bluuebunny/crossref_metadata_2025 huggingface dataset:
Lysozyme resistance inC. difficileis dependent on two peptidoglycan deacetylases
Authors: Kaus Gabriela M. , Snyder Lindsey F. , Müh Ute , Flores Matthew J. , Popham David L. , Ellermeier Craig D.
Published: July 2020

Clostridioides (Clostridium) difficileis a major cause of hospital-acquired infections leading to antibiotic-associated diarrhea.C. difficileexhibits a very high level of resistance to lysozyme. Bacteria commonly resist lysozyme through modification of the cell wall. InC. difficileσVis required for lysozyme resistance and σVis activated in response to lysozyme. Once activated σV, encoded bycsfV, directs transcription of genes necessary for lysozyme resistance. Here we analyze the contribution of individual genes in thecsfVregulon to lysozyme resistance. Using CRISPR-Cas9 mediated mutagenesis we constructed in-frame deletions of single genes in thecsfVoperon. We findpdaV, which encodes a peptidoglycan deacetylase, is partially responsible for lysozyme resistance. We then performed CRISPR inhibition (CRISPRi) to identify a second peptidoglycan deacetylase,pgdA, that is important for lysozyme resistance. Deletion of eitherpgdAorpdaVresulted in modest decreases in lysozyme resistance. However, deletion of bothpgdAandpdaVresulted in a 1000-fold decrease in lysozyme resistance. Further, muropeptide analysis revealed loss of either PgdA or PdaV had modest effects on peptidoglycan deacetylation but loss of both PgdA and PdaV resulted in almost complete loss of peptidoglycan deacetylation. This suggests that PgdA and PdaV are redundant peptidoglycan deacetylases. We also use CRISPRi to compare other lysozyme resistance mechanisms and conclude that peptidoglycan deacetylation is the major mechanism of lysozyme resistance inC. difficile.

Importance

Clostridioides difficileis the leading cause of hospital-acquired diarrhea.C. difficileis highly resistant to lysozyme. We previously showed that thecsfVoperon is required for lysozyme resistance. Here we use CRISPR-Cas9 mediated mutagenesis and CRISPRi knockdown to show that peptidoglycan deacetylation is necessary for lysozyme resistance and is the major lysozyme resistance mechanism inC. difficile. We show that two peptidoglycan deacetylases inC. difficileare partially redundant and are required for lysozyme resistance. PgdA provides an intrinsic level of deacetylation and PdaV, encoded as part of thecsfVoperon, provides lysozyme-induced peptidoglycan deacetylation.
Unpicking the mysterious symbiosis ofMycoplasmain salmonids
Authors: Cheaib B , Yang P , Kazlauskaite R , Lindsay E , Heys C , De Noa M , Schaal Patrick , Dwyer T , Sloan W , Ijaz UZ , Llewellyn MS
Published: July 2020

Lacking a peptidoglycan cell wall, mycoplasmas are the smallest self-replicating life forms. Members of this bacterial genus are known to parasitise a wide array of metazoans including vertebrates. Whilst much research has been significant targeted at parasitic mammalian mycoplasmas, very little is known about their role in other vertebrates. In the current study, we aim to explore the biology and evolution ofMycoplasmain salmonids, including cellular niche, genome size structure and gene content. Using Fluorescencein-situhybridisation (FISH),mycoplasmaswere identified in epithelial tissues across the digestive tract (stomach, pyloric caecum and midgut) during the developmental stages (eggs, parr, subadult) of farmed Atlantic salmon (Salmo salar), showing a high abundance in acidic compartments. With high throughput sequencing from subadults farmed Atlantic salmon, we assembled a nearly complete genome (~0.57 MB) via shotgun-metagenomics. The phylogenetic inference from the recovered genome revealed successful taxonomic proximity toMycoplasma penetrans(~1.36 Mb) from the recovered genome. Although, no significant correlation between genome size and its phylogeny was observed, we recovered functional signatures, especially, riboflavin encoding genes pathway and sugars transporters, suggesting a symbiotic relationship betweenMycoplasmaand the host. Though 247 strains ofMycoplasmaare available in public databases, to the best of our knowledge, this is the first study to demonstrate ecological and functional association betweenMycoplasmaandSalmo salarwhich delineates symbiotic reductive evolution and genome erosion primarily and also serves as a proxy for salmonid health in aquaculture processes (cell lines,in vitrogut models).
The transcriptional landscape of a hepatoma cell line grown on scaffolds of extracellular matrix proteins
Authors: Ghosh Souvik , Börsch Anastasiya , Zavolan Mihaela
Published: July 2020

The behavior of cells in vivo is complex and highly dynamic, as it results from an interplay between components of the intercellular matrix proteins with surface receptor and other microenvironmental cues. Although the effects of the cellular niche have been investigated for a number of cell types using different molecular approaches, comprehensive assessments of how the global transcriptome responds to 3D scaffolds composed of various extracellular matrix (ECM) constituents are still lacking. In this study, we explored the effect of the biomechanical parameters of Collagen I and Matrigel (ECM) on transcriptional gene regulation in a cell culture system. Using Huh-7 cells cultured on traditional cell culture plates or on the components of the ECM at different concentrations to modulate microenvironment properties, we have generated transcriptome sequencing data that may be further explored to understand the differentiation and growth potential of this cell for the development of 3D cultures. Assessment of the hepatocyte phenotype in relation to our transcriptomic data set would be very useful for the development of systems mimicking the in vivo structure and function of liver cells which still remains a challenge.
Respiratory Syncytial Virus Infections Induce Hypermetabolism in Pediatric Upper Airways
Authors: Rezinciuc Svetlana , Bezavada Lavanya , Bahadoran Azadeh , Ingels Jesse F. , Kim Young-Yin , Cormier Stephania A. , Devincenzo John P. , Shulkin Barry L. , Smallwood Heather S.
Published: July 2020

