bluuebunny/crossref_metadata_2025 huggingface dataset:
Longitudinal changes in diet cause repeatable and largely reversible shifts in gut microbial communities of laboratory mice and are observed across segments of the entire intestinal tract
Authors: Low Adrian , Soh Melissa , Miyake Sou , Jie Aw Vanessa Zhi , Feng Jian , Wong Adeline , Seedorf Henning
Published: May 2021

Dietary changes are known to alter the composition of the gut microbiome. However, it is less understood how repeatable and reversible these changes are and how diet-switches affect the microbiota in the various segments of the gastrointestinal tract. Here, a treatment group of conventionally raised laboratory mice is subjected to two periods of western diet (WD) interrupted by a period of standard diet (SD) of the same duration. Beta-diversity analyses show that diet-induced microbiota changes are largely reversible (q= 0.1501; PERMANOVA, weighted-UniFrac comparison of the treatment-SD group to the control-SD group) and repeatable (q= 0.032; PERMANOVA, weighted-UniFrac comparison of both WD treatments). Furthermore, we report that diet switches alter the gut microbiota composition along the length of the intestinal tract in a segment-specific manner, leading to gut segment-specific Firmicutes/Bacteroidota ratios. We identified prevalent and distinct Amplicon Sequencing Variants (ASVs), particularly in genera of the recently describedMuribaculaceae, along the gut as well as ASVs that are differentially abundant between segments of treatment and control groups. Overall, this study provides insights into the reversibility of diet-induced microbiota changes and highlights the importance to expand sampling efforts beyond the collections of fecal samples to characterize diet-dependent and segment-specific microbiome differences.
FliW and CsrA govern flagellin (FliC) synthesis and play pleiotropic roles in virulence and physiology ofClostridioides difficileR20291
Authors: Zhu Duolong , Wang Shaohui , Sun Xingmin
Published: May 2021

Clostridioides difficileis a Gram-positive, spore-forming, and toxin-producing anaerobe that can cause nosocomial antibiotic-associated intestinal disease. InC. difficile, the expression of flagellar genes is coupled to toxin gene regulation and bacterial colonization and virulence. The flagellin FliC is responsible for pleiotropic gene regulation duringin vivoinfection. However, howfliCexpression is regulated is unclear. InBacillus subtilis, flagellin homeostasis and motility are coregulated by flagellar assembly factor FliW, Flagellin Hag (FliC homolog), and CsrA (Carbon storage regulator A), which is referred to as partner-switching mechanism “FliW-CsrA-Hag”. In this study, we characterized FliW and CsrA functions by deleting or overexpressingfliW, csrA, andfliW-csrAinC. difficileR20291. We showed that bothfliWdeletion orcsrAoverexpression in R20291, andcsrAcomplementation in R20291ΔWA (fliW-csrAcodeletion) dramatically decreased FliC production, however,fliCgene transcription was unaffected. While suppression offliCtranslation bycsrAoverexpression was mostly relieved whenfliWwas coexpressed, and no significant difference in FliC production was detected when onlyfliWwas complemented in R20291ΔWA. Further, loss offliWled to increased biofilm formation, cell adhesion, toxin production, and pathogenicity in a mouse model ofC. difficileinfection (CDI), whilefliW-csrAcodeletion decreased toxin production and mortalityin vivo. Taken together, these data suggest that CsrA negatively modulatesfliCexpression and FliW indirectly affectsfliCexpression through inhibition of CsrA post-transcriptional regulation, which seems similar to the “FliW-CsrA-Hag” switch inB. subtilis. Our data also suggest that “FliW-CsrA-fliC/FliC” can regulate many facets ofC. difficileR20291 pathogenicity.

IMPORTANCE

C. difficileflagellin FliC is associated with toxin gene expression, bacterial colonization and virulence, and is also involved in pleiotropic gene regulation duringin vivoinfection. However, how fliCexpression is regulated remains unclear. In light of “FliW-CsrA-Hag” switch coregulation mechanism reported inB. subtilis, we showed thatfliWandcsrAplay an important role in flagellin synthesis which affectsC. difficilemotility directly. Our data also suggest that FliW-CsrA-fliC/FliC” can regulate many facets ofC. difficileR20291 pathogenicity. These findings further aid us in understanding the virulence regulation inC. difficile.
Host bioenergetic parameters reveal cytotoxicity of anti-tuberculosis drugs undetected using conventional viability assays
Authors: Cumming Bridgette M. , Baig Zainab , Addicott Kelvin W. , Chen D , Steyn AJC
Published: May 2021

