic DNA, and reassembled in one step inSaccharomyces cerevisiaeusing transformation associated recombination (TAR) cloning to maintain the genome as a yeast artificial chromosome (YAC). T7-RNA polymerase has been used to generate infectious RNA, which was then used to rescue viable virus. Based on this platform we have been able to engineer and resurrect chemically-synthetized clones of the recent epidemic SARS-CoV-24in only a week after receipt of the synthetic DNA fragments. The technical advance we describe here allows to rapidly responding to emerging viruses as it enables the generation and functional characterization of evolving RNA virus variants - in real-time - during an outbreak.
Silencing branched-chain ketoacid dehydrogenase or treatment with branched-chain ketoacids ex vivo inhibits muscle insulin signaling
Authors: Biswas Dipsikha , Dao Khoi T. , Mercer Angella , Cowie Andrew , Duffley Luke , El Hiani Yassine , Kienesberger Petra C. , Pulinilkunnil Thomas
Published: February 2020

Branched-chain α-keto acids (BCKAs) are downstream catabolites of branched-chain amino acids (BCAAs). Mitochondrial oxidation of BCKAs is catalyzed by branched-chain ketoacid dehydrogenase (BCKDH), an enzyme sensitive to inhibitory phosphorylation by BCKD kinase (BCKDK). Emerging studies show that defective BCAA catabolism and elevated BCKAs levels correlate with glucose intolerance and cardiac dysfunction. However, if/how BCKDH and BCKDK exert control on the availability and flux of intramyocellular BCKAs and if BCKA reprograms nutrient metabolism by influencing insulin action remains unexplored. We observed altered BCAA catabolizing enzyme expression in the murine heart and skeletal muscle during physiological fasting and diet-induced obesity and after ex vivo exposure of C2C12 cells to increasing concentration of saturated fatty acid, palmitate. BCKAs per se impaired insulin-induced AKT phosphorylation and AKT activity in skeletal myotubes and cardiomyocytes. In skeletal muscle cells, mTORC1 and protein translation signaling was enhanced by BCKA with concomitant suppression of mitochondrial respiration. Lowering intracellular BCKA levels by genetic and pharmacological activation of BCKDHA enhanced insulin signaling and activated pyruvate dehydrogenase, an effector of glucose oxidation and substrate metabolism. Our findings suggest that BCKAs profoundly influence muscle insulin function, providing new insight into the molecular nexus of BCAA metabolism and signaling with cellular insulin action and respiration.
Reprogramming of Human Cells to Pluripotency Induces CENP-A Chromatin Depletion
Authors: Milagre Inês , Pereira Carolina , Oliveira Raquel , Jansen Lars E.T.
Published: February 2020

Pluripotent stem cells (PSCs) are central to development as they are the precursors of all cell types in the embryo. Therefore, maintaining a stable karyotype is essential, both for their physiological role as well as for use in regenerative medicine. In culture, an estimated 10-30% of PSC lines present karyotypic abnormalities, but the underlying causes remain unknown. To gain insight into the mitotic capacity of human embryonic stem cells and induced pluripotent stem cells, we explore the structure of the centromere and kinetochore. Centromere function depends on CENP-A nucleosome-defined chromatin. We show that while PSCs maintain abundant pools of CENP-A, CENP-C and CENP-T, these essential centromere components are strongly reduced at stem cell centromeres. Outer kinetochore recruitment is also impaired to a lesser extent, indicating an overall weaker kinetochore. This impairment is specific for the kinetochore forming centromere complex while the inner centromere protein Aurora B remains unaffected. We further show that, similar to differentiated human cells, CENP-A chromatin assembly in PSCs requires transition into G1 phase. Finally, reprogramming experiments indicate that reduction of centromeric CENP-A levels is an early event during dedifferentiation, coinciding with global chromatin remodelling. Our characterisation of centromeres in human stem cells drives new hypotheses including a possible link between impaired centromere function and stem cell aneuploidies.
Automated continuous evolution of proteinsin vivo
Authors: Zhong Ziwei , Wong Brandon G. , Ravikumar Arjun , Arzumanyan Garri A. , Khalil Ahmad S. , Liu Chang C.
Published: February 2020

We present automated continuous evolution (ACE), a platform for the hands-free directed evolution of biomolecules. ACE pairs OrthoRep, a genetic system for continuous targeted mutagenesis of user-selected genesin vivo, with eVOLVER, a scalable and automated continuous culture device for precise, multi-parameter regulation of growth conditions. By implementing real-time feedback-controlled tuning of selection stringency with eVOLVER, genes of interest encoded on OrthoRep autonomously traversed multi-mutation adaptive pathways to reach desired functions, including drug resistance and improved enzyme activity. The durability, scalability, and speed of biomolecular evolution with ACE should be broadly applicable to protein engineering as well as prospective studies on how selection parameters and schedules shape adaptation.