title: A machine-learning approach for detection of local brain networks and marginally weak signals identifies novel AD/MCI differentiating connectomic neuroimaging biomarkers

abstract:
IntroductionA computationally fast machine learning method is introduced for uncovering the wholebrain voxel-level connectomic spectra that differentiates different status of Alzheimers disease (AD). The method is applied to the Alzheimers Disease Neuroimaging Initiative (ADNI) Fluorinefluorodeoxyglucose Positron Emission Tomography (FDG-PET) imaging and clinical data and identified novel AD/MCI differentiating connectomic neuroimaging biomarkers.

MethodsA divide-and-conquer algorithm is introduced for detect informative local brain networks at voxel level and whole-brain scale. The connection information within the local networks is integrated into the node voxels, which makes detection of the marginally weak signals possible. Prediction accuracy is significantly improved by incorporating the local brain networks and marginally weak signals.

ResultsBrain connectomic structures differentiating AD and mild cognitive impairment (MCI), AD and healthy, and MIC and healthy were discovered. We identified novel AD/MCI-associated neuroimaging biomarkers by integrating local brain networks and marginally weak signals. For example, networkbased signals in paracentral lobule (p-value=6.1e-5), olfactory cortex (p-value=4.6e-5), caudate nucleus (1.8e-3) and precentral gyrus (1.8e-3) are informative in differentiating AD and MCI. Connections between calcarine sulcus and lingual gyrus (p-value=0.049), between parahippocampal gyrus and Amygdala (p-value=0.025), between rolandic opercula and insula lobes (p-values=0.0028 and 0.0026). An overall prediction accuracy of 95.3% was achieved by integrating the selected local brain networks and marginally weak signals, compared to 84.0% by not considering the inter-voxel connections and using marginally strong signals only.

Conclusion(i) The connectomic structures differentiating AD and MCI are significantly different to that differentiating MCI and healthy, which may indicate different neuronal etiology for AD and MCI. (ii) Many neuroimaging biomarkers exert their effects on the outcome diseases through their connections to other markers. Integrating such connections can help identify novel neuroimaging biomarkers and improve disease prediction accuracy.


title: Sleep spindle dynamics suggest over-consolidation in post-traumatic stress disorder

abstract: Devastating and persisting traumatic memories are a central symptom of post-traumatic stress disorder (PTSD). Sleep problems are highly co-occurrent with PTSD and intertwined with its etiology. Notably, sleep hosts memory consolidation processes, supported by sleep spindles (11-16 Hz). Here we assess the hypothesis that intrusive memory symptoms in PTSD may arise from excessive memory consolidation, reflected in exaggerated spindling. We use a newly developed spindle detection method, entailing minimal assumptions regarding spindle phenotype, to assess spindle activity in PTSD patients and traumatized controls (n=2x14, matched on gender). Our results show increased spindle activity in PTSD, which positively correlates with daytime intrusive memory symptoms. Together, these findings provide a putative mechanism through which profound sleep disturbance in PTSD may contribute to memory problems. Due to its uniform and unbiased approach, the new, minimal assumption spindle detection method seems a promising tool to detect aberrant spindling in psychiatric disorders.

category: neuroscience


title: Large herbivorous wildlife and livestock differentially influence the relative importance of different sources of carbon for riverine food webs

abstract: In many regions around the world, large populations of native wildlife have declined or been replaced by livestock grazing areas and farmlands, with consequences on terrestrial-aquatic ecosystems connectivity and trophic resources supporting food webs in aquatic ecosystems. The river continuum concept (RCC) and the riverine productivity model (RPM) predict a shift of carbon supplying aquatic food webs along the river: from terrestrial inputs in low-order streams to autochthonous production in mid-sized rivers. Here, we studied the influence of replacing large wildlife (mainly hippos) with livestock on the relative importance of C3 vegetation, C4 grasses and periphyton on macroinvertebrates in the Mara River, which is an African montane-savanna river known to receive large subsidy fluxes of terrestrial carbon and nutrients mediated by LMH, both wildlife and livestock. Using stable carbon ({delta}13C) and nitrogen ({delta}15N) isotopes, we identified spatial patterns of the relative importance of allochthonous carbon from C3 and C4 plants (woody vegetation and grasses, respectively) and autochthonous carbon from periphyton for macroinvertebrates at various sites of the Mara River and its tributaries. Potential organic carbon sources and invertebrates were sampled at 80 sites spanning stream orders 1 to 7, various catchment land uses (forest, agriculture and grasslands) and different loading rates of organic matter and nutrients by LMH (livestock and wildlife, i.e., hippopotamus). The importance of different sources of carbon along the river did not follow predictions of RCC and RPM. First, the importance of C3 and C4 carbon was not related to river order or location along the fluvial continuum but to the loading of organic matter (dung) by both wildlife and livestock. Notably, C4 carbon was important for macroinvertebrates even in large river sections inhabited by hippos. Second, even in small 1st -3rd order forested streams, autochthonous carbon was a major source of energy for macroinvertebrates, and this was fostered by livestock inputs fuelling aquatic primary production throughout the river network. Importantly, our results show that replacing wildlife (hippos) with livestock shifts river systems towards greater reliance on autochthonous carbon through an algae-grazer pathway as opposed to reliance on allochthonous inputs of C4 carbon through a detrital pathway.

category: ecology


title: GCAT dotplots characterize precisely and imprecisely defined topological features of DNA

abstract: Two-dimensional graphical dotplotting is adopted to identify sequence elements and their variants in lengths of DNA of up to 10 kb. Named GCAT for identification of precisely defined short sequences and their variants, its use complements the precise matching of many computational programs, including BLAST. Short reiterated "search" sequences are entered in the Y-axis of the dotplot program to be matched at their identical and near identical (variant) sites in a sequence of interest entered in the X-axis. The result is a barcode-like representation of the identified sequence elements along the X-axis of the dotplot. Alignments of searches and sequence landmarks provide visualization of composition and juxtapositions. The method is described here by example of characterizations of three distinctive sequences available in the annotated Drosophila melanogaster reference genome (www.flybase.org): the Jonah 99C gene region, the transcript of Dipeptidase B and the transposable element roo. Surprising observations emerging from these explorations include in-frame STOP codons in the large exonic intron of Dip-B, high A-content of the replicative strand of roo as TE example and similarities of its ORF and the large intron of Dip-B.

category: genetics


title: Species-wide phylogenomics of the Staphylococcus aureus agr operon reveals convergent evolution of frameshift mutations

abstract:
Staphylococcus aureus is a prominent nosocomial pathogen that causes several life-threatening diseases such as pneumonia and bacteremia. S. aureus modulates expression of its arsenal of virulence factors through sensing and integrating responses to environmental signals. The agr (accessory gene regulator) quorum sensing (QS) system is a major regulator of virulence phenotypes in S. aureus. There are four agr specificity groups each with a different autoinducer peptide sequence (encoded by the agrD gene). Though agr is critical for expression of many toxins, paradoxically, S. aureus strains often have non-functional agr activity due to loss-of-function mutations in the four-gene agr operon. To understand patterns in agr variability across S. aureus, we undertook a species-wide genomic investigation. We developed a software tool (AgrVATE; https://github.com/VishnuRaghuram94/AgrVATE) for typing and detecting frameshift mutations in the agr operon. In an analysis of over 40,000 S. aureus genomes, we showed close association between agr type and S. aureus clonal complex. We also found strong linkage between agrBDC alleles (encoding the peptidase, the autoinducing peptide itself, and the peptide sensor respectively) but not agrA (encoding the -response regulator). More than five percent of genomes were found to have frameshift mutations in the agr operon. Though most mutations occur only once in the entire species, we observed a small number of recurring mutations evolving convergently across different clonal lineages. Phylogenetic patterns suggested that strains with agr frameshifts were evolutionary dead ends. Overall, genomic analysis of agr operon suggests evolution through multiple processes with functional consequences that are not fully understood.

ImportanceStaphylococcus aureus is a globally pervasive pathogen that produces a plethora of toxic molecules that can degrade, evade, or inhibit the host immune cells. Production of these toxins is mainly controlled by an active agr quorum sensing system, which senses and responds to bacterial cell density. However, there are many reports of S. aureus strains with genetic changes leading to impaired agr activity, often found during chronic bloodstream infections, and may be associated with increased disease severity. We developed an open-source software called AgrVATE to type agr systems and identify putative frameshifts. We used AgrVATE for a species-wide genomic survey of S. aureus, finding that more than 5 % of strains in the public database had non-functional agr systems. We also provide new insights into the evolution of these genetic mutations in the agr system. Overall, this study contributes to our understanding of a common but relatively understudied means of virulence regulation in S. aureus.

category: genomics


title: Discovering molecular features of intrinsically disordered regions by using evolution for contrastive learning

abstract: A major challenge to the characterization of intrinsically disordered regions (IDRs), which are widespread in the proteome, but relatively poorly understood, is the identification of molecular features that mediate functions of these regions, such as short motifs, amino acid repeats and physicochemical properties. Here, we introduce a proteome-scale feature discovery approach for IDRs. Our approach, which we call "reverse homology", exploits the principle that important functional features are conserved over evolution. We use this as a contrastive learning signal for deep learning: given a set of homologous IDRs, the neural network has to correctly choose a held-out homologue from another set of IDRs sampled randomly from the proteome. We pair reverse homology with a simple architecture and standard interpretation techniques, and show that the network learns conserved features of IDRs that can be interpreted as motifs, repeats, or bulk features like charge or amino acid propensities. We also show that our model can be used to produce visualizations of what residues and regions are most important to IDR function, generating hypotheses for uncharacterized IDRs. Our results suggest that feature discovery using unsupervised neural networks is a promising avenue to gain systematic insight into poorly understood protein sequences.

category: bioinformatics


title: Discovery of small molecule pathway regulators by image profile matching

abstract:
Identifying chemical regulators of biological pathways is a time-consuming bottleneck in developing therapeutics and research compounds. Typically, thousands to millions of candidate small molecules are tested in target-based biochemical screens or phenotypic cell-based screens, both expensive experiments customized to each disease. Here, our uncustomized, virtual profile-based screening approach instead matches compounds to pathways based on phenotypic information in public cell image data, created using the Cell Painting assay. Our simple correlation-based computational strategy efficiently uncovered the expected small molecule regulators for 32% of positive control gene queries. In discovery mode, we identified compounds related to three of seven tested query genes, validated in subsequent gene-relevant assays. This image profile-based approach could replace many customized labor- and resource-intensive screens and accelerate the discovery of biologically and therapeutically useful compounds.