To determine whether respiratory syncytial virus (RSV) regulates human metabolism, we used positron emission tomography (PET) of patient lungs along with bioenergetics and metabolomics of patient upper airway cells and fluids. We previously found a significant negative monotonic relationship between glucose uptake and respiratory viral infection in 20 pediatric patients (e.g., 70% of infected patients had glucose uptake within 0–3 days). In our recent study, 3 out of 4 patients positive for glucose uptake at later times (>5 days) were positive for RSV infection. At present, the bioenergetics of upper respiratory cells (URCs) from nasal pharyngeal aspirates have not been investigated, andin vitrostudies indicate RSV reduces metabolism in cell lines. To define metabolic changes in RSV-infected pediatric patients, we acquired fresh aspirates from 6 pediatric patients. Immediately following aspiration of URCs, we measured the two major energy pathways using an XFe flux analyzer. Glycolysis and mitochondrial respiration were significantly increased in URCs from RSV-infected patients, and mitochondrial respiration was operating at near maximal levels, resulting in loss of cellular capacity to increase respiration with impaired coupling efficiency. Metabolomics analysis of metabolites flushed from the upper airways confirmed a significant increase in TCA cycle intermediates. Taken together, these studies demonstrate RSV induces significant hypermetabolism in pediatric patients’ lungs and respiratory tract. Thus, hypermetabolism is a potential anti-viral drug target and reveals RSV can regulate human metabolism.

Contributions to the field

Metabolic changes in humans in response to viral infection are largely unknown. In this brief clinical report, we find metabolism is markedly increased in live upper respiratory cells from infants infected with respiratory syncytial virus (RSV) concomitant to changes in metabolites in their upper airway fluids. This sheds light on viral induced hypermetabolism in the airways and offers potential biomarkers for RSV. In addition, this identifies potential therapeutic targets for host directed therapies of aberrant metabolism in RSV. This work has clinical impact as biomarkers and therapeutics for RSV are needed for this pervasive virus that causes infections with long term consequence for some children. Further, advancements in molecular mechanisms underpinning RSV infection biology are constrained by the difficulties in translating model systems to humans as well as relating human studies in adults to infants (Mestas and Hughes, 2004; Papin et al., 2013).
Estimation of stroke outcomes based on time to thrombolysis and thrombectomy
Authors: Allen Michael , Pearn Kerry , Stein Ken , James Martin
Published: July 2020

Background & Motivation

Stroke outcomes following revascularization therapy (intravenous thrombolysis, IVT, and/or mechanical thrombectomy, MT) depend critically on time from stroke onset to treatment. Different service configurations may prioritise time to IVT or time to MT. In order to evaluate alternative acute stroke care configurations, it is necessary to estimate clinical outcomes given differing times to IVT and MT.

Method

Model using an algorithm coded in Python. This is available at https://github.com/MichaelAllen1966/stroke_outcome_algorithm

Results

We demonstrate how the code may be used to estimate clinical outcomes given varying times to IVT and MT.

Conclusion

Python code has been developed and shared to enable estimation of clinical outcome given times to IVT and MT. Here we share pseudocode and links to full Python code.
The association between Internet Usage and Overweight/Obesity modified by Gender: Evidence from a nationally representative survey in Nepal
Authors: Rana Juwel , Islam Md. Momin , Oldroyd John , Samad Nandeeta , Islam Rakibul M.
Published: July 2020

Objective

Using a nationally representative data, we examined the associations between internet use and overweight/obesity in people aged 15-49 years in Nepal, and the extent to which these associations vary by gender.

Materials and methods

The study analyzed the nationally representative Nepal Demographic and Health Survey (NDHS) 2016 data, collected between June 2016 and January 2017. The outcome was overweight/obesity. Exposures were internet use (IU) in the last twelve months and internet use frequency (FIU) in the last month. Multivariable ordinal logistic regression models were fitted to estimate the total effects of IU and FIU on overweight/obesity adjusted for minimal sufficient adjustment set of potential confounders. P-difference was extracted using a Wald test for the models with interaction terms.

Results

Of the 10,380 participants, 33.9% used internet in the last 12 months, and 13.1 % used less than/at least once in a week, and 17.5% used internet almost every day. The prevalence of overweight/obesity by IU was 38% (95% CI: 35.9%, 40.1%) for male and 44.1% (95% CI: 41.6%, 46.6) for female. The risk of overweight and obesity was significantly 1.55 times higher (aOR: 1.55; 95% CI: 1.40, 1.73; p < 0.001) among those participants who used the internet compared to the individual who did not use the internet in the last 12 months or earlier of the interview. Similar associations were observed when using the augmented measure of exposure-FIU. We observed modification effect of gender in the associations of IU (p-difference<0.001) and FIU (p-difference<0.002) with overweight and obesity in Nepal.