High attrition rates in tuberculosis (TB) drug development have been largely attributed to safety, which is likely due to the use of endpoint assays measuring cell viability to detect drug cytotoxicity. In drug development of cancer, metabolic and neurological disorders, and antibiotics, cytotoxicity is increasingly being assessed using extracellular flux (XF) analysis, which measures cellular bioenergetic metabolism in real-time. Here, we adopt the XF platform to investigate the cytotoxicity of drugs currently used in TB treatment on the bioenergetic metabolism of HepG2 cells, THP-1 macrophages, and human monocyte derived macrophages (hMDM). We found that the XF analysis reveals earlier drug-induced effects on the cells’ bioenergetic metabolism prior to cell death, measured by conventional viability assays. Furthermore, each cell type has a distinct response to drug treatment, suggesting that more than one cell type should be considered to examine cytotoxicity in TB drug development. Interestingly, chemically unrelated drugs with different modes of action onMycobacterium tuberculosishave similar effects on the bioenergetic parameters of the cells, thus, discouraging the prediction of potential cytotoxicity based on chemical structure and mode of action of new chemical entities. The clustering of the drug-induced effects on the hMDM bioenergetic parameters are reflected in the clustering of the effects of the drugs on cytokine production in hMDMs, demonstrating concurrence between the effects of the drugs on the metabolism and functioning of the macrophages. These findings can be used as a benchmark to establish XF analysis as a new tool to assay cytotoxicity in TB drug development.
Inhibitory concentrations of ciprofloxacin induce an adaptive response promoting the intracellular survival ofSalmonellaTyphimurium
Authors: Sridhar Sushmita , Forrest Sally , Pickard Derek , Cormie Claire , Lees Emily , Thomson Nicholas R , Dougan Gordon , Baker Stephen
Published: May 2021

Antimicrobial resistance (AMR) is a pressing global health crisis, which has been fuelled by the sustained use of certain classes of antimicrobials, including fluoroquinolones. While the genetic mutations responsible for decreased fluoroquinolone (ciprofloxacin) susceptibility are known, the implications of ciprofloxacin exposure on bacterial growth, survival, and interactions with host cells are not well described. Aiming to understand the influence of inhibitory concentrations of ciprofloxacinin vitro, we subjected three clinical isolates ofS.Typhimurium to differing concentrations of ciprofloxacin, dependent on their minimum inhibitory concentrations (MIC), and assessed the impact on bacterial growth, morphology, and transcription. We further investigated the differential morphology and transcription that occurred following ciprofloxacin exposure and measured the ability of ciprofloxacin-treated bacteria to invade and replicate in host cells. We found that ciprofloxacin-exposedS.Typhimurium are able to recover from inhibitory concentrations of ciprofloxacin, and that the drug induces specific morphological and transcriptional signatures associated with the bacterial SOS response, DNA repair, and intracellular survival. In addition, ciprofloxacin-treatedS.Typhimurium have increased capacity for intracellular replication in comparison to untreated organisms. These data suggest thatS.Typhimurium undergoes an adaptive response under ciprofloxacin perturbation that promotes cellular survival, a consequence that may justify more measured use of ciprofloxacin forSalmonellainfections. The combination of multiple experimental approaches provides new insights into the collateral effects that ciprofloxacin and other antimicrobials have on invasive bacterial pathogens.

Importance

Antimicrobial resistance is a critical concern in global health. In particular, there is rising resistance to fluoroquinolones, such as ciprofloxacin, a first-line antimicrobial for many Gram-negative pathogens. We investigated the adaptive response of clinical isolates ofSalmonellaTyphimurium to ciprofloxacin, finding that the bacteria adapt in short timespans to high concentrations of ciprofloxacin in a way that promotes intracellular survival during early infection. Importantly, by studying three clinically relevant isolates, we were able to show that individual isolates respond differently to ciprofloxacin, and for each isolate, there was a heterogeneous response under ciprofloxacin treatment. The heterogeneity that arises from ciprofloxacin exposure may drive survival and proliferation ofSalmonelladuring treatment and lead to drug resistance.
Disentangling cadherin-mediated cell-cell interactions in collective cancer cell migration
Authors: Zisis Themistoklis , Brückner David B. , Brandstätter Tom , d’Alessandro Joseph , Vollmar Angelika M. , Broedersz Chase P. , Zahler Stefan
Published: May 2021

Cell dispersion from a confined area is fundamental in a number of biological processes, including cancer metastasis. To date, a quantitative understanding of the interplay of single cell motility, cell proliferation, and intercellular contacts remains elusive. In particular, the role of E- and N-Cadherin junctions, central components of intercellular contacts, is still controversial. Combining theoretical modeling within vitroobservations, we investigate the collective spreading behavior of colonies of human cancer cells (T24). Inhibition of E- and N-Cadherin junctions decreases colony spreading and average spreading velocities, without affecting the strength of correlations in spreading velocities of neighboring cells. Based on a biophysical simulation model for cell migration, we show that the behavioral changes upon disruption of these junctions can be explained by reduced repulsive excluded volume interactions between cells. This suggests that cadherin-based intercellular contacts sharpen cell boundaries leading to repulsive rather than cohesive interactions between cells, thereby promoting efficient cell spreading during collective migration.
Network analysis of large phospho-signalling datasets: application to Plasmodium-erythrocyte interactions
Authors: Adderley Jack D. , O’Donoghue Finn , Doerig Christian , Davis Stephen
Published: May 2021

