title: PalaCell2D: A framework for detailed tissue morphogenesis

abstract:
In silico, cell based approaches for modeling biological morphogenesis are used to test and validate our understanding of the biological and mechanical process that are at work during the growth and the organization of multi-cell tissues. As compared to in vivo experiments, computer based frameworks dedicated to tissue modeling allow us to easily test different hypotheses, and to quantify the impact of various biophysically relevant parameters.

Here, we propose a formalism based on a detailed, yet simple, description of cells that accounts for intra-, inter- and extra-cellular mechanisms. More precisely, the cell growth and division is described through the space and time evolution of the membrane vertices. These vertices follow a Newtonian dynamics, meaning that their evolution is con-trolled by different types of forces: a membrane force (spring and bending), an adherence force (inter-cellular spring), external and internal pressure forces. Different evolution laws can be applied on the internal pressure, depending on the intra-cellular mechanism of interest. In addition to the cells dynamics, our formalism further relies on a lattice Boltzmann method, using the Palabos library, to simulate the diffusion of chemical signals. The latter aims at driving the growth and migration of a tissue by simply changing the state of the cells.

All of this leads to an accurate description of the growth and division of cells, with realistic cell shapes and where membranes can have different properties. While this work is mainly of methodological nature, we also propose to validate our framework through simple, yet biologically relevant benchmark tests at both single-cell and full tissue scales. This includes free and chemically controlled cell tissue growth in an unbounded domain. The ability of our framework to simulate cell migration, cell compression and morphogenesis under external constraints is also investigated in a qualitative manner.


title: AhNGE : A Database for Arachis hypogaea Nodule Developmental Gene Expression

abstract: Symbiotic nitrogen fixation (SNF) inside root-nodules is a primary and sustainable source of soil nitrogen. Understanding nodule development and metabolism in crop legumes may lead to more effective SNF in agriculture. Peanut (Arachis hypogaea) is an economically important allotetraploid legume with non-canonical nodule developmental features. Recent genome sequencing of peanut has opened the possibility of making peanut a model for studying atypical nodule development. To help the community of nodule biologists, we have developed a database called AhNGE (Arachis hypogaea Nodule Developmental Gene Expression: http://nipgr.ac.in/AhNGE/index.php). AhNGE contains RNAseq data from six data points of nodule development in A. hypogagea cv. Tifrunner. This data represents a dynamic view of gene expression during peanut nodule development. Research in model legumes has generated a huge knowledgebase in the last twenty years. To streamline comparative genomics among legumes, we performed ortholog analysis among four legumes (Cicer, Glycine, Lotus, and Medicago) and one non-legume (Arabidopsis). This will facilitate the integration of existing knowledge in nodule development with the Arachis transcriptome. The available data can be retrieved using a single or batch query or searching using gene ID, from above mentioned five species. The output displays the gene expression pattern in graphical as well as tabular form, along with further options to download the sequence data. The database is linked with PeanutBase, the main genomic resource of peanut. Additionally, the expression level of different splicing variants can be retrieved from the database. In summary, AhNGE serves as an important resource for the scientific community working on nodule development.

category: bioinformatics


title: Thermodynamic modeling reveals widespread multivalent binding by RNA-binding proteins

abstract:
MotivationUnderstanding how proteins recognize their RNA targets is essential to elucidate regulatory processes in the cell. Many RNA-binding proteins (RBPs) form complexes or have multiple domains that allow them to bind to RNA in a multivalent, cooperative manner. They can thereby achieve higher specificity and affinity than proteins with a single RNA-binding domain. However, current approaches to de-novo discovery of RNA binding motifs do not take multivalent binding into account.

ResultsWe present Bipartite Motif Finder (BMF), which is based on a thermodynamic model of RBPs with two cooperatively binding RNA-binding domains. We show that bivalent binding is a common strategy among RBPs, yielding higher affinity and sequence specificity. We furthermore illustrate that the spatial geometry between the binding sites can be learned from bound RNA sequences. These discovered bipartite motifs are consistent with previously known motifs and binding behaviors. Our results demonstrate the importance of multivalent binding for RNA-binding proteins and highlight the value of bipartite motif models in representing the multivalency of protein-RNA interactions.

AvailabilityBMF source code is available at https://github.com/soedinglab/bipartite_motif_finder under a GPL license. The BMF web server is accessible at https://bmf.soedinglab.org.


title: Tools enabling flexible approaches to high-resolution subtomogram averaging

abstract: Cryo-electron tomography and subtomogram averaging are increasingly used for macromolecular structure determination in situ. Here we introduce a set of computational tools and resources designed to enable flexible approaches to subtomogram averaging. In particular, our tools simplify metadata handling, increase automation, and interface the Dynamo software package with the Warp-Relion-M pipeline. We provide a framework for ab initio and geometrical approaches to subtomogram averaging combining tools from these packages. We illustrate the power of working within the framework enabled by our developments by applying it to EMPIAR-10164, a publicly available dataset containing immature HIV-1 virus-like particles, and a challenging in situ dataset containing chemosensory arrays in bacterial minicells. Additionally, we establish an open and collaborative online platform for sharing knowledge and tools related to cryo-electron tomography data processing. To this platform, we contribute a comprehensive guide to obtaining state-of-the-art results from EMPIAR-10164.

category: biophysics


title: Membrane barrels are taller, fatter, inside-out soluble barrels

abstract: Up-and-down {beta}-barrel topology exists in both the membrane and soluble environment. However, {beta}-barrels are virtually the only topology that exist in the outer membrane. By comparing features of these structurally similar proteins, we can determine what features are particular to the environment rather than the fold. Here we compare structures of membrane {beta}-barrels to soluble {beta}-barrels and evaluate their relative size, shape, amino acid composition, hydrophobicity, and periodicity. We find that membrane {beta}-barrels are generally larger than soluble {beta}-barrels in with more strands per barrel and more amino acids per strand, making them wider and taller. We also find that membrane {beta}-barrels are inside-out soluble {beta}-barrels. The inward region of membrane {beta}-barrels have similar hydrophobicity to the outward region of soluble {beta}-barrels, and the outward region of membrane {beta}-barrels has similar hydrophobicity to the inward region of the soluble {beta}-barrels. Moreover, even though both types of {beta}-barrel have been assumed to have strands with amino acids that alternate in direction and hydrophobicity, we find that the membrane {beta}-barrels have more regular alternation than soluble {beta}-barrels. These features give insight into how membrane barrels maintain their fold and function in the membrane.


title: Water as a reactant in the first step of triosephosphate isomerase catalysis

abstract: The enzyme triosephosphate isomerase (TIM) performs a crucial role in the extraction of energy from glucose, doing so by converting dihydroxyacetone phosphate (DHAP) into glyceraldehyde phosphate, thereby doubling the yield of ATP molecules during glycolysis. The initial step of the mechanism is the seemingly unlikely abstraction of the pro-R methylene hydrogen from C1 by a conserved glutamate (Glu165), an assignment that has been both universally accepted yet a much-studied phenomenon for decades. In this work we introduce an alternative mechanism in which water as a strong general base abstracts the carbon proton acting effectively as hydroxide. We posit that strong electric fields associated with the substrate phosphate promote facile autoionization of water trapped near the phosphate dianion of DHAP and Glu165, an example of substrate assisted catalysis. Classical molecular dynamics simulations assert that the closest water oxygen atom is consistently closer to the pro-R H than the carboxylate oxygen atoms of the accepted base Glu165. Our proposal is further supported by quantum computations that confirm the implausibility of abstraction of the methylene hydrogen by glutamate and the ease with which it is abstracted by hydroxide. The necessity of Glu165 for efficient catalysis is attributed to its crucial involvement in trapping the vital water in an environment of high electric fields which promote ionization far more rapidly than in bulk solvent.


title: Multicellular immune hubs and their organization in MMRd and MMRp colorectal cancer

abstract: Immune responses to cancer are highly variable, with mismatch repair-deficient (MMRd) tumors exhibiting more anti-tumor immunity than mismatch repair-proficient (MMRp) tumors. To understand the rules governing these varied responses, we transcriptionally profiled 371,223 cells from colorectal tumors and adjacent normal tissues of 28 MMRp and 34 MMRd patients. Analysis of 88 cell subsets and their 204 associated gene expression programs revealed extensive transcriptional and spatial remodeling across tumors. To discover hubs of interacting malignant and immune cells, we identified expression programs in different cell types that co-varied across patient tumors and used spatial profiling to localize coordinated programs. We discovered a myeloid cell-attracting hub at the tumor-luminal interface associated with tissue damage, and an MMRd-enriched immune hub within the tumor, with activated T cells together with malignant and myeloid cells expressing T-cell-attracting chemokines. By identifying interacting cellular programs, we thus reveal the logic underlying spatially organized immune-malignant cell networks.