Conclusions

Our findings suggest that it is imperative for future overweight/obesity interventions in LMICs, including Nepal, to discourage unnecessary internet use, particularly among males.
Combined short and long-read sequencing reveals a complex transcriptomic architecture of African swine fever virus
Authors: Torma Gábor , Tombácz Dóra , Csabai Zsolt , Moldován Norbert , Mészáros István , Zádori Zoltán , Boldogkői Zsolt
Published: July 2020

African swine fever virus (ASFV) is a large DNA virus belonging to theAsfarviridaefamily. Despite its agricultural importance, little is known about the fundamental molecular mechanisms of this pathogen. Understanding of genetic regulation provides new insights into the virus pathogenicity, which can help prevent epidemics. Short-read sequencing (SRS) is able to produce a huge amount of high-precision sequencing reads for transcriptomic profiling, but it is inefficient for the comprehensive annotation of transcriptomes. Long-read sequencing (LRS) is able to overcome some of the limitations of SRS, but they also have drawbacks, such as low-coverage and high error rate. The limitations of the two approaches can be surmounted by the combined use of these techniques. In this study, we used Illumina SRS and Oxford Nanopore Technologies LRS platforms with multiple library preparation methods (amplified and direct cDNA sequencings and native RNA sequencing) for constructing the transcriptomic atlas of ASFV. This work identified a large number of novel genes, transcripts and RNA isoforms, and annotated the precise termini of previously described RNA molecules. In contrast to the current view that the ASFV transcripts are monocistronic, we detected a significant extent of polycistronism. A multifaceted meshwork of transcriptional overlaps is also discovered.
The landscape of SARS-CoV-2 RNA modifications
Authors: Miladi Milad , Fuchs Jonas , Maier Wolfgang , Weigang Sebastian , Pedrosa Núria Díaz i , Weiss Lisa , Lother Achim , Nekrutenko Anton , Ruzsics Zsolt , Panning Marcus , Kochs Georg , Gilsbach Ralf , Grüning Björn
Published: July 2020

In 2019 the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the first documented cases of severe lung disease COVID-19. Since then, SARS-CoV-2 has been spreading around the globe resulting in a severe pandemic with over 500.000 fatalities and large economical and social disruptions in human societies. Gaining knowledge on how SARS-Cov-2 interacts with its host cells and causes COVID-19 is crucial for the intervention of novel therapeutic strategies. SARS-CoV-2, like other coronaviruses, is a positive-strand RNA virus. The viral RNA is modified by RNA-modifying enzymes provided by the host cell. Direct RNA sequencing (DRS) using nanopores enables unbiased sensing of canonical and modified RNA bases of the viral transcripts. In this work, we used DRS to precisely annotate the open reading frames and the landscape of SARS-CoV-2 RNA modifications. We provide the first DRS data of SARS-CoV-2 in infected human lung epithelial cells. From sequencing three isolates, we derive a robust identification of SARS-CoV-2 modification sites within a physiologically relevant host cell type. A comparison of our data with the DRS data from a previous SARS-CoV-2 isolate, both raised in monkey renal cells, reveals consistent RNA modifications across the viral genome. Conservation of the RNA modification pattern during progression of the current pandemic suggests that this pattern is likely essential for the life cycle of SARS-CoV-2 and represents a possible target for drug interventions.
Haloferax volcanii immersed liquid biofilms develop independently of known biofilm machineries and exhibit rapid honeycomb pattern formation
Authors: Schiller Heather , Schulze Stefan , Mutan Zuha , de Vaulx Charlotte , Runcie Catalina , Schwartz Jessica , Rados Theopi , Bisson Filho Alexandre W. , Pohlschroder Mechthild
Published: July 2020

The ability to form biofilms is shared by many microorganisms, including archaea. Cells in a biofilm are encased in extracellular polymeric substances that typically include polysaccharides, proteins, and extracellular DNA, conferring protection while providing a structure that allows for optimal nutrient flow. In many bacteria, flagella and evolutionarily conserved type IV pili are required for the formation of biofilms on solid surfaces or floating at the air-liquid interface of liquid media. Similarly, in many archaea it has been demonstrated that type IV pili and, in a subset of these species, archaella are required for biofilm formation on solid surfaces. Additionally, in the model archaeon Haloferax volcanii, chemotaxis and AglB-dependent glycosylation play important roles in this process. H. volcanii also forms immersed biofilms in liquid cultures poured into Petri dishes. This study reveals that mutants of this haloarchaeon that interfere with the biosynthesis of type IV pili or archaella, as well as a chemotaxis-targeting transposon and aglB-deletion mutants, lack obvious defects in biofilms formed in liquid cultures. Strikingly, we have observed that these liquid-based biofilms are capable of rearrangement into honeycomb-like patterns that rapidly form upon removal of the Petri dish lid, a phenomenon that is not dependent on changes in light or oxygen concentration but can be induced by controlled reduction of humidity. Taken together, this study demonstrates that H. volcanii requires novel, as-yet-unidentified strategies for immersed liquid biofilm formation and also exhibits rapid structural rearrangements.