One sentence summaryIf a genetic perturbation impacts cell morphology, a computational query can reveal compounds whose morphology "matches".


title: Virtual screening for small molecule pathway regulators by image profile matching


title: OperonSEQer: A set of machine-learning algorithms with threshold voting for detection of operon pairs using short-read RNA-sequencing data

abstract:
Operon prediction in prokaryotes is critical not only for understanding the regulation of endogenous gene expression, but also for exogenous targeting of genes using newly developed tools such as CRISPR-based gene modulation. A number of methods have used transcriptomics data to predict operons, based on the premise that contiguous genes in an operon will be expressed at similar levels. While promising results have been observed using these methods, most of them do not address uncertainty caused by technical variability between experiments, which is especially relevant when the amount of data available is small. In addition, many existing methods do not provide the flexibility to determine whether the stringency with which genes should be evaluated for being in an operon pair. We present OperonSEQer, a set of machine learning algorithms that uses the statistic and p-value from a non-parametric analysis of variance test (Kruskal-Wallis) to determine the likelihood that two adjacent genes are expressed from the same RNA molecule. We implement a voting system to allow users to choose the stringency of operon calls depending on whether your priority is high coverage of operons or high accuracy of the calls. In addition, we provide the code so that users can retrain the algorithm and re-establish hyperparameters based on any data they choose, allowing for this method to be expanded on as additional data is generated and incorporated. We show that our approach detects operon pairs that are missed by current methods by comparing our predictions to publicly available long-read sequencing data. OperonSEQer therefore improves on existing methods in terms of accuracy, flexibility and adaptability.

Author SummaryBacteria and archaea, single-cell organisms collectively known as prokaryotes, live in all imaginable environments and comprise the majority of living organisms on this planet. Prokaryotes play a critical role in the homeostasis of multicellular organisms (such as animals and plants) and ecosystems. In addition, bacteria can be pathogenic, and cause a variety of diseases in these same hosts and ecosystems. In short, understanding the biology and molecular functions of bacteria and archaea and devising mechanisms to engineer and optimize their properties are critical scientific endeavors with significant implications in healthcare, agriculture, manufacturing and climate science among others. One major molecular difference between unicellular and multicellular organisms is the way the express genes - rather than making individual RNA molecules like multicellular organisms, prokaryotes express genes in long contiguous RNA molecules known as operons, which are subsequently processed. Understanding which genes exist within operons is critical for elucidating basic biology and for engineering organisms. In this work, we use a combination of statistical and machine learning-based methods to use next-generation sequencing data to predict operon structure across a range of prokaryotes. Our method provides a easily implemented, robust, accurate and flexible way to determine operon structure in an organism-agnosic manner using readily-available data.


title: Temporal variation in lymphocyte proteomics

abstract: Chronic Lymphocytic Leukemia (CLL) is a slow progressing disease, characterized by a long asymptomatic stage followed by a symptomatic stage during which patients receive treatment. While proteomic studies have discovered differential pathways in CLL, the proteomic evolution of CLL during the asymptomatic stage has not been studied. In this pilot study, we show that by using small sample sizes comprising ~145 cells, we can detect important features of CLL necessary for studying tumor evolution. Our small samples are collected at two time points and reveal large proteomic changes in healthy individuals over time. A meta-analysis of two CLL proteomic papers showed little commonality in differentially expressed proteins and demonstrates the need for larger control populations sampled over time. To account for proteomic variability between time points and individuals, large control populations sampled at multiple time points are necessary for understanding CLL progression. Data is available via ProteomeXchange with identifier PXD027429.


title: TRPM2 deficiency protects against atherosclerosis by inhibiting TRPM2-CD36 inflammatory axis in macrophages

abstract:
Atherosclerosis is the major cause of ischemic heart diseases and ischemic brain stroke, which are the leading causes of mortality worldwide. The central pathological features of atherosclerosis include macrophage infiltration and foam cell formation. However, the detailed mechanisms regulating these two processes remain unclear. Here we show that oxidative stress-activated Ca2+-permeable TRPM2 plays a key role in the pathogenesis of atherosclerosis. Trpm2 deletion produces a potent protective effect against atherosclerosis in ApoE-/- mice fed with a high-fat diet (HFD), as evidenced by reduced atherosclerotic plaque burden, decreased macrophage load and suppressed inflammasome activation in the vessel wall. Moreover, we show that Trpm2 deletion or inhibition reduces oxidized low-density lipoprotein (oxLDL) uptake by macrophages, suppresses macrophage infiltration induced by monocyte chemoattractant protein-1 (MCP1), and prevents the impairment of macrophage emigration caused by oxLDL. Intriguingly, we uncover that activation of CD36, an oxLDL receptor, can promote the activation of TRPM2, and vice versa, the CD36-mediated inflammatory cascade in atherosclerosis is dependent on TRPM2. In transfected HEK293T cells, CD36 ligands oxLDL and TSP1 induce TRPM2 activation in a CD36-dependent manner. Deleting Trpm2 or inhibiting TRPM2 activity in cultured macrophages suppresses the CD36 signaling cascade induced by oxLDL and TSP1. Our studies establish TRPM2-CD36 axis as a new mechanism underlying atherogenesis, and suggest TRPM2 as an effective therapeutic target for atherosclerosis.

HIGHLIGHTSO_LITrpm2 deletion protects against atherosclerosis in ApoE-/- mice fed with a high-fat diet (HFD)
C_LIO_LITrpm2 deficiency reduces atherosclerotic lesions by minimizing foam cell formation, inhibiting macrophage infiltration and preserving macrophage emigration
C_LIO_LITRPM2 activation is required for CD36-induced oxLDL uptake and subsequent inflammatory responses
C_LIO_LIThe ligands of CD36, oxLDL and TSP1, activate TRPM2, thereby perpetuating TRPM2-CD36 inflammatory cycle in atherogenesis cascade
C_LIO_LIOur data establish TRPM2-CD36 axis as a new atherogenesis mechanism and TRPM2 as a novel therapeutic target for atherosclerosis
C_LI

O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY TRPM2-mediated Ca2+ signal is essential for CD36 induced oxLDL uptake and atherosclerosis in ApoE-/- mice fed with a high-fat diet (HFD). The activation of CD36 and TRPM2 form a positive feedback loop in atherogenesis.

C_FIG_DISPLAY

category: physiology


title: Carbon nanotube biocompatibility in plants is determined by their surface chemistry

abstract:
Agriculture faces significant global challenges including climate change and an increasing food demand due to a growing population. Addressing these challenges will require the adoption of transformative innovations into biotechnology practice, such as nanotechnology. Recently, nanomaterials have emerged as unmatched tools for their use as biosensors, or as biomolecule delivery vehicles. Despite their increasingly prolific use, plant-nanomaterial interactions remain poorly characterized, drawing into question the breadth of their utility and their broader environmental compatibility. Herein, we characterize Arabidopsis thaliana transcriptional response to single walled carbon nanotubes (SWNTs) with two different surface chemistries commonly used for biosensing and nucleic acid delivery: oligonucleotide adsorbed-pristine SWNTs, and polyethyleneimine-SWNTs loaded with plasmid DNA (PEI-SWNTs), both introduced by leaf infiltration. We observed that SWNTs elicit a mild stress response almost undistinguishable from the infiltration process, indicating that these nanomaterials are well-tolerated by the plant. However, PEI-SWNTs induce a much larger transcriptional reprogramming that involves stress, immunity, and senescence responses. PEI-SWNT-induced transcriptional profile is very similar to that of mutant plants displaying a constitutive immune response or treated with stress-priming agrochemicals. We selected molecular markers from our transcriptomic analysis and identified PEI as the main cause of this reaction. We show that PEI-SWNT response is concentration-dependent and, when persistent over time, leads to cell death. We probed a panel of PEI variant-functionalized SWNTs across two plant species and identified biocompatible SWNT surface functionalizations. Our results highlight the importance of nanoparticle surface chemistry on their biocompatibility and will facilitate the use of functionalized nanomaterials for agricultural improvement.

Significance statementNanomaterials can be used in agriculture as biosensors to monitor plant health, as fertilizers or growth regulators, and as delivery vehicles for genome engineering reagents to improve crops. However, the interactions between nanoparticles and plant cells are not well understood. Here, we characterize the plant transcriptomic response to single-walled carbon nanotubes (SWNTs) commonly used for sensing and nucleic acid delivery. While SWNTs themselves are well tolerated by plants, SWNTs surface-functionalized with positively charged polymers become toxic and produce cell death. We identify molecular markers of this toxic response to create biocompatible SWNT formulations. These results highlight the significance of nanoparticle surface chemistry, perhaps more than the nanoparticles themselves, on downstream interactions of nanoparticles with the environment.

category: plant biology


title: Robust adaptive distance functions for approximate Bayesian inference on outlier-corrupted data

abstract:
Approximate Bayesian Computation (ABC) is a likelihood-free parameter inference method for complex stochastic models in systems biology and other research areas. While conceptually simple, its practical performance relies on the ability to efficiently compare relevant features in simulated and observed data via distance functions. Complications can arise particularly from the presence of outliers in the data, which can severely impair the inference. Thus, robust methods are required that provide reliable estimates also from outlier-corrupted data.

We illustrate how established ABC distance functions are highly sensitive to outliers, and can in practice yield erroneous or highly uncertain parameter estimates and model predictions. We introduce self-tuned outlier-insensitive distance functions, based on a popular adaptive distance weighting concept, complemented by a simulation-based online outlier detection and downweighting routine. We evaluate and compare the presented methods on six test models covering different model types, problem features, and outlier scenarios. Our evaluation demonstrates substantial improvements on outlier-corrupted data, while giving at least comparable performance on outlier-free data.