category: cancer biology


title: MYC controls metastatic heterogeneity in pancreatic cancer

abstract: The degree of metastatic disease varies widely amongst cancer patients and impacts clinical outcomes. However, the biological and functional differences that drive the extent of metastasis are poorly understood. We analyzed primary tumors and paired metastases using a multi-fluorescent lineage-labeled mouse model of pancreatic ductal adenocarcinoma (PDAC) - a tumor type where most patients present with metastases. Genomic and transcriptomic analysis revealed an association between metastatic burden and gene amplification or transcriptional upregulation of MYC and its downstream targets. Functional experiments showed that MYC promotes metastasis by recruiting tumor associated macrophages (TAMs), leading to greater bloodstream intravasation. Consistent with these findings, metastatic progression in human PDAC was associated with activation of MYC signaling pathways and enrichment for MYC amplifications specifically in metastatic patients. Collectively, these results implicate MYC activity as a major determinant of metastatic burden in advanced PDAC.


title: Vasoconstriction in isolated goat aorta does not increase mean aortic pressure

abstract:
Vasoconstriction in small arteries and arterioles is known to increase resistance to flow, while vasoconstriction in large arteries and aorta is known to decrease their compliance. Besides this general understanding, there is no systematic documentation on what happens to small artery compliance and large artery resistance during vasoconstriction and the corresponding alterations in vascular pressure. The aim of the study is to assess the effect of adrenaline on goat aortae and small arteries in terms of resistance and compliance.

Isolated goat aortae and small arteries were perfused with a pulsatile pump and lumen pressure was recorded before and after addition of adrenaline. In the aortae, systolic pressure increased, diastolic pressure decreased, pulse pressure increased (p = 0.018, WSR); but the mean pressure remained the same (p = 0.357, WSR). Small artery vasoconstriction caused an increase in systolic, diastolic and mean pressures (p = 0.028, WSR). Using length, radius, and thickness data from the tissues and the tubes of the experimental set-up, electrical models were simulated to understand the biological data. The simulations allow us to infer that vasoconstriction in aorta leads to a reduction in compliance, but an increase in resistance if any, is not sufficient to change the mean aortic pressure. On the other hand, vasoconstriction in small arteries increases resistance, but a decrease in compliance if any, does not affect any of the four pressure parameters measured. Vasoconstriction in aorta decreases compliance and therefore increases pulse pressure but does not change resistance significantly enough to alter mean pressure.

Key Points SummaryO_LIThe main aim of the study is to understand where exactly resistance (R) and compliance (C) components of the vasculature occur. There is no definitive evidence for the effect of large artery vasoconstriction on resistance and hence the mean arterial pressure.
C_LIO_LIThe manuscript presents biological experiments studying the pressure response of goat aorta and small arteries to adrenaline (invitro) and the interpretations using equivalent electrical models.
C_LIO_LIThe study shows that in aorta and large arteries, vasoconstriction does not lead to a reduction in lumen diameter sufficient to cause a rise in resistance and mean pressure as compared to small arteries.
C_LIO_LIKnowledge of exact location of R and C in the arterial tree enables re-assessment of the differential action of vasoactive drugs on resistance versus compliance vessels once we resolve beat-to-beat R and C changes in response to a drug. This way antihypertensive therapy can be tailored to address the specific cause of the type of hypertension.
C_LI

category: physiology


title: Surviving hypothermia by ferritin-mediated iron detoxification

abstract: How animals rewire cellular programs to survive cold is a fascinating problem with potential biomedical implications, ranging from emergency medicine to space travel. Studying a hibernation-like response in the free-living nematode Caenorhabditis elegans, we uncovered a regulatory axis that enhances the natural resistance of nematodes to severe cold. This axis involves conserved transcription factors, DAF-16/FoxO and PQM-1, which jointly promote cold survival by upregulating FTN-1, a protein related to mammalian Fth1/ferritin. Moreover, we show that inducing expression of Fth1 also promotes cold survival of mammalian neurons, a cell type particularly sensitive to deterioration in hypothermia. Our findings in both animals and cells suggest that FTN-1/Fth1 facilitates cold survival by detoxifying ROS-generating iron species. We finally show that mimicking the effects of FTN-1/Fth1 with drugs protects neurons from cold-induced degeneration, opening a potential avenue to improved treatments of hypothermia.


title: Evolution of the bread wheat D-subgenome and enriching it with diversity from Aegilops tauschii

abstract: Aegilops tauschii, the diploid wild progenitor of the D-subgenome of bread wheat, constitutes a reservoir of genetic diversity for improving bread wheat performance and environmental resilience. To better define and understand this diversity, we sequenced 242 Ae. tauschii accessions and compared them to the wheat D-subgenome. We characterized a rare, geographically-restricted lineage of Ae. tauschii and discovered that it contributed to the wheat D-subgenome, thereby elucidating the origin of bread wheat from at least two independent hybridizations. We then used k-mer-based association mapping to identify discrete genomic regions with candidate genes for disease and pest resistance and demonstrated their functional transfer into wheat by transgenesis and wide crossing, including the generation of a library of  synthetic hexaploids incorporating diverse Ae. tauschii genomes. This pipeline permits rapid trait discovery in the diploid ancestor through to functional genetic validation in a hexaploid background amenable to breeding.

category: plant biology


title: Regulation of lateral root development by shoot-sensed far-red light via HY5 is nitrate-dependent and involves the NRT2.1 nitrate transporter.

abstract: Plants are very effective in responding to environmental changes during competition for light and nutrients. Low Red:Far-Red (low R:FR)-mediated neighbor detection allows plants to compete successfully with other plants for available light. This above-ground signal can also reduce lateral root growth by inhibiting lateral root emergence, a process that might help the plant invest resources in shoot growth. Nitrate is an essential nutrient for plant growth and Arabidopsis thaliana responds to low nitrate conditions by enhancing nutrient uptake and reducing lateral and main root growth. There are indications that low R:FR signaling and low nitrate signaling can affect each other. It is unknown which response is prioritized when low R:FR light- and low nitrate signaling co-occur. We investigated the effect of low nitrate conditions on the low R:FR response of the A. thaliana root system in agar plate media, combined with the application of supplemental Far-Red (FR) light to the shoot. We observed that under low nitrate conditions main and lateral root growth was reduced, but more importantly, that the response of the root system to low R:FR was suppressed. Consistently, a loss-of-function mutant of a nitrate transporter gene NRT2.1 lacked low R:FR-induced lateral root reduction and its root growth was hypersensitive to low nitrate. ELONGATED HYPOCOTYL5 (HY5) plays an important role in the root response to low R:FR and we found that it was less sensitive to low nitrate conditions with regards to lateral root growth. In addition, we found that low R:FR increases NRT2.1 expression and that low nitrate enhances HY5 expression. HY5 also affects NRT2.1 expression, however, it depended on the presence of ammonium in which direction this effect was. Replacing part of the nitrogen source with ammonium also removed the effect of low R:FR on the root system, showing that changes in nitrogen sources can be crucial for root plasticity. Together our results show that nitrate signaling can repress low R:FR responses and that this involves signaling via HY5 and NRT2.1.


title: Robust, sensitive, and quantitative single-cell proteomics based on ion mobility filtering

abstract: Unbiased single-cell proteomics (scProteomics) promises to advance our understanding of cell functions within complex biological systems. However, a major challenge for current methods is their ability to identify and provide accurate quantitative information for low abundance proteins. Herein, we describe an ion mobility-enhanced mass spectrometry acquisition and peptide identification method, TIFF (Transferring Identification based on FAIMS Filtering), designed to improve the sensitivity and accuracy of label-free scProteomics. TIFF significantly extends the ion accumulation times for peptide ions by filtering out singly charged background ions. The peptide identities are then assigned by a 3-dimensional MS1 feature matching approach (retention time, accurate mass, and FAIMS compensation voltage). The TIFF method enabled unbiased proteome analysis to a depth of >1,700 proteins in single HeLa cells with >1,100 proteins consistently quantified. As a demonstration, we applied the TIFF method to obtain temporal proteome profiles of >150 single murine macrophage cells during a lipopolysaccharide stimulation experiment and identified time-dependent proteome profiles.