Importance

This first molecular biological study of archaeal immersed liquid biofilms advances our basic-biological understanding of the model archaeon Haloferax volcanii. Data gleaned from this study also provide an invaluable foundation for future studies to uncover components required for immersed liquid biofilms in this haloarchaeon and also potentially for liquid biofilm formation in general, which is poorly understood compared to the formation of biofilms on surfaces. Moreover, this first description of rapid honeycomb pattern formation is likely to yield novel insights into the underlying structural architecture of extracellular polymeric substances and cells within immersed liquid biofilms.
Understanding photothermal interactions will help expand production range and increase genetic diversity of lentil (Lens culinarisMedik.)
Authors: Wright Derek M. , Neupane Sandesh , Heidecker Taryn , Haile Teketel A. , Coyne Clarice J. , McGee Rebecca J. , Udupa Sripada , Henkrar Fatima , Barilli Eleonora , Rubiales Diego , Gioia Tania , Logozzo Giuseppina , Marzario Stefania , Mehra Reena , Sarker Ashutosh , Dhakal Rajeev , Anwar Babul , Sarkar Debashish , Vandenberg Albert , Bett Kirstin E.
Published: July 2020

Lentil (Lens culinarisMedik.) is cultivated under a wide range of environmental conditions, which led to diverse phenological adaptations and resulted in a decrease in genetic variability within breeding programs due to reluctance in using genotypes from other environments.

We phenotyped 324 genotypes across nine locations over three years to assess their phenological response to the environment of major lentil production regions and to predict days from sowing to flowering (DTF) using a photothermal model.

DTF was highly influenced by the environment and is sufficient to explain adaptation. We were able to predict DTF reliably in most environments using a simple photothermal model, however, in certain site-years, results suggest there may be additional environmental factors at play. Hierarchical clustering of principal components revealed the presence of eight groups based on the responses of DTF to contrasting environments. These groups are associated with the coefficients of the photothermal model and revealed differences in temperature and photoperiod sensitivity.

Expanding genetic diversity is critical to the success of a breeding program; understanding adaptation will facilitate the use of exotic germplasm. Future climate change scenarios will result in increase temperature and/or shifts in production areas, we can use the photothermal model to identify genotypes most likely to succeed in these new environments.
RNAseq analysis of rodent spaceflight experiments is confounded by sample collection techniques
Authors: Polo San-Huei Lai , Saravia-Butler Amanda M. , Boyko Valery , Dinh Marie T. , Chen Yi-Chun , Fogle Homer , Reinsch Sigrid S. , Ray Shayoni , Chakravarty Kaushik , Marcu Oana , Chen Rick B. , Costes Sylvain V. , Galazka Jonathan M.
Published: July 2020

To understand the physiological changes that occur in response to spaceflight, mice are transported to the International Space Station (ISS) and housed for variable periods of time before euthanasia on-orbit or return to Earth. Sample collection under such difficult conditions introduces confounding factors that need to be identified and addressed. We found large changes in the transcriptome of mouse tissues dissected and preserved on-orbit compared to tissues from mice euthanized on-orbit, preserved, and dissected after return to Earth. Changes due to preservation method eclipsed those between flight and ground samples making it difficult to identify spaceflight-specific changes. Follow-on experiments to interrogate the roles of euthanasia methods, tissue and carcass preservation protocols, and library preparation methods suggested that differences due to preservation protocols are exacerbated when coupled with polyA selection. This has important implications for the interpretation of existing datasets and the design of future experiments.
The KDEL Trafficking Receptor Exploits pH to Tune the Strength of an Unusual Short Hydrogen Bond
Authors: Wu Zhiyi , Newstead Simon , Biggin Philip C.
Published: July 2020

The endoplasmic reticulum (ER) is the main site of protein synthesis in eukaryotic cells and requires a high concentration of luminal chaperones to function. During protein synthesis, ER luminal chaperones are swept along the secretory pathway and must be retrieved to maintain cell viability. ER protein retrieval is achieved by the KDEL receptor, which recognises a C-terminal Lys-Asp-Glu-Leu (KDEL) sequence. Recognition of ER proteins by the KDEL receptor is pH dependent, with binding occurring under acidic conditions in the Golgi and release under conditions of higher pH in the ER. Recent crystal structures of the KDEL receptor in the apo and peptide bound state suggested that peptide binding drives the formation of a short-hydrogen bond that locks the KDEL sequence in the receptor and activates the receptor for COPI binding in the cytoplasm. Using quantum mechanical calculations we demonstrate that the strength of this short hydrogen bond is reinforced following protonation of a nearby histidine, linking receptor protonation to high affinity peptide binding. Protonation also controls the wetting of a cavity adjacent to the peptide binding site, leading to a conformational change that ultimately allows the complex to be recognized by the COPI system.
Metformin chlorination byproducts in drinking water exhibit marked toxicity to nematode worms, human cells, and mice
Authors: Zhang Runshuai , He Yuanzhen , Yao Luxia , Chen Jie , Zhu Shihao , Rao Xinxin , Tang Peiyuan , You Jia , Hua Guoqiang , Zhang Lu , Ju Feng , Wu Lianfeng
Published: July 2020

Metformin (MET), a worldwide used drug for type 2 diabetes, has been found with the largest amount by weight among all drugs in aquatic environment, including the drinking water sources where chlorination inevitably transforms MET into chlorination byproducts. Although MET has health-promoting properties, whether or how its chlorination byproducts affect health remains largely unknown. Here we reveal that MET chlorination byproducts Y (C4H6ClN5) and C (C4H6ClN3) exhibit marked toxicity, even higher than that of the well-known poisonous arsenic, to live worms and human cells. Moreover, both byproducts are harmful to mice and Y at 250 ng/L destroys the mouse small intestine integrity. Strikingly, we detected MET and byproduct C in worldwide drinking water. Both byproducts are increasingly produced with more MET present during chlorination process. Unprecedentedly, we unveil boiling and activated carbon adsorption as effective solutions that are in urgent demand globally for removing these byproducts from water.
Long-term Reconstruction of Human Airway Epithelium-like Structurein Vivowith hESCs-derived organoid cells
Authors: Chen Yong , Han Le , Zhao Shanshan , Feng Jianqi , Li Lian , Rong Zhili , Lin Ying
Published: July 2020

