title: Liver Endothelium Microenvironment Promotes HER3-mediated Cell Survival in KRAS Wild-type and Mutant Colorectal Cancer

abstract: We previously showed that liver endothelial cells (ECs) secreted soluble factors in a paracrine fashion and activated human epidermal growth factor receptor 3 (HER3, also known as ERBB3) for promoting colorectal cancer (CRC) growth and chemoresistance. However, RAS proteins play a critical role in receptor tyrosine kinase signaling pathways, and KRAS mutations mediate CRC resistance to therapies targeting EGFR, another HER protein. Therefore, the role of KRAS mutation status in EC-induced HER3 activation and CRC survival was investigated as it has therapeutic implications. We used CRC cell lines and patient-derived xenografts harboring KRAS wild-type or mutant genes and demonstrated that liver EC-secreted factors promoted HER3-mediated CRC cell growth independent of KRAS mutation status. Also, blocking HER3 in CRC cells by siRNAs or a HER3 antibody seribantumab blocked EC-induced CRC survival. Our findings highlight the potential of utilizing HER3 targeted therapies for treating patients with mCRC independent of RAS mutational status.


title: Liver Endothelium Promotes HER3-mediated Cell Survival in KRAS Wild-type and Mutant Colorectal Cancer Cells

category: cancer biology


title: PodoCount: A robust, fully automated whole-slide podocyte quantification tool

abstract:
BackgroundPodocyte depletion is an established indicator of glomerular injury and predicts clinical outcomes. The semi-quantitative nature of existing podocyte estimation methods or podometrics hinders incorporation of such analysis into experimental and clinical pathologic workflows. Computational image analysis offers a robust approach to automate podometrics through objective quantification of cell and tissue structure. Toward this goal, we developed PodoCount, a computational tool for quantitative analysis of podocytes, and validated the generalizability of the tool across a diverse dataset.

MethodsPodocyte nuclei and glomerular boundaries were labeled in murine whole kidney sections, n = 135, from six disease models and human kidney biopsies, n = 45, from diabetic nephropathy (DN) patients. Digital whole slide images (WSIs) of tissues were then acquired. Classical image analysis was applied to obtain podocyte nuclear and glomerular morphometrics. Statistically significant morphometric features, which correlated with each murine disease, were identified. Engineered features were also assessed for their ability to predict outcomes in human DN. PodoCount has been disbursed for other researchers as an open-source, cloud-based computational tool.

ResultsPodoCount offers highly accurate quantification of podocytes. Engineered podometric features were benchmarked against routine glomerular histopathology and were found to be significant predictors of disease diagnosis, proteinuria level, and clinical outcomes.

ConclusionsPodoCount offers high quantification performance in diverse murine disease models as well as in human DN. Resultant podometric features offers significant correlation with associated metadata as well as outcome. Our cloud-based end-user tool will provide a standardized approach for podometric analysis from gigapixel size WSIs in basic research and clinical practice.

category: pathology


title: GDF8 Contributes to the Adverse Effects of Liver Injury on Skeletal Muscle Homeostasis and Regeneration

abstract:
BackgroundAn emerging clinical phenomenon in patients with end stage liver disease is progressive skeletal muscle atrophy. This loss in lean mass predicts poor survival outcomes for liver disease patients and highlights an underappreciated crosstalk between injured liver and muscle that lacks defined mediators. The purpose of our study was to identify potential liver-muscle mediator(s) in pre-clinical in vivo models of liver injury which may contribute to the muscle loss observed in liver disease.

MethodsUtilizing a mouse model of carbon tetrachloride CCl4-induced liver injury in the presence or absence of cardiotoxin-induced muscle injury, we evaluated whether neutralizing Activin type IIB receptor (ActRIIB) ligands, or specifically growth differentiation factor 8 (Gdf8), could preserve or reverse muscle atrophy associated with liver disease.

ResultsWe found that hepatic injury via CCl4 or bile duct ligation (BDL) similarly caused significant muscle atrophy along with decreased gene expression in key myogenesis markers. This adverse effect of injured liver on muscle were completely prevented and reversed by the intervention of Activin type IIB receptor (ActRIIB)-Fc fusion protein, which neutralizes the ActRIIB ligands, including Activins and growth differentiation factor 8 (Gdf8 or myostatin). The results indicate that ActRIIB ligands promoted muscle atrophy which was manifested in response to hepatic injury/disease and conferred the negative communication of injured liver with muscle. Indeed, direct injection of exogenous Gdf8 protein into muscle along with acute focal muscle injury recapitulated similar dysregulated muscle regeneration as observed with liver injury. Furthermore, we found that hepatocytes produced Gdf8 in response to liver injury in rodents and in patients with end stage liver disease. A neutralizing antibody to Gdf8 attenuated muscle atrophy and unexpectedly ameliorated liver fibrosis in both CCl4 and BDL models. Following this observation, we demonstrated Gdf8s ability to induce fibrogenesis in stellate cells, potentially identifying a novel hepatic role for this protein. Moreover, hepatic Gdf8 promoted muscle wasting in response to liver damage and hindered skeletal muscle regeneration.

ConclusionOur findings identified Gdf8 as a novel hepatomyokine contributing to injured liver-muscle negative crosstalk and liver injury progression. Moreover, we demonstrated a promising therapeutic strategy for muscle atrophy accompanying liver diseases.


title: PEGylated isoprenaline reveals distinct functions of cardiac β-adrenergic receptors located in the T-tubule vs. outer surface membrane

abstract: Membrane proteins are present in both cardiac T-tubule (TTM) and outer surface membrane (OSM), although at a different density. Classical pharmacology does not allow to explore the function of a membrane protein separately in OSM vs. TTM. Here, we developed a technology based on size exclusion to explore the function of {beta}-adrenergic receptors ({beta}-ARs) located in the OSM. We synthetized a PEG-Iso molecule by covalent linking between isoprenaline (Iso) and a 5000 Da PolyEthylene-Glycol (PEG). Using confocal microscopy, we show that PEGylation constrains molecules outside the T-tubule network. PEG-Iso produced similar effects as Iso on Ica,L, sarcomere shortening and Ca2+ transients. However, PEG-Iso increased [cAMP]i with a lower efficacy than Iso, produced a much lower stimulation of nuclear PKA activity than Iso but a larger stimulation of cytosolic PKA at equivalent levels of [cAMP]i. Our results show that activation of OSM {beta}-ARs is sufficient to activate cytosolic PKA and excitation-contraction coupling, but insufficient to activate nuclear PKA or nuclear protein phosphorylation for which additional activation of TTM {beta}-ARs is needed.

category: physiology


title: Loss of lamin-B1 and defective nuclear morphology are hallmarks of astrocyte senescence in vitro and in the aging human hippocampus

abstract: The increase in senescent cells in tissues, including the brain, is a general feature of normal aging and age-related pathologies. Senescent cells exhibit a specific phenotype, which includes an altered nuclear morphology and transcriptomic changes. Astrocytes undergo senescence in vitro and in age-associated neurodegenerative diseases, but little is known about whether this process also occurs in physiological aging. Here, we investigated astrocyte senescence in vitro, in old mouse brains and in post-mortem human brain tissue of elderly. We identified a significant loss of lamin-B1, a major component of the nuclear lamina, as a hallmark of senescent astrocytes. We showed a severe reduction of lamin-B1 in the dentate gyrus of aged mice, including in hippocampal astrocytes, and in the granular cell layer of the hippocampus of post-mortem human tissue from non-demented elderly. Interestingly, the lamin-B1 reduction was associated with nuclear deformations, represented by an increased incidence of invaginated nuclei and loss of nuclear circularity in senescent astrocytes in vitro and in the aging human hippocampus. In conclusion, our findings show that reduction of lamin-B1 is a conserved hallmark of astrocyte aging, as well as shed light on significant defects in nuclear lamina structure, which may impact astrocyte function during human aging.

category: neuroscience


title: Holo-Hilbert Spectral-based Removal of Gamma-Band Noise from Simultaneous EEG-fMRI Recordings

abstract: Simultaneous EEG-fMRI is a growing and promising field, as it has great potential to further our understanding of the spatiotemporal dynamics of brain function in health and disease. In particular, there is much interest in understanding the fMRI correlates of brain activity in the gamma band (> 30 Hz), as these frequencies are thought to be associated with cognitive processes involving perception, attention, and memory, as well as with disorders such as schizophrenia and autism. However, progress in this area has been limited due to issues such as MR-induced artifacts in EEG recordings, which seem to be more problematic for gamma frequencies. This paper presents a noise removal method for the gamma band of EEG that is based on the Holo-Hilbert spectral analysis (HHSA), but with a new implementation strategy. HHSA uses a nested empirical mode decomposition (EMD) to identify amplitude and frequency modulations (AM and FM, respectively) by averaging over frequencies with high and significant powers. Our method examines gamma band by applying two layers of EMD to the FM and AM components, removing components with very low power based on the power-instantaneous frequency spectrum, and subsequently reconstructs the denoised gamma-band signal from the remaining components. Simulations demonstrate that our proposed method efficiently reduces artifacts while preserving the original gamma signal which is especially critical for simultaneous EEG/fMRI studies.


title: Holo-Hilbert Spectral-based Noise Removal Method for EEG High-Frequency Bands


title: Transcriptomic encoding of sensorimotor transformation in the midbrain

abstract: Sensorimotor transformation, a process that converts sensory stimuli into motor actions, is critical for the brain to initiate behaviors. Although the circuitry involved in sensorimotor transformation has been well delineated, the molecular logic behind this process remains poorly understood. Here, we performed high-throughput and circuit-specific single-cell transcriptomic analyses of neurons in the superior colliculus (SC), a midbrain structure implicated in early sensorimotor transformation. We found that SC neurons in distinct laminae express discrete marker genes. Of particular interest, Cbln2 and Pitx2 are key markers that define glutamatergic projection neurons in the optic nerve (Op) and intermediate gray (InG) layers, respectively. The Cbln2+ neurons responded to visual stimuli mimicking cruising predators, while the Pitx2+ neurons encoded prey-derived vibrissal tactile cues. By forming distinct input and output connections with other brain areas, these neuronal subtypes independently mediate behaviors of predator avoidance and prey capture. Our results reveal that, in the midbrain, sensorimotor transformation for different behaviors may be performed by separate circuit modules that are molecularly defined by distinct transcriptomic codes.