category: systems biology


title: Pupillary aperture is a potential biomarker ofmovement preparation

abstract: In response to variable light intensity, the pupils reflexively constrict or dilate to maintain a uniform retinal illumination. The pupillary light reflex (PLR) pathway receives projections from two important areas in primates brain that plan rapid saccadic eye-movement - frontal eye field (FEF) and superior colliculus (SC). The speed with which neurons in these areas increase firing rate to a threshold determines latency of a saccade. Micro-stimulation of FEF/SC neurons below this threshold modulates the magnitude of PLR. Nonetheless, how the saccade latency and pupil dynamics are related remains unknown. Our study shows that the appearance of a bright stimulus evokes pupil constriction at higher rate when the latency of impending saccade to the stimulus is shorter. This inverse relationship between the rate of pupil constriction and the saccade latency is robust irrespective of the reward outcome. In a homeomorphic biomechanical model of pupil, we have projected build-up signal similar to FEF and SC activity to the parasympathetic and sympathetic divisions of the PLR pathway, respectively. Model simulation mimics the observed data to indicate that the FEF and SC activity for eye movement modulates autonomic input to the pupillary muscle plant. A striking similarity between the dynamics of pupil constriction and stochastic rise in neural activity for saccade elicitation suggests that PLR is a potential proxy of movement preparation, and not mere an indicator of attentional orientation. Our study suggests a mechanism of how the retinal luminosity is timely regulated to aid perception by minimizing visual transients due to gaze orientation.

category: neuroscience


title: Ribosome Profiling Reveals a Dichotomy Between Ribosome Occupancy of Nuclear-Encoded and Mitochondrial-Encoded OXPHOS mRNA Transcripts in a Striatal Cell Model of Huntington Disease

abstract: Huntington disease (HD) is caused by an expanded polyglutamine mutation in huntingtin (mHTT), which promotes a prominent atrophy in the striatum and subsequent psychiatric, cognitive, and choreiform movements. Multiple lines of evidence point to an association between HD and aberrant striatal mitochondrial functions. However, present knowledge about whether (or how) mitochondrial mRNA translation is differentially regulated in HD remains unclear. We have recently applied ribosome profiling (Ribo-Seq), a technique based on the high-throughput sequencing of ribosome-protected mRNA fragments, to analyze detailed snapshots of ribosome occupancy of the mitochondrial mRNA transcripts in control and HD striatal cells. Ribo-seq data revealed almost unaltered ribosome occupancy on the nuclear-encoded mitochondrial transcripts involved in oxidative phosphorylation (OXPHOS) and only a mild reduction in ribosome occupancy on a few selected transcripts (SHDA, Ndufv1, Timm23, Tomm5, and Mrps22) in HD cells. By contrast, ribosome occupancy of mitochondrially encoded OXPHOS mRNAs (mtNd-1, mtNd-2, mtNd-4, mtNd-4l, mtNd-5, mtNd-6, mt-Co1, mtCyt b, and mt-ATP8) was dramatically increased, implying widespread dichotomous effects on ribosome occupancy and OXPHOS mRNA translation in HD. Thus, mHTT may command signals that specifically regulate translation of the mitochondrial OXPHOS transcripts and influence HD pathogenesis.


title: Prefrontal-motor and somatosensory-motor cortical network interactions during reactive balance are associated with distinct aspects of balance behavior in older adults

abstract: Heightened reliance on the cerebral cortex for postural stability with aging is well-known, yet the cortical dynamics of balance control, particularly in relationship to balance function, is unclear. Here we aimed to investigate motor cortical activity in relationship to the level of balance challenge presented during reactive balance recovery, and identify circuit-specific interactions between motor cortex and prefrontal or somatosensory regions to metrics of balance function that predict fall risk. Using electroencephalography, we assessed motor cortical beta power, and beta coherence during balance reactions to perturbations in older adults. We found that individuals with greater somatosensory-motor beta coherence at baseline and lower beta power evoked over motor regions following perturbations demonstrated higher general clinical balance function. At the group-level, beta coherence between prefrontal-motor regions reduced during balance reactions. Older adults with the highest post-perturbation prefrontal-motor coherence showed greater cognitive dual-task interference and elicited stepping reactions at lower perturbation magnitudes. Our results support motor cortical beta activity as a potential biomarker for individual level of balance challenge and implicate prefrontal-and somatosensory-motor cortical networks in different aspects of balance control in older adults. Cortical network activity during balance may provide a neural target for precision-medicine efforts aimed at fall-prevention with aging.


title: 7-Dehydrocholesterol-derived oxysterols cause neurogenic defects in Smith-Lemli-Opitz syndrome

abstract: Defective 3{beta}-hydroxysterol-{Delta}7 -reductase (DHCR7) in the developmental disorder, Smith-Lemli-Opitz syndrome (SLOS), results in deficiency in cholesterol and accumulation of its precursor, 7-dehydrocholesterol (7-DHC). Here, we show that loss of DHCR7 causes accumulation of 7-DHC-derived oxysterol metabolites, premature neurogenesis, and perturbation of neuronal localization in developing murine or human cortical neural precursors, both in vitro and in vivo. We found that a major oxysterol, 3{beta},5-dihydroxycholest-7-en-6-one (DHCEO), mediates these effects by initiating crosstalk between glucocorticoid receptor (GR) and neurotrophin receptor kinase TrkB. Either loss of DHCR7 or direct exposure to DHCEO causes hyperactivation of GR and TrkB and their downstream MEK-ERK-C/EBP signaling pathway in cortical neural precursors. Moreover, direct inhibition of GR activation with an antagonist or inhibition of DHCEO accumulation with antioxidants rescues the premature neurogenesis phenotype caused by the loss of DHCR7. These results suggest that GR could be a new therapeutic target against the neurological defects observed in SLOS.


title: Moving outside the lab: The viability of conducting sensorimotor learning studies online

abstract: Collecting data online via crowdsourcing platforms has proven to be a very efficient way to recruit a large and diverse sample. Studies of motor learning, however, have been largely confined to the lab due to the need for special equipment to record movement kinematics and, as such, are typically only accessible to specific participants (e.g., college students). As a first foray to make motor learning studies accessible to a larger and more diverse audience, we developed an online, web-based platform (OnPoint) to collect kinematic data, serving as a template for researchers to create their own online sensorimotor control and learning experiments. As a proof-of-concept, we asked if fundamental motor learning phenomena discovered in the lab could be replicated online. In a series of three experiments, we observed a close correspondence between the results obtained online with those previously reported from research conducted in the laboratory. This web-based platform paired with online crowdsourcing can serve as a powerful new method for the study of motor control and learning.


title: Cortical Microcircuitry of Executive Control and Source of Associated Event-Related Potentials

abstract: Medial frontal cortex enables executive control by monitoring relevant information and using it to adapt behavior. In macaques performing a saccade countermanding (stop-signal) task, we recorded EEG over and neural spiking across all layers of the supplementary eye field (SEF). We report the laminar organization of concurrently activated neurons monitoring the conflict between incompatible responses and the timing of events serving goal maintenance and executive control. We also show their relation to coincident event-related potentials (ERP). Neurons signaling response conflict were largely broad-spiking found across all layers. Neurons signaling the interval until specific task events were largely broad-spiking neurons concentrated in L3 and L5. Neurons predicting the duration of control and sustaining the task goal until the release of operant control were a mix of narrow- and broad-spiking neurons confined to L2/3. We complement these results with the first report of a monkey homologue of the N2/P3 ERP complex associated with response inhibition. N2 polarization varied with error likelihood and P3 polarization varied with the duration of expected control. The amplitude of the N2 and P3 were predicted by the spike rate of different classes of neurons located in L2/3 but not L5/6. These findings reveal important, new features of the cortical microcircuitry supporting executive control and producing associated ERP.


title: Functional Architecture of Executive Control and Associated Event-Related Potentials


title: CDSeqR: fast complete deconvolution for gene expression data from bulk tissues

abstract:
BackgroundBiological tissues consist of heterogenous populations of cells. Because gene expression patterns from bulk tissue samples reflect the contributions from all cells in the tissue, understanding the contribution of individual cell types to the overall gene expression in the tissue is fundamentally important. We recently developed a computational method, CDSeq, that can simultaneously estimate both sample-specific cell-type proportions and cell-type-specific gene expression profiles using only bulk RNA-Seq counts from multiple samples. Here we present an R implementation of CDSeq (CDSeqR) with significant performance improvement over the original implementation in MATLAB and an added new function to aid cell type annotation. The R package would be of interest for the broader R community.

ResultWe developed a novel strategy to substantially improve computational efficiency in both speed and memory usage. In addition, we designed and implemented a new function for annotating the CDSeq estimated cell types using single-cell RNA sequencing (scRNA-seq) data. This function allows users to readily interpret and visualize the CDSeq estimated cell types. In addition, this new function further allows the users to annotate CDSeq-estimated cell types using marker genes. We carried out additional validations of the CDSeqR software using synthetic, real cell mixtures, and real bulk RNA-seq data from the Cancer Genome Atlas (TCGA) and The Genotype-Tissue Expression (GTEx) project.