The developed methods have been made available as part of the open-source Python package pyABC (https://github.com/icb-dcm/pyabc).

category: systems biology


title: Large-scale dendritic spine extraction and analysis through petascale computing

abstract: The synapse is a central player in the nervous system serving as the key structure that permits the relay of electrical and chemical signals from one neuron to another. The anatomy of the synapse contains important information about the signals and the strength of signal it transmits. Because of their small size, however, electron microscopy (EM) is the only method capable of directly visualizing synapse morphology and remains the gold standard for studying synapse morphology. Historically, EM has been limited to small fields of view and often only in 2D, but recent advances in automated serial EM ("connectomics") have enabled collecting large EM volumes that capture significant fractions of neurons and the different classes of synapses they receive (i.e. shaft, spine, soma, axon). However, even with recent advances in automatic segmentation methods, extracting neuronal and synaptic profiles from these connectomics datasets are difficult to scale over large EM volumes. Without methods that speed up automatic segmentation over large volumes, the full potential of utilizing these new EM methods to advance studies related to synapse morphologies will never be fully realized. To solve this problem, we describe our work to leverage Argonne leadership-scale supercomputers for segmentation of a 0.6 terabyte dataset using state of the art machine learning-based segmentation methods on a significant fraction of the 11.69 petaFLOPs supercomputer Theta at Argonne National Laboratory. We describe an iterative pipeline that couples human and machine feedback to produce accurate segmentation results in time frames that will make connectomics a more routine method for exploring how synapse biology changes across a number of biological conditions. Finally, we demonstrate how dendritic spines can be algorithmically extracted from the segmentation dataset for analysis of spine morphologies. Advancing this effort at large compute scale is expected to yield benefits in turnaround time for segmentation of individual datasets, accelerating the path to biology results and providing population-level insight into how thousands of synapses originate from different neurons; we expect to also reap benefits in terms of greater accuracy from the more compute-intensive algorithms these systems enable.


title: Training a spiking neuronal network model of visual-motor cortex to play a virtual racket-ball game using reinforcement learning

abstract:
Recent models of spiking neuronal networks have been trained to perform behaviors in static environments using a variety of learning rules, with varying degrees of biological realism. Most of these models have not been tested in dynamic visual environments where models must make predictions on future states and adjust their behavior accordingly. The models using these learning rules are often treated as black boxes, with little analysis on circuit architectures and learning mechanisms supporting optimal performance.

Here we developed visual/motor spiking neuronal network models and trained them to play a virtual racket-ball game using several reinforcement learning algorithms inspired by the dopaminergic reward system. We systematically investigated how different architectures and circuit-motifs (feed-forward, recurrent, feedback) contributed to learning and performance. We also developed a new biologically-inspired learning rule that significantly enhanced performance, while reducing training time.

Our models included visual areas encoding game inputs and relaying the information to motor areas, which used this information to learn to move the racket to hit the ball. Neurons in the early visual area relayed information encoding object location and motion direction across the network. Neuronal association areas encoded spatial relationships between objects in the visual scene. Motor populations received inputs from visual and association areas representing the dorsal pathway. Two populations of motor neurons generated commands to move the racket up or down. Model-generated actions updated the environment and triggered reward or punishment signals that adjusted synaptic weights so that the models could learn which actions led to reward.

Here we demonstrate that our biologically-plausible learning rules were effective in training spiking neuronal network models to solve problems in dynamic environments. We used our models to dissect the circuit architectures and learning rules most effective for learning. Our model shows that learning mechanisms involving different neural circuits produce similar performance in sensory-motor tasks. In biological networks, all learning mechanisms may complement one another, accelerating the learning capabilities of animals. Furthermore, this also highlights the resilience and redundancy in biological systems.


title: Direct and indirect interactions promote complexes of the lipoprotein LbcA, the CtpA protease and its substrates, and other cell wall proteins in Pseudomonas aeruginosa

abstract:
The Pseudomonas aeruginosa lipoprotein LbcA was discovered because it copurified with and promoted the activity of CtpA, a carboxyl-terminal processing protease (CTP) required for type III secretion system function, and for virulence in a mouse model of acute pneumonia. In this study we explored the role of LbcA by determining its effect on the proteome and its participation in protein complexes. lbcA and ctpA null mutations had strikingly similar effects on the proteome, suggesting that facilitating CtpA might be the most impactful role of LbcA in the bacterial cell. Independent complexes containing LbcA and CtpA, or LbcA and substrate, were isolated from P. aeruginosa cells, indicating that LbcA facilitates proteolysis by recruiting the protease and its substrates independently. An unbiased examination of proteins that copurified with LbcA revealed an enrichment for proteins associated with the cell wall. One of these copurification partners was found to be a new CtpA substrate, and the first substrate that is not a peptidoglycan hydrolase. Many of the other LbcA copurification partners are known or predicted peptidoglycan hydrolases. However, some of these LbcA copurification partners were not cleaved by CtpA, and an in vitro assay revealed that while CtpA and all of its substrates bound to LbcA directly, these non-substrates did not. Subsequent experiments suggested that the non substrates might co-purify with LbcA by participating in multi-enzyme complexes containing LbcA-binding CtpA substrates.

IMPORTANCECarboxyl-terminal processing proteases (CTPs) are widely conserved and associated with the virulence of several bacteria, including CtpA in Pseudomonas aeruginosa. CtpA copurifies with the uncharacterized lipoprotein, LbcA. This study shows that the most impactful role of LbcA might be to promote CtpA-dependent proteolysis, and that it achieves this as a scaffold for CtpA and its substrates. It also reveals that LbcA copurification partners are enriched for cell wall-associated proteins, one of which is a novel CtpA substrate. Some of the other LbcA copurification partners are not cleaved by CtpA, but might copurify with LbcA because they participate in multi-enzyme complexes containing CtpA substrates. These findings are important, given the links between CTPs, their associated proteins, peptidoglycan remodeling, and virulence.

category: microbiology


title: The Diguanylate Cyclase YfiN of Pseudomonas aeruginosa Regulates Biofilm Maintenance in Response to Peroxide

abstract:
Pseudomonas aeruginosa forms surface-attached communities that persist in the face of antimicrobial agents and environmental perturbation. Published work has found extracellular polysaccharide (EPS) production, regulation of motility and induction of stress response pathways as contributing to biofilm tolerance during such insults. However, little is known regarding the mechanism(s) whereby biofilm maintenance is regulated when exposed to such environmental challenges. Here, we provide evidence that the diguanylate cyclase YfiN is important for the regulation of biofilm maintenance when exposed to peroxide. We find that, compared to the wild type (WT), static biofilms of the {Delta}yfiN mutant exhibit a maintenance defect, which can be further exacerbated by exposure to peroxide (H2O2); this defect can be rescued through genetic complementation. Additionally, we found that the {Delta}yfiN mutant biofilms produce less c-di-GMP than WT, and that H2O2 treatment enhanced motility of surface-associated bacteria and increased cell death for the {Delta}yfiN mutant grown as a biofilm compared to WT biofilms. These data provide evidence that YfiN is required for biofilm maintenance by P. aeruginosa, via c-di-GMP signaling, to limit motility and protect viability in response to peroxide stress. These findings add to the growing recognition that biofilm maintenance by P. aeruginosa is an actively regulated process that is controlled, at least in part, by the wide array of c-di-GMP metabolizing enzymes found in this microbe.

ImportanceWe build on previous findings that suggest that P. aeruginosa utilizes c-di-GMP metabolizing enzymes to actively maintain a mature biofilm. Here, we explore how the diguanylate cyclase YfiN contributes to the regulation of biofilm maintenance during peroxide exposure. We find that mature P. aeruginosa biofilms require YfiN to synthesize c-di-GMP, regulate motility and to insure viability during peroxide stress. These findings provide further evidence that the modulation of c-di-GMP in response to environmental signals is an important mechanism by which biofilms are maintained.


title: Exogenous pigments shield microorganisms from spaceflight-induced changes

abstract: Research has indicated that pigments commonly produced by microorganisms may be protective against the environmental stresses inherent to spaceflight. However, few studies have directly tested the protective capabilities of microbial pigments applied externally as shielding materials. In this study, liquid cultures of Bacillus subtilis were shielded by various pigment solutions, and solid media cultures of Bacillus subtilis were co-inoculated with the highly pigmented microorganisms Aspergillus niger and Neurospora crassa. These experiments were conducted in a compact, automated payload aboard the International Space Station (ISS) interior for 30 days. Post-flight phenotypic analyses of liquid cultures showed that solutions of carotenoid pigments were effective at minimizing detrimental effects of spaceflight. Elevated growth rate was observed for solid cultures, and distinct morphology changes were identified in both liquid and solid samples and quantified as markers of spaceflight-induced stress. These findings collectively progress our understanding of microbial pigments for the development of space-related applications.


title: Non-coding circular RNAs repertoire and expression profile during Brassica rapa pollen development

abstract: Circular RNAs (circRNAs) are covalently closed long non-coding RNA (lncRNA) molecules generated by the back-splicing of exons from linear precursor mRNAs. Though linear lncRNAs have been shown to play important regulatory roles in diverse biological and developmental processes, little is known about the role of their circular counterparts. In this study, we have performed high-throughput RNA sequencing to delineate the expression profile and potential function of circRNAs during the five stages of pollen development in Brassica rapa. A total of 1180 circRNAs were detected in pollen development, of which 367 showed stage-specific expression patterns. Functional enrichment and metabolic pathway analysis showed that the parent genes of circRNAs were mainly involved in pollen-related molecular and biological processes such as mitotic and meiosis cell division, DNA processes, protein synthesis, protein modification, and polysaccharide biosynthesis. Moreover, by predicting the circRNA-miRNA network from our differentially expressed circRNAs, we found 88 circRNAs with potential miRNA binding sites suggesting their role in post-transcriptional regulation of the genes. Finally, we confirmed the back-splicing sites of 9 randomly selected circRNAS using divergent primers and Sanger sequencing. Our study presents the first systematic analysis of circular RNAs during pollen development and forms the basis of future studies for unlocking complex gene regulatory networks underpinning reproduction in flowering plants.

category: molecular biology


title: Habit formation viewed as structural change in the behavioral network

abstract: Habit formation is a process in which an action becomes involuntary. While goal-directed behavior is driven by its consequences, habits are elicited by a situation rather than its consequences. Existing theories have proposed that actions are controlled by corresponding two distinct systems. Although canonical theories based on such distinctions are starting to be challenged, there is no theoretical framework that implements goal-directed behavior and habits within a single system. Here, we propose a novel theoretical framework by hypothesizing that behavior is a network composed of several responses. With this framework, we have shown that the transition of goal-directed actions to habits is caused by a change in a single network structure. Furthermore, we confirmed that the proposed network model behaves in a manner consistent with the existing experimental results reported in animal behavioral studies. Our results revealed that habit could be formed under the control of a single system rather than two distinct systems. By capturing the behavior as a single network change, this framework provides a new perspective on studying the structure of the behavior for experimental and theoretical research.