Human embryonic stem cells (hESCs) derived lung organoids (HLOs) provide a promising model to study human lung development and disease. However, whether HLO cells could reconstitute airway epithelial structurein vivoremains unclear. Here we established an orthotopic xenograft system for hESCs-derived HLOs, enabling stable reconstruction of human airway epitheliumin vivo. Removal of the mouse airway epithelium by naphthalene (NA) treatment enabled xenografted organoid cells survival, differentiation, and reconstruction of airway pseudostratified epithelium in immune-compromised NSG mice. Compared to unsorted pool cells, CD47highcells generated more ciliated cells and possessed thicker pseudostratified epithelium. RNA-seq data revealed that CD47highcells highly expressed epithelial cell, lung progenitor, lung proximal cell and embryonic lung development associated genes. These data reveal that HLOs hold cell therapy potential in regenerative medicine by long-term reconstituting airway epithelium.
Myofibril and mitochondria morphogenesis are coordinated by a mechanical feedback mechanism in muscle
Authors: Avellaneda Jerome , Rodier Clement , Daian Fabrice , Rival Thomas , Luis Nuno Miguel , Schnorrer Frank
Published: July 2020

Complex animals build specialised muscle to match specific biomechanical and energetic needs. Hence, composition and architecture of sarcomeres as well as mitochondria are muscle type specific. However, mechanisms coordinating mitochondria with sarcomere morphogenesis are elusive. Here we useDrosophilamuscles to demonstrate that myofibril and mitochondria morphogenesis are intimately linked. In flight muscles, the muscle selectorspaltinstructs mitochondria to intercalate between myofibrils, which in turn mechanically constrain mitochondria into elongated shapes. Conversely in cross-striated muscles, mitochondria networks surround myofibril bundles, contacting myofibrils only with thin extensions. To investigate the mechanism causing these differences, we manipulated mitochondrial dynamics and found that increased mitochondrial fusion during myofibril assembly prevents mitochondrial intercalation in flight muscles. Strikingly, this coincides with the expression of cross-striated muscle specific sarcomeric proteins. Consequently, flight muscle myofibrils convert towards a partially cross-striated architecture. Together, these data suggest a biomechanical feedback mechanism downstream ofspaltsynchronizing mitochondria with myofibril morphogenesis.
A multipass membrane protein interacts with the cGMP-dependent protein kinase to regulate critical calcium signals in malaria parasites
Authors: Balestra Aurélia C. , Koussis Konstantinos , Klages Natacha , Howell Steven A. , Flynn Helen R. , Bantscheff Marcus , Pasquarello Carla , Perrin Abigail J. , Brusini Lorenzo , Arboit Patrizia , Sanz Olalla , Castaño Laura Peces-Barba , Withers-Martinez Chrislaine , Hainard Alexandre , Ghidelli-Disse Sonja , Snijders Ambrosius P. , Baker David A. , Blackman Michael J. , Brochet Mathieu
Published: July 2020

In malaria parasites, all cGMP-dependent signalling is mediated through a single cGMP-dependent protein kinase (PKG), a major function of which is to control essential calcium signals. However, how PKG transmits these signals in the absence of known second messenger-dependent calcium channels or scaffolding proteins is unknown. Here we identify a polytopic membrane protein, ICM1, with homology to transporters and calcium channels that is tightly-associated with PKG in bothPlasmodium falciparumasexual blood stages andP. bergheigametocytes. Phosphoproteomic analyses in bothPlasmodiumspecies reveal multiple ICM1 phosphorylation events dependent upon PKG activity. Stage-specific depletion ofP. bergheiICM1 blocks gametogenesis due to the inability of mutant parasites to mobilise intracellular calcium upon PKG activation, whilst conditional loss ofP. falciparumICM1 results in reduced calcium mobilisation, defective egress and lack of invasion. Our findings provide new insights into atypical calcium homeostasis in malaria parasites essential for pathology and disease transmission.
Inhibition of CRISPR-Cas12a DNA Targeting by Nucleosomes and Chromatin
Authors: Strohkendl Isabel , Saifuddin Fatema A. , Gibson Bryan A. , Rosen Michael K. , Russell Rick , Finkelstein Ilya J.
Published: July 2020

Genome engineering nucleases, including CRISPR-Cas12a, must access chromatinized DNA. Here, we investigate howAcidaminococcus sp. Cas12a cleaves DNA within human nucleosomes and phase-condensed nucleosome arrays. Using quantitative kinetics approaches, we show that dynamic nucleosome unwrapping regulates DNA target accessibility to Cas12a. Nucleosome unwrapping determines the extent to which both steps of Cas12a binding–PAM recognition and R-loop formation–are inhibited by the nucleosome. Nucleosomes inhibit Cas12a binding even beyond the canonical core particle. Relaxing DNA wrapping within the nucleosome by reducing DNA bendability, adding histone modifications, or introducing a target-proximal nuclease-inactive Cas9 enhances DNA cleavage rates over 10-fold. Surprisingly, Cas12a readily cleaves DNA linking nucleosomes within chromatin-like phase separated nucleosome arrays—with DNA targeting reduced only ~4-fold. This work provides a mechanism for the observation that on-target cleavage within nucleosomes occurs less often than off-target cleavage within nucleosome-depleted regions of cells. We conclude that nucleosome wrapping restricts accessibility to CRISPR-Cas nucleases and anticipate that increasing nucleosome breathing dynamics will improve DNA binding and cleavage in eukaryotic cells.
Inhibitors of VPS34 and lipid metabolism suppress SARS-CoV-2 replication
Authors: Silvas Jesus A. , Jureka Alexander S. , Nicolini Anthony M. , Chvatal Stacie A. , Basler Christopher F.
Published: July 2020