ConclusionsThe existing bulk RNA-seq repositories, such as TCGA and GTEx, provide enormous resources for better understanding changes in transcriptomics and human diseases. They are also potentially useful for studying cell-cell interactions in the tissue microenvironment. Bulk level analyses neglect tissue heterogeneity, however, and hinder investigation of a cell-type-specific expression. The CDSeqR package may aid in silico dissection of bulk expression data, enabling researchers to recover cell-type-specific information.


title: Trial-to-trial variability of spiking delay activity in prefrontal cortex constrains burst-coding models of working memory

abstract:
A hallmark neuronal correlate of working memory (WM) is stimulus-selective spiking activity of neurons in prefrontal cortex (PFC) during mnemonic delays. These observations have motivated an influential computational modeling framework in which WM is supported by persistent activity. Recently this framework has been challenged by arguments that observed persistent activity may be an artifact of trial-averaging, which potentially masks high variability of delay activity at the single-trial level. In an alternative scenario, WM delay activity could be encoded in bursts of selective neuronal firing which occur intermittently across trials. However, this alternative proposal has not been tested on single-neuron spike-train data. Here, we developed a framework for addressing this issue by characterizing the trial-to-trial variability of neuronal spiking quantified by Fano factor (FF). By building a doubly stochastic Poisson spiking model, we first demonstrated that the burst-coding proposal implies a significant increase in FF positively correlated with firing rate, and thus loss of stability across trials during the delay. Simulation of spiking cortical circuit WM models further confirmed that FF is a sensitive measure that can well dissociate distinct WM mechanisms. We then tested these predictions on datasets of single-neuron recordings from macaque prefrontal cortex during three WM tasks. In sharp contrast to the burst-coding model predictions, we only found a small fraction of neurons showing increased WM-dependent burstiness, and stability across trials during delay was strengthened in empirical data. Therefore, reduced trial-to-trial variability during delay provides strong constraints on the contribution of single-neuron intermittent bursting to WM maintenance.

Significance StatementThere are diverging classes of theoretical models explaining how information is maintained in working memory by cortical circuits. In an influential model class, neurons fire exhibit persistent elevated memorandum-selective firing, whereas a recently developed class of burst-coding models suggests that persistent activity is an artifact of trial-averaging, and spiking is sparse in each single trial, subserved by brief intermittent bursts. However, this alternative picture has not been characterized or tested on empirical spike-train data. Here we combine mathematical analysis, computational model simulation and experimental data analysis to test empirically theses two classes of models and show that the trial-to-trial variability of empirical spike trains is not consistent with burst coding. These findings provide constraints for theoretical models of working memory.


title: TMEM97 increases in synapses and is a potential synaptic Aβ binding partner in human Alzheimer's disease

abstract:
Synapse loss correlates with cognitive decline in Alzheimers disease (AD), and soluble amyloid beta (A{beta}) is implicated in synaptic dysfunction and loss. An important knowledge gap is the lack of understanding of how synaptic accumulation of A{beta} leads to synapse degeneration. In particular, there has been difficulty in determining whether there is a synaptic receptor that binds A{beta} and mediates toxicity. While many candidate synaptic binding partners have been observed in model systems, their relevance to human AD brain remains unknown. This is in part due to methodological limitations preventing visualization of A{beta} binding at individual synapses. To overcome this limitation, we combined two high resolution microscopy techniques: array tomography and Forster resonance energy transfer (FRET) to image over 1 million individual synaptic terminals in temporal cortex from AD (n=9) and age matched control cases (n=6). Within postsynaptic densities, A{beta} generates a FRET signal with transmembrane protein 97 (TMEM97), recently discovered to be the Sigma-2 receptor, cellular prion protein, and postsynaptic density 95 (PSD95). TMEM97 is also present in a higher proportion of postsynapses in AD brain compared to control. Further, we inhibited A{beta}-TMEM97 interaction in the APP/PS1+Tau mouse model of AD by treating with the Sigma-2 receptor complex allosteric antagonist CT1812 (n=20) or vehicle (n=20). CT1812 drug concentration correlated negatively with synaptic FRET signal between TMEM97 and A{beta}. These data support a role for TMEM97 in the synaptic binding of A{beta} in human Alzheimers disease brain where it may mediate synaptotoxicity.

One Sentence SummaryIn Alzheimers disease, TMEM97 was present in a higher proportion of synapses and close enough to amyloid beta to be a potential synaptic binding partner.


title: Discovery of re-purposed drugs that slow SARS-CoV-2 replication in human cells

abstract: COVID-19 vaccines based on the Spike protein of SARS-CoV-2 have been developed that appear to be largely successful in stopping infection. However, vaccine escape variants might arise leading to a re-emergence of COVID. In anticipation of such a scenario, the identification of repurposed drugs that stop SARS-CoV-2 replication could have enormous utility in stemming the disease. Here, using a nano-luciferase tagged version of the virus (SARS-CoV-2- DOrf7a-NLuc) to quantitate viral load, we evaluated a range of human cell types for their ability to be infected and support replication of the virus, and performed a screen of 1971 FDA-approved drugs. Hepatocytes, kidney glomerulus, and proximal tubule cells were particularly effective in supporting SARS-CoV-2 replication, which is in- line with reported proteinuria and liver damage in patients with COVID-19. We identified 35 drugs that reduced viral replication in Vero and human hepatocytes when treated prior to SARS-CoV-2 infection and found amodiaquine, atovaquone, bedaquiline, ebastine, LY2835219, manidipine, panobinostat, and vitamin D3 to be effective in slowing SARS-CoV-2 replication in human cells when used to treat infected cells. In conclusion, our study has identified strong candidates for drug repurposing, which could prove powerful additions to the treatment of COVID.

category: immunology


title: Expression of human ACE2 N-terminal domain, part of the receptor for SARS-CoV-2, in fusion with maltose binding protein, E. coli ribonuclease I and human RNase A

abstract:
The SARS-CoV-2 viral genome contains a positive-strand single-stranded RNA of ~30 kb. Human ACE2 protein is the receptor for SARS-CoV-2 virus attachment and initiation of infection. We propose to use ribonucleases (RNases) as antiviral agents to destroy the viral genome in vitro. In the virions the RNA is protected by viral capsid proteins, membrane proteins and nucleocapsid proteins. To overcome this protection we set out to construct RNase fusion with human ACE2 receptor N-terminal domain (ACE2NTD). We constructed six proteins expressed in E. coli cells: 1) MBP-ACE2NTD, 2) ACE2NTD-GFP, 3) RNase I (6xHis), 4) RNase III (6xHis), 5) RNase I-ACE2NTD (6xHis), and 6) human RNase A-ACE2NTD150 (6xHis). We evaluated fusion expression in different E. coli strains, partially purified MBP-ACE2NTD protein from the soluble fraction of bacterial cell lysate, and refolded MBP-ACE2NTD protein from inclusion body. The engineered RNase I-ACE2NTD (6xHis) and hRNase A-ACE2NTD (6xHis) fusions are active in cleaving COVID-19 RNA in vitro. The recombinant RNase I (6xHis) and RNase III (6xHis) are active in cleaving RNA and dsRNA in test tube. This study provides a proof-of-concept for construction of fusion protein between human cell receptor and nuclease that may be used to degrade viral nucleic acids in our environment.

Graphical Abstract

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category: microbiology


title: Mn2+ coordinates Cap-0-RNA to align substrates for efficient 2'-O-methyl transfer by SARS-CoV-2 nsp16

abstract: Capping viral messenger RNAs is essential for efficient translation and prevents their detection by host innate immune responses. For SARS-CoV-2, RNA capping includes 2'-O-methylation of the first ribonucleotide by methyltransferase nsp16 in complex with activator nsp10. The reaction requires substrates, a short RNA and SAM, and is catalyzed by divalent cations, with preference for Mn2+. Crystal structures of nsp16-nsp10 with capped RNAs revealed a critical role of metal ions in stabilizing interactions between ribonucleotides and nsp16, resulting in precise alignment of the substrates for methyl transfer. An aspartate residue that is highly conserved among coronaviruses alters the backbone conformation of the capped RNA in the binding groove. This aspartate is absent in mammalian methyltransferases and is a promising site for designing coronavirus-specific inhibitors.


title: Sequential delivery of LAIV and SARS-CoV-2 in the ferret model can reduce SARS-CoV-2 shedding and does not result in enhanced lung pathology.