category: animal behavior and cognition


title: A small-molecule fluorescent probe for live-cell imaging of endocytosis

abstract: Endocytosis involves plasma membrane-derived vesicles for the recycling of intra- and extracellular components. Increasing evidence suggests that endocytosis is related to maintaining intracellular homeostasis and defense against disease. Consequently, investigation of the endocytic pathway attracts considerable scientific interest. This study reports live-cell imaging of endocytosis using the newly-developed fluorescent probe ECGreen. We demonstrate that ECGreen is not membrane permeable and its fluorescence signal increases in acidic conditions. Because of these characteristics, ECGreen remains on the plasma membrane, and then shows increased fluorescence when it is internalized into the acidic vesicles formed in the endocytic process. ECGreen allows direct observation of the internalized vesicle; it is a valuable new probe for endocytic imaging.

category: biochemistry


title: Removing the ceiling on droplet microfluidics

abstract: We developed an open channel droplet microfluidic system that autonomously generates droplets at low Ca (~10-4-10-3) by leveraging competing hydrostatic and capillary pressure. With only our open channel polytetrafluoroethylene (PTFE) device, pipettes, and commercially available carrier fluid, we produce hundreds of microliter droplets; tubing, electronics, or pumps are not required, making droplet technology feasible for research labs without external flow generators. Furthermore, we demonstrated conceptual applications that showcase the process of droplet generation, splitting, transport, incubation, mixing, and sorting in our system. Unlike conventional droplet microfluidics, the open nature of the device enables the use of physical tools such as tweezers and styli to directly access the system; with this, we developed a new method of droplet sorting and transfer that capitalizes on the Cheerios effect, the aggregation of buoyant objects along a liquid interface. Our platform offers enhanced usability, direct access to the droplet contents, easy manufacturability, compact footprint, and high customizability. This design is a first step in exploring the space of power-free open droplet microfluidic systems and provide design rules for similar channel designs.

category: bioengineering


title: Interfacial tension driven open droplet microfluidics


title: MOSGA 2: Comparative genomics and validation tools

abstract: Due to the highly growing number of available genomic information, the need for accessible and easy-to-use analysis tools is increasing. To facilitate eukaryotic genome annotations, we created MOSGA. In this work, we show how MOSGA 2 is developed by including several advanced analyses for genomic data. Since the genomic data quality greatly impacts the annotation quality, we included multiple tools to validate and ensure high-quality user-submitted genome assemblies. Moreover, thanks to the integration of comparative genomics methods, users can benefit from a broader genomic view by analyzing multiple genomic data sets simultaneously. Further, we demonstrate the new functionalities of MOSGA 2 by different use-cases and practical examples. MOSGA 2 extends the already established application to the quality control of the genomic data and inte-grates and analyzes multiple genomes in a larger context, e.g., by phylogenetics.


title: miRSCAPE - Inferring miRNA expression in single-cell clusters

abstract: Micro-RNAs (miRNA) are critical in development, homeostasis, and diseases, including cancer. However, our understanding of miRNA function at cellular resolution is thwarted by the inability of the standard single cell RNA-seq protocols to capture miRNAs. Here we introduce a machine learning tool -- miRSCAPE -- to infer miRNA expression in a sample from its RNA-seq profile. We establish miRSCAPEs accuracy separately in 10 tissues comprising ~10,000 tumor and normal bulk samples and demonstrate that miRSCAPE accurately infers cell type-specific miRNA activities (predicted vs observed fold-difference correlation ~ 0.81) in two independent datasets where miRNA profiles of specific cell types are available (HEK-GBM, Kidney-Breast-Skin). When trained on human hematopoietic cancers, miRSCAPE can identify active miRNAs in 8 hematopoietic cell lines in mouse with a reasonable accuracy (auROC = 0.67). Finally, we apply miRSCAPE to infer miRNA activities in scRNA clusters in Pancreatic and Lung cancers, as well as in 56 cell types in the Human Cell Landscape (HCL). Across the board, miRSCAPE recapitulates and provides a refined view of known miRNA biology. miRSCAPE is freely available and promises to substantially expand our understanding of gene regulatory networks at cellular resolution.


title: Surface Electromyography for Identification of Pre-Phonatory Activity

abstract: Surface electrode EMG is an established method for studying biomechanical activity. It has not been well studied in detecting laryngeal biomechanical activity of pre-phonatory onset. Our aims were to compare the sensitivity of surface EMG in identifying pre-phonatory laryngeal activity to needle electrode laryngeal EMG and to compare the pre-phonatory period in patients with adductor laryngeal dystonia (ADLD) with that in controls. ADLD patients (n = 10) undergoing needle LEMG prior to Botox(R) injection and participants with normal voices (n = 6) were recruited. Surface EMG electrodes were placed over the cricoid ring and thyrohyoid membrane. Needle EMG electrodes were inserted into the thyroarytenoid muscle. EMG and auditory output samples were collected during phonation onset. Tracings were de-identified and evaluated. Measurements of time from onset in change of the amplitude and motor unit frequency on the interference pattern to onset of phonation were calculated by two blinded raters. 42 of 71 patient and 40 of 50 control tracings were available for analysis. Correlation for pre-phonatory time between electrode configuration was 0.70 for patients, 0.64 for controls and 0.79 for all the data combined. Interrater correlation was 0.97 for needle and 0.96 for surface electrodes. ADLD patients had a longer pre-phonatory time than control subjects by 169.48ms with surface electrode and 140.23ms with needle electrode (p < 0.001). Surface EMG demonstrates equal reliability as Needle EMG in detecting pre-phonatory activity in controls and subjects. Patients with ADLD have a significantly prolonged pre-phonatory period when compared with controls.


title: A semisynthetic glycoconjugate provides expanded cross-serotype protection against Streptococcus pneumoniae

abstract: Streptococcus pneumoniae infections are the leading cause of child mortality globally. Current vaccines fail to induce a protective immune response towards a conserved part of the pathogen, resulting in new serotypes causing disease. Therefore, new vaccine strategies are urgently needed. Described is a two-pronged approach combining S.pneumoniae proteins, pneumolysin and PspA, with a precisely defined synthetic oligosaccharide, whereby the carrier protein acts as a serotype-independent antigen to provide additional protection. Proof of concept in mice and swine models revealed that the conjugates inhibit colonization of the nasopharynx, decrease the bacterial load and reduce disease severity in the bacteria challenged model. Immunization of piglets provided the first evidence for the immunogenicity and protective potential of synthetic glycoconjugate vaccine in a large animal model. A combination of synthetic oligosaccharides with proteins from the target pathogen opens the path to create broadly cross-protective ("universal") pneumococcal vaccines.

category: immunology


title: Neural-specific alterations in glycosphingolipid biosynthesis and cell signaling associated with two human ganglioside GM3 Synthase Deficiency variants

abstract: GM3 Synthase Deficiency (GM3SD) is a neurodevelopmental disorder resulting from pathogenic variants in the ST3GAL5 gene, which encodes GM3 synthase, a glycosphingolipid (GSL)-specific sialyltransferase. This enzyme adds a single 3-linked sialic acid to the terminal galactose of lactosylceramide (LacCer) to produce the monosialylated ganglioside GM3. In turn, GM3 is extended by other glycosyltransferases to generate nearly all the complex gangliosides enriched in neural tissue. Pathogenic mechanisms that account for neural phenotypes associated with GM3SD are not known. To explore how loss of GM3 impacts neural-specific glycolipid glycosylation and cell signaling, GM3SD patient fibroblasts bearing one of two different ST3GAL5 variants were reprogrammed to induced pluripotent stem cells (iPSCs) and then differentiated to neural crest cells (NCCs). GM3 and GM3-derived gangliosides were undetectable in iPSCs and NCCs from both variants, while LacCer precursor levels were elevated compared to wildtype (WT). NCCs of both variants synthesized elevated levels of neutral lacto- and globo-series, as well as minor alternatively sialylated, GSLs compared to WT. Shifts in ceramide profiles associated with iPSC and NCC GSLs were also detected in GM3SD variants. Altered GSL profiles in the GM3SD cells were accompanied by dynamic changes in the cell surface proteome, protein O-GlcNAcylation, and receptor tyrosine kinase abundance. GM3SD cells also exhibited increased apoptosis and sensitivity to erlotnib, an inhibitor of epidermal growth factor receptor signaling. Pharmacologic inhibition of O-GlcNAcase increased protein O-GlcNAcylation and significantly rescued baseline and erlotnib-induced apoptosis. Collectively, these findings indicate broad effects on cell signaling during differentiation of GM3SD patient-derived iPSCs to NCCs. Thus, human GM3SD cells provide a novel platform to investigate structure/function relationships that connect GSL diversity to cell signaling, cell survival, and neural differentiation.


title: Photosensitized Electrospun Nanofibrous Filters for Capturing and Killing Airborne Coronaviruses under Visible Light Irradiation

abstract:
To address the challenge of the airborne transmission of SARS-CoV-2, photosensitized electrospun nanofibrous membranes were fabricated to effectively capture and inactivate coronavirus aerosols. With an ultrafine fiber diameter ([~] 200 nm) and a small pore size ([~] 1.5 {micro}m), the optimized membranes caught 99.2% of the aerosols of the murine hepatitis virus A59 (MHV-A59), a coronavirus surrogate for SARS-CoV-2. In addition, rose bengal was used as the photosensitizer for the membranes because of its excellent reactivity in generating virucidal singlet oxygen, and the membranes rapidly inactivated 98.9% of MHV-A59 in virus-laden droplets only after 15 min irradiation of simulated reading light. Singlet oxygen damaged the virus genome and impaired virus binding to host cells, which elucidated the mechanism of disinfection at a molecular level. Membrane robustness was also evaluated, and no efficiency reduction for filtering MHV-A59 aerosols was observed after the membranes being exposed to both indoor light and sunlight for days. Nevertheless, sunlight exposure photobleached the membranes, reduced singlet oxygen production, and compromised the performance of disinfecting MHV-A59 in droplets. In contrast, the membranes after simulated indoor light exposure maintained their excellent disinfection performance. In summary, photosensitized electrospun nanofibrous membranes have been developed to capture and kill airborne environmental pathogens under ambient conditions, and they hold promise for broad applications as personal protective equipment and indoor air filters.