Phosphorylation based signalling is a complicated and intertwined series of pathways critical to all domains of life. This interconnectivity, though essential to life, makes understanding and decoding the interactions difficult. Large datasets of phosphorylation interactions through the activity of kinases on their numerous effectors are now being generated, however interpretation of the network environment remains challenging. In humans, many phosphorylation interactions have been identified across published works to form the known phosphorylation interaction network. We overlayed phosphorylation datasets onto this network which provided information to each of the connections. To analyse the datasets now mapped into a network, we designed a pathway analysis that uses random walks to identify chains of phosphorylation events occurring much more or much less frequently than expected. This analysis highlights pathways of phosphorylation that work synergistically, providing a rapid interpretation of the most critical pathways in a given dataset. Here we used datasets of human red blood cells infected with the notable stages ofPlasmodium falciparumasexual development. The analysis identified several known signalling interactions, and additional interactions which could form the basis of numerous future studies. The network analysis designed here is widely applicable to any comparative phosphorylation dataset across infection and disease and can provide a rapid and reliable analysis to guide validation studies.
Infection, recovery and re-infection of farmed mink with SARS-CoV-2
Authors: Rasmussen Thomas Bruun , Fonager Jannik , Sværke Jørgensen Charlotte , Lassaunière Ria , Hammer Anne Sofie , Quaade Michelle Lauge , Boklund Anette , Lohse Louise , Strandbygaard Bertel , Rasmussen Morten , Michaelsen Thomas Yssing , Mortensen Sten , Fomsgaard Anders , Belsham Graham J. , Bøtner Anette
Published: May 2021

Mink, on a farm with about 15,000 animals, became infected with SARS-CoV-2. Over 75% of tested animals were positive for SARS-CoV-2 RNA in throat swabs and 100% of tested animals were seropositive. The virus responsible had a deletion of nucleotides encoding residues H69 and V70 within the spike protein gene. The infected mink recovered and after free-testing of the mink, the animals remained seropositive. During follow-up studies, after a period of more than 2 months without virus detection, over 75% of tested animals scored positive again for SARS-CoV-2 RNA. Whole genome sequencing showed that the virus circulating during this re-infection was most closely related to the virus identified in the first outbreak on this farm but additional sequence changes had occurred. Animals had much higher levels of anti-SARS-CoV-2 antibodies after re-infection than at free-testing. Thus, following recovery from an initial infection, seropositive mink rapidly became susceptible to re-infection by SARS-CoV-2.

Article Summary Line

Following widespread infection with SARS-CoV-2 of mink on a farm, all tested animals had seroconverted and the farm was then tested free of infection; however, less than 3 months later, a further round of infection affected more than 75% of tested animals.
Salvaging complete and high-quality genomes of novel microbial species from a meromictic lake using a workflow combining long- and short-read sequencing platforms
Authors: Chen Yu-Hsiang , Chiang Pei-Wen , Rogozin Denis Yu , Degermendzhy Andrey G. , Chiu Hsiu-Hui , Tang Sen-Lin
Published: May 2021

Background

Most of Earth’s bacteria have yet to be cultivated. The metabolic and functional potentials of these uncultivated microorganisms thus remain mysterious, and the metagenome-assembled genome (MAG) approach is the most robust method for uncovering these potentials. However, MAGs discovered by conventional metagenomic assembly and binning methods are usually highly fragmented genomes with heterogeneous sequence contamination, and this affects the accuracy and sensitivity of genomic analyses. Though the maturation of long-read sequencing technologies provides a good opportunity to fix the problem of highly fragmented MAGs as mentioned above, the method’s error-prone nature causes severe problems of long-read-alone metagenomics. Hence, methods are urgently needed to retrieve MAGs by a combination of both long- and short-read technologies to advance genome-centric metagenomics.

Results

In this study, we combined Illumina and Nanopore data to develop a new workflow to reconstruct 233 MAGs—six novel bacterial orders, 20 families, 66 genera, and 154 species—from Lake Shunet, a secluded meromictic lake in Siberia. Those new MAGs were underrepresented or undetectable in other MAGs studies using metagenomes from human or other common organisms or habitats. Using this newly developed workflow and strategy, the average N50 of reconstructed MAGs greatly increased 10–40-fold compared to when the conventional Illumina assembly and binning method were used. More importantly, six complete MAGs were recovered from our datasets, five of which belong to novel species. We used these as examples to demonstrate many novel and intriguing genomic characteristics discovered in these newly complete genomes and proved the importance of high-quality complete MAGs in microbial genomics and metagenomics studies.

Conclusions

The results show that it is feasible to apply our workflow with a few additional long reads to recover numerous complete and high-quality MAGs from short-read metagenomes of high microbial diversity environment samples. The unique features we identified from five complete genomes highlight the robustness of this method in genome-centric metagenomic research. The recovery of 154 novel species MAGs from a rarely explored lake greatly expands the current bacterial genome encyclopedia and broadens our knowledge by adding new genomic characteristics of bacteria. It demonstrates a strong need to recover MAGs from diverse unexplored habitats in the search for microbial dark matter.
Exploring the cytotoxic mechanisms of Pediocin PA-1 towards HeLa and HT29 cells by comparison to known bacteriocins
Authors: Buss George P. , Wilson Cornelia
Published: May 2021