abstract: Co-circulation of SARS-CoV-2 and influenza viruses could pose unpredictable risks to health systems globally, with recent studies suggesting more severe disease outcomes in co-infected patients. The lack of a readily available COVID-19 vaccine has reinforced the importance of influenza vaccine programmes during the COVID-19 pandemic. Live Attenuated Influenza Vaccine (LAIV) is an important tool in protecting against influenza, particularly in children. However, it is unknown whether LAIV administration might influence the outcomes of acute SARS-CoV-2 infection or disease. To investigate this, quadrivalent LAIV (QLAIV) was administered to ferrets 3 days pre- or post-SARS-CoV-2 infection. LAIV administration did not exacerbate SARS-CoV-2 disease course or lung pathology with either regimen. Additionally, LAIV administered prior to SARS-CoV-2 infection significantly reduced SARS-CoV-2 replication and shedding in the upper respiratory tract (URT). We conclude that LAIV administration in close proximity to SARS-CoV-2 infection does not exacerbate mild disease and can reduce SARS-CoV-2 shedding.


title: Nelfinavir markedly improves lung pathology in SARS-CoV-2-infected Syrian hamsters despite a lack of an antiviral effect

abstract: In response to the ongoing COVID-19 pandemic, repurposing of drugs for the treatment of SARS-CoV-2 infections is being explored. The HIV protease inhibitor Nelfinavir, widely prescribed in combination with other HIV inhibitors, has been shown to inhibit in vitro SARS-CoV-2 replication. We here report on the effect of Nelfinavir in the Syrian hamster SARS-CoV-2 infection model. Although treatment of infected hamsters with either 15 or 50 mg/kg BID Nelfinavir [for four consecutive days, initiated on the day of infection] does not reduce viral RNA loads nor infectious virus titres in the lungs compared to the vehicle control, the drug reduced virus-induced lung pathology to nearly the baseline scores of healthy animals. A substantial interstitial infiltration of neutrophils is observed in the lungs of treated (both infected and uninfected) animals. The protective effect of Nelfinavir on SARS-CoV-2-induced lung pathology (at doses that are well tolerated and that result in exposures nearing those observed in HIV-infected patients) may lay the foundation for clinical studies with this widely used drug.


title: Nelfinavir markedly improves lung pathology in SARS-CoV-2-infected Syrian hamsters despite lack of an antiviral effect


title: Kinesin-3 and kinesin-1 motors direct basement membrane protein secretion to a basal sub-region of the basolateral plasma membrane in epithelial cells

abstract:
Basement membranes (BMs) are sheet-like extracellular matrices that line the basal surfaces of all epithelia. Since BM proteins form networks, they likely need to be secreted near the basal surface. However, the location of their secretion site and how it is selected are unknown. Working in the Drosophila follicular epithelium, we identified two kinesins essential for normal BM formation. Our data suggest the two kinesins work together to transport Rab10+ BM protein-filled secretory vesicles towards the basal surface along the polarized microtubule array common to epithelia. This kinesin transport biases BM protein secretion basally. When kinesins are depleted, BM proteins are mis-secreted to more apical regions of the lateral membrane, creating ectopic BM protein networks between cells that disrupt cell movements and tissue architecture. These results introduce a new transport step in the BM protein secretion pathway and highlight the importance of controlling the sub-cellular exocytic site of network-forming proteins.

HighlightsO_LIA kinesin-3 and a kinesin-1 are required for normal basement membrane (BM) assembly
C_LIO_LIKinesins move Rab10+ BM secretory vesicles basally on polarized microtubule arrays
C_LIO_LITransport biases BM exocytosis to basal subregions of the basolateral membrane
C_LIO_LILoss of kinesins creates ectopic BM networks that disrupt tissue architecture
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category: cell biology


title: Environmentally-induced mdig is a major contributor to the severity of COVID-19 through fostering expression of SARS-CoV-2 receptor NRPs and glycan metabolism

abstract: The novel {beta}-coronavirus, SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19), has infected more than 101 million people and resulted in 2.2 million death worldwide. Recent epidemiological studies suggested that some environmental factors, such as air pollution, might be the important contributors to the mortality of COVID-19. However, how environmental exposure enhances the severity of COVID-19 remains to be fully understood. In the present report, we provide evidence showing that mdig, a previously reported environmentally-induced oncogene that antagonizes repressive trimethylation of histone proteins, is a master regulator for SARS-CoV-2 receptors neuropilin-1 (NRP1) and NRP2, cathepsins, glycan metabolism and inflammation, key determinants for viral infection and cytokine storm of the patients. Depletion of mdig in bronchial epithelial cells by CRISPR-Cas-9 gene editing resulted in a decreased expression of NRP1, NRP2, cathepsins, and genes involved in protein glycosylation and inflammation, largely due to a substantial enrichment of lysine 9 and/or lysine 27 trimethylation of histone H3 (H3K9me3/H3K27me3) on these genes as determined by ChIP-seq. These data, accordingly, suggest that mdig is a key mediator for the severity of COVID-19 in response to environmental exposure and targeting mdig may be one of the effective strategies in ameliorating the symptom and reducing the mortality of COVID-19.


title: Identification of a conserved neutralizing epitope present on spike proteins from highly pathogenic coronaviruses

abstract: Three pathogenic human coronaviruses have emerged within the last 20 years, with SARS-CoV-2 causing a global pandemic. Although therapeutic antibodies targeting the SARS-CoV-2 spike currently focus on the poorly conserved receptor-binding domain, targeting essential neutralizing epitopes on the more conserved S2 domain may provide broader protection. We report an antibody binding an epitope conserved in the pre-fusion core of MERS-CoV, SARS-CoV and SARS-CoV-2 spike S2 domains. Antibody 3A3 binds a conformational epitope with ~2.5 nM affinity and neutralizes spike from SARS-CoV, SARS-CoV-2 and variants of concern in in vitro pseudovirus assays. Hydrogen-deuterium exchange mass spectrometry identified residues 980-1006 in the flexible hinge region at the S2 apex as the 3A3 epitope, suggesting 3A3 prevents the S2 conformational rearrangements required for conversion to the spike post-fusion state and virus-host cell fusion. This work defines a conserved vulnerable site on the SARS-CoV-2 S2 domain and guides the design of pan-protective spike immunogens.

category: biochemistry


title: Identification of a conserved neutralizing epitope present on spike proteins from all highly pathogenic coronaviruses


title: Cell-type specific expression and behavioral impact of galanin and GalR1 in the locus coeruleus during opioid withdrawal

abstract: The neuropeptide galanin is reported to attenuate opioid withdrawal symptoms, potentially by reducing neuronal hyperactivity in the noradrenergic locus coeruleus (LC) via galanin receptor 1 (GalR1). We evaluated this mechanism by using RNAscope in situ hybridization to characterize GalR1 mRNA distribution in the dorsal pons and to compare galanin and GalR1 mRNA expression in tyrosine hydroxylase-positive (TH+) LC cells at baseline and following chronic morphine or precipitated withdrawal. We then used genetically altered mouse lines and pharmacology to test whether noradrenergic galanin (NE-Gal) modulates withdrawal symptoms. RNAscope revealed that, while GalR1 signal was abundant in the dorsal pons, 80.7% of the signal was attributable to TH-neurons outside the LC. Galanin and TH mRNA were abundant in LC cells at baseline and were further increased by withdrawal, whereas low basal GalR1 mRNA expression was unaltered by chronic morphine or withdrawal. Naloxone-precipitated withdrawal symptoms in mice lacking NE-Gal (GalcKO-Dbh) were largely similar to WT littermates, indicating that loss of NE-Gal does not exacerbate withdrawal. Complimentary experiments using NE-Gal overexpressor mice (NE-Gal OX) and systemic administration of the galanin receptor agonist galnon revealed that increasing galanin signaling also failed to alter behavioral withdrawal, while suppressing noradrenergic transmission with the alpha-2 adrenergic receptor agonist clonidine attenuated multiple symptoms. These results indicate that galanin does not acutely attenuate precipitated opioid withdrawal via an LC-specific mechanism, which has important implications for the general role of galanin in regulation of somatic and affective opioid responses and LC activity.


title: Drosophila Evolution over Space and Time (DEST) - A New Population Genomics Resource

abstract: Drosophila melanogaster is a leading model in population genetics and genomics, and a growing number of whole-genome datasets from natural populations of this species have been published over the last 20 years. A major challenge is the integration of these disparate datasets, often generated using different sequencing technologies and bioinformatic pipelines, which hampers our ability to address questions about the evolution and population structure of this species. Here we address these issues by developing a bioinformatics pipeline that maps pooled sequencing (Pool-Seq) reads from D. melanogaster to a hologenome consisting of fly and symbiont genomes and estimates allele frequencies using either a heuristic (PoolSNP) or a probabilistic variant caller (SNAPE-pooled). We use this pipeline to generate the largest data repository of genomic data available for D. melanogaster to date, encompassing 271 population samples from over 100 locations in >20 countries on four continents based on a combination of 121 unpublished and 150 previously published genomic datasets. Several of these locations have been sampled at different seasons across multiple years. This dataset, which we call Drosophila Evolution over Space and Time (DEST), is coupled with sampling and environmental meta-data. A web-based genome browser and web portal provide easy access to the SNP dataset. Our aim is to provide this scalable platform as a community resource which can be easily extended via future efforts for an even more extensive cosmopolitan dataset. Our resource will enable population geneticists to analyze spatio-temporal genetic patterns and evolutionary dynamics of D. melanogaster populations in unprecedented detail.