SynopsisPhotosensitized electrospun nanofibrous filters with excellent capture-and-kill performance against coronaviruses were designed and implemented to prevent the airborne transmission of COVID-19.

Table of Contents

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title: A method for the generation of pseudotyped virus particles bearing SARS coronavirus spike protein in high yields

abstract: The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has threatened human health and the global economy. Development of additional vaccines and therapeutics is urgently required, but such development with live virus must be conducted with biosafety level 3 confinement. Pseudotyped viruses have been widely adopted for studies of virus entry and pharmaceutical development to overcome this restriction. Here we describe a modified protocol to generate vesicular stomatitis virus (VSV) pseudotyped with SARS-CoV or SARS-CoV-2 Spike protein in high yield. We found that pseudovirions produced with the conventional transient expression system lacked coronavirus Spike protein at their surface as a result of inhibition of parental VSV infection by overexpression of this protein. Establishment of stable cell lines with an optimal expression level of coronavirus Spike protein allowed the efficient production of progeny pseudoviruses decorated with Spike protein. This improved VSV pseudovirus production method should facilitate studies of coronavirus entry and development of antiviral agents.


title: 3D printed cobalt-chromium-molybdenum porous superalloy with superior antiviral activity

abstract:
COVID-19 pandemic and associated supply-chain disruptions emphasise the requirement for antimicrobial materials for on-demand manufacturing. Besides aerosol transmission, SARS-CoV-2 is also propagated through contact with virus-contaminated surfaces. As such, the development of effective biofunctional materials that can inactivate SARS-CoV-2 are critical for pandemic preparedness. Such materials will enable the rational development of antiviral devices with prolonged serviceability reducing the environmental burden of disposable alternatives. This research reveals the novel use of Laser Powder Bed Fusion (LPBF) to 3D print porous Cobalt-Chromium-Molybdenum (Co-Cr-Mo) superalloy with potent antiviral activity (100% viral inactivation in 30 mins). The porous material was rationally conceived using a multi-objective surrogate model featuring track thickness (tt) and pore diameter ({phi}d) as responses. The regression analysis found the most significant parameters for Co-Cr-Mo track formation to be the interaction effects of scanning rate (Vs) and laser power (Pl) in the order PlVs > Vs > Pl. Contrastively, the pore diameter was found to be primarily driven by the hatch spacing (Sh). The study is the first to demonstrate the superior antiviral properties of 3D printed Co-Cr-Mo superalloy against an enveloped virus used as biosafe viral model of SARS-CoV-2. The material significantly outperforms the viral inactivation time of other broadly used antiviral metals such as copper and silver from 5 hours to 30 minutes. As such the study goes beyond the current state-of-the-art in antiviral alloys to provide extra-protection to combat the SARS-COV-2 viral spread. The evolving nature of the COVID-19 pandemic brings new and unpredictable challenges where on-demand 3D printing of antiviral materials can achieve rapid solutions while reducing the environmental impact of disposable devices.

Graphical abstract

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title: Identification and Characterization of novel mutants of Nsp13 Protein among Indian SARS-CoV-2 isolates.

abstract: SARS-CoV-2, the causative agent of COVID-19 has mutated rapidly which enabled them to adapt and evade the immune system of the host. Emerging SARS-CoV-2 variants with crucial mutations pose a global challenge in context of therapeutic drugs and vaccines being developed globally. There are currently no specific therapeutics or vaccines available to combat SARS-CoV-2 devastation. In view of this, the current study aimed to identify and characterize the mutations found in the Nsp13 of SARS-CoV-2 in Indian isolates. Non-structural protein, Nsp13 protein sequences from Indian isolates were analyzed by comparing with the first reported Severe acute respiratory syndrome Corona Virus-2 (SARS-CoV-2) protein sequence from Wuhan, China. Out of 825 Nsp13 protein sequences, a total of 38 mutations were observed among Indian isolates. Our data show that mutations in Nsp13 at various positions (H164Y, A237T, T214I, C309Y, S236I, P419S, V305E, G54S, H290Y, P53S, A308Y, and A308Y) have a significant impact on the proteins stability and flexibility. Also, the impact of Nsp13 mutations on the protein function were predicted based on PROVEAN score that includes 15 mutants as neutral and 23 mutants as deleterious effect. Furthermore, B-cell epitopes contributed by Nsp13 were identified using various predictive immunoinformatic tools. Immunological Parameters of Nsp13 such as antigenicity, allergenicity and toxicity were evaluated to predict the potential B-cell epitopes. The predicted peptide sequences were correlated with the observed mutants. Our predicted data showed that there are seven high rank linear epitopes as well as 18 discontinuous B-cell epitopes based on immunoinformatic tools. Moreover, it was observed that out of total 38 identified mutations among Indian SARS-CoV-2 Nsp13 protein, four mutant residues at position 142 (E142), 245 (H245), 247 (V247) and 419 (P419) are localised in the predicted B cell epitopic region. Altogether, the results of the present in-silico study might help to understand the impact of the identified mutations in Nsp13 protein on its stability, flexibility and function.


title: Mice with High FGF21 Serum Levels Had a Reduced Preference for Morphine and an Attenuated Development of Acute Antinociceptive Tolerance and Physical Dependence

abstract: Because of increased opioid misuse, there is a need to identify new targets for minimizing opioid tolerance, and physical and psychological dependence. Previous studies showed that fibroblast growth factor 21 (FGF21) decreased alcohol and sweet preference in mice. In this study, FGF21-transgenic (FGF21-Tg) mice, expressing high FGF21 serum levels, and wildtype (WT) C57BL/6J littermates were treated with morphine and saline to determine if differences exist in their physiological and behavioral responses to opioids. FGF21-Tg mice displayed reduced preference for morphine in the conditioned place preference assay compared to WT littermates. Similarly, FGF21-Tg mice had an attenuation of the magnitude and rate of acute morphine antinociceptive tolerance development, and acute and chronic morphine physical dependence, but exhibited no change in chronic morphine antinociceptive tolerance. The ED50 values for morphine-induced antinociception in the 55{degrees}C hot plate and the 55{degrees}C warm-water tail withdrawal assays were similar in both strains of mice. Likewise, FGF21-Tg and WT littermates had comparable responses to morphine-induced respiratory depression. Overall, FGF21-Tg mice had a decrease in the development of acute analgesic tolerance, and the development of physical dependence, and morphine preference. FGF21 and its receptor have therapeutic potential for reducing opioid withdrawal symptoms and craving, and augmenting opioid therapeutics for acute pain patients to minimize tolerance development.


title: Structural basis for the therapeutic advantage of dual and triple agonists at the human GIP, GLP-1 or GCG receptors

abstract: Glucose homeostasis, regulated by glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1) and glucagon (GCG) is critical to human health. Several multi-targeting agonists at GIPR, GLP-1R or GCGR, developed to maximize metabolic benefits with reduced side-effects, are in clinical trials to treat type 2 diabetes and obesity. To elucidate the molecular mechanisms by which tirzepatide, a GIPR/GLP-1R dualagonist, and peptide 20, a GIPR/GLP-1R/GCGR triagonist, manifest their superior efficacies over monoagonist such as semaglutide, we determined cryo-electron microscopy structures of tirzepatide-bound GIPR and GLP-1R as well as peptide 20-bound GIPR, GLP-1R and GCGR The structures reveal both common and unique features for the dual and triple agonism by illustrating key interactions of clinical relevance at the atomic level. Retention of glucagon function is required to achieve such an advantage over GLP-1 monotherapy. Our findings provide valuable insights into the structural basis of functional versatility and therapeutic supremacy of tirzepatide and peptide 20.


title: SARS-CoV-2 introduction and lineage dynamics across three epidemic peaks in Southern Brazil: massive spread of P.1

abstract:
BackgroundGenomic surveillance of SARS-CoV-2 is paramount for understanding viral dynamics, contributing to disease control. This study analyzed SARS-CoV-2 genomic diversity in Rio Grande do Sul (RS), Brazil, including the first case of each Regional Health Coordination and cases from three epidemic peaks.

MethodsNinety SARS-CoV-2 genomes from RS were sequenced and analyzed against SARS-CoV-2 datasets available in GISAID for phylogenetic inference and mutation analysis.

ResultsSARS-CoV-2 lineages among the first cases in RS were B.1 (33.3%), B.1.1.28 (26.7%), B.1.1 (13.3%), B.1.1.33 (10.0%), and A (6.7%), evidencing SARS-CoV-2 introduction by both international origin and community-driven transmission. We found predominance of B.1.1.33 (50.0%) and B.1.1.28 (35.0%) during the first epidemic peak (July-August, 2020), emergence of P.2 (55.6%) in the second peak (November-December, 2020), and massive spread of P.1 and related sequences (78.4%), such as P.1-like-II, P.1.1 and P.1.2 in the third peak (February-April, 2021). Eighteen novel mutation combinations were found among P.1 genomes, and 22 different spike mutations and/or deletions among P.1 and related sequences.

ConclusionsThis study shows the dispersion of SARS-CoV-2 lineages in Southern Brazil, and describes SARS-CoV-2 diversity during three epidemic peaks, highlighting the spread of P.1 and the high genetic diversity of currently circulating lineages. Genomic monitoring of SARS-CoV-2 is essential to guide health authorities decisions to control COVID-19 in Brazil.