Therapeutics targeting replication of SARS coronavirus 2 (SARS-CoV-2) are urgently needed. Coronaviruses rely on host membranes for entry, establishment of replication centers and egress. Compounds targeting cellular membrane biology and lipid biosynthetic pathways have previously shown promise as antivirals and are actively being pursued as treatments for other conditions. Here, we tested small molecule inhibitors that target membrane dynamics or lipid metabolism. Included were inhibitors of the PI3 kinase VPS34, which functions in autophagy, endocytosis and other processes; Orlistat, an inhibitor of lipases and fatty acid synthetase, is approved by the FDA as a treatment for obesity; and Triacsin C which inhibits long chain fatty acyl-CoA synthetases. VPS34 inhibitors, Orlistat and Triacsin C inhibited virus growth in Vero E6 cells and in the human airway epithelial cell line Calu-3, acting at a post-entry step in the virus replication cycle. Of these the VPS34 inhibitors exhibit the most potent activity.
Membrane voltage-dependent activation of the flagellar protein export engine
Authors: Minamino Tohru , Morimoto Yusuke V. , Kinoshita Miki , Namba Keiichi
Published: July 2020

Ion motive force (IMF) consists of the electric potential difference (ΔΨ) and the ion concentration difference (ΔpI) across the cytoplasmic membrane. The flagellar protein export machinery is an ion/protein antiporter utilizing IMF to drive ion-coupled protein export, but it remains unknown how. Here, we report a ΔΨ-dependent activation mechanism of the transmembrane export gate complex. Depletions of both H+and Na+gradients nearly diminished flagellar protein export in the absence of the cytoplasmic ATPase complex, but an increase in ΔΨ by an upward shift of external pH from 7.5 to 8.5 dramatically recovered it. An increase in the cytoplasmic level of export substrates and gain-of-function mutations in FlhA enhanced protein export at external pH 7.5 in the absence of Na+in a similar manner to ΔΨ increase. We propose that the export gate complex has a voltage-gated mechanism to activate the ion/protein antiporter of the flagellar protein export engine.
Structural and biophysical correlation of anti-NANP antibodies within vivoprotection againstP. falciparum
Authors: Pholcharee Tossapol , Oyen David , Flores-Garcia Yevel , Gonzalez-Paez Gonzalo , Han Zhen , Williams Katherine L. , Volkmuth Wayne , Emerling Daniel , Locke Emily , King C. Richter , Zavala Fidel , Wilson Ian A.
Published: July 2020

The most advancedP. falciparumcircumsporozoite protein (PfCSP)-based malaria vaccine, RTS,S/AS01 (RTS,S), confers partial protection but with antibody titers that wane relatively rapidly, highlighting the need to elicit more potent and durable antibody responses. Here, we elucidate crystal structures, binding affinities and kinetics, andin vivoprotection of eight anti-NANP antibodies (Abs) derived from an RTS,S phase 2a trial and encoded by three different heavy-chain germline genes. The structures reinforce the importance of homotypic Fab-Fab interactions in protective Abs and the overwhelmingly dominant preference for a germline-encoded aromatic residue for recognition of the NANP motif. A number of biophysical properties were analyzed and antibody affinity correlated best with protection in anin vivomouse model, with the more potent antibodies also recognizing epitopes with repeating secondary structural motifs of type I β- and Asn pseudo 310turns. Such insights can be incorporated into design of more effective immunogens as well as antibodies for passive immunization.
Haplotype Explorer: an infection cluster visualization tool for spatiotemporal dissection of the COVID-19 pandemic
Authors: Kawano-Sugaya Tetsuro , Yatsu Koji , Sekizuka Tsuyoshi , Itokawa Kentaro , Hashino Masanori , Tanaka Rina , Kuroda Makoto
Published: July 2020

The worldwide eruption of COVID-19 that began in Wuhan, China in late 2019 reached 10 million cases by late June 2020. In order to understand the epidemiological landscape of the COVID-19 pandemic, many studies have attempted to elucidate phylogenetic relationships between collected viral genome sequences using haplotype networks. However, currently available applications for network visualization are not suited to understand the COVID-19 epidemic spatiotemporally, due to functional limitations That motivated us to develop Haplotype Explorer, an intuitive tool for visualizing and exploring haplotype networks. Haplotype Explorer enables people to dissect epidemiological consequences via interactive node filters to provide spatiotemporal perspectives on multimodal spectra of infectious diseases, including introduction, outbreak, expansion, and containment, for given regions and time spans. Here, we demonstrate the effectiveness of Haplotype Explorer by showing an example of its visualization and features. The demo using SARS-CoV-2 genome sequences is available athttps://github.com/TKSjp/HaplotypeExplorer