category: genomics


title: Monitoring mitochondrial translation by pulse SILAC

abstract: Mitochondrial ribosomes are specialized to translate the 13 membrane proteins encoded in the mitochondrial genome, but it is challenging to quantify mitochondrial translation products due to their hydrophobic nature. Here, we introduce a proteomic method that combines biochemical isolation of mitochondria with pulse stable isotope labeling by amino acids in cell culture (pSILAC). Our method provides the highest protein coverage (quantifying 12 out of the 13 inner-membrane proteins; average 2-fold improvement over previous studies) with the shortest measurement time. We applied this method to uncover the global picture of (post)translational regulation of both mitochondrial- and nuclear-encoded proteins involved in the assembly of protein complexes that mediate oxidative phosphorylation (OXPHOS). The results allow us to infer the assembly order of complex components and/or partners, as exemplified by complex III. This method should be applicable to study mitochondrial translation programs in many contexts, including oxidative stress and mitochondrial disease.


title: Nanotraps for the containment and clearance of SARS-CoV-2

abstract:
SARS-CoV-2 enters host cells through its viral spike protein binding to angiotensin-converting enzyme 2 (ACE2) receptors on the host cells. Here we show functionalized nanoparticles, termed "Nanotraps", completely inhibited SARS-CoV-2 infection by blocking the interaction between the spike protein of SARS-CoV-2 and the ACE2 of host cells. The liposomal-based Nanotrap surfaces were functionalized with either recombinant ACE2 proteins or anti-SARS-CoV-2 neutralizing antibodies and phagocytosis-specific phosphatidylserines. The Nanotraps effectively captured SARS-CoV-2 and completely blocked SARS-CoV-2 infection to ACE2-expressing human cell lines and primary lung cells; the phosphatidylserine triggered subsequent phagocytosis of the virus-bound, biodegradable Nanotraps by macrophages, leading to the clearance of pseudotyped and authentic virus in vitro. Furthermore, the Nanotraps demonstrated excellent biosafety profile in vitro and in vivo. Finally, the Nanotraps inhibited pseudotyped SARS-CoV-2 infection in live human lungs in an ex vivo lung perfusion system. In summary, Nanotraps represent a new nanomedicine for the inhibition of SARS-CoV-2 infection.

HighlightsO_LINanotraps block interaction between SARS-CoV-2 spike protein and host ACE2 receptors
C_LIO_LINanotraps trigger macrophages to engulf and clear virus without becoming infected
C_LIO_LINanotraps showed excellent biosafety profiles in vitro and in vivo
C_LIO_LINanotraps blocked infection to living human lungs in ex vivo lung perfusion system
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Progress and PotentialTo address the global challenge of creating treatments for SARS-CoV-2 infection, we devised a nanomedicine termed "Nanotraps" that can completely capture and eliminate the SARS-CoV-2 virus. The Nanotraps integrate protein engineering, immunology, and nanotechnology and are effective, biocompatible, safe, stable, feasible for mass production. The Nanotraps have the potential to be formulated into a nasal spray or inhaler for easy administration and direct delivery to the respiratory system, or as an oral or ocular liquid, or subcutaneous, intramuscular or intravenous injection to target different sites of SARS-CoV-2 exposure, thus offering flexibility in administration and treatment. More broadly, the highly versatile Nanotrap platform could be further developed into new vaccines and therapeutics against a broad range of diseases in infection, autoimmunity and cancer, by incorporating with different small molecule drugs, RNA, DNA, peptides, recombinant proteins, and antibodies.

category: bioengineering


title: Outer Hair Cell Function is Normal in βV Spectrin Knockout Mice

abstract: Reports have proposed a putative role for {beta}V spectrin in outer hair cells (OHCs) of the cochlea. In an ongoing investigation of the role of the cytoskeleton in electromotility, we tested mice with a targeted exon deletion of {beta}V spectrin (Spnb5), and unexpectedly find that Spnb5(-/-) animals auditory thresholds are unaffected. Similarly, these mice have normal OHC electromechanical activity (otoacoustic emissions) and non-linear capacitance. In contrast, magnitudes of auditory brainstem response (ABR) wave 1-amplitudes are significantly reduced. Evidence of a synaptopathy was absent with normal hair cell CtBP2 counts. In Spnb5(-/-) mice, the number of afferent and efferent nerve fibers is decreased. Consistent with this data, Spnb5 mRNA is present in Type I and II spiral ganglion neurons, but undetectable in OHCs. Together, these data establish that {beta}V spectrin is important for hearing, affecting neuronal structure and function. Significantly, these data exclude {beta}V spectrin as functionally important to OHCs as has been previously suggested.


title: Novel EGFRvIII-CAR transgenic mice for rigorous preclinical studies in syngeneic mice

abstract:
BackgroundRigorous preclinical studies of chimeric antigen receptor (CAR) immunotherapy will require large quantities of consistent and high-quality CAR-transduced T (CART)-cells that can be used in syngeneic mouse glioblastoma (GBM) models. To this end, we developed a novel transgenic (Tg) mouse strain with a fully murinized CAR targeting epidermal growth factor receptor variant III (EGFRvIII).

MethodsWe first established the murinized version of EGFRvIII-CAR and validated its function using a retroviral vector (RV) in C57BL/6J mice bearing syngeneic SB28 GBM expressing EGFRvIII. Next, we created C57BL/6J-background Tg mice carrying the anti-EGFRvIII-CAR downstream of a Lox-Stop-Lox cassette in the Rosa26 locus. We bred these mice with CD4-Cre Tg mice to allow CAR expression on T-cells and evaluated the function of the CART-cells both in vitro and in vivo. In order to inhibit immunosuppressive myeloid cells within SB28 GBM, we also evaluated a combination approach of CART and an anti-EP4 compound (ONO-AE3-208).

ResultsBoth RV- and Tg-CART-cells demonstrated specific cytotoxic activities against SB28-EGFRvIII cells. A single intravenous infusion of EGFRvIII-CART-cells prolonged the survival of glioma-bearing mice when preceded by a lymphodepletion regimen with recurrent tumors displaying profound EGFRvIII loss. The addition of ONO-AE3-208 resulted in long-term survival in a fraction of CART-treated mice and those survivors demonstrated delayed growth of subcutaneously re-challenged both EGFRvIII+ and parental EGFRvIII- SB28.

ConclusionOur new syngeneic CAR Tg mouse model can serve as a useful tool to address clinically relevant questions and develop future immunotherapeutic strategies.

Importance of studyThe majority of preclinical studies evaluating CART therapy for GBM have utilized xenografts implanted into immunocompromised mice. Because the successful development of these strategies will depend on the understanding of critical interactions between therapeutic cells and the endogenous immune environment, it is essential to develop a novel immunocompetent system which allows us to study these interactions in a robust and reproducible manner. To this end, we created a Tg mouse strain in which all T-cells express a murinized EGFRvIII-CAR. T-cells derived from these mice demonstrated consistent CAR expression and EGFRvIII-specific cytotoxicity while traditional transduction with a CAR vector showed batch-to-batch variability. The syngeneic system also gave us the opportunity to evaluate a combination regimen with blockade of myeloid-derived suppressor cells. The Tg-CART mice represent a novel system for robust, and reproducible preclinical investigations.