title: Inter-Subject Correlation during New Music Listening: A Study of Electrophysiological and Behavioral Responses to Steve Reich's Piano Phase

abstract: Musical minimalism utilizes the temporal manipulation of restricted collections of rhythmic, melodic, and/or harmonic materials. One example, Steve Reichs Piano Phase, offers listeners readily audible formal structures containing unpredictable events at local levels. Pattern recurrences may generate strong expectations which are violated by small temporal and pitch deviations. A hyper-detailed listening strategy prompted by these minute deviations stands in contrast to the type of listening engagement typically cultivated around functional tonal Western music. Recent research has suggested that the inter-subject correlation (ISC) of electroencephalographic (EEG) responses to natural audio-visual stimuli objectively indexes a state of "engagement", demonstrating the potential of this approach for analyzing music listening. But can ISCs capture engagement with minimal music, which features less obvious expectation formation and has historically received a wide range of reactions? To approach this question, we collected EEG and continuous behavioral (CB) data while 30 adults listened to an excerpt from Steve Reichs Piano Phase, as well as three controlled manipulations and a popular-music remix of the work. Our analyses reveal that EEG and CB ISC are highest for the remix stimulus and lowest for our most repetitive manipulation. In addition, we found no statistical differences in overall EEG ISC between our most musically meaningful manipulations and Reichs original piece. We also found that aesthetic evaluations corresponded well with overall EEG ISC. Finally we highlight co-occurrences between stimulus events and time-resolved EEG and CB ISC. We offer the CB paradigm as a useful analysis measure and note the value of minimalist compositions as a limit case for studying music listening using EEG ISC. We show that ISC is less effective at measuring engagement with this minimalist stimulus than with popular music genres and argue that this may be due to a difference between the type of engagement measured by ISC and the particular engagement patterns associated with minimalism.


title: Inter-Subject Correlation during New Music Listening: A Study of Electrophysiological and Behavioral Responses to Steve Reich`s Piano Phase


title: Exogenous attention generalizes perceptual learning in adults with amblyopia

abstract: Visual perceptual learning (VPL) is a behavioral manifestation of brain neuroplasticity. However, its practical effectiveness is limited because improvements are often specific to the trained conditions and require significant time and effort. It is critical to understand the conditions that promote learning and transfer. Covert endogenous (voluntary) and exogenous (involuntary) spatial attention help overcome VPL location specificity in neurotypical adults, but whether they also do so for people with atypical visual development is unknown. This is the first study to investigate the role of exogenous attention during VPL in adults with amblyopia, an ideal population given their asymmetrically developed, but highly plastic, visual cortex. Here we show that training on a discrimination task leads to improvements in foveal contrast sensitivity, acuity, and stereoacuity. Remarkably, exogenous attention helps generalize learning beyond trained spatial locations. Our findings reveal that attention can enhance the effectiveness of perceptual learning during rehabilitation of visual disorders.


title: Exogenous attention generalizes location transfer of perceptual learning in adults with amblyopia


title: Helios is a marker, not a driver, of human Treg stability

abstract: Regulatory T cell (Treg) therapy holds promise as a potentially curative approach to establish immune tolerance in transplantation and autoimmune disease. An outstanding question is whether therapeutic Tregs have the potential to transdifferentiate into effector T cells and thus exacerbate rather than suppress immune responses. In mice, the transcription factor Helios is thought to promote Tregs lineage stability in a range of inflammatory contexts. In humans, the role of Helios in Tregs is less clear, in part due to the inability to enrich and study subsets of Helios-positive versus Helios-negative Tregs. Using an in vitro expansion system, we found that loss of high Helios expression and emergence of an intermediate Helios (Heliosmid)-expressing population correlated with Treg destabilization. We then used CRISPR/Cas9 to genetically ablate Helios expression in human naive or memory Tregs and found that Helios-knockout and unedited Tregs were equivalent in their suppressive function and stability in inflammation. Thus, high Helios expression is a marker, but not a driver, of human Treg stability in vitro. These data highlight the importance of monitoring Helios expression in therapeutic Treg manufacturing and provide new insight into the biological function of this transcription factor in human T cells.

category: immunology


title: Laser facilitated epicutaneous peptide immunization using dry patch technology

abstract: The skin has been intensely investigated as a target tissue for immunization because it is populated by multiple types of antigen presenting cells. Directly addressing dendritic cells or Langerhans cells in vivo represents an attractive strategy for inducing T cell responses in cancer immunotherapy. We and others have studied fractional laser ablation as a novel method combining efficient delivery of macromolecules to the skin with an inherent adjuvant effect of laser illumination. In this proof of concept study, we demonstrate the feasibility of peptide delivery to the skin using the P.L.E.A.S.E. professional Erb:YAG fractional infrared laser together with EPIMMUN patches. In an ovalbumin mouse model we demonstrate that a dry patch formulation of SIINFEKL peptide in combination with CpG-ODN1826, but not imiquimod or polyI:C, induces potent cytotoxic T cell responses, which can be further boosted by co-delivery of the pan-helper T cell epitope PADRE.


title: A Novel Family of RNA-Binding Proteins Regulate Polysaccharide Metabolism in Bacteroides thetaiotaomicron

abstract:
Human gut microbiome composition is constantly changing, and diet is a major driver of these changes. Gut microbial species that persist in mammalian hosts for long periods of time must possess mechanisms for sensing and adapting to nutrient shifts to avoid being outcompeted. Global regulatory mechanisms mediated by RNA-binding proteins (RBPs) that govern responses to nutrient shifts have been characterized in Proteobacteria and Firmicutes but remain undiscovered in the Bacteroidetes. Here we report the identification of RBPs that are broadly distributed across the Bacteroidetes, with many genomes encoding multiple copies. Genes encoding these RBPs are highly expressed in many Bacteroides species. A purified RBP, RbpB, from Bacteroides thetaiotaomicron binds to single-stranded RNA in vitro with an affinity similar to other characterized regulatory RBPs. B. thetaiotaomicron mutants lacking RBPs show dramatic shifts in expression of polysaccharide utilization and capsular polysaccharide loci, suggesting that these RBPs may act as global regulators of polysaccharide metabolism. A B. thetaiotaomicron {Delta}rbpB mutant shows a growth defect on dietary sugars belonging to the raffinose family of oligosaccharides (RFOs). The {Delta}rbpB mutant had reduced expression of BT1871, encoding a predicted RFO-degrading melibiase, compared to the wild-type strain. Mutation of BT1871 confirmed that the enzyme it encodes is essential for growth on melibiose and promotes growth on the RFOs raffinose and stachyose. Our data reveal that RbpB is required for optimal expression of BT1871 and other polysaccharide-related genes, suggesting that we have identified an important new family of global regulatory proteins in the Bacteroidetes.

ImportanceThe human colon houses hundreds of bacterial species, including many belonging to the genus Bacteroides, that aid in breaking down our food to keep us healthy. Bacteroides have many genes responsible for breaking down different dietary carbohydrates and complex regulatory mechanisms ensure that specific genes are only expressed when the right carbohydrates are available. In this study, we discovered that Bacteroides use a family of RNA-binding proteins as global regulators to coordinate expression of carbohydrate utilization genes. The ability to turn different carbohydrate utilization genes on and off in response to changing nutrient conditions is critical for Bacteroides to live successfully in the gut, and thus the new regulators we have identified may be important for life in the host.

category: microbiology


title: A broadly neutralizing antibody protects against SARS-CoV, pre-emergent bat CoVs, and SARS-CoV-2 variants in mice

abstract: SARS-CoV in 2003, SARS-CoV-2 in 2019, and SARS-CoV-2 variants of concern (VOC) can cause deadly infections, underlining the importance of developing broadly effective countermeasures against Group 2B Sarbecoviruses, which could be key in the rapid prevention and mitigation of future zoonotic events. Here, we demonstrate the neutralization of SARS-CoV, bat CoVs WIV-1 and RsSHC014, and SARS-CoV-2 variants D614G, B.1.1.7, B.1.429, B1.351 by a receptor-binding domain (RBD)-specific antibody DH1047. Prophylactic and therapeutic treatment with DH1047 demonstrated protection against SARS-CoV, WIV-1, RsSHC014, and SARS-CoV-2 B1.351infection in mice. Binding and structural analysis showed high affinity binding of DH1047 to an epitope that is highly conserved among Sarbecoviruses. We conclude that DH1047 is a broadly neutralizing and protective antibody that can prevent infection and mitigate outbreaks caused by SARS-like strains and SARS-CoV-2 variants. Our results argue that the RBD conserved epitope bound by DH1047 is a rational target for pan Group 2B coronavirus vaccines.


title: Therapeutic effect of CT-P59 against SARS-CoV-2 South African variant

abstract:
The global circulation of newly emerging variants of SARS-CoV-2 is a new threat to public health due to their increased transmissibility and immune evasion. Moreover, currently available vaccines and therapeutic antibodies were shown to be less effective against new variants, in particular, the South African (SA) variant, termed 501Y.V2 or B.1.351. To assess the efficacy of the CT-P59 monoclonal antibody against the SA variant, we sought to perform as in vitro binding and neutralization assays, and in vivo animal studies. CT-P59 neutralized B.1.1.7 variant to a similar extent as to wild type virus. CT-P59 showed reduced binding affinity against a RBD (receptor binding domain) triple mutant containing mutations defining B.1.351 (K417N/E484K/N501Y) also showed reduced potency against the SA variant in live virus and pseudovirus neutralization assay systems. However, in vivo ferret challenge studies demonstrated that a therapeutic dosage of CT-P59 was able to decrease B.1.351 viral load in the upper and lower respiratory tracts, comparable to that observed for the wild type virus. Overall, although CT-P59 showed reduced in vitro neutralizing activity against the SA variant, sufficient antiviral effect in B.1.351-infected animals was confirmed with a clinical dosage of CT-P59, suggesting that CT-P59 has therapeutic potential for COVID-19 patients infected with SA variant.