A lot of software for network visualization are available, but existing software have not been optimized to infection cluster visualization against the current worldwide invasion of COVID-19 started since 2019. To reach the spatiotemporal understanding of its epidemics, we developed Haplotype Explorer. It is superior to other applications in the point of generating HTML distribution files with metadata searches which interactively reflects GISAID IDs, locations, and collection dates. Here, we introduce the features and products of Haplotype Explorer, demonstrating the time-dependent snapshots of haplotype networks inferred from total of 4,282 SARS-CoV-2 genomes.
A systematic review and meta-analysis protocol on the association of malaria in pregnancy and adverse birth outcomes
Authors: Yadav Vikas , Bairwa Mohan , Dabar Deepti , Goel Akhil Dhanesh , Palepu Sarika , Joshi Ankur
Published: July 2020

Introduction

Malaria in pregnancy contributes to significant adverse birth outcomes. This study is aimed to quantify the relationship between malaria in pregnancy and occurrence of adverse birth outcomes, including preterm delivery, low birth weight, small for gestational age, miscarriages, and stillbirth.

Methods and analysis

Observational studies and Randomised controlled trials reporting data on selected birth outcomes separately for pregnancies, with or without malaria will be included. We will search for studies over various information sources and data extraction will be done from included studies. Pooled odds ratio (OR) will be calculated for each birth outcomes using fixed effect model or random effects models, based on the level of heterogeneity. Forest plot will be prepared with effect size (with 95 percent confidence interval) of each study and pooled effect size. The methodological quality will be assessed for included observational studies using the Newcastle-Ottawa scale (NOS). Cochrane Risk of Bias tool will be used to evaluate bias in randomised controlled trials. For publication bias, funnel plot will be prepared and assessed for asymmetry, along with Egger’s test.

Discussion

This study will provide an estimate of the risk of adverse birth outcomes in pregnancies with malaria. Results of this study will contribute towards planning effective service delivery in areas with a higher risk of malaria transmission.

Ethics and dissemination

The current study is a review of published literature, and it does not require ethical committee approval. Results of this review will be published in a peer-reviewed journal.

PROSPERO registration number

CRD42020153009

Article summary

Strengths and limitations of this study

To the best of reviewers’ knowledge, this will be a first-ever comprehensive review on the relationship between malaria in pregnancy and occurrence of most frequent adverse birth outcomes, i.e. preterm delivery, low birth weight, small for gestational age and miscarriages.

The finding of this review will be very crucial for the governments of malaria-endemic countries.

All the leading information sources will be included in this study to perform searches.

The protocol is prepared in adherence to Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols guidelines (PRISMA-P).

A limitation of this study will be, various definition criteria used to define adverse birth outcomes among the included studies. Due to which, it will be challenging to interpret and extrapolate the findings of this study.
A satellite-based spatio-temporal machine learning model to reconstruct daily PM2.5 concentrations across Great Britain
Authors: Santos Rochelle Schneider dos , Vicedo-Cabrera Ana M. , Sera Francesco , Masselot Pierre , Stafoggia Massimo , de Hoogh Kees , Kloog Itai , Reis Stefan , Vieno Massimo , Gasparrini Antonio
Published: July 2020

Epidemiological studies on health effects of air pollution usually rely on measurements from fixed ground monitors, which provide limited spatio-temporal coverage. Data from satellites, reanalysis and chemical transport models offer additional information used to reconstruct pollution concentrations at high spatio-temporal resolution. The aim of this study is to develop a multi-stage satellite-based machine learning model to estimate daily fine particulate matter (PM2.5) levels across Great Britain during 2008-2018. This high-resolution model consists of random forest (RF) algorithms applied in four stages. Stage-1 augments monitor-PM2.5 series using co-located PM10 measures. Stage-2 imputes missing satellite aerosol optical depth observations using atmospheric reanalysis models. Stage-3 integrates the output from previous stages with spatial and spatiotemporal variables to build a prediction model for PM2.5. Stage-4 applies Stage-3 models to estimate daily PM2.5 concentrations over a 1 km grid. The RF architecture performed well in all stages, with results from Stage-3 showing an average cross-validated R2 of 0.767 and minimal bias. The model performed better over the temporal scale when compared to the spatial component, but both presented good accuracy with an R2 of 0.795 and 0.658, respectively. The high spatio-temporal resolution and relatively high precision allows this dataset (approximately 950 million points) to be used in epidemiological analyses to assess health risks associated with both short- and long-term exposures to PM2.5.
The genetic architecture of human infectious diseases and pathogen-induced cellular phenotypes
Authors: Hale Andrew T. , Zhou Dan , Sale Rebecca L. , Bastarache Lisa , Wang Liuyang , Zinkel Sandra S. , Schiff Steven J. , Ko Dennis C. , Gamazon Eric R.
Published: July 2020

Here, we develop a genetics-anchored framework to decipher mechanisms of infectious disease (ID) risk and infer causal effect on potential complications. We perform transcriptome-wide association studies (TWAS) of 35 ID traits in 27,615 individuals in a broad collection of human tissues, identifying 70 gene-level associations with 26 ID traits, with replication in two large-scale biobanks. A phenome-scale scan and Mendelian Randomization of the 70 gene-level associations across 197 traits proposes a molecular basis for known complications of the ID traits. This rich resource of host genetic associations with pathogen cultures and 16S-rRNA-based microbiome variation provides a platform to investigate host-pathogen interactions. To identify relevant cellular processes, we develop a TWAS repository of 79 pathogen-exposure induced cellular phenotypes. Our study will facilitate mechanistic insights into the role of host genetic variation on ID risk and pathophysiology, with important implications for our molecular understanding of severe phenotypic outcomes.
Concerns with the use of imputation to assign HLA allele-level typing in research predicting transplant outcomes
Authors: Tambur Anat R , Gmeiner Michael , Manski Charles F
Published: July 2020