The purpose of this study was to explore potential mechanisms of cytotoxicity towards HeLa and HT29 cells displayed by Pediocin PA-1. We did this by carrying out sequence alignments and 3D modelling of related bacteriocins which have been studied in greater detail: Microcin E492, Eneterocin AB heterodimer and Divercin V41. Microcin E492 interacts with Toll-Like Receptor 4 in order to activate an apoptosis reaction, sequence alignment showed a high homology between Pediocin PA-1 and Microcin E492 and 3D modelling showed Pediocin PA-1 interacting with TLR-4 in a way reminiscent of Microcin E492. Furthermore, Pediocin PA-1 had the highest homology with the Enterocin heterodimer, particularly chain A; Enterocin has also shown to cause an apoptotic response in cancer cells. Based on this we are led to strongly believe Pediocin PA-1 interacts with TLRs in order to cause cell death. If this is the case it would explain the difference in cytotoxicity towards HeLa over HT29 cells, due to difference in expression of particular TLRs. Overall, we believe Pediocin PA-1 exhibits a dual effect which is dose dependant, like that of Microcin. Unfortunately, the COVID-19 pandemic meant that we were unable to carry out experiments in the lab, and the unavailability of important data meant we were unable to make solid conclusions but rather suggestions. However despite this we have still been able to highlight interesting findings and how these could be translated into future research and therapeutics in order to improve the quality of treatment and life of cancer patients.
Hydration layer of only few molecules controls lipid mobility in biomimetic membranes
Authors: Chattopadhyay Madhurima , Krok Emilia , Orlikowska Hanna , Schwille Petra , Franquelim Henri G. , Piatkowski Lukasz
Published: May 2021

Self-assembly of biomembranes results from the intricate interactions between water and the lipids’ hydrophilic head groups. Therefore, the lipid-water interplay strongly contributes to modulating membranes architecture, lipid diffusion, and chemical activity. Here, we introduce a new method of obtaining dehydrated, phase-separated, supported lipid bilayers (SLBs) solely by controlling the decrease of their environment’s relative humidity. This facilitates the study of the structure and dynamics of SLBs over a wide range of hydration states. We show that the lipid domain structure of phase-separated SLBs is largely insensitive to the presence of the hydration layer. In stark contrast, lipid mobility is drastically affected by dehydration, showing a 6-fold decrease in lateral diffusion. At the same time, the diffusion activation energy increases approximately twofold for the dehydrated membrane. The obtained results, correlated with the hydration structure of a lipid molecule, revealed that about 6-7 water molecules directly hydrating the phosphocholine moiety play a pivotal role in modulating lipid diffusion. These findings could provide deeper insights into the fundamental reactions where local dehydration occurs, for instance during cell-cell fusion, and help us better understand the survivability of anhydrobiotic organisms. Finally, the strong dependence of lipid mobility on the number of hydrating water molecules opens up an application potential for SLBs as very precise, nanoscale hydration sensors.
poreCov - an easy to use, fast, and robust workflow for SARS-CoV-2 genome reconstruction via nanopore sequencing
Authors: Brandt Christian , Krautwurst Sebastian , Spott Riccardo , Lohde Mara , Jundzill Mateusz , Marquet Mike , Hölzer Martin
Published: May 2021

In response to the SARS-CoV-2 pandemic, a highly increased sequencing effort has been established worldwide to track and trace ongoing viral evolution. Technologies such as nanopore sequencing via the ARTIC protocol are used to reliably generate genomes from raw sequencing data as a crucial base for molecular surveillance. However, for many labs that perform SARS-CoV-2 sequencing, bioinformatics is still a major bottleneck, especially if hundreds of samples need to be processed in a recurring fashion. Pipelines developed for short-read data cannot be applied to nanopore data. Therefore, specific long-read tools and parameter settings need to be orchestrated to enable accurate genotyping and robust reference-based genome reconstruction of SARS-CoV-2 genomes from nanopore data. Here we present poreCov, a highly parallel workflow written in Nextflow, using containers to wrap all the tools necessary for a routine SARS-CoV-2 sequencing lab into one program. The ease of installation, combined with concise summary reports that clearly highlight all relevant information, enables rapid and reliable analysis of hundreds of SARS-CoV-2 raw sequence data sets or genomes. poreCov is freely available on GitHub under the GNUv3 license: github.com/replikation/poreCov.
Elucidating Microscopic Events Driven by GTP Hydrolysis Reaction in Ras-GAP System with Semi-reactive Molecular Dynamics Simulation: Alternative Role of Phosphate Binding Loop as Mechanical Energy Storage
Authors: Kurisaki Ikuo , Tanaka Shigenori
Published: May 2021

ATPase and GTPase have been widely found as chemical energy-mechanical work transducers, whereas the physicochemical mechanisms are not satisfactorily understood. We addressed the problem by examining John Ross’ conjecture that repulsive Coulomb interaction between ADP/GDP and inorganic phosphate (Pi) does the mechanical work upon the system. We effectively simulated the consequence of GTP hydrolysis reaction in a complex system of Rat sarcoma (Ras) and GTPase activation protein (GAP) in the framework of classical molecular dynamics by switching force field parameters between the reactant and product systems. We then observed ca. 5 kcal/mol raise of potential energy about the phosphate-binding loop (P-loop) in Ras protein, indicating that the mechanical work generated via the GTP hydrolysis is converted into the local interaction energy and stored in the P-loop. Interestingly, this local energy storage in the P-loop depends on neither impulsive nor consecutive collisions of GDP and Pi with P-loop. Instead, GTP-GDP conversion itself does work on the Ras system, elevating the potential energy. These observations encourage us to challenge a conjecture previously given by Ross. We assert that triphosphate nucleotide hydrolyses do mechanical work by producing emergent steric interaction accompanied with relaxation, namely, a shift of biomolecular system to non-equilibrium state on the reshaped potential energy landscape. Recalling the universality of the P-loop motif among GTPases and ATPases, the observations that we obtained through this study would progress physicochemical understanding for the operating principles of GTP/ATP hydrolysis-driven biological nano-machines.
Perturbation of alphavirus and flavivirus infectivity by components of the bacterial cell wall
Authors: Langendries Lana , Jacobs Sofie , Abdelnabi Rana , Verwimp Sam , Kaptein Suzanne , Baatsen Pieter , Van Mellaert Lieve , Delang Leen
Published: May 2021