HighlightsO_LICT-P59 significantly inhibit B.1.1.7 variant to a similar extent as to wild type virus
C_LIO_LICT-P59 showed reduced potency against the B.1.351 variant in in vitro studies
C_LIO_LITherapeutic dosage of CT-P59 showed in vivo neutralizing potency against B.1.351 in ferret challenge study.
C_LI


title: Distinct gene expression dynamics in germ line and somatic tissue during ovariole morphogenesis in Drosophila melanogaster

abstract: The survival and evolution of a species is a function of the number of offspring it can produce. In insects the number of eggs that an ovary can produce is a major determinant of reproductive capacity. Insect ovaries are made up of tubular egg-producing subunits called ovarioles, whose number largely determines the number of eggs that can be potentially laid. Ovariole number is directly determined by the number of cellular structures called terminal filaments, which are stacks of cells that assemble in the larval ovary. Elucidating the developmental and regulatory mechanisms of terminal filament formation is thus key to understanding the regulation of insect reproduction through ovariole number regulation. We systematically measured mRNA expression of all cells in the larval ovary at the beginning, middle and end of terminal filament formation. We also separated somatic and germ line cells during these stages and assessed their tissue-specific gene expression during larval ovary development. We found that the number of differentially expressed somatic genes is highest during late stages of terminal filament formation and includes many signaling pathways that govern ovary development. We also show that germ line tissue, in contrast, shows greater differential expression during early stages of terminal filament formation, and highly expressed germ line genes at these stages largely control cell division and DNA repair. We provide a tissue-specific and temporal transcriptomic dataset of gene expression in the developing larval ovary as a resource to study insect reproduction.

category: developmental biology


title: Highly variable fidelity drives symbiont community composition in an obligate symbiosis

abstract: Many animals are obligately associated with microbial symbionts that provide essential services such as nutrition or protection against predators. It is assumed that in such obligate associations fidelity between the host and its symbionts must be high to ensure the evolutionary success of the symbiosis. We show here that this is not the case in marine oligochaete worms, despite the fact that they are so dependent on their bacterial symbionts for their nutrition and waste recycling that they have lost their digestive and excretory systems. Our metagenomic analyses of 64 gutless oligochaete species from around the world revealed highly variable levels of fidelity not only across symbiont lineages, but also within symbiont clades. We hypothesize that in gutless oligochaetes, selection within host species for locally adapted and temporally stable symbiont communities leads to varying levels of symbiont fidelity and shuffles the composition of symbiont assemblages across geographic and evolutionary scales.

category: evolutionary biology


title: Transcriptome diversity is a systematic source of bias in RNA-sequencing data

abstract:
BackgroundRNA sequencing has been widely used as an essential tool to probe gene expression. While standard practices have been established to analyze RNA-seq data, it is still challenging to detect and remove artifactual signals. Several factors such as sex, age, and sequencing technology have been found to bias these estimates. Probabilistic estimation of expression residuals (PEER) has been used to account for some systematic effects, but it has remained challenging to interpret these PEER factors.

ResultsHere we show that transcriptome diversity - a simple metric based on Shannon entropy - explains a large portion of variability in gene expression, and is a major factor detected by PEER. We then show that transcriptome diversity has significant associations with multiple technical and biological variables across diverse organisms and datasets. This prevalent confounding factor provides a simple explanation for a major source of systematic biases in gene expression estimates.

ConclusionsOur results show that transcriptome diversity is a metric that captures a systematic bias in RNA-seq and is the strongest known factor encoded in PEER covariates.

category: genomics


title: Massively Multiplexed Affinity Characterization of Therapeutic Antibodies Against SARS-CoV-2 Variants

abstract:
Antibody therapies represent a valuable tool to reduce COVID-19 deaths and hospitalizations. Multiple antibody candidates have been granted emergency use authorization by the FDA and many more are in clinical trials. Most antibody therapies for COVID-19 are engineered to bind to the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein and disrupt its interaction with ACE2. Notably, several SARS-CoV-2 strains have accrued mutations throughout the RBD that improve ACE2 binding affinity, enhance viral transmission, and escape some existing antibody therapies. Here, we measure the binding affinity of 33 therapeutic antibodies against a large panel of SARS-CoV-2 variants and related strains of clinical significance to determine epitopic residues, determine which mutations result in loss of binding, and predict how future RBD variants may impact antibody efficacy.

One-Sentence SummaryBy measuring protein binding in vitro, we identify which clinical antibodies retain binding to various mutant SARS-CoV-2 strains.

category: bioengineering


title: RFRSN: Improving protein fold recognition by siamese network

abstract: Protein fold recognition is the key to study protein structure and function. As a representative pattern recognition task, there are two main categories of approaches to improve the protein fold recognition performance: 1) extracting more discriminative descriptors, and 2) designing more effective distance metrics. The existing protein fold recognition approaches focus on the first category to finding a robust and discriminative descriptor to represent each protein sequence as a compact feature vector, where different protein sequence is expected to be separated as much as possible in the fold space. These methods have brought huge improvements to the task of protein fold recognition. However, so far, little attention has been paid to the second category. In this paper, we focus not only on the first category, but also on the second point that how to measure the similarity between two proteins more effectively. First, we employ deep convolutional neural network techniques to extract the discriminative fold-specific features from the potential protein residue-residue relationship, we name it SSAfold. On the other hand, due to different feature representation usually subject to varying distributions, the measurement of similarity needs to vary according to different feature distributions. Before, almost all protein fold recognition methods perform the same metrics strategy on all the protein feature ignoring the differences in feature distribution. This paper presents a new protein fold recognition by employing siamese network, we named it PFRSN. The objective of PFRSN is to learns a set of hierarchical nonlinear transformations to project protein pairs into the same fold feature subspace to ensure the distance between positive protein pairs is reduced and that of negative protein pairs is enlarged as much as possible. The experimental results show that the results of SSAfold and PFRSN are highly competitive.

category: bioinformatics


title: Prediction of RNA-protein interactions using a nucleotide language model

abstract:
MotivationThe accumulation of sequencing data has enabled researchers to predict the interactions between RNA sequences and RNA-binding proteins (RBPs) using novel machine learning techniques. However, existing models are often difficult to interpret and require additional information to sequences. Bidirectional encoder representations from Transformer (BERT) is a language-based deep learning model that is highly interpretable. Therefore, a model based on BERT architecture can potentially overcome such limitations.

ResultsHere, we propose BERT-RBP as a model to predict RNA-RBP interactions by adapting the BERT architecture pre-trained on a human reference genome. Our model outperformed state-of-the-art prediction models using the eCLIP-seq data of 154 RBPs. The detailed analysis further revealed that BERT-RBP could recognize both the transcript region type and RNA secondary structure only from sequence information. Overall, the results provide insights into the fine-tuning mechanism of BERT in biological contexts and provide evidence of the applicability of the model to other RNA-related problems.

AvailabilityPython source codes are freely available at https://github.com/kkyamada/bert-rbp.

Contactmhamada@waseda.jp


title: Post-outbreak surveillance strategies to support proof of freedom from foot-and-mouth disease

abstract:
Whilst emergency vaccination may help contain foot-and-mouth disease in a previously FMD-free country, its use complicates post-outbreak surveillance and the recovery of FMD-free status. A structured surveillance program is required that can distinguish between vaccinated and residually infected animals, and provide statistical confidence that the virus is no longer circulating in previously infected areas.

Epidemiological models have been well-used to investigate the potential benefits of emergency vaccination during a control progam and when/where/whom to vaccinate in the face of finite supplies of vaccine and personnel. Less well studied are post-outbreak issues such as the management of vaccinated animals and the implications of having used vaccination during surveillance regimes to support proof-of-freedom. This paper presents enhancements to the Australian Animal Disease Model (AADIS) that allow comparisons of different post-outbreak surveillance sampling regimes for establishing proof-of-freedom from FMD.

A case study is provided that compares a baseline surveillance sampling regime (derived from current OIE guidelines), with an alternative less intensive sampling regime. It was found that when vaccination was not part of the control program, a reduced sampling intensity significantly reduced the number of samples collected and the cost of the post-outbreak surveillance program, without increasing the risk of missing residual infected herds.


title: DTI-Voodoo: machine learning over interaction networks and ontology-based background knowledge predicts drug-target interactions

abstract:
MotivationIn silico drug-target interaction (DTI) prediction is important for drug discovery and drug repurposing. Approaches to predict DTIs can proceed indirectly, top-down, using phenotypic effects of drugs to identify potential drug targets, or they can be direct, bottom-up and use molecular information to directly predict binding potentials. Both approaches can be combined with information about interaction networks.

ResultsWe developed DTI-Voodoo as a computational method that combines molecular features and ontology-encoded phenotypic effects of drugs with protein-protein interaction networks, and uses a graph convolutional neural network to predict DTIs. We demonstrate that drug effect features can exploit information in the interaction network whereas molecular features do not. DTI-Voodoo is designed to predict candidate drugs for a given protein; we use this formulation to show that common DTI datasets contain intrinsic biases with major affects on performance evaluation and comparison of DTI prediction methods. Using a modified evaluation scheme, we demonstrate that DTI-Voodoo improves significantly over state of the art DTI prediction methods.

AvailabilityDTI-Voodoo source code and data necessary to reproduce results are freely available at https://github.com/THinnerichs/DTI-VOODOO.

Contacttilman.hinnerichs@kaust.edu.sa

Supplementary informationSupplementary data are available at https://github.com/THinnerichs/DTI-VOODOO.


title: TReNCo: Topologically associating domain (TAD) aware regulatory network construction

abstract: There has long been a desire to understand, describe, and model gene regulatory networks controlling numerous biologically meaningful processes like differentiation. Despite many notable improvements to models over the years, many models do not accurately capture subtle biological and chemical characteristics of the cell such as high-order chromatin domains of the chromosomes. Topologically Associated Domains (TAD) are one of these genomic regions that are enriched for contacts within themselves. Here we present TAD-aware Regulatory Network Construction or TReNCo, a memory-lean method utilizing epigenetic marks of enhancer and promoter activity, and gene expression to create context-specific transcription factor-gene regulatory networks. TReNCo utilizes common assays, ChIP-seq, RNA-seq, and TAD boundaries as a hard cutoff, instead of distance based, to efficiently create context-specific TF-gene regulatory networks. We used TReNCo to define the enhancer landscape and identify transcription factors (TFs) that drive the cardiac development of the mouse. Our results show that we are able to build specialized adjacency regulatory network graphs containing biologically relevant connections and time dependent dynamics.


title: Immune microenvironment subtypes and association with tumor cell mutations and antigen expression in follicular lymphoma.

abstract:
Follicular lymphoma (FL) is a B-cell lymphoma with a complex tumor microenvironment that is rich in non-malignant immune cells. We applied single-cell RNA-sequencing to characterize the diverse tumor and immune cell populations of FL and identified major phenotypic subsets of FL T-cells including a novel cytotoxic CD4 T-cell population. Their relative proportions of T-cells defined four major FL subtypes, characterized by differential representation or relative depletion of distinct T-cell subsets. By integrating exome sequencing, we observed that somatic mutations are associated with, but not definitive for, reduced antigen presentation on FL cells. In turn, expression of MHC class II genes by FL cells was associated with significant differences in the proportions and targetable immunophenotypic characteristics. This provides a classification framework of the FL microenvironment, their association with FL genotypes and antigen presentation, and informs different potential immunotherapeutic strategies based upon tumor cell MHC class II expression.