title: Enhanced immunogenicity of a synthetic DNA vaccine expressing consensus SARS-CoV-2 Spike protein using needle-free immunization

abstract: The ongoing global pandemic of Coronavirus Disease 2019 (COVID-19) calls for an urgent development of effective and safe prophylactic and therapeutic measures. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein is a major immunogenic and protective protein, and plays a crucial role in viral pathogenesis. In this study, we successfully constructed a synthetic codon-optimized DNA-based vaccine as a countermeasure against SARS-CoV-2; denoted as VIU-1005. The design was based on the synthesis of codon-optimized coding sequence for optimal mammalian expression of a consensus full-length S glycoprotein. The successful construction of the vaccine was confirmed by restriction digestion and sequencing, and the protein expression of the S protein was confirmed by western blot and immunofluorescence staining in mammalian cells. The immunogenicity of the vaccine was tested in two mouse models (BALB/c and C57BL/6J). Th1-skewed systemic S-specific IgG antibodies and neutralizing antibodies (nAbs) were significantly induced in both models four weeks post three injections with 100 g of the VIU-1005 vaccine via intramuscular needle injection but not intradermal or subcutaneous routes. Importantly, such immunization induced long-lasting IgG response in mice that lasted for at least 6 months. Interestingly, using a needle-free system, we showed an enhanced immunogenicity of VIU-1005 in which lower doses such as 25-50 g or less number of doses were able to elicit significantly high levels of Th1-biased systemic S-specific IgG antibodies and nAbs via intramuscular immunization compared to needle immunization. Compared to the intradermal needle injection which failed to induce any significant immune response, intradermal needle-free immunization elicited robust Th1-biased humoral response similar to that observed with intramuscular immunization. Furthermore, immunization with VIU-1005 induced potent S-specific cellular response as demonstrated by the significantly high levels of IFN-{gamma}, TNF and IL-2 cytokines production in memory CD8+ and CD4+ T cells in BALB/c mice. Together, our results demonstrate that the synthetic VIU-1005 candidate DNA vaccine is highly immunogenic and capable of inducing long-lasting and Th1-skewed immune response in mice. Furthermore, we show that the use of needle-free system could enhance the immunogenicity and minimize doses needed to induce protective immunity in mice, supporting further preclinical and clinical testing of this candidate vaccine.


title: Metagenomic screening for lipolytic genes reveals an ecology-clustered distribution pattern

abstract: Lipolytic enzymes are one of the most important enzyme types for application in various industrial processes. Despite the continuously increasing demand, only a small portion of the so far encountered lipolytic enzymes exhibit adequate stability and activities for biotechnological applications. To explore novel and/or extremophilic lipolytic enzymes, microbial consortia in two composts at thermophilic stage were analyzed using function-driven and sequence-based metagenomic approaches. Analysis of community composition by amplicon-based 16S rRNA genes and transcripts, and direct metagenome sequencing revealed that the communities of the compost samples were dominated by members of the phyla Actinobacteria, Proteobacteria, Firmicutes, Bacteroidetes and Chloroflexi. Function-driven screening of the metagenomic libraries constructed from the two samples yielded 115 unique genes encoding lipolytic enzymes. The family assignment of these enzymes was conducted by analyzing the phylogenetic relationship and generation of a protein sequence similarity network according to an integral classification system. The sequence-based screening was performed by using a newly developed database, containing a set of profile Hidden Markov models, highly sensitive and specific for detection of lipolytic enzymes. By comparing the lipolytic enzymes identified through both approaches, we demonstrated that the activity-directed complements sequence-based detection, and vice versa. The sequence-based comparative analysis of lipolytic genes regarding diversity, function and taxonomic origin derived from 175 metagenomes indicated significant differences between habitats. Analysis of the prevalent and distinct microbial groups providing the lipolytic genes revealed characteristic patterns and groups driven by ecological factors. The here presented data suggests that the diversity and distribution of lipolytic genes in metagenomes of various habitats are largely constrained by ecological factors.


title: The conserved serine transporter SdaC moonlights to enable self recognition

abstract:
Cells can use self recognition to achieve cooperative behaviors. Self-recognition genes principally evolve in tandem with partner self-recognition alleles. However, other constraints on protein evolution could exist. Here, we have identified an interaction outside of self-recognition loci that could constrain the sequence variation of a self-recognition protein. We show that during collective swarm expansion in Proteus mirabilis, self-recognition signaling co-opts SdaC, a serine transporter. Serine uptake is crucial for bacterial survival and colonization. Single-residue variants of SdaC reveal that self recognition requires an open conformation of the protein; serine transport is dispensable. A distant ortholog from Escherichia coli is sufficient for self recognition; however, a homologous serine transporter, YhaO, is not. Thus, SdaC couples self recognition and serine transport, likely through a shared molecular interface. Understanding molecular and ecological constraints on self-recognition proteins can provide insights into the evolution of self recognition and emergent collective behaviors.

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title: Genome-scale metabolic modelling of P. thermoglucosidasius NCIMB 11955 reveals metabolic bottlenecks in anaerobic metabolism.

abstract: Parageobacillus thermoglucosidasius represents a thermophilic, facultative anaerobic bacterial chassis, with several desirable traits for metabolic engineering and industrial production. To further optimize strain productivity, a systems level understanding of its metabolism is needed, which can be facilitated by a genome-scale metabolic model. Here, we present p-thermo, the most complete, curated and validated genome-scale model (to date) of Parageobacillus thermoglucosidasius NCIMB 11955. It spans a total of 890 metabolites, 1175 reactions and 917 metabolic genes, forming an extensive knowledge base for P. thermoglucosidasius NCIMB 11955 metabolism. The model accurately predicts aerobic utilization of 22 carbon sources, and the predictive quality of internal fluxes was validated with previously published 13C-fluxomics data. In an application case, p-thermo was used to facilitate more in-depth analysis of reported metabolic engineering efforts, giving additional insight into fermentative metabolism. Finally, p-thermo was used to resolve a previously uncharacterised bottleneck in anaerobic metabolism, by identifying the minimal required supplemented nutrients (thiamin, biotin and iron(III)) needed to sustain anaerobic growth. This highlights the usefulness of p-thermo for guiding the generation of experimental hypotheses and for facilitating data-driven metabolic engineering, expanding the use of P. thermoglucosidasius as a high yield production platform.


title: CSF metabolites associate with CSF tau and improve prediction of Alzheimer's disease status

abstract:
INTRODUCTIONCerebrospinal fluid (CSF) total tau (t-tau) and phosphorylated tau (p-tau) are biomarkers of Alzheimers disease (AD), yet much is unknown about AD-associated changes in tau metabolism and tau tangle etiology.

METHODSWe assessed the variation of t-tau and p-tau explained by 38 previously identified CSF metabolites using linear regression models in middle-age controls from the Wisconsin Alzheimers Disease Research Center, and predicted AD/mild cognitive impairment (MCI) vs. an independent set of older controls using metabolites selected by the least absolute shrinkage and selection operator (LASSO).

RESULTSThe 38 CSF metabolites explained 70.3% and 75.7% of the variance in t-tau and p-tau. Of these, 7 LASSO-selected metabolites improved the prediction ability of AD/MCI vs. older controls (AUC score increased from 0.92 to 0.97 and 0.78 to 0.93) compared to the base model.

DISCUSSIONThese tau-correlated CSF metabolites increase AD/MCI prediction accuracy and may provide insight into tau tangle etiology.

category: molecular biology


title: Arylsulfonamide mediated RBM39 degradation causes aberrant splicing of mitotic kinesins

abstract: Certain arylsulfonamides (ArSulfs) induce an interaction between the E3 ligase substrate adaptor DCAF15 and the critical splicing factor RBM39, ultimately causing its degradation. Although molecules like the ArSulfs, which interfere with splicing decisions, are exciting potential medicines, the molecular glue mechanism of RBM39 degradation introduces complex pleiotropic effects that are difficult to untangle. For example, DCAF15 inhibition, RBM39 degradation, and the downstream proteome effects of splicing changes will all cause different yet overlaid effects. As such the precise cell-killing mechanism by RBM39 loss is largely unknown. By overlaying transcriptome and proteome datasets, we distinguish transcriptional from post-transcriptional effects, pinpointing those proteins most impacted by splicing changes. Our proteomic profiling of several ArSulfs suggests a selective DCAF15/ArylSulf/RBM39RRM2 interaction with a narrow degradation profile. We identify two mitotic kinesin motor proteins that are aberrantly spliced upon RBM39 degradation, and we demonstrate that these are likely contributors to the antiproliferative activity of ArSulfs.


title: Mitochondrial energy dysfunction induces remodeling of the cardiac mitochondrial protein acylome

abstract: Mitochondria are increasingly recognized as signaling organelles because, under conditions of stress, mitochondria can trigger various signaling pathways to coordinate the cells response. The specific pathway(s) engaged by mitochondria in response to defects in mitochondrial energy production in vivo and in high-energy tissues like the heart are not fully understood. Here, we investigated cardiac pathways activated in response to mitochondrial energy dysfunction by studying mice with cardiomyocyte-specific loss of the mitochondrial phosphate carrier (SLC25A3), an established model that develops cardiomyopathy as a result of defective mitochondrial ATP synthesis. In heart tissue from these mice, mitochondrial energy dysfunction induced a striking pattern of acylome remodeling, with significantly increased post-translational acetylation and malonylation. Mass spectrometry-based proteomics further revealed that energy dysfunction-induced remodeling of the acetylome and malonylome preferentially impacts mitochondrial proteins. Acetylation and malonylation modified a highly interconnected interactome of mitochondrial proteins, and both modifications were present on the enzyme isocitrate dehydrogenase 2 (IDH2). Intriguingly, IDH2 activity was enhanced in SLC25A3-deleted mitochondria, and further study of IDH2 sites targeted by both acetylation and malonylation revealed that these modifications can have site-specific and distinct functional effects. Finally, we uncovered a novel crosstalk between the two modifications, whereby mitochondrial energy dysfunction-induced acetylation of sirtuin 5 (SIRT5), inhibited its function. Because SIRT5 is a mitochondrial deacylase with demalonylase activity, this finding suggests that acetylation can modulate the malonylome. Together, our results position acylations as an arm of the mitochondrial response to energy dysfunction and suggest a mechanism by which focal disruption to the mitochondrial energy production machinery can have an expanded impact on global mitochondrial function.