The impact of the host microbiota on arbovirus infections is currently not well understood. Arboviruses are viruses transmitted through the bites of infected arthropods, predominantly mosquitoes or ticks. The first site of arbovirus inoculation is the biting site in the host skin, which is colonized by a complex microbial community that could possibly influence arbovirus infection. We demonstrated that pre-incubation of arboviruses with certain components of the bacterial cell wall, including lipopolysaccharides (LPS) of some Gram-negative bacteria and lipoteichoic acids or peptidoglycan of certain Gram-positive bacteria, significantly reduced arbovirus infectivityin vitro. This inhibitory effect was observed for arboviruses of different virus families, including chikungunya virus of theAlphavirusgenus and Zika virus of theFlavivirusgenus, showing that this is a broad phenomenon. A modest inhibitory effect was observed following incubation with a panel of heat-inactivated bacteria, including bacteria residing on the skin. No viral inhibition was observed after pre-incubation of cells with LPS. Furthermore, a virucidal effect of LPS on viral particles was noticed by electron microscopy. Therefore, the main inhibitory mechanism seems to be due to a direct effect on the virus particles. Together, these results suggest that bacteria are able to decrease the infectivity of alphaviruses and flaviviruses.

Importance

During the past decades the world has experienced a vast increase in epidemics of alphavirus and flavivirus infections. These viruses can cause severe diseases such as hemorrhagic fever, encephalitis and arthritis. Several alpha- and flaviviruses, such as chikungunya virus, Zika virus and dengue virus, are significant global health threats because of their high disease burden, their widespread (re-)emergence and the lack of (good) anti-arboviral strategies. Despite the clear health burden, alphavirus and flavivirus infection and disease are not fully understood. A knowledge gap in the interplay between the host and the arbovirus is the potential interaction with host skin bacteria. Therefore, we studied the effect of (skin) bacteria and bacterial cell wall components on alphavirus and flavivirus infectivity in cell culture. Our results show that certain bacterial cell wall components markedly reduced viral infectivity by directly interacting with the virus particle.
Mutations in conserved residues of the myosin chaperone UNC-45 result in both reduced stability and chaperoning activity
Authors: Moncrief Taylor , Matheny Courtney J. , Gaziova Ivana , Miller John , Qadota Hiroshi , Benian Guy M. , Oberhauser Andres F.
Published: May 2021

Proper muscle development and function depends on myosin being properly folded and integrated into the thick filament structure. For this to occur the myosin chaperone UNC-45, or UNC-45B, must be present and able to chaperone myosin. Here we use a combination ofin vivo C. elegansexperiments andin vitrobiophysical experiments to analyze the effects of six missense mutations in conserved regions of UNC-45/UNC-45B. We found that the phenotype of paralysis and disorganized thick filaments in 5/6 of the mutant nematode strains can likely be attributed to both reduced steady state UNC-45 protein levels and reduced chaperone activity. Interestingly, the biophysical assays performed on purified proteins show that all of the mutations result in reduced myosin chaperone activity but not overall protein stability. This suggests that these mutations only cause protein instability in thein vivosetting and that these conserved regions may be involved in UNC-45 protein stability/ regulation via post translational modifications, protein-protein interactions, or some other unknown mechanism.
Structure and mechanistic features of the prokaryotic minimal RNase P
Authors: Feyh Rebecca , Wäber Nadine B. , Prinz Simone , Giammarinaro Pietro Ivan , Bange Gert , Hochberg Georg , Hartmann Roland K. , Altegoer Florian
Published: May 2021

Endonucleolytic removal of 5’-leader sequences from tRNA precursor transcripts (pre-tRNAs) by RNase P is essential for protein synthesis. Beyond RNA-based RNase P enzymes, protein-only versions of the enzyme exert this function in various Eukarya (there termed PRORPs) and in some bacteria (Aquifex aeolicusand close relatives); both enzyme types belong to distinct subgroups of the PIN domain metallonuclease superfamily. Homologs ofAquifexRNase P (HARPs) are also expressed in some other bacteria and many archaea, where they coexist with RNA-based RNase P and do not represent the main RNase P activity. Here we solved the structure of the bacterial HARP fromHalorhodospira halophilaby cryo-EM revealing a novel screw-like dodecameric assembly. Biochemical experiments demonstrate that oligomerization is required for RNase P activity of HARPs. We propose that the tRNA substrate binds to an extended spike-helix (SH) domain that protrudes from the screw-like assembly to position the 5’-end in close proximity to the active site of the neighboring dimer subunit. The structure suggests that eukaryotic PRORPs and prokaryotic HARPs recognize the same structural elements of pre-tRNAs (tRNA elbow region and cleavage site). Our analysis thus delivers the structural and mechanistic basis for pre-tRNA processing by the prokaryotic HARP system.
Single-cell transcriptome analysis of the zebrafish embryonic trunk
Authors: Metikala Sanjeeva , Chetty Satish Casie , Sumanas Saulius
Published: May 2021