Incomplete information on HLA allele typing is a persistent problem when analyzing the role of Human Leukocyte Antigen (HLA) in transplantation. To refine the predictions possible with partial knowledge of HLA typing, some researchers use HaploStats statistics on the frequencies of haplotypes within specified ethnic/national populations to impute complete HLA allele typing. We evaluated methods that use imputation to predict patient outcomes after organ transplantation, with focus on prediction of graft survival conditional on typing information of the donor and recipient. Logical arguments show that imputation yields no predictive power when predictions are conditioned on all observed HLA typing data. Computational experiments indicate that imputation does not have predictive power when applied to risk-assessment models that make predictions conditional on only part of the observable HLA data. We therefore caution against reliance on imputation to overcome incomplete measurement. We encourage high-resolution typing of HLA antigens to improve prediction of transplant outcomes and matching of donors with recipients. Similar considerations should likely apply in other clinical settings.
The different functions of exercise, supplementary resveratrol, and the combination of exercise and supplementary resveratrol in young and old SAMP8 mice liver
Authors: Suchkov Sergey , Wu Jia-Ping
Published: July 2020

The senescence-accelerated mouse prone 8 (SAMP8) has about half the normal lifespan of a rodent. Therefore, we determined whether the different age degrees has rapid physiological senescence with the same interaction function and mechanism of exercise and supplementary resveratrol intake by SAMP8 mice liver. Histological of aging related liver disease was examined SAMP8 mice by hematoxylin-eosin and Masson’s trichrome staining.

Apoptosis was determined by TUNEL staining. The mechanism proteins expression of p-PI3K/PI3K, p-Akt/Akt, Bad, cytochrome c, Bcl 2, ERK1, IL-6, STAT3, MEK5, p-ERK5/ERK5, FGF2, and MMP2/9 were examined by western blot. Results showed that the 3- and 6-month-old SAMP8 mice liver, the senescence-accelerated liver cross-sections observed adipocytes, collagen, and apoptosis cells appear, but lower occurs by exercise, supplementary resveratrol, and their combination. Combination exercise and supplementary resveratrol at the 6-month-old SAMP8 mice has significant increase ratio by p-PI3K/PI3K compared to 3-month-old SAMP8 mice liver (p<0.01), and compared with without treatment SAMP8 mice liver. Exercise, supplementary resveratrol intake, and combined of exercise and resveratrol has facilitated p-Akt/Akt ratio increases, and ERK1/Bcl2 increases, but Bad/Cytochrome c decreases at the 6-month-old SAMP8 by western blot. IL-6/STAT3/MEK5 and p-ERK5/ERK5 ratio have good functions at 6-month-old SAMP8 mice better than 3-month-old SAMP8. FGF2/MMP2 decreases and MMP9 increases observed at the 3-month-old SAMP8 by exercise, supplementary resveratrol, and combination. Indeed, we suggest supplementary resveratrol can help exercise therapy ageing related liver diseases.
microRNAs (miR 9, 124, 155 and 224) transdifferentiate macrophages to neurons
Authors: Challagundla Naveen , Agrawal-Rajput Reena
Published: July 2020

Development is an irreversible process of differentiating the undifferentiated cells to functional cells. Brain development involves generation of cells with varied phenotype and functions, which is limited during adulthood, stress, damage/degeneration. Cellular reprogramming makes differentiation reversible process with reprogramming somatic/stem cells to alternative fate with/without stem cells. Exogenously expressed transcription factors or small molecule inhibitors have driven reprogramming of stem/somatic cells to neurons providing alternative approach for pre-clinical/clinical testing and therapeutics. Here in, we report a novel approach of microRNA (miR)-induced trans-differentiation of macrophages (CD11b high) to induced neuronal cells (iNCs) (neuronal markers high-Nestin, Nurr1, Map2, NSE, Tubb3 and Mash1) without exogenous use of transcription factors. miR 9, 124, 155 and 224 successfully transdifferentiated macrophages to neurons with transient stem cell-like phenotype. We report trans differentiation efficacy 18% and 21% with miR 124 and miR 155.in silico(String 10.0, miR gator, mESAdb, TargetScan 7.0) and experimental analysis indicate that the reprogramming involves alteration of pluripotency genes likeOct4, Sox2, Klf4, Nanogand pluripotency miR,miR 302. iNCs also shifted to G0 phase indicating manipulation of cell cycle by these miRs. Further, CD133+ intermediate cells obtained during current protocol could be differentiated to iNCs using miRs. The syanpsin+neurons were functionally active and displayed intracellular Ca+2evoke on activation. miRs could also transdifferentiate bone marrow-derived macrophages and peripheral blood mononuclear cells to neuronal cells. The current protocol could be employed for directin vivoreprogramming of macrophages to neurons without teratoma formation for transplantation and clinical studies.

Highlights

miR 9, miR 124 and miR155 could reprogramme macrophages to mature neurons.

miR-induced neuronal reprogramming involves stem cell like intermediate phenotype.

Graphical Abstract

Macrophages transfected with miR 9, 124, 155 and 224 alter pluripotency genes and neuronal differentiation genes via various mechanisms as elucidated. NIM components may also manipulate driving neuronal differentiation gene expression inducing formation of neuronal cells.