Statement of significanceWe have characterized the FL-infiltrating T-cells, identified cytotoxic CD4 T-cells as an important component, showed that the abundance of these T-cell populations is associated with tumor-cell-intrinsic characteristics, and identified sets of targetable immune checkpoints on T-cells that differed between FLs with normal versus low antigen presentation.


title: A selective glucocorticoid receptor modulator attenuates lung inflammation and improves alveolarization in a neonatal rat model of bronchopulmonary dysplasia.

abstract:
BackgroundBronchopulmonary dysplasia (BPD) is a major problem for extremely preterm infants. Glucocorticoids effectively treat BPD; however, they have many side effects. Compound A (Cpd A) is a nonsteroidal Selective Glucocorticoid Receptor Modulator (SEGRM) that acts as a glucocorticoid receptor ligand without inducing the expression of glucocorticoid-response element-driven genes. Cpd A reportedly has anti-inflammatory properties with fewer side effects than glucocorticoids.

MethodsUsing a bleomycin (Bleo)-induced BPD model, we evaluated the therapeutic effects of Cpd A. 0-day-old Sprague-Dawley rats were administered Bleo for 10 days and treated with dexamethasone (Dex) or Cpd A from day 0 to 13. We evaluated lung pathology by histology and the mRNA levels of interleukin (IL)-1{beta}, transforming growth factor (TGF)-{beta}1 and chemokines, CXCL1 and CCL2.

ResultsBleo-treated mice had lungs with impaired alveolarization. Dex and Cpd A treatments improved the alveolar structure, attenuating the lung injury. Bleo-exposed lungs had increased inflammatory cells recruitment and inflammatory mediator mRNA levels. Cpd A treatment reduced inflammatory cells infiltration and CXCL1, CCL2 and TGF-{beta}1 expression.

ConclusionCpd A improved lung inflammation and alveolar maturation arrest, and restored histological and biochemical changes in a model of BPD. SEGRMs, including Cpd A, are promising candidates for the therapy of BPD.

Impact Statement{circ} What is the key message of your article?
Compound A decreased lung inflammation and improved lung morphometric changes in Bleomycin-exposed lungs.
{circ}What does it add to the existing literature?
Compound A has anti-inflammatory effects in an experimental model of BPD.
{circ}What is the impact?
SEGRMs, including Cpd A, may be promising candidates for the therapy of BPD.


title: Soluble adenylyl cyclase inhibition prevents human sperm functions essential for fertilization

abstract: Soluble adenylyl cyclase (sAC: ADCY10) is essential for activating dormant sperm. Studies of freshly dissected mouse sperm identified sAC as needed for initiating capacitation and activating motility. We now use an improved sAC inhibitor, TDI-10229, for a comprehensive analysis of sAC function in human sperm. Unlike dissected mouse sperm, human sperm are collected post-ejaculation, after sAC activity has already been stimulated. Even in ejaculated human sperm, TDI-10229 interrupts stimulated motility and capacitation, and it prevents acrosome reaction in capacitated sperm. At present, there are no non-hormonal, pharmacological methods for contraception. Because sAC activity is required post-ejaculation at multiple points during the sperms journey to fertilize the oocyte, sAC inhibitors define candidates for non-hormonal, on-demand contraceptives suitable for delivery via intravaginal devices in females.

category: pharmacology and toxicology


title: Regulation and impact of cardiac lymphangiogenesis in pressure-overload-induced heart failure

abstract:
RationaleLymphatics are essential for cardiac health, and insufficient lymphatic expansion (lymphangiogenesis) contributes to development of heart failure (HF) after myocardial infarction. However, the regulation and impact of lymphatics in non-ischemic cardiomyopathy induced by pressure-overload remains to be determined.

ObjectiveInvestigate cardiac lymphangiogenesis following transverse aortic constriction (TAC) in adult male or female C57Bl/6J or Balb/c mice, and in patients with end-stage HF.

Methods & ResultCardiac function was evaluated by echocardiography, and cardiac hypertrophy, lymphatics, inflammation, edema, and fibrosis by immunohistochemistry, flow cytometry, microgravimetry, and gene expression analysis, respectively. Treatment with neutralizing anti-VEGFR3 antibodies was applied to inhibit cardiac lymphangiogenesis in mice.

The gender- and strain-dependent mouse cardiac hypertrophic response to TAC, especially increased ventricular wall stress, led to lymphatic expansion in the heart. Our experimental findings that ventricular dilation triggered cardiac lymphangiogenesis was mirrored by observations in clinical HF samples, with increased lymphatic density found in patients with dilated cardiomyopathy. Surprisingly, the striking lymphangiogenesis observed post-TAC in Balb/c mice, linked to increased cardiac Vegfc, did not suffice to resolve myocardial edema, and animals progressed to dilated cardiomyopathy and HF. Conversely, selective inhibition of the essentially Vegfd-driven capillary lymphangiogenesis observed post-TAC in male C57Bl/6J mice did not significantly aggravate cardiac edema. However, cardiac immune cell levels were increased, notably myeloid cells at 3 weeks and T lymphocytes at 8 weeks. Moreover, while the TAC-triggered development of interstitial cardiac fibrosis was unaffected by anti-VEGFR3, inhibition of lymphangiogenesis increased perivascular fibrosis and accelerated the development of left ventricular dilation and cardiac dysfunction.

ConclusionsWe demonstrate for the first time that endogenous cardiac lymphangiogenesis limits pressure-overload-induced cardiac inflammation and perivascular fibrosis, thus delaying HF development. While these findings remain to be confirmed in a larger study of HF patients, we propose that under settings of pressure-overload poor cardiac lymphangiogenesis may accelerate HF development.


title: SARS-COV-2 induced Diarrhea is inflammatory, Ca2+ Dependent and involves activation of calcium activated Cl channels

abstract: Diarrhea occurs in 2-50% of cases of COVID-19 ([~]8% is average across series). The diarrhea does not appear to account for the disease mortality and its contribution to the morbidity has not been defined, even though it is a component of Long Covid or post-infectious aspects of the disease. Even less is known about the pathophysiologic mechanism of the diarrhea. To begin to understand the pathophysiology of COVID-19 diarrhea, we exposed human enteroid monolayers obtained from five healthy subjects and made from duodenum, jejunum, and proximal colon to live SARS-CoV-2 and virus like particles (VLPs) made from exosomes expressing SARS-CoV-2 structural proteins (Spike, Nucleocapsid, Membrane and Envelope). Results: 1) Live virus was exposed apically for 90 min, then washed out and studied 2 and 5 days later. SARS-Cov-2 was taken up by enteroids and live virus was present in lysates and in the apical>>basolateral media of polarized enteroids 48 h after exposure. This is the first demonstration of basolateral appearance of live virus after apical exposure. High vRNA concentration was detected in cell lysates and in the apical and basolateral media up to 5 days after exposure. 2) Two days after viral exposure, cytokine measurements of media showed significantly increased levels of IL-6, IL-8 and MCP-1. 3) Two days after viral exposure, mRNA levels of ACE2, NHE3 and DRA were reduced but there was no change in mRNA of CFTR. NHE3 protein was also decreased. 4) Live viral studies were mimicked by some studies with VLP exposure for 48 h. VLPs with Spike-D614G bound to the enteroid apical surface and was taken up; this resulted in decreased mRNA levels of ACE2, NHE3, DRA and CFTR. 4) VLP effects were determined on active anion secretion measured with the Ussing chamber/voltage clamp technique. S-D614G acutely exposed to apical surface of human ileal enteroids did not alter the short-circuit current (Isc). However, VLPS-D614G exposure to enteroids that were pretreated for [~]24 h with IL-6 plus IL-8 induced a concentration dependent increase in Isc indicating stimulated anion secretion, that was delayed in onset by [~]8 min. The anion secretion was inhibited by apical exposure to a specific calcium activated Cl channel (CaCC) inhibitor (AO1) but not by a specific CFTR inhibitor (BP027); was inhibited by basolateral exposure to the K channel inhibit clortimazole; and was prevented by pretreatment with the calcium buffer BAPTA-AM. 5) The calcium dependence of the VLP-induced increase in Isc was studied in Caco-2/BBe cells stably expressing the genetically encoded Ca2+ sensor GCaMP6s. 24 h pretreatment with IL-6/IL-8 did not alter intracellular Ca2+. However, in IL-6/IL-8 pretreated cells, VLP S-D614G caused appearance of Ca2+waves and an overall increase in intracellular Ca2+ with a delay of [~]10 min after VLP addition. We conclude that the diarrhea of COVID-19 appears to an example of a calcium dependent inflammatory diarrhea that involves both acutely stimulated Ca2+ dependent anion secretion (stimulated Isc) that involves CaCC and likely inhibition of neutral NaCl absorption (decreased NHE3 protein and mRNA and decreased DRA mRNA).