During embryonic development, cells differentiate into a variety of distinct cell types and subtypes with diverse transcriptional profiles. To date, transcriptomic signatures of different cell lineages that arise during development have been only partially characterized. Here we used single-cell RNA-seq to perform transcriptomic analysis of over 20,000 cells disaggregated from the trunk region of zebrafish embryos at the 30 hpf stage. Transcriptional signatures of 27 different cell types and subtypes were identified and annotated during this analysis. This dataset will be a useful resource for many researchers in the fields of developmental and cellular biology and facilitate the understanding of molecular mechanisms that regulate cell lineage choices during development.
Corticothalamic Projections Deliver Enhanced-Responses to Medial Geniculate Body as a Function of the Temporal Reliability of the Stimulus
Authors: Kommajosyula Srinivasa P , Bartlett Edward L. , Cai Rui , Ling Lynne , Caspary Donald
Published: May 2021

Aging and challenging signal-in-noise conditions are known to engage use of cortical resources to help maintain speech understanding. Extensive corticothalamic projections are thought to provide attentional, mnemonic and cognitive-related inputs in support of sensory inferior colliculus (IC) inputs to the medial geniculate body (MGB). Here we show that a decrease in modulation depth, a temporally less distinct periodic acoustic signal, leads to a jittered ascending temporal code, changing MGB unit responses from adapting responses to responses showingrepetition-enhancement, posited to aid identification of important communication and environmental sounds. Young-adult male Fischer Brown Norway rats, injected with the inhibitory opsin archaerhodopsin T (ArchT) into the primary auditory cortex (A1), were subsequently studied using optetrodes to record single-units in MGB. Decreasing the modulation depth of acoustic stimuli significantly increased repetition-enhancement. Repetition-enhancement was blocked by optical inactivation of corticothalamic terminals in MGB. These data support a role for corticothalamic projections in repetition-enhancement, implying that predictive anticipation could be used to improve neural representation of weakly modulated sounds.

Key points

Aging has been shown to increase temporal jitter in the ascending acoustic code prompting use of cognitive/attentional mechanisms to help better understand communication-like signals.

Auditory thalamus receives extensive projections from cortex that are implicated in delivering higher-order cortical computations to enhance thalamic responses.

The present study modeled aging in young rats by using temporally less distinct stimuli shown to alter the pattern of MGB unit responses from response adaptation to repetition-enhancement. Enhanced responses to repeating less temporally distinct modulated stimuli were reversed when inputs from cortex to auditory thalamus were blocked. Collectively, these data argue that low salience temporal signals engage cortical processes to enhance coding of weakly modulated signals in auditory thalamus.
Insulin mRNA is stored in RNA granules in resting beta cells
Authors: Vasiljević Jovana , Vasiljević Djordje , Ganß Katharina , Sönmez Anke , Wegbrod Carolin , Quezada Esteban , Münster Carla , Schöniger Eyke , Friedland Daniela , Kipke Nicole , Distler Marius , Selbach Matthias , Solimena Michele
Published: May 2021

The glucose-stimulated biosynthesis of insulin in pancreatic islet beta cells is post-transcriptionally regulated. Several RNA-binding proteins (RBPs) that regulateInsulinmRNA stability and translation also bind mRNAs coding for other insulin secretory granule (ISG) proteins. However, an overview of these interactions and their glucose-induced remodelling is still missing. Here we identify two distinct sets of RBPs that were preferentially pulled down with the 5’-UTRs of mouseIns1,Ins2, splicedIns2,Ica512/PtprnandPc2/Pcsk2mRNAs from extracts of either resting or stimulated mouse insulinoma MIN6 cells. Among RBPs binding to all tested transcripts in resting conditions was hnRNP A2/B1.Hnrnpa2b1KO MIN6 cells contained lower levels ofIns1mRNA, proinsulin and insulin, and had reduced insulin secretion. In resting cells, both hnRNP A2/B1 andInsulinmRNAs localized to stress granules, which dissolved upon glucose stimulation.InsulinmRNA-positive RNA granules were also found in human pancreatic beta cellsin situ. Our results suggest that resting beta cells store mRNAs for insulin secretory granule proteins in stress granules through specific RNA protein interactions. Glucose stimulation remodels these interactions, releasing the transcripts, and another set of RBPs coordinates their translation.
Influenza A M2 Channel Oligomerization is Sensitive to its Chemical Environment
Authors: Townsend Julia A. , Sanders Henry M. , Rolland Amber D. , Park Chad K. , Horton Nancy C. , Prell James S. , Wang Jun , Marty Michael T.
Published: May 2021

Viroporins are small viral ion channels that play important roles in the viral infection cycle and are proven antiviral drug targets. Matrix protein 2 from influenza A (AM2) is the best characterized viroporin, and the current paradigm is that AM2 forms monodisperse tetramers. Here, we used native mass spectrometry and other techniques to characterize the oligomeric state of both the full-length and transmembrane domain (TM) of AM2 in a variety of different pH and detergent conditions. Unexpectedly, we discovered that AM2 formed a range of different oligomeric complexes that were strongly influenced by the local chemical environment. Native mass spectrometry of AM2 in nanodiscs with different lipids showed that lipids also affected the oligomeric states of AM2. Finally, nanodiscs uniquely enabled measurement of amantadine binding stoichiometries to AM2 in the intact lipid bilayer. These unexpected results reveal that AM2 can form a wider range of oligomeric states than previously thought possible, which may provide new potential mechanisms of influenza pathology and pharmacology.