SummaryNinety SARS-CoV-2 genomes from Rio Grande do Sul, Brazil, were sequenced, including the first cases from 15 State Health Coordination regions and samples from three epidemic peaks. Phylogenomic inferences showed SARS-CoV-2 lineages spread, revealing its genomic diversity.


title: COVID-ONE-humoral immune: The One-stop Database for COVID-19-specific Antibody Responses and Clinical Parameters

abstract: Coronavirus disease 2019 (COVID-19), which is caused by SARS-CoV-2, varies with regard to symptoms and mortality rates among populations. Humoral immunity plays critical roles in SARS-CoV-2 infection and recovery from COVID-19. However, differences in immune responses and clinical features among COVID-19 patients remain largely unknown. Here, we report a database for COVID-19-specific IgG/IgM immune responses and clinical parameters (COVID-ONE humoral immune). COVID-ONE humoral immunity is based on a dataset that contains the IgG/IgM responses to 21 of 28 known SARS-CoV-2 proteins and 197 spike protein peptides against 2,360 COVID-19 samples collected from 783 patients. In addition, 96 clinical parameters for the 2,360 samples and information for the 783 patients are integrated into the database. Furthermore, COVID-ONE humoral immune provides a dashboard for defining samples and a one-click analysis pipeline for a single group or paired groups. A set of samples of interest is easily defined by adjusting the scale bars of a variety of parameters. After the "START" button is clicked, one can readily obtain a comprehensive analysis report for further interpretation. COVID-ONE-humoral immune is freely available at www.COVID-ONE.cn.


title: Lipid flippase dysfunction as a novel therapeutic target for endosomal anomalies in Alzheimer's disease

abstract: {beta}-amyloid precursor protein (APP) and their metabolites are deeply involved in the development of Alzheimers disease (AD). Upon the upregulation of {beta}-site APP cleaving enzyme 1 (BACE1), its product, the {beta}-carboxyl-terminal fragment of APP ({beta}CTF), is accumulated in the early stage of sporadic AD brains. {beta}CTF accumulation is currently considered the trigger for endosomal anomalies to form enlarged endosomes, one of the earliest pathologies in AD. However, the details of the underlying mechanism remain largely unclear. In this study, using BACE1 stably-overexpressing cells, we describe that lipid flippase subcomponent TMEM30A interacts with accumulated {beta}CTF. Among the lipid flippases in endosomes, those composed of TMEM30A and active subcomponent ATP8A1 transports phospholipid, phosphatidylserine (PS), to the cytosolic side of the endosomes. The lipid flippase activity and cytosolic PS distribution are critical for membrane fission and vesicle transport. Intriguingly, accumulated {beta}CTF in model cells impaired lipid flippase physiological formation and activity, along with endosome enlargement. Moreover, in the brains of AD model mice before the amyloid-{beta} (A{beta}) deposition, the TMEM30A/{beta}CTF complex formation occurred, followed by lipid flippase dysfunction. Importantly, our novel A{beta}/{beta}CTF interacting TMEM30A-derived peptide "T-RAP" improved endosome enlargement and reduced {beta}CTF levels. These T-RAP effects could result from the recovery of lipid flippase activity. Therefore, we propose lipid flippase dysfunction as a key pathogenic event and a novel therapeutic target for AD.


title: Development of a compact alkynyl-enrichable crosslinker for in-depth in-vivo crosslinking analysis

abstract: Chemical crosslinking coupled with mass spectrometry (CXMS) has emerged as a powerfl technique to capture the dynamic information of protein complexes with high sensitivity, throughput and sample universality. To advance the study of in-vivo protein structures and protein-protein interactions on the large scale, a new alkynyl-enrichable crosslinker was developed with high efficiency of membrane penetration, reactivity and enrichment. The crosslinker was successfully used for in-vivo crosslinking of intact human cells, reslting in 6820 non-redundant crosslinks identified at a false discovery rate (FDR) of 1% using pLink 2.0, which 4898 (71.8%) of the cross-links were assigned as intraprotein and 1922 (28.2%) were interprotein links. To our knowledge, this is also the first time to realize the in-vivo crosslinking with a non-cleavable crosslinker for homo species cells.


title: PERK inhibition blocks metastasis initiation by limiting UPR-dependent survival of dormant disseminated cancer cells

abstract: The unfolded protein response (UPR) kinase PERK has been shown to serve as a survival factor for HER2-driven breast and prostate cancers as well as for dormant cancer cells. However, its role in metastasis is not understood. Here we found in the MMTV-HER2 mouse model that quiescent HER2+ disseminated cancer cells (DCCs) displayed unresolved ER stress as revealed by high expression of the PERK-inducible GADD34 gene. Single cell gene expression profiling and imaging confirmed that a significant proportion of DCCs in lungs were dormant and displayed an active UPR. In human breast cancer metastasis biopsies, GADD34 expression and quiescence were also positively correlated. Importantly, PERK inhibition with a specific inhibitor (HC4) blunted metastasis development by selectively killing UPRhigh quiescent but not proliferative DCCs. We also show that PERK inhibition altered optimal HER2 activity in primary tumors as a result of sub-optimal HER2 trafficking and phosphorylation in response to EGF. Our data identify PERK as a unique "Achilles heel" in dormant DTCs, supporting a requisite role for PERK in DTCs. Taken together, these data identify novel strategies to eliminate quiescent DCCs in patients with disseminated cancer disease.

category: cancer biology


title: PROTECTIVE ROLE FOR SMOOTH MUSCLE CELL HEPCIDIN IN ABDOMINAL AORTIC ANEURYSM

abstract:
RationaleHepcidin (HAMP) is a hormone produced primarily in the liver. It controls systemic iron homeostasis by inhibiting the iron exporter ferroportin (FPN) in the gut and spleen, respective sites of iron absorption and recycling. HAMP and FPN are also found ectopically in tissues not involved in systemic iron homeostasis. The physiological functions of ectopic HAMP and FPN are only just beginning to be uncovered. We observed that HAMP expression is markedly increased in smooth muscle cells (SMCs) of abdominal aortic aneurysms (AAA), both in patients and in an experimental mouse model of AAA.

ObjectiveTo understand the role of SMC-derived HAMP in the pathophysiology of AAA.

Methods and ResultsWe generated mice harbouring an inducible, SMC-specific deletion of the hamp gene. We then applied the experimental model of AAA and simultaneously induced deletion of hamp in SMCs. We found that these mice developed large aneurysms and had greater incidences of rupture and of fatal dissection than mice with intact hamp in SMCs. A similar phenotype was observed in mice harbouring an inducible SMC-specific knock-in of HAMP-resistant FPNC326Y. Additionally, we observed that expression of Lipocalin-2 (LCN2), a protein known to promote AAA, was suppressed in AAA tissue from patients and from mice with intact hamp in SMCs, but not in mice lacking hamp in SMCs. Treatment of these mice with a LCN2-neutralising antibody protected them from the otherwise detrimental effects of loss of hamp in SMCs.

ConclusionsThe present study demonstrates that the rise in SMC-derived HAMP within the aneurysm tissue is protective in the setting of AAA, and that such protection involves the cell-autonomous action of HAMP, and suppression of local LCN2. These findings are the first example of a protective role for ectopic HAMP in disease. They expand understanding of the multifaceted functions of HAMP outside the liver.


title: Activation of M1 muscarinic receptors reduce pathology and slow progression of neurodegenerative disease.

abstract: The most prevalent types of dementias, including Alzheimers disease, are those that are propagated via the spread of "prion-like" misfolded proteins. Despite considerable effort no treatments are available to slow or stop the progression of these dementias. Here we investigate the possibility that activation of the M1-muscarinic receptor (M1-receptor), which is highly expressed in the brain and that shows pro-cognitive properties, might present a novel disease modifying target. We demonstrate that the progression of murine prion disease, which we show here displays many of the pathological, behavioural and biochemical hallmarks of human neurodegenerative disease, is slowed and normal behaviour maintained by the activation of the M1-receptor with a highly tolerated positive allosteric modulator (VU846). This correlates with a reduction in both neuroinflammation and indicators of mitochondrial dysregulation, as well as a normalisation in the expression of markers associated with neurodegeneration and Alzheimers disease. Furthermore, VU846 preserves expression of synaptic proteins and post-synaptic signalling components that are altered in disease. We conclude that allosteric regulation of M1-receptors has the potential to reduce the severity of neurodegenerative diseases caused by the prion-like propagation of misfolded protein in a manner that extends life span and maintains normal behaviour.


title: Regeneration of humoral immunity from pluripotent stem cells by defined transcription factors

abstract: Regeneration of humoral immunity from pluripotent stem cells (PSCs) is a crucial aim in translational medicine. However, reconstitution of complete, sustained, and functional B lymphopoiesis from PSCs has not yet been developed. Here, we successfully achieved regenerative B lymphopoiesis in B-cell deficient animals transplanted with PSC-derived hematopoietic progenitors (iHPCs) guided by synergistic expression of Runx1, Hoxa9, and Lhx2. Upon transplantation, the iHPCs immediately gave rise to pro/pre-B cells in recipients bone marrow, which were able to further differentiate into the entire B cell lineages, including innate B-1a, B-1b, MZ B cells, as well as adaptive FO B cells. In responding to antigen stimuli, the regenerative B cells produced adaptive humoral immune responses, sustained a prolonged antigen-specific antibody production, and formed immune-memory. Particularly, the regenerative B cells in spleen showed developing patterns of immunoglobulin chain-switch and hyper-mutation via a cross-talk with the host T follicular helper cells, which eventually formed T cell-dependent humoral responses. This study provides de novo evidence that B lymphopoiesis can be regenerated from PSCs via a HSC-independent approach, which provides insights into treating B-cell related humoral deficiencies using PSCs as unlimited cell resource.


title: CD49a Identifies Polyfunctional Memory CD8 T cell Subsets that Persist in the Lungs after Influenza Infection

abstract:
CD8 T cell memory offers critical antiviral protection, even in the absence of neutralizing antibodies. The paradigm is that CD8 T cell memory within the lung tissue consists of a mix of circulating TEM cells and non-circulating TRM cells. However, based on our analysis, the heterogeneity within the tissue is much higher, identifying TCM, TEM, TRM, and a multitude of populations which do not perfectly fit these classifications. Further interrogation of the populations shows that TRM cells that express CD49a, both with and without CD103, have increased and diverse effector potential compared with CD49a negative populations. These populations function as a one-man band, displaying antiviral activity, chemokine production, release of GM-CSF, and the ability to kill specific targets in vitro with delayed kinetics compared with effector CD8 T cells. Together, this study establishes that CD49a defines multiple polyfunctional CD8 memory subsets after clearance of influenza infection, which act to eliminate virus in the absence of direct killing, recruit and mature innate immune cells, and destroy infected cells if the virus persists.