title: The Boundary-Expressed EPIDERMAL PATTERNING FACTOR-LIKE2 Gene Encoding a Signaling Peptide Promotes Cotyledon Growth during Arabidopsis thaliana Embryogenesis

abstract: The shoot organ boundaries have important roles in plant growth and morphogenesis. It has been reported that a gene encoding a cysteine-rich secreted peptide of the EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family, EPFL2, is expressed in the boundary domain between the two cotyledon primordia of Arabidopsis thaliana embryo. However, its developmental functions remain unknown. This study aimed to analyze the role of EPFL2 during embryogenesis. We found that cotyledon growth was reduced in its loss-of-function mutants, and this phenotype was associated with the reduction of auxin response peaks at the tips of the primordia. The reduced cotyledon size of the mutant embryo recovered in germinating seedlings, indicating the presence of a factor that acted redundantly with EPFL2 to promote cotyledon growth in late embryogenesis. Our analysis indicates that the boundary domain between the cotyledon primordia acts as a signaling center that organizes auxin response peaks and promotes cotyledon growth.

category: plant biology


title: Bioremediation of industrial pollutants by insects expressing a fungal laccase

abstract: Inadequate management of household and industrial wastes pose major challenges to human and environmental health. Advances in synthetic biology may help address these challenges by engineering biological systems to perform new functions such as biomanufacturing of high-value compounds from low-value waste streams and bioremediation of industrial pollutants. The current emphasis on microbial systems for biomanufacturing, which often require highly pre-processed inputs and sophisticated infrastructure, is not feasible for many waste streams. Concerns about transgene biocontainment have limited the release of engineered microbes or plants for bioremediation. Engineering animals may provide opportunities for utilizing various waste streams that are not suitable for microbial biomanufacturing while effective transgene biocontainment options should enable in situ bioremediation. Here, we engineer the model insect Drosophila melanogaster to express a functional laccase from the fungus Trametes trogii. Laccase expressing flies reduced concentrations of the endocrine disruptor bisphenol A by more than 50% when present in their growth media. A lyophilized powder made from engineered adult flies retained substantial enzymatic activity, degrading more than 90% of bisphenol A and the textile dye indigo carmine in aqueous solutions. Our results demonstrate that transgenic animals may be used to bioremediate environmental contaminants in vivo and serve as novel production platforms for industrial enzymes. These results support further development of insects, and possibly other animals, as bioproduction platforms and their potential use in bioremediation.

category: synthetic biology


title: Microbiota links to neural dynamics supporting threat processing

abstract: There is growing recognition that the composition of the gut microbiota influences behaviour, including responses to threat. The cognitive-interoceptive appraisal of threat-related stimuli relies on dynamic neural computations between the anterior insular (AIC) and the dorsal anterior cingulate (dACC) cortices. If, to what extent, and how microbial consortia influence the activity of this cortical threat processing circuitry is unclear. We addressed this question by combining a threat processing task, neuroimaging, 16S rRNA profiling, and computational modelling in healthy participants. Results showed interactions between high-level ecological indices with threat-related AIC-dACC neural dynamics. At finer taxonomic resolutions, the abundance of Ruminococcus was differentially linked to connectivity between, and activity within the AIC and dACC during threat updating. Functional inference analysis provides a strong rationale to motivate future investigations of microbiota-derived metabolites in the observed relationship with threat-related brain processes.


title: Differential tracking of linguistic vs. mental state content in naturalistic stimuli by language and Theory of Mind (ToM) brain networks

abstract: Language and social cognition, especially the ability to reason about mental states, known as Theory of Mind (ToM), are deeply related in development and everyday use. However, whether these cognitive faculties rely on distinct, overlapping, or the same mechanisms remains debated. Some evidence suggests that, by adulthood, language and ToM draw on largely distinct--though plausibly interacting--cortical networks. However, the broad topography of these networks is similar, and some have emphasized the importance of social content / communicative intent in the linguistic signal for eliciting responses in the language areas. Here, we combine the power of individual-subjects functional localization with the naturalistic-cognition inter-subject correlation approach to illuminate the language-ToM relationship. Using fMRI, we recorded neural activity as participants (n=43) listened to stories and dialogs with mental state content (+linguistic, +ToM), viewed silent animations and live action films with mental state content but no language (-linguistic, +ToM), or listened to an expository text (+linguistic, -ToM). The ToM network robustly tracked stimuli rich in mental state information regardless of whether mental states were conveyed linguistically or non-linguistically, while tracking a +linguistic/-ToM stimulus only weakly. In contrast, the language network tracked linguistic stimuli more strongly than a) non-linguistic stimuli, and than b) the ToM network, and showed reliable tracking even for the linguistic condition devoid of mental state content. These findings suggest that in spite of their indisputably close links, language and ToM dissociate robustly in their neural substrates--and thus plausibly cognitive mechanisms--including during the processing of rich naturalistic materials.


title: OTX2 homeoprotein functions in adult choroid plexus

abstract: Choroid plexus secretes cerebrospinal fluid important for brain development and homeostasis. The OTX2 homeoprotein is critical for choroid plexus development and remains highly expressed in adult choroid plexus. Through RNA sequencing analyses of constitutive and conditional knockdown adult mouse models, we reveal putative roles for OTX2 in choroid plexus function, including cell signaling and adhesion, and show that it regulates the expression of factors secreted into cerebrospinal fluid, notably transthyretin. We show that Otx2 expression impacts choroid plexus immune and stress responses, and also affects splicing which leads to changes in mRNA isoforms of proteins implicated in oxidative stress response and DNA repair. Through mass spectrometry analysis of OTX2 protein partners in the choroid plexus, and in known non-cell autonomous target regions such as visual cortex and ventricular-subventricular zone, we identified putative targets involved in cell adhesion, chromatin structure and RNA processing. Thus, OTX2 retains important roles in choroid plexus function and brain homeostasis throughout life.


title: Microglial ablation does not affect opioid-induced hyperalgesia in rodents

abstract: Opioids are the frontline analgesics in pain management. However, chronic use of opioid analgesics causes paradoxical pain that contributes to the decrease of their efficacy in pain control and the escalation of dose in long-term management of pain. The underling pathogenic mechanism is not well understood. Microglia have been commonly thought to play a critical role in the expression of opioid-induced hyperalgesia (OIH) in animal models. We performed microglial ablation experiments using either a genetic (CD11b-diphtheria toxin receptor transgenic mouse) or pharmacological (colony-stimulating factor 1 receptor inhibitor PLX5622) approaches. Surprisingly, ablating microglia using these specific and effective approaches did not cause detectable impairment in the expression of hyperalgesia induced by morphine. We confirmed this conclusion with behavioral test of mechanical and thermal hyperalgesia, in male and female mice, and with different species (mouse and rat). These findings raise caution about the widely assumed contribution of microglia to the development of OIH.


title: A spike-ferritin nanoparticle vaccine induces robust innate immune activity and drives polyfunctional SARS-CoV-2-specific T cells

abstract:
Potent cellular responses to viral infections are pivotal for long -lived protection. Evidence is growing that these responses are critical in SARS -CoV-2 immunity. Assessment of a SARS -CoV-2 spike ferritin nanoparticle (SpFN) immunogen paired with two distinct adjuvants, Alhydrogel(R) (AH) or Army Liposome Formulation containing QS-21 (ALFQ) demonstrated unique vaccine evoked immune signatures. SpFN+ALFQ enhanced recruitment of highly activated classical and non -classical antigen presenting cells (APCs) to the vaccine-draining lymph nodes of mice. The multifaceted APC response of SpFN+ALFQ vaccinated mice was associated with an increased frequency of polyfunctional spike -specific T cells with a bias towards TH1 responses and more robust SARS-CoV-2 spike-specific recall response. In addition, SpFN+ALFQ induced Kb spike(539-546)-specific memory CD8+ T cells with effective cytolytic function and distribution to the lungs. This epitope is also present in SARS-CoV, thus suggesting that generation of cross-reactive T cells may provide protection against other coronavirus strains. Our study reveals that a nanoparticle vaccine, combined with a potent adjuvant, generates effective SARS-CoV-2 specific innate and adaptive immune T cell responses that are key components to inducing long-lived immunity.

One Sentence SummarySpFN vaccine generates multifactorial cellular immune responses.


title: Th17 T cells and immature dendritic cells are the preferential initial targets after rectal challenge with an SIV-based replication-defective dual-reporter vector

abstract:
Understanding the earliest events of HIV sexual transmission is critical to develop and optimize HIV prevention strategies. To gain insights into the earliest steps of HIV rectal transmission, including cellular targets, rhesus macaques were intra-rectally challenged with a single-round SIV-based dual reporter that expresses luciferase and iRFP670 upon productive transduction. The vector was pseudotyped with the HIV-1 envelope JRFL. Regions of tissue containing foci of luminescent, transduced cells were identified macroscopically using an in vivo imaging system, and individual transduced cells expressing fluorescent protein were identified and phenotyped microscopically. This system revealed that anal and rectal tissues are both susceptible to transduction 48 hours after the rectal challenge. Detailed phenotypic analysis revealed that on average, 62% of transduced cells are CCR6+ T cells--the vast majority of which express ROR{gamma}T, a Th17 lineage-specific transcription factor. The second most common target cells were immature dendritic cells at 20%. These two cell types were transduced at the rates that are four to five times higher than their relative abundances indicate. Our work demonstrates that Th17 T and immature dendritic cells are preferential initial targets of HIV/SIV rectal transmission.

IMPORTANCEMen and women who participate in unprotected receptive anal intercourse are at high risk for acquiring HIV. While in vitro data have developed a framework for understanding HIV cell tropism, the initial target cells in the rectal mucosa have not been identified. In this study, we identify these early host cells by using an innovative rhesus macaque rectal challenge model and methodology, which we previously developed. Thus, by shedding light on these early HIV/SIV transmission events, this study provides a specific cellular target for future prevention strategies.


title: Sewage Promotes Vibrio vulnificus Growth and Alters Gene Expression

abstract:
Vibrio vulnificus is a naturally-occurring, potentially lethal pathogen found in coastal waters, fish, and shellfish. Sewage spills in coastal waters occur when infrastructure fails due to severe storms or age, and may affect bacterial populations by altering nutrient levels. This study investigated effects of sewage on clonal and natural V. vulnificus populations in microcosms. Addition of 1% sewage to estuarine water caused the density of a pure culture of V. vulnificus CMCP6 and a natural V. vulnificus population to increase significantly, whether measured by qPCR or culture. Changes in the transcription of six virulence- and survival-associated genes in response to sewage were assessed using continuous culture. Exposure to sewage affected transcription of genes that may be associated with virulence. Specifically, sewage modulated the oxidative stress response by altering superoxide dismutase transcription, significantly increasing sodB transcription while repressing sodA. Sewage also repressed transcription of nptA, which encodes a sodium-phosphate cotransporter. Sewage had no effect on sodC transcription or the putative virulence-associated genes hupA or wza. The effects of environmentally relevant levels of sewage on V. vulnificus populations and gene transcription suggest that sewage spills that impact warm coastal waters could lead to an increased risk of V. vulnificus infections.

ImportanceVibrio vulnificus infections have profound impacts such as limb amputation and death for individuals with predisposing conditions. The warming climate is contributing to rising V. vulnificus prevalence in waters that were previously too cold to support high levels of the pathogen. Climate change is also expected to increase precipitation in many regions, which puts more pressure on wastewater infrastructure and will result in more frequent sewage spills. The finding that 1% wastewater in estuarine water leads to tenfold to 1000-fold greater V. vulnificus concentrations suggests that human exposure to oysters and estuarine water could have greater health impacts in the future. Further, wastewater had a significant effect on gene transcription and has the potential to affect virulence during the initial environment-to-host transition.


title: Clonal inactivation of telomerase promotes accelerated stem cell differentiation

abstract: Telomerase is intimately associated with stem cells and upregulated in cancer, where it serves essential roles through its catalytic action in elongating telomeres, nucleoprotein caps that protect chromosome ends1. Overexpression of the telomerase reverse transcriptase (TERT) enhances cell proliferation through telomere-independent means, yet definitive evidence for such a direct role in stem cell function has yet to be revealed through loss-of-function studies. Here, we show that conditional deletion of TERT in spermatogonial stem cells (SSCs) markedly impairs competitive clone formation. Using lineage-tracing from the Tert locus, we find that TERT-expressing SSCs yield long-lived clones, but that selective TERT-inactivation in SSCs causes accelerated stem cell differentiation thereby disrupting clone formation. This requirement for TERT in clone formation is bypassed by expression of a catalytically inactive TERT transgene and occurs independently of the canonical telomerase complex. TERT inactivation induces a genome-wide reduction in open chromatin evident in purified SSCs, but not in committed progenitor cells. Loss of TERT causes reduced activity of the MYC oncogene and transgenic expression of MYC in TERT-deleted SSCs efficiently rescues clone formation. These data reveal a required catalytic activity-independent role for TERT in preventing stem cell differentiation, forge a genetic link between TERT and MYC and suggest new means by which TERT may promote tumorigenesis.


title: Direct observation of adaptive tracking on ecological timescales in Drosophila

abstract:
Direct observation of evolution in response to natural environmental change can resolve fundamental questions about adaptation including its pace, temporal dynamics, and underlying phenotypic and genomic architecture. We tracked evolution of fitness-associated phenotypes and allele frequencies genome-wide in ten replicate field populations of Drosophila melanogaster over ten generations from summer to late fall. Adaptation was evident over each sampling interval (1-4 generations) with exceptionally rapid phenotypic adaptation and large allele frequency shifts at many independent loci. The direction and basis of the adaptive response shifted repeatedly over time, consistent with the action of strong and rapidly fluctuating selection. Overall, we find clear phenotypic and genomic evidence of adaptive tracking occurring contemporaneously with environmental change, demonstrating the temporally dynamic nature of adaptation.

One sentence summaryRapid environmental change drives continuous phenotypic and polygenic adaptation, demonstrating the temporal dynamism of adaptation.


title: Substrate ligand density modulates gap junction intercellular communication during mesenchymal cell condensation

abstract:
To unveil the influence of cell-matrix adhesions in the establishment of gap junction intercellular communication (GJIC) during cell condensation in chondrogenesis.

Materials & MethodsPreviously developed nanopatterns of the cell adhesive ligand arginine-glycine-aspartic acid (RGD) were used as cell culture substrates to control cell adhesion at the nanoscale. We conducted in vitro chondrogenesis of mesenchymal stem cells on the nanopatterns. We evaluated cohesion and GJIC in cell condensates.

ResultsMechanical stability and GJIC are enhanced by a nanopattern configuration in which 90% of the surface area presents adhesion sites separated less than 70 nm, thus providing an onset for cell signaling.

ConclusionCell-matrix adhesions regulate GJIC of mesenchymal cell condensates during in vitro chondrogenesis from a threshold configuration at the nanoscale.


title: Nanoscale ligand density modulates gap junction intercellular communication of cell condensates during chondrogenesis


title: DeepRegFinder: Deep Learning-Based Regulatory Elements Finder

abstract:
MotivationEnhancers and promoters are important classes of DNA regulatory elements that control gene expression. Identifying them at the genomic scale is a critical and challenging task in bioinformatics. The most successful method so far is to train machine learning models on known enhancer and promoter sites and predict them at other genomic regions using ChIP-seq and related data.

ResultsWe have developed a highly customizable program called DeepRegFinder which automates data processing, model training and genome-wide prediction of enhancers and promoters using convolutional and recurrent neural networks. Our program further classifies the enhancers and promoters into active and poised states to facilitate downstream analysis. Based on mean average precision scores of different classes across multiple cell types, our method significantly outperforms the existing algorithms.

Availabilityhttps://github.com/shenlab-sinai/DeepRegFinder


title: Development of a transboundary model of livestock disease in Europe

abstract:
Epidemiological models of notifiable livestock disease are typically framed at a national level and targeted for specific diseases. There are inherent difficulties in extending models beyond national borders as details of the livestock population, production systems and marketing systems of neighbouring countries are not always readily available. It can also be a challenge to capture heterogeneities in production systems, control policies, and response resourcing across multiple countries, in a single transboundary model.

In this paper we describe EuFMDiS, a continental-scale modelling framework for transboundary animal disease, specifically designed to support emergency animal disease planning in Europe. EuFMDiS simulates the spread of livestock disease within and between countries and allows control policies to be enacted and resourced on per-country basis. It provides a sophisticated decision support tool that can be used to look at the risk of disease introduction, establishment and spread; control approaches in terms of effectiveness and costs; resource management; and post-outbreak management issues.


title: Recombination patterns in coronaviruses

abstract: As shown during the SARS-CoV-2 pandemic, phylogenetic and phylodynamic methods are essential tools to study the spread and evolution of pathogens. One of the central assumptions of these methods is that the shared history of pathogens isolated from different hosts can be described by a branching phylogenetic tree. Recombination breaks this assumption. This makes it problematic to apply phylogenetic methods to study recombining pathogens, including, for example, coronaviruses. Here, we introduce a Markov chain Monte Carlo approach that allows inference of recombination networks from genetic sequence data under a template switching model of recombination. Using this method, we first show that recombination is extremely common in the evolutionary history of SARS-like coronaviruses. We then show how recombination rates across the genome of the human seasonal coronaviruses 229E, OC43 and NL63 vary with rates of adaptation. This suggests that recombination could be beneficial to fitness of human seasonal coronaviruses. Additionally, this work sets the stage for Bayesian phylogenetic tracking of the spread and evolution of SARS-CoV-2 in the future, even as recombinant viruses become prevalent.


title: Regulation of Lysosome-Associated Membrane Protein 3 (LAMP3) in Lung Epithelial Cells by Coronaviruses (SARS-CoV-1/2) and Type I Interferon Signaling

abstract: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is a major risk factor for mortality and morbidity in critical care hospitals around the world. Lung epithelial type II cells play a major role in several physiological processes, including recognition and clearance of respiratory viruses as well as repair of lung injury in response to environmental toxicants. Gene expression profiling of lung epithelial type II-specific genes led to the identification of lysosomal-associated membrane protein 3 (LAMP3). Intracellular locations of LAMP3 include plasma membrane, endosomes, and lysosomes. These intracellular organelles are involved in vesicular transport and facilitate viral entry and release of the viral RNA into the host cell cytoplasm. In this study, regulation of LAMP3 expression in human lung epithelial cells by several respiratory viruses and type I interferon signaling was investigated. Coronaviruses including SARS-CoV-1 and SARS-CoV-2 significantly induced LAMP3 expression in lung epithelial cells within 24 hours after infection that required the presence of ACE2 viral entry receptor. Time-course experiments revealed that the induced expression of LAMP3 by SARS-CoV-2 was correlated with the induced expression of interferon-beta1 (IFNB1) and signal transducers and activator of transcription 1 (STAT1) mRNA levels. LAMP3 was also induced by direct IFN-beta treatment or by infection with influenza virus lacking the non-structural protein1(NS1) in NHBE bronchial epithelial cells. LAMP3 expression was induced in human lung epithelial cells by several respiratory viruses, including respiratory syncytial virus (RSV) and the human parainfluenza virus 3 (HPIV3). Location in lysosomes and endosomes as well as induction by respiratory viruses and type I Interferon suggests that LAMP3 may have an important role in inter-organellar regulation of innate immunity and a potential target for therapeutic modulation in health and disease. Furthermore, bioinformatics revealed that a subset of lung type II cell genes were differentially regulated in the lungs of COVID-19 patients.


title: Microbiota-dependent inflammation promotes metabolic disorder via NF-kB-mediated inhibition of SREBP in Drosophila adipocytes

abstract:
Bacteria that colonize eukaryotic gut have profound influences on the physiology of their host. In Drosophila, many of these effects are mediated by the adipocytes that combine immune and metabolic functions. We show here that gut colonization by specific bacteria species stimulate lipogenesis in surrounding enterocytes but also in remote fat body cells and ovaries. This bacteria-dependent lipid production is mediated by SREBP and requires a functional insulin signaling. However, it is antagonized by microbiota-born peptidoglycan which by activating NF-{kappa}B signaling, cell-autonomously represses SREPP activation in adipocytes but not in enterocytes. We finally show that by reducing microbiota-derived PGN, the gut-produced PGRP-LB amidase balances host immune and metabolic responses of the fat body to gut-associated bacteria. In the absence of such modulation, uncontrolled immune pathway activation prevents lipid production by the fat body resulting in infection-dependent host death.

BulletsO_LIGut microbiota activates lipogenesis locally in enterocytes and remotely in adipocytes
C_LIO_LIBacteria-dependent activation of SREBP in adipocytes is mediated by insulin signaling
C_LIO_LIActivation of lipid synthesis by microbiota is antagonized by NF-{kappa}B signaling triggered by gut-born peptidoglycan
C_LIO_LIBy reducing peptidoglycan circulating levels, PGRP-LB maintains a balance between immune and metabolic response to microbiota
C_LI


title: LPMO-oxidized cellulose oligosaccharides evoke immunity in Arabidopsis conferring resistance towards necrotrophic fungus B. cinerea

abstract: Lytic Polysaccharide Monooxygenases (LPMOs) are powerful redox enzymes able to oxidatively cleave cellulose polymers. Widely conserved across biological kingdoms, LPMOs of the AA9 family are deployed by phytopathogens during necrotrophic attack of plant cell wall. In response, plants have evolved sophisticated mechanisms to sense cell wall damage and thus self-triggering Damage Triggered Immunity (DTI) responses. Here, we show that Arabidopsis plants exposed to LPMO products responds by activating the innate immunity ultimately leading to increased resistance to pathogenic fungus Botrytis cinerea. We demonstrated with microarray hybridization that plants undergo a deep transcriptional reprogramming upon elicitation with AA9 derived cellulose- or cello-oligosaccharides (AA9_COS). To decipher the specific effects of native and oxidized LPMO-generated cello-oligosaccharides, a pairwise comparison with cellobiose, the smallest non-oxidized unit constituting cellulose, is presented. Moreover, we identified two leucine-rich repeat receptor-like kinases, namely STRESS INDUCED FACTOR 2 and 4, playing a crucial role in signaling the AA9_COS-dependent responses such as camalexin production. We observed an increased production of ethylene, jasmonic and salicylic acid hormones, and finally deposition of callose in cell wall. Collectively, our data reveal that LPMOs might play a crucial role in plant-pathogen interactions.


title: Nucleic acid delivery of immune-focused SARS-CoV-2 nanoparticles drive rapid and potent immunogenicity capable of single-dose protection

abstract: Antibodies from SARS-CoV-2 vaccines may target epitopes which reduce durability or increase the potential for escape from vaccine-induced immunity. Using a novel synthetic vaccinology pipeline, we developed rationally immune focused SARS-CoV-2 Spike-based vaccines. N-linked glycans can be employed to alter antibody responses to infection and vaccines. Utilizing computational modeling and comprehensive in vitro screening, we incorporated glycans into the Spike Receptor-Binding Domain (RBD) and assessed antigenic profiles. We developed glycan coated RBD immunogens and engineered seven multivalent configurations. Advanced DNA delivery of engineered nanoparticle vaccines rapidly elicited potent neutralizing antibodies in guinea pigs, hamsters and multiple mouse models, including human ACE2 and human B cell repertoire transgenics. RBD nanoparticles encoding wild-type and the P.1 SARS-CoV-2 variant induced high levels of cross-neutralizing antibodies. Single, low dose immunization protected against a lethal SARS-CoV-2 challenge. Single-dose coronavirus vaccines via DNA-launched nanoparticles provide a platform for rapid clinical translation of novel, potent coronavirus vaccines.


title: Cross-tissue immune cell analysis reveals tissue-specific adaptations and clonal architecture across the human body

abstract:
Despite their crucial role in health and disease, our knowledge of immune cells within human tissues remains limited. Here, we surveyed the immune compartment of 15 tissues of six deceased adult donors by single-cell RNA sequencing and paired VDJ sequencing. To systematically resolve immune cell heterogeneity across tissues, we developed CellTypist, a machine learning tool for rapid and precise cell type annotation. Using this approach, combined with detailed curation, we determined the tissue distribution of 45 finely phenotyped immune cell types and states, revealing hitherto unappreciated tissue-specific features and clonal architecture of T and B cells. In summary, our multi-tissue approach lays the foundation for identifying highly resolved immune cell types by leveraging a common reference dataset, tissue-integrated expression analysis and antigen receptor sequencing.

One Sentence SummaryWe provide an immune cell atlas, including antigen receptor repertoire profiling, across lymphoid and non-lymphoid human tissues.


title: Physiological relevance, localization and substrate specificity of the alternative (type II) mitochondrial NADH dehydrogenases of Ogataea parapolymorpha

abstract:
Mitochondria from Ogataea parapolymorpha harbor a branched electron-transport chain containing a proton-pumping Complex I NADH dehydrogenase and three alternative (type II) NADH dehydrogenases (NDH2s). To investigate the physiological role, localization and substrate specificity of these enzymes, growth of various NADH dehydrogenase mutants was quantitatively characterized in shake-flask and chemostat cultures, followed by oxygen-uptake experiments with isolated mitochondria. Furthermore, NAD(P)H:quinone oxidoreduction of the three NDH2s were individually assessed. Our findings show that the O. parapolymorpha respiratory chain contains an internal NADH-accepting NDH2 (Ndh2-1/OpNdi1), at least one external NAD(P)H-accepting enzyme and likely additional mechanisms for respiration-linked oxidation of cytosolic NADH. Metabolic regulation appears to prevent competition between OpNdi1 and Complex I for mitochondrial NADH. With the exception of OpNdi1, the respiratory chain of O. parapolymorpha exhibits metabolic redundancy and tolerates deletion of multiple NADH-dehydrogenase genes without compromising fully respiratory metabolism.

ImportanceTo achieve high productivity and yields in microbial bioprocesses, efficient use of the energy substrate is essential. Organisms with branched respiratory chains can respire via the energy-efficient proton-pumping Complex I, or make use of alternative NADH dehydrogenases (NDH2s). The yeast Ogataea parapolymorpha contains three uncharacterized, putative NDH2s which were investigated in this work. We show that O. parapolymorpha contains at least one  internal NDH2, which provides an alternative to Complex I for mitochondrial NADH oxidation, albeit at a lower efficiency. The use of this NDH2 appeared to be limited to carbon excess conditions and the O. parapolymorpha respiratory chain tolerated multiple deletions without compromising respiratory metabolism, highlighting opportunities for metabolic (redox) engineering. By providing a more comprehensive understanding of the physiological role of NDH2s, including insights into their metabolic capacity, orientation and substrate specificity this study also extends our fundamental understanding of respiration in organisms with branched respiratory chains.


title: Refined quantification of infection bottlenecks and pathogen dissemination with STAMPR

abstract:
Pathogen population dynamics during infection are critical determinants of infection susceptibility and define patterns of dissemination. However, deciphering these dynamics, particularly founding population sizes in host organs and patterns of dissemination between organs, is difficult because measuring bacterial burden alone is insufficient to observe these patterns. Introduction of allelic diversity into otherwise identical bacteria using DNA barcodes enables sequencing-based measurements of these parameters, in a method known as STAMP (Sequence Tag-Based Analysis of Microbial Population dynamics). However, bacteria often undergo unequal expansion within host organs, resulting in marked differences in the frequencies of barcodes in input and output libraries. Here, we show that these differences confound STAMP-based analyses of founding population sizes and dissemination patterns. We present STAMPR, a successor to STAMP that accounts for such population expansions. Using data from systemic infection of barcoded Extraintestinal Pathogenic E. coli we show that this new framework along with the metrics it yields enhances the fidelity of measurements of bottlenecks and dissemination patterns. STAMPR was also validated on an independent, barcoded Pseudomonas aeruginosa dataset, uncovering new patterns of dissemination within the data. This framework (available at https://github.com/hullahalli/stampr_rtisan), when coupled with barcoded datasets, enables a more complete assessment of within-host bacterial population dynamics.

ImportanceBarcoded bacteria are often employed to monitor pathogen population dynamics during infection. The accuracy of these measurements is diminished by unequal bacterial expansion rates. Here, we develop computational tools to circumvent this limitation and establish additional metrics that collectively enhance the fidelity of measuring within-host pathogen founding population sizes and dissemination patterns. These new tools will benefit future studies of the dynamics of pathogens and symbionts within their respective hosts and may have additional barcode-based applications beyond host-microbe interactions.


title: Five years of GenoTyphi: updates to the global Salmonella Typhi genotyping framework

abstract: In 2016 a whole genome sequence (WGS) based genotyping framework (GenoTyphi) was developed providing a phylogenetically informative nomenclature for lineages of Salmonella Typhi, the aetiological agent of typhoid fever. Subsequent surveillance studies have revealed additional epidemiologically important subpopulations, necessitating the definition of new genotypes and extension of associated software to facilitate the detection of antimicrobial resistance (AMR) mutations. Analysis of 4,632 WGS provide an updated overview of the global S. Typhi population structure and genotyping framework, revealing the widespread nature of H58 (4.3.1) genotypes and the diverse range of genotypes carrying AMR mutations.


title: Micrarchaeota are covered by a proteinaceous S-Layer

abstract: In previous publications, it was hypothesized that Micrarchaeota cells are covered by two individual membrane systems. This study proofs that at least the recently cultivated "Candidatus Micrarchaeum harzensis A_DKE" possesses an S-layer covering its cytoplasmic membrane. The potential S-layer protein was found to be among the proteins with the highest abundance in A_DKE and in silico characterization of its primary structure indicated homologies to other known S-layer proteins. Homologs of this protein were found in other Micrarchaeota genomes, which raises the question, whether the ability to form an S-layer is a common trait within this phylum. The S-layer protein seems to be glycosylated and the Micrarchaeum expresses genes for N-glycosylation under cultivation conditions, despite not being able to synthesize carbohydrates. Electron micrographs of freeze-etched samples of a previously described co-culture, containing Micrarchaeum A_DKE and a Thermoplasmatales member as its host organism, verified the hypothesis of an S-layer on the surface of A_DKE. Both organisms are clearly distinguishable by cell size, shape and surface structure.


title: Co-Infection of cattle in Virginia with Theileria orientalis ikeda genotype and Anaplasma marginale

abstract: Theileria orientalis ikeda is a newly identified agent of bovine infectious anemia in the United States. Although it is transmitted by separate tick hosts than Anaplasma marginale - a bacterial etiology of bovine infectious anemia -the geographic distributions of these two infectious organisms overlap, with co-infection reported in some cattle. Only anaplasmosis has approved effective treatment in the United States. To provide rapid diagnostic information for producers with anemic animals, we developed a duplex qPCR for A. marginale and T. orientalis. With a cut-off of 38 cycles, the duplex assay has a sensitivity of 96.97% and a specificity of 100% for A. marginale; with a cut-off of 45 cycles, the duplex assay has a sensitivity and a specificity of 100% for T. orientalis. In addition to providing a tool for improved clinical decision-making for veterinarians and producers, this qPCR facilitates the study of co-infection rate of cattle in Virginia. Of 1,359 blood samples analyzed, 174 were positive for the presence of T. orientalis, 125 were positive for the presence of A. marginale, and 12 samples were positive for both T. orientalis and A. marginale. This indicated that co-infection of both of these etiologies of bovine infectious anemia does occur within the state of Virginia. It is likely that this pattern of infection will be seen in regions where T. orientalis and A. marginale are endemic, despite the difference in tick vectors.


title: The evolution of regulatory elements in the emerging promoter variant strains of HIV-1

abstract:
In a multicentric, observational, investigator-blinded, and longitudinal clinical study of 764 ART-naive subjects, we identified nine different promoter-variant strains of HIV-1 subtype C (HIV-1C) emerging in the Indian population, with some of these variants being reported for the first time. Unlike several previous studies, our work here focuses on the evolving viral regulatory elements, not coding sequences. The emerging viral strains contain additional copies of the existing transcription factor binding sites (TFBS), including TCF-1/LEF-1, RBEIII, AP-1, and NF-{kappa}B, created by sequence duplication. The additional TFBS are genetically diverse and may blur the distinction between the modulatory region of the promoter and the viral enhancer. In a follow-up analysis, we found trends, but not significant associations between any specific variant promoter and prognostic markers, probably because the emerging viral strains might not have established mono infections yet. Illumina sequencing of four clinical samples containing a co-infection indicated the domination of one strain over the other and establishing a stable ratio with the second strain at the follow-up time-points. Since a single promoter regulates viral gene expression and constitutes the master regulatory circuit with Tat, the acquisition of additional and variant copies of the TFBS may significantly impact viral latency and latent reservoir characteristics. Further studies are urgently warranted to understand how the diverse TFBS profiles of the viral promoter may modulate the characteristics of the latent reservoir, especially following the initiation of antiretroviral therapy.

Significance StatementA unique conglomeration of TFBS enables the HIV-1 promoter to accomplish two diametrically opposite functions - transcriptional activation and transcriptional silencing. The various phases of viral latency -establishment, maintenance, and reversal -collectively determine the replication fitness of individual viral strains. A profound variation in the TFBS composition of the viral promoter may significantly alter the viral latency properties and the latent reservoir characteristics. Although the duplication of certain TFBS remains a quality unique to HIV-1C, the high-level genetic recombination of HIV-1 may promote the transfer of such molecular properties to the other HIV-1 subtypes. The emergence of several promoter-variant viral strains may make the task of a  functional cure more challenging in HIV-1C.


title: Suboptimal proteome allocation during changing environments constrains bacterial response and growth recovery

abstract: To sustain growth in fluctuating environments microbial organisms must respond appropriately. The response generally requires the synthesis of novel proteins, but this synthesis can be impeded due to the depletion of biosynthetic precursors when growth conditions vary. Microbes must thus devise effective response strategies to manage depleting precursors. To better understand these strategies, we here investigate the active response of Escherichia coli to changes in nutrient conditions, connecting transient gene-expression behavior to growth phenotypes. By synthetically modifying the gene expression during changing growth conditions, we show how the competition by genes for the limited protein synthesis capacity constrains the cellular response. Despite this constraint, cells substantially express genes that are not required, severely slowing down the response. These findings highlight that cells do not optimize growth and recovery in every encountered environment but rather exhibit hardwired response strategies that may have evolved to promote growth and fitness in their native environment and include the regulation of multiple genes. The constraint and the suboptimality of the cellular response uncovered in this study provides a conceptual framework relevant for many research applications, from the prediction of evolution and adaptation to the improvement of gene circuits in biotechnology.


title: Prophylactic protection against respiratory viruses conferred by a prototype live attenuated influenza virus vaccine.

abstract: The influenza A non-structural protein 1 (NS1) is known for its ability to hinder the synthesis of type I interferon (IFN) during viral infection. Influenza viruses lacking NS1 ({Delta}NS1) are under clinical development as live attenuated human influenza virus vaccines and induce potent influenza virus-specific humoral and cellular adaptive immune responses. Attenuation of {Delta}NS1 influenza viruses is due to their high IFN inducing properties, that limit their replication in vivo. This study demonstrates that pre-treatment with a {Delta}NS1 virus results in an immediate antiviral state which prevents subsequent replication of homologous and heterologous viruses, preventing disease from virus respiratory pathogens, including SARS-CoV-2. Our studies suggest that {Delta}NS1 influenza viruses could be used for the prophylaxis of influenza, SARS-CoV-2 and other human respiratory viral infections, and that an influenza virus vaccine based on {Delta}NS1 live attenuated viruses would confer broad protection against influenza virus infection from the moment of administration, first by non-specific innate immune induction, followed by specific adaptive immunity.


title: DAXX safeguards pericentromeric heterochromatin formation in embryonic stem cells

abstract: DNA methylation is essential for heterochromatin formation and repression of DNA repeat transcription, both of which are essential for genome integrity. Loss of DNA methylation is associated with disease, including cancer, but is also required for development. Alternative pathways to maintain heterochromatin are thus needed to limit DNA damage accumulation. Here, we find that DAXX, an H3.3 chaperone, protects pericentromeric heterochromatin and is essential for embryonic stem cells (ESCs) maintenance in the ground-state of pluripotency. Upon DNA demethylation-mediated damage, DAXX relocalizes to pericentromeric regions, and recruits PML and SETDB1, thereby promoting heterochromatin formation. In the absence of DAXX, the 3D-architecture and physical properties of pericentric heterochromatin are disrupted, resulting in derepression of major satellite DNA. Using epigenome editing tools, we demonstrate that H3.3, and specifically H3.3K9 modification, directly contribute to maintaining pericentromeric chromatin conformation. Altogether, our data reveal that DAXX and H3.3 unite DNA damage response and heterochromatin maintenance in ESCs.

category: molecular biology


title: Benchmarking a highly selective USP30 inhibitor for enhancement of mitophagy and pexophagy.

abstract: The deubiquitylase USP30 is an actionable target considered for treatment of conditions associated with defects in the PINK1/Parkin pathway leading to mitophagy. These include Parkinsons disease and pulmonary fibrosis. We provide a detailed cell biological characterisation of a benzenesulphonamide molecule, compound 39, that has previously been reported to inhibit USP30 in an in vitro enzymatic assay. The current compound offers increased selectivity over previously described inhibitors. It enhances mitophagy and generates a signature response for USP30 inhibition following mitochondrial depolarisation. This includes enhancement of TOM20 and SYNJ2BP ubiquitylation and phosphoubiquitin accumulation, alongside increased mitophagy. In dopaminergic neurons, generated from Parkinsons disease patients carrying loss of function Parkin mutations, compound 39 could significantly restore mitophagy to a level approaching control values. USP30 is located on both mitochondria and peroxisomes and has also been linked to the PINK1 independent pexophagy pathway. Using a fluorescence reporter of pexophagy expressed in U20S cells, we observe increased pexophagy upon application of compound 39 that recapitulates the previously described effect for USP30 depletion. This provides the first pharmacological intervention with a synthetic molecule to enhance peroxisome turnover.

category: cell biology


title: Cdc48 influence on separase levels is independent of mitosis and suggests translational sensitivity of separase

abstract: Cdc48 (p97/VCP) is a AAA-ATPase that can extract ubiquitinated proteins from their binding partners and channel substrates to the proteasome. A fission yeast cdc48 mutant (cdc48-353) shows low levels of the cohesin protease, separase, and pronounced chromosome segregation defects in mitosis. Separase initiates chromosome segregation when its binding partner securin is ubiquitinated and degraded. The low separase levels in the cdc48-353 mutant have been attributed to a failure to extract ubiquitinated securin from separase resulting in co-degradation of separase along with securin. If true, this establishes Cdc48 as a key regulator of mitosis. In contrast, we show here that low separase levels in the cdc48-353 mutant are independent of mitosis. Moreover, we find no evidence of enhanced separase degradation in the mutant. Instead, we suggest that the cdc48-353 mutant uncovers specific requirements for separase translation. Our results highlight a need to better understand how this key mitotic enzyme is synthesized.


title: Met-signaling controls Dendritic Cell migration by regulating podosome formation and function

abstract: Signaling by the HGF receptor/Met in skin-resident Langerhans cells (LC) and dermal dendritic cells (dDC) is essential for their emigration toward draining lymph nodes upon inflammation-induced activation. Here we addressed the role of Met-signaling in distinct steps of LC/dDC emigration from skin by employing a conditional Met-deficient mouse model (Metflox/flox). We found that Met deficiency severely impaired podosome formation in DC and concomitantly decreased proteolytic degradation of gelatin. Accordingly, Met-deficient LC failed to efficiently cross the extracellular matrix (ECM) rich basement membrane between epidermis and dermis. We further observed that Met-signaling by HGF reduced adhesion of bone marrow-derived LC to various ECM factors and enhanced the motility of Met-signaling competent DC in a 3D collagen matrix, which was not the case for Met-deficient LC/DC. We found no impact of Met-signaling on the integrin-independent amoeboid migration of DC in response to the CCR7 chemokine CCL19. Collectively, our data show that the Met-signaling pathway regulates the migratory properties of DC in HGF-dependent and HGF-independent manners.


title: Box C/D Small Nucleolar Ribonucleoproteins Regulate Mitochondrial Surveillance and Innate Immunity

abstract:
Monitoring of mitochondrial functions is crucial for organismal survival. This task is performed by mitochondrial surveillance or quality control pathways, which are activated by signals originating from mitochondria and relayed to the nucleus (retrograde response) to start the transcription of protective genes. In Caenorhabditis elegans, several systems exist, including the UPRmt, MAPKmt, and the ESRE pathway. These pathways are highly conserved and their loss results in compromised survival following mitochondrial stress.

In this study, we found a novel interaction between the box C/D snoRNA core proteins (snoRNPs) and mitochondrial surveillance and innate immunity pathways. We showed that C/D snoRNPs are required for the full expressions of UPRmt and ESRE upon stress. Meanwhile, we found that the loss of C/D snoRNPs increased immune responses. Understanding the "molecular switch" mechanisms of interplay between these pathways may be important for understanding of multifactorial processes, including response to infection or aging.