Significance Statement

Many viruses contain small ion channels called viroporins that play diverse roles in viral infections. Influenza A M2 (AM2) is the best characterized viroporin and the target of the antivirals amantadine and rimantadine. Although past structural studies showed AM2 was a monodisperse tetramer, we discovered that AM2 can form polydisperse and dynamic oligomers that are sensitive to their local chemical environment. Our findings provide a new perspective on the structure and mechanisms of AM2 that may extend to other viroporins.
Breathing on Chip: Dynamic flow and stretch tune cellular composition and accelerate mucociliary maturation of airway epitheliumin vitro
Authors: Nawroth Janna C. , Roth Doris , van Schadewijk Annemarie , Ravi Abilash , Maulana Tengku Ibrahim , Senger Christiana N. , van Riet Sander , Ninaber Dennis K. , de Waal Amy M. , Kraft Dorothea , Hiemstra Pieter S. , Ryan Amy L , van der Does Anne M.
Published: May 2021

Human lung function is intricately linked to blood flow and breathing cycles, but it remains unknown how these dynamic cues shape human airway epithelial biology. Here we report a state-of-the-art protocol for studying effects of physiological airflow and stretch on differentiation, cellular composition, and mucociliary clearance of human primary airway epithelial cells cultured on a perfused airway chip. Perfused epithelial tissue cultures developed a large airway-like cellular composition with accelerated maturation and polarization of mucociliary clearance when compared to traditional (static) culture methods. Additional application of airflow and stretch to the airway chip resulted in a cellular composition more comparable to the small(er) airways, reduced baseline secretion of interleukin-8 and other inflammatory proteins, and reduced gene expression of matrix metalloproteinase (MMP) 9, fibronectin, and other extracellular matrix factors. These results indicate that breathing-like mechanical stimuli are important modulators of airway epithelial cell differentiation and homeostasis and that their fine-tuned application could generate models of specific epithelial regions, pathologies, and mucociliary (dys)function.
Glycocalyx crowding with synthetic mucin mimetics strengthens interactions between soluble and virus-associated lectins and cell surface glycan receptors
Authors: Honigfort Daniel J. , Altman Meghan O. , Gagneux Pascal , Godula Kamil
Published: May 2021

Membrane-associated mucins protect epithelial cell surfaces against pathogenic threats by serving as non-productive decoys that capture infectious agents and clear them from the cell surface and by erecting a physical barrier that restricts their access to target receptors on host cells. However, the mechanisms through which mucins function are still poorly defined due to a limited repertoire of tools available for tailoring their structure and composition in living cells with molecular precision. Using synthetic glycopolymer mimetics of mucins, we modeled the mucosal glycocalyx on red blood cells (RBC) and evaluated its influence on lectin (SNA) and virus (H1N1) adhesion to endogenous sialic acid receptors. The glycocalyx inhibited the rate of SNA and H1N1 adhesion in a size- and density-dependent manner, consistent with current view of the mucins as providing a protective shield against pathogens. Counterintuitively, increasing density of the mucin mimetics enhanced the retention of bound lectins and viruses. Careful characterization of SNA behavior at the RBC surface using a range of biophysical and imaging techniques revealed lectin-induced crowding and reorganization of the glycocalyx with concomitant enhancement in lectin clustering, presumably through the formation of a more extensive glycan receptor patch at the cell surface. Our findings indicate that glycan-targeting pathogens may exploit the biophysical and biomechanical properties of mucins to overcome the mucosal glycocalyx barrier.

Significance

Like other animal hosts, humans are constantly challenged by pathogens. This has led to an evolution of physical barriers coating all mucosal tissues, which are most vulnerable to infection. An important part of this defense is a dense brush of large proteins, called mucins, which are heavily decorated with sugars and keep pathogens at bay. Deciphering how pathogens overcome the mucin barrier is necessary to understand early stages of infection and to develop more effective treatments. By artificially installing the mucin-like shield on the surfaces of cells using synthetic sugar-bearing polymers, we have discovered a new physical mechanism by which proteins and viruses can exploit this barrier to more strongly adhere to their targets.
Quantitative comparison ofin vitro and in vivoembryogenesis at a single cell resolution
Authors: Tan Yuqi , Spangler Abby , Farid Michael , Peng Da , Cahan Patrick
Published: May 2021

Stem cell engineering is a powerful platform to address numerous challenges in regenerative medicine and developmental biology. Typically, engineered populations are derived by exposing pluripotent stem cells to a series of signaling cues meant to recapitulate developmental milestones, such as the induction of the primitive streak. Morphologic, genetic, transcriptomic, and functional differences between fully differentiated in vivo and engineered populations have long been recognized. However, the correspondence between engineered and native embryonic progenitors has been relatively less well characterized, largely due to their transient and highly heterogenous nature, and therefore many studies have relied on expression of a few canonical markers to ensure that their cells are on the correct path. To address this challenge, we first generated anin vivogastrulation mouse single cell RNA sequencing (scRNA-seq) reference data set and used it to train a collection of computational tools for comparing cell types, states, regulators, and trajectories across studies. Then we used these tools to evaluate a commonly used protocol designed to promote mesoderm derivation, as well as four previously published biomimetic protocols. Finally, we diversified our toolkits to reach a broader scientific community by implementing our primary analysis tool in Python and as an interactive web application.
Visualization of trigeminal ganglion sensory neuronal signaling regulated by Cdk5
Authors: Hu Minghan , Doyle Andrew , Yamada Kenneth M. , Kulkarni Ashok B.
Published: May 2021

The mechanisms underlying facial and oral pain are still incompletely understood, posing major therapeutic challenges. Cyclin-dependent kinase 5 (Cdk5) is a key neuronal kinase involved in pain signaling. However, the regulatory roles of Cdk5 in orofacial pain signaling and the possibility of therapeutic intervention at the level of mouse trigeminal ganglion primary neurons remain elusive. In this study, we used optimized intravital imaging to directly compare trigeminal neuronal activities after mechanical, thermal, and chemical stimulation. We then tested whether facial inflammatory pain in mice could be alleviated by the Cdk5 inhibitor peptide TFP5. We demonstrated regulation of total Ca2+intensities by Cdk5 activity using transgenic and knockout mouse models. In mice with orofacial inflammation, application of TFP5 specifically decreased total Ca2+intensities in response to noxious stimuli. It also alleviated inflammation-induced allodynia by inhibiting activation of trigeminal peripheral sensory neurons. Cdk5 inhibitors may provide promising non-opioid candidates for pain treatment.
ATRX promotes heterochromatin formation to protect cells from G-quadruplex DNA-mediated stress
Authors: Teng Yu-Ching , Sundaresan Aishwarya , O’Hara Ryan , Gant Vincent U. , Li Minhua , Martire Sara , Warshaw Jane N. , Basu Amrita , Banaszynski Laura A.
Published: May 2021

ATRX is a tumor suppressor that has been associated with protection from DNA replication stress, purportedly through resolution of difficult-to-replicate G-quadruplex (G4) DNA structures. While several studies demonstrate that loss of ATRX sensitizes cells to chemical stabilizers of G4 structures, the molecular function of ATRX at G4 regions during replication remains unknown. Here, we demonstrate that ATRX associates with the MCM replication complex and that loss of ATRX leads to G4 structure accumulation at newly synthesized DNA. We show that both the helicase domain of ATRX and its H3.3 chaperone function are required to protect cells from G4-induced replicative stress. Furthermore, these activities are upstream of heterochromatin formation mediated by the histone methyltransferase, ESET, which is the critical molecular event that protects cells from G4-mediated stress. In support, tumors carrying mutations in either ATRX or ESET show increased mutation burden at G4-enriched DNA sequences. Overall, our study provides new insights into mechanisms by which ATRX promotes genome stability with important implications for understanding impacts of its loss on human disease.
Corrosion inhibition of AA3003 aluminum alloy by self-assembled layers of myristic acid
Authors: Puc-Oxté María V. , Pech-Canul Máximo A.
Published: May 2021

Purpose

This paper aims to prepare highly hydrophobic films on aluminum AA3003 using myristic acid (MA) and evaluate its corrosion protection efficiency in a low-chloride solution.

Design/methodology/approach

The aluminum surface was initially treated with boiling water to develop a porous nanostructure, and then surface modification was carried out in ethanolic solutions with different concentrations of MA. The surface morphology, wetting behavior and film composition were first characterized, and then, the corrosion behavior was evaluated with electrochemical techniques.

Findings

The best hydrophobicity and corrosion resistance were obtained with 50 mM of MA. For such concentration, a water contact angle of 140° and protective efficiency of 96% were achieved. A multilayer structure was revealed by scanning electron microscope and X-ray photoelectron spectroscopy.

Originality/value

The results of this work shed light on the anticorrosion performance of fatty acid self-assembled multilayers on the surface of Al–Mn alloys.
Social performance and social media activity in times of pandemic: evidence from COVID-19-related Twitter activity
Authors: Baboukardos Diogenis , Gaia Silvia , She Chaoyuan
Published: May 2021

Purpose

The purpose of this study is to examine corporate disclosure of stakeholder-oriented actions on Twitter in response to COVID-19 during the pandemic outbreak and to empirically investigate whetherfirms’ social performance and their financial resilience impact on their engagement in, and communication of, stakeholder-oriented COVID-19 actions.

Design/methodology/approach

This study scrapes a sample of tweets communicated by major global listed firms between March 1, 2020 and April 30, 2020 and identifies disclosures that mention firm engagement in stakeholder-oriented actions in response to the COVID-19 pandemic. Cross-sectional regression analysis is used to examine the relationship between firms’ social performance and the number of tweets they post about stakeholder-oriented COVID-19 actions. Further, firms’ financial resilience is examined as a moderating factor of this relationship.

Findings

The results show that firms with better social performance are more likely to engage in and, hence, communicate stakeholder-oriented actions for the COVID-19 pandemic on Twitter. Moreover, it is evident that firms with better social performance communicate more stakeholder-oriented actions only when they belong to industries that have not been severely impacted by the pandemic.

Originality/value

This study has two important contributions. First, this study provides contemporary evidence of corporate disclosure of firms and their stakeholder-oriented actions on Twitter in response to the COVID-19 pandemic during the initial outbreak period. Second, it reveals insights into what characteristics drive firms to engage in costly corporate social responsibility (CSR) activities, and promote them on social media, in a period characterized by high economic uncertainty.