Contribution to the fieldProtection from previously seen infections requires specialized immune memory cells properly positioned throughout the body to combat the newly invading pathogen. In the case of re-exposure to influenza virus, CD8 T cells resident within the respiratory tract (TRM) are critical for eliminating the virus. Previously, TRM were viewed as mostly homogenous, with a limited range of immune functions. In this study, lung TRM were compared with circulating memory CD8 T cells transiently present within the lung, to define the breadth of their effector capabilities. Using TRM defining surface proteins CD49a and CD103 to identify different memory CD8 T cell subsets, gene and protein expression were evaluated. In addition to demonstrating higher levels of diversity than previously reported, multiple polyfunctional subsets were identified. This polyfunctionality was primarily associated with cell populations expressing CD49a, and these cells produced multiple antiviral factors, chemokines to recruit other immune cells, a growth factor associated with improved antigen presenting cell function, and cytolytic granules. Functional assays further demonstrated killing of target cells by TRM. This study paints a more holistic, complete picture of the phenotype and functions of lung CD8 T cells after viral infection, revealing CD49a as a marker of cells with high effector capacity.


title: The effects of a synthetic and biological surfactant on the community composition and metabolic activity of a freshwater biofilm

abstract: Surfactants are used to control microbial biofilms in industrial and medical settings. Their known toxicity on aquatic biota and their longevity in the environment has encouraged research on biodegradable alternatives such as rhamnolipids. While previous research has investigated the effects of biological surfactants on single species biofilms, there remains a lack of information regarding the effects of synthetic and biological surfactants in freshwater ecosystems. We conducted a mesocosm experiment to test how the surfactant sodium dodecyl sulphate (SDS) and the biological surfactant rhamnolipid altered community composition and metabolic activity of freshwater biofilms. Biofilms were cultured in the flumes using lake water from Lake Lunz in Austria, under high (300 ppm) and low (150 ppm) concentrations of either surfactant over a four-week period. Our results show that both surfactants significantly affected microbial diversity. Up to 36% of microbial operational taxonomic units were lost after surfactant exposure. Rhamnolipid exposure also increased the production of the extracellular enzymes, leucine aminopeptidase and glucosidase, while SDS exposure reduced leucine aminopeptidase and glucosidase. This study demonstrates that exposure of freshwater biofilms to chemical and biological surfactants caused a reduction of microbial diversity and changes in biofilm metabolism, exemplified by shifts in extracellular enzyme activities.


title: Atp{Theta} is an inhibitor of F0F1 ATP synthase to arrest ATP hydrolysis during low-energy conditions in cyanobacteria

abstract: Biological processes in all living cells are powered by ATP, a nearly universal molecule of energy transfer. ATP synthases produce ATP utilizing proton gradients that are usually generated by either respiration or photosynthesis. However, cyanobacteria are unique in combining photosynthetic and respiratory electron transport chains in the same membrane system, the thylakoids. How cyanobacteria prevent the futile reverse operation of ATP synthase under unfavorable conditions pumping protons while hydrolyzing ATP is mostly unclear. Here, we provide evidence that the small protein Atp{Theta}, which is widely conserved in cyanobacteria, is mainly fulfilling this task. The expression of Atp{Theta} becomes induced under conditions such as darkness or heat shock, which can lead to a weakening of the proton gradient. Translational fusions of Atp{Theta} to the green fluorescent protein revealed targeting to the thylakoid membrane. Immunoprecipitation assays followed by mass spectrometry and far Western blots identified subunits of ATP synthase as interacting partners of Atp{Theta}. ATP hydrolysis assays with isolated membrane fractions as well as purified ATP synthase complexes demonstrated that Atp{Theta} inhibits ATPase activity in a dose-dependent manner similar to the F0F1-ATP synthase inhibitor N,N-dicyclohexylcarbodimide. The results show that, even in a well-investigated process, crucial new players can be discovered if small proteins are taken into consideration and indicate that ATP synthase activity can be controlled in surprisingly different ways.


title: Roscovitine exacerbates Mycobacterium abscessus infection by reducing NADPH oxidase-dependent neutrophil trafficking

abstract:
Persistent neutrophilic inflammation associated with chronic pulmonary infection causes progressive lung injury and eventually death in individuals with cystic fibrosis (CF), a genetic disease caused by bi-allelic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

We therefore examined whether Roscovitine, a cyclin-dependent kinase inhibitor that (in other conditions) reduces inflammation while promoting host defence, might provide a beneficial effect in the context of CF.

Herein, using CFTR-depleted zebrafish larvae as an innovative vertebrate model of CF immuno-pathophysiology, combined with murine and human approaches, we sought to determine the effects of Roscovitine on innate immune responses to tissue injury and pathogens in CF condition.

We show that Roscovitine exerts anti-inflammatory and pro-resolution effects in neutrophilic inflammation induced by infection or tail amputation in zebrafish. Roscovitine reduces overactive epithelial ROS-mediated neutrophil trafficking, by reducing DUOX2/NADPH-oxidase activity, and accelerates inflammation resolution by inducing neutrophil apoptosis and reverse migration. Importantly, while Roscovitine efficiently enhances intracellular bacterial killing of Mycobacterium abscessus in human CF macrophages ex vivo, we found that treatment with Roscovitine results in worse infection in mouse and zebrafish models. By interfering with DUOX2/NADPH oxidase-dependent ROS production, Roscovitine reduces the number of neutrophils at infection sites, and consequently compromises granuloma formation and maintenance, favouring extracellular multiplication of M. abscessus and more severe infection.

Our findings bring important new understanding of the immune-targeted action of Roscovitine and have significant therapeutic implications for safety targeting inflammation in CF.


title: Mechanism of Antiviral Activity of Nitazoxanide against the Influenza Virus: Effect of Tizoxanide on AdenosineTriphosphate in Influenza-virus Infected Madin Darby Canine Kidney Cells

abstract:
BackgroundNitazoxanide (NTZ) is a broad-spectrum antiviral undergoing clinical development for treating influenza and other viral respiratory infections such as those caused by rhinovirus/enterovirus and coronavirus including the emerging SARS-CoV-2.

MethodsNitazoxanide is a mild uncoupler of oxidative phosphorylation, which is modulating the ATP production in cells. ATP is an essential component of viral replication, and we have evaluated the effect of tizoxanide (TIZ), the active circulating metabolite of NTZ, on ATP in Madin-Darby canine kidney (MDCK) cells and in MDCK cells infected with influenza A and B viruses.

ResultsTIZ decreased cellular ATP in a dose-dependent manner in MDCK cells and in MDCK cells infected with influenza A and B viruses. Maximum inhibition of ATP in influenza infected or uninfected MDCK cells reached up to 45% after 6 and 24 hours of exposure to 100{micro}M TIZ. The decrease in cellular ATP did not affect cell viability and was reversible after eliminating TIZ from the culture.

ConclusionThe concentrations of TIZ required to decrease cellular ATP levels were similar to those reported to inhibit replication of influenza A and B viruses in our laboratory. A decrease in ATP triggers activation of AMP-activated protein kinase, which is known to suppress the secretion of pro-inflammatory cytokines. Additional studies are warranted to evaluate the effect of TIZ on mitochondrial function.


title: Genomic and chemical decryption of the Bacteroidetes phylum for its potential to biosynthesize natural products

abstract:
With progress in genome sequencing and data sharing, 1000s of bacterial genomes are publicly available. Genome mining - using bioinformatics tools in terms of biosynthetic gene cluster (BGC) identification, analysis and rating - has become a key technology to explore the capabilities for natural product (NP) biosynthesis. Comprehensively, analyzing the genetic potential of the phylum Bacteroidetes revealed Chitinophaga as the most talented genus in terms of BGC abundance and diversity. Guided by the computational predictions, we conducted a metabolomics and bioactivity driven NP discovery program on 25 Chitinophaga strains. High numbers of peerless strain-specific metabolite buckets confirmed the upfront predicted biosynthetic potential and revealed a tremendous uncharted chemical space. Sourcing this dataset, we isolated the new iron chelating nonribosomally-synthesized cyclic tetradeca- and pentadecalipodepsipeptide antibiotics chitinopeptins with activity against Candida, produced by C. eiseniae DSM 22224 and C. flava KCTC 62435, respectively.

TeaserCombination of omics-technologies revealed taxonomical hotspots for specialized metabolites within Bacteroidetes.


title: Identification and functional characterization of transcriptional activators in human cells

abstract: Transcription is orchestrated by thousands of transcription factors and chromatin-associated proteins, but how these are causally connected to transcriptional activation or repression is poorly understood. Here, we conduct an unbiased proteome-scale screen to systematically uncover human proteins that activate transcription in a natural chromatin context. We also identify potent transactivation domains among the hits. By combining interaction proteomics and chemical inhibitors, we delineate the preference of both known and novel transcriptional activators for specific co-activators, highlighting how even closely related TFs can function via distinct co-factors. Finally, we show that many novel activators are partners in fusion events in tumors and functionally characterize a myofibroma-associated fusion between SRF and C3orf62, a potent activator. SRF-C3orf62 activates transcription in a CBP/p300-dependent manner and promotes proliferative and myogenic transcriptional programs. Our work provides a functional catalogue of potent transactivators in the human proteome and a platform for discovering transcriptional regulators at genome scale.


title: A new paradigm for leprosy diagnosis based on host gene expression

abstract:
Transcriptional profiling is a powerful tool to investigate and detect human diseases. In this study, we used bulk RNA-sequencing (RNA-Seq) to compare the transcriptomes in skin lesions of leprosy patients or controls affected by other dermal conditions such as granuloma annulare, a confounder for paucibacillary leprosy. We identified five genes capable of accurately distinguishing multibacillary and paucibacillary leprosy from other skin conditions. Indoleamine 2,3-dioxygenase 1 (IDO1) expression alone was highly discriminatory, followed by TLR10, BLK, CD38, and SLAMF7, whereas the HS3ST2 and CD40LG mRNA separated multi- and paucibacillary leprosy. Finally, from the main differentially expressed genes (DEG) and enriched pathways, we conclude that paucibacillary disease is characterized by epithelioid transformation and granuloma formation, with an exacerbated cellular immune response, while multibacillary leprosy features epithelial-mesenchymal transition with phagocytic and lipid biogenesis patterns in the skin. These findings will help catalyze the development of better diagnostic tools and potential host-based therapeutic interventions. Finally, our data may help elucidate host-pathogen interplay driving disease clinical manifestations.

Author SummaryDespite effective treatment, leprosy is still a significant public health issue in more than 120 countries, with more than 200 000 new cases yearly. The disease is caused mainly by Mycobacterium leprae, a slow-growing bacillus still uncultivable in axenic media. This limitation has hampered basic research into host-pathogen interaction and the development of new diagnostic assays. Currently, leprosy is diagnosed clinically, with no standalone diagnostic assay accurate enough for all clinical forms. Here, we use RNA-seq transcriptome profiling in leprosy lesions and granuloma annulare to identify mRNA biomarkers with potential diagnostic applications. Also, we explored new pathways that can be useful in further understanding the host-pathogen interaction and how the bacteria bypass host immune defenses. We found that IDO1, a gene involved with tryptophan catabolism, is an excellent candidate for distinguishing leprosy lesions from other dermatoses. Additionally, we observed that a previous signature of keratinocyte development and cornification negatively correlates with epithelial-mesenchymal transition genes in the skin, suggesting new ways in which the pathogen may subvert its host to survive and spread throughout the body. Our study identifies new mRNA biomarkers that can improve leprosy diagnostics and describe new insights about host-pathogen interactions in human skin.


title: Localization of infection in neonatal rhesus macaques after oral viral challenge

abstract:
While vertical transmission of human immunodeficiency virus (HIV) can occur in utero and during delivery and through breastfeeding. We utilized Positron Emission Tomography (PET) imaging coupled with fluorescent microscopy of 64Cu-labeled photoactivatable-GFP-HIV (PA-GFP-BaL) to determine how HIV virions distribute and localize in neonatal rhesus macaques two and four hours after oral viral challenge. Our results show that by four hours after oral viral exposure, HIV virions localize to and penetrate the rectal mucosa. We also used a dual viral challenge with a non-replicative viral vector and a replication competent SHIV-1157ipd3N4 to examine viral transduction and dissemination at 96 hours. Our data show that while SHIV-1157ipd3N4 infection can be found in the oral cavity and upper gastrointestinal (GI) tract, the small and large intestine contained the largest number of infected cells. Moreover, we found that T cells were the biggest population of infected immune cells. Thus, thanks to these novel technologies, we are able to visualize and delineate of viral distribution and infection throughout the entire neonatal GI tract during acute viral infection.

Author SummaryApproximately 1.8 million children are currently living with human immunodeficiency virus (HIV). While mother-to-child HIV transmission can occur in utero and during delivery, it most commonly occurs through breastfeeding, creating the need to understand how the virus moves throughout the body and infects the infant once breast milk is consumed. Here, we used multiple imaging techniques and PCR to determine how HIV distributes throughout the gastrointestinal tract after oral viral exposure and in which tissues and cell types become acutely infected. We found that HIV rapidly spreads throughout and penetrates the entire gastrointestinal tract as early as four hours after exposure. We also found that the intestine contained the largest number of infected cells at 96 hours and that most cells infected were T cells. Our study shows that these imaging technologies allow for the examination of viral distribution and infection in a rhesus macaque model.

category: cell biology


title: Role of glutathione on cell adhesion and volume.

abstract: Glutathione is the most abundant thiol in animal cells. Reduced glutathione (GSH) is a major intracellular antioxidant neutralizing free radicals and detoxifying electrophiles. It plays important roles in many cellular processes, including cell differentiation, proliferation, and apoptosis. In the present study we demonstrate that extracellular concentration of reduced glutathione markedly increases cell volume within few hours, in a dose-response manner. Pre-incubation of cells with BSO, the inhibitor of {gamma}-glutamylcysteine synthetase, responsible for the first step in intracellular glutathione synthesis did not change the effect of reduced glutathione on cell volume suggesting a mechanism limited to the interaction of extracellular reduced glutathione on cell membrane. Similarly, inhibition of {gamma}-glutamylcyclotransferase involved in intracellular glutamate production had no effect on the action of reduced glutathione. Oxidized glutathione exerted no effect on cell volume. Results show that reduced GSH decreases cell adhesion resulting in an increased cell volume. Since many cell types are able to export GSH, the present results suggest that this could be a fundamental self-regulation of cell volume, giving the cells a self-control on their adhesion proteins.


title: Reduced glutathione decreases cell adhesion and increases cell volume.


title: The genetic origin of Daunians and the Pan-Mediterranean southern Italian Iron Age context

abstract: The geographical location and shape of Apulia, a narrow land stretching out in the sea at the South of Italy, made this region a Mediterranean crossroads connecting Western Europe and the Balkans. Such movements culminated at the beginning of the Iron Age with the Iapygian civilization which consisted of three cultures: Peucetians, Messapians and Daunians. Among them, the Daunians left a peculiar cultural heritage, with one-of-a-kind stelae and pottery, but, despite the extensive archaeological literature, their origin has been lost to time. In order to shed light on this and to provide a genetic picture of Iron Age Southern Italy, we collected and sequenced human remains from three archaeological sites geographically located in Northern Apulia (the area historically inhabited by Daunians) and radiocarbon dated between 1157 and 275 calBCE. We find that Iron Age Apulian samples are still distant from the genetic variability of modern-day Apulians, they show a remarkable genetic heterogeneity, even though a few kilometers and centuries separate them, and they are well inserted into the Iron Age Pan-Mediterranean genetic landscape. Our study provides for the first time a window on the genetic make-up of pre-imperial Southern Italy, whose increasing connectivity within the Mediterranean landscape, would have contributed to laying the foundation for modern genetic variability. In this light, the genetic profile of Daunians may be compatible with an autochthonous origin, with plausible contributions from the Balkan peninsula.


title: Epigenetic Divergence during Early Stages of Speciation in an African Crater Lake Cichlid Fish

abstract:
Epigenetic variation can alter transcription and promote phenotypic divergence between populations facing different environmental challenges. Here we assess the epigenetic basis of diversification during the early stages of speciation. We focus on the extent and functional relevance of DNA methylome divergence between two Astatotilapia calliptera ecomorphs in crater Lake Masoko, southern Tanzania. We report extensive genome-wide methylome divergence between populations linked to key biological processes, including transcriptional activity of ecologically-relevant genes. These include genes involved in steroid metabolism, haemoglobin composition and erythropoiesis, consistent with divergent habitat occupancy of the ecomorphs. Using a common garden experiment, we found that global methylation profiles are rapidly remodelled across generations, but ecomorph-specific differences can be inherited. Collectively, our study suggests an epigenetic contribution to early stages of vertebrate speciation.

One sentence summaryInheritance and plasticity of epigenetic divergence characterise early stages of speciation in an incipient cichlid species of an African crater lake.


title: Molecular dissection of Carbapenem-resistant Acinetobacter baumannii circulating in Indian hospitals usingWhole Genome Sequencing

abstract:
ObjectivesCarbapenem-resistant Acinetobacter baumannii (CRAB) has acquired worldwide recognition as a serious nosocomial infection. It poses a concern to hospitalized patients because of the limited therapeutic options available. Thus, we investigated the molecular epidemiology and antibiotic resistance profiles of A. baumannii isolates in India.

Materials and MethodsWe characterized 306 retrospective A. baumannii clinical isolates collected from 18 centers across 10 states and 1 Union Territory of India between 2015 and 2019. Molecular epidemiology, and carbapenem resistance were studied by Whole Genome Sequencing.

ResultsA total of 105 different Sequence Types (STs) were identified including 48 reported STs and 57 Novel STs. 99 isolates were classified into Clonal Complex 451 (CC451) among which ST848 and ST1956 were the common STs. Carbapenemase resistance was confirmed in all the isolates with the presence of intrinsic blaOXA-51-like genes, and the acquired blaOXA-23 and blaNDM-1 genes.

ConclusionMost of the isolates were grouped under clonal complex 451. ST1053 caused an outbreak in Northern India during 2018 and 2019. Novel MLST alleles and STs were also detected, underlining an evolutionary divergence in India. The carbapenem-resistance was dominated by OXA-type carbapenemases and further surveillance of these carbapenem-resistant A. baumannii and antimicrobial stewardship should be strengthened.


title: Directed biosynthesis of fluorinated polyketides

abstract: Modification of polyketides with fluorine offers a promising approach to develop new pharmaceuticals. While synthetic chemical methods for site-specific incorporation of fluorine in complex molecules have improved in recent years, approaches for the direct biosynthetic fluorination of natural compounds are still rare. Herein, we present a broadly applicable approach for site-specific, biocatalytic derivatization of polyketides with fluorine. Specifically, we exchanged the native acyltransferase domain (AT) of a polyketide synthase (PKS), which acts as the gatekeeper for selection of extender units, with an evolutionarily related but substrate tolerant domain from metazoan type I fatty acid synthase (FAS). The resulting PKS/FAS hybrid can utilize fluoromalonyl coenzyme A and fluoromethylmalonyl coenzyme A for polyketide chain extension, introducing fluorine or fluoro-methyl disubstitutions in polyketide scaffolds. Addition of a fluorine atom is often a decisive factor toward developing superior properties in next-generation antibiotics, including the macrolide solithromycin. We demonstrate the feasibility of our approach in the semisynthesis of a fluorinated derivative of the macrolide antibiotic YC-17.


title: Phosphoproteomic analysis of chondrocytes after short-term exposure to inorganic polyphosphate

abstract: Osteoarthritis is a debilitating disease of the joint that affects over 230 million people worldwide. Currently there are no treatments that slow the progression of this disease. For these reasons, new biological treatment options are currently being explored. Inorganic polyphosphates are naturally occurring biological molecules that have an anabolic effect on chondrocytes grown in vitro in the presence of Ca2+. We hypothesized that when examining significant changes in protein phosphorylation, key candidates would emerge that could help to elucidate the anabolic effects of polyphosphate on chondrocytes. Therefore, we conducted a large-scale quantitative proteomic and phosphoproteomic study of bovine primary articular chondrocytes after 30-minute treatment with inorganic polyphosphate and Ca2+. Mass spectrometry identified more than 6000 phosphorylation sites on [~]1600 chondrocyte phosphoproteins while proteomic analysis detected approximately 4100 proteins. Analysis of the data revealed a swift and dynamic response to polyphosphate after 30 minutes. What emerged from the list of proteins most affected by the treatment were proteins with key roles in chondrogenesis including TNC, IGFBP-5, and CTGF, indicating that polyphosphate plays an important role in chondrocyte metabolism. This phosphoproteome serves as a meaningful resource to help elucidate the molecular events that contribute to extracellular matrix production in cartilage.