title: Viral evolutionary dynamics predict Influenza-Like-Illnesses in patients

abstract: Viral infections such as those caused by the influenza virus can put a strain on healthcare systems. However, such a burden is typically difficult to predict. In order to improve such predictions, we hypothesize that the severity of epidemics can be linked to viral evolutionary dynamics. More specifically, we posit the existence of a negative association between patients health and the stability of coevolutionary networks at key viral proteins. To test this, we performed a thorough evolutionary analysis of influenza viruses circulating in continental US between 2010 and 2019, assessing how measures of the stability of these coevolutionary networks correlate with clinical data based on outpatient healthcare visits showing Influenza-Like Illness (ILI) symptoms. We first show evidence of a significant correlation between viral evolutionary dynamics and increased influenza activity during seasonal epidemics, and then show that these dynamics closely follow the progression of epidemics through each season, providing us with predictive power based on genetic data collected between week 20 and week 40/52, that is one to fifteen weeks prior to peak ILI. Viral evolutionary dynamics may hence be used by health authorities to further guide non-pharmaceutical interventions.

category: evolutionary biology


title: Independent origins of the Golgi phosphoprotein 3 oncoprotein gene

abstract: Golgi phosphoprotein 3 (GOLPH3) was the first reported oncoprotein of the Golgi apparatus. It was identified as an evolutionarily conserved protein upon its discovery about 20 years ago, but its function remains puzzling in normal and cancer cells. The GOLPH3 gene is part of a group of genes that also includes the GOLPH3L gene. Because cancer has deep roots in multicellular evolution, studying the evolution of the GOLPH3 gene family in non-model species represents an opportunity to identify new model systems that could help better understand the biology behind this group of genes. The main goal of this study is to explore the evolution of the GOLPH3 gene family in birds as a starting point to understand the evolutionary history of this oncoprotein. We identified a repertoire of three GOLPH3 genes in birds. We found duplicated copies of the GOLPH3 gene in all main groups of birds other than paleognaths, and a single copy of the GOLPH3L gene. We suggest there were at least three independent origins for GOLPH3 duplicates. Amino acid divergence estimates show that most of the variation is located in the N-terminal region of the protein. Our transcript abundance estimations show that one paralog is highly and ubiquitously expressed, and the others were variable. Our results are an example of the significance of understanding the evolution of the GOLPH3 gene family, especially for unraveling its structural and functional attributes.


title: Independent duplications of the Golgi phosphoprotein 3 oncogene in birds


title: Non-essential ribosomal proteins in bacteria and archaea identified using COGs

abstract:
Ribosomal proteins (RPs) are highly conserved across the bacterial and archaeal domains. Although many RPs are essential for survival, genome analysis demonstrates the absence of some RP genes in many bacterial and archaeal genomes. Furthermore, global transposon mutagenesis and/or targeted deletion showed that elimination of some RP genes had only a moderate effect on the bacterial growth rate. Here, we systematically analyze the evolutionary conservation of RPs in prokaryotes by compiling the list of the ribosomal genes that are missing from the one or more genomes in the recently updated version of the Clusters of Orthologous Genes (COG) database. Some of these absences occurred because the respective genes carried frameshifts, presumably, resulting from sequencing errors, while others were overlooked and not translated during genome annotation. Apart from these annotation errors, we identified multiple genuine losses of RP genes in a variety of bacteria and archaea. Some of these losses are clade-specific, whereas others occur in symbionts and parasites with dramatically reduced genomes. The lists of computationally and experimentally defined non-essential ribosomal genes show a substantial overlap, revealing a common trend in prokaryote ribosome evolution that could be linked to the architecture and assembly of the ribosomes. Thus, RPs that are located at the surface of the ribosome and/or are incorporated at a late stage of ribosome assembly are more likely to be non-essential and to be lost during microbial evolution, particularly, in the course of genome compaction.

IMPORTANCEIn many prokaryote genomes, one or more ribosomal protein (RP) genes are missing. Analysis of 1,309 prokaryote genomes included in the COG database shows that only about half of the RPs are universally conserved in bacteria and archaea. In contrast, up to 21 other RPs are missing in some genomes, primarily, tiny (<1 Mb) genomes of host-associated bacteria and archaea. Ten universal and nine archaea-specific ribosomal proteins show clear patterns of lineage-specific gene loss. Most of the RPs that are frequently lost from bacterial genomes are located on the ribosome periphery and are non-essential in Escherichia coli and Bacillus subtilis. These results reveal general trends and common constraints in the architecture and evolution of ribosomes in prokaryotes.


title: Effect of Silicon Application on Leaf mineral Composition of Eucalyptus

abstract: Eucalyptus is a commonly planted tree in North India and its cultivation area is increasing. Leaves of Eucalyptus are used for tea which has healing properties and many other economic uses. Silicon is the second most abundant element in the earths crust next to oxygen. Silicon (Si) can improve photosynthesis, decrease transpiration, increase water use efficiency and provide resistance against biotic and abiotic stress. The clear information was not available regarding Silicon nourishment in tree species like Eucalyptus. Although a positive in plant development and overall dry matter production was noticed in Eucalyptus with Si application. The amount of nitrogen (N), phosphorus (P), and potassium (K) were inclined leaf whereas Calcium (Ca) and magnesium (Mg) content was not affected by Si application potassium silicate (K2SiO3). Finally results revealed that upon Si application significant increase in the leaf mineral content in almost all genotypes was recorded. Correlation studies revealed that leaf N and P were highly and significantly correlated with leaf copper (Cu) and iron (Fe) content, leaf Mg and leaf Cu were positively and significantly correlated with leaf N, manganese (Mn) and Fe content, leaf Mn and Fe content were highly and significantly correlated with leaf N, Mg and Cu content. To our detailed knowledge this is the first ever study conducted in Eucalyptus. Here the influence of potassium silicate on popular North Indian cultivars leaf mineral content Nevertheless, more studies are required to analyze the effect of Si application in tree species like Eucalyptus.


title: SCOPE: Flexible targeting and stringent CARF activation enables type III CRISPR-Cas diagnostics

abstract: Characteristic properties of type III CRISPR-Cas systems include recognition of target RNA (rather than DNA) and the subsequent induction of a multifaceted immune response. This involves sequence-specific cleavage of a target RNA and production of cyclic oligoadenylate (cOA) second messenger molecules that may trigger dormancy or cell death. In this study, we discovered that a largely exposed seed region at the 3 end of the crRNA is essential for target RNA binding and cleavage, whereas base pairing at a unique region at the 5 end of the guide is required to trigger cOA production. Moreover, we uncovered that the natural variation in the composition of type III complexes within a single host results in different guide lengths, and hence variable seed regions. This shifting seed may prevent escape by invading genetic elements, while controlling cOA production very tightly to prevent unnecessary damage to the host. Lastly, we used these findings to develop a new diagnostic tool, named SCOPE, which was used for the specific detection of SARS-CoV-2 from human nasal swab samples, showing sensitivities in the atto-molar range.


title: Deep learning integration of molecular and interactome data for protein-compound interaction prediction

abstract:
MotivationVirtual screening, which can computationally predict the presence or absence of protein-compound interactions, has attracted attention as a large-scale, low-cost, and short-term search method for seed compounds. Existing machine learning methods for predicting protein-compound interactions are largely divided into those based on molecular structure data and those based on network data. The former utilize information on proteins and compounds, such as amino acid sequences and chemical structures, while the latter utilize interaction network data, such as data on protein-protein interactions and compound-compound interactions. However, few attempts have been made to combine both types of data in molecular information and interaction networks.

ResultsWe developed a deep learning-based method that integrates protein features, compound features, and multiple types of interactome data to predict protein-compound interactions. We designed three benchmark datasets with different difficulties and evaluated the performance on them. The performance evaluations show that our deep learning framework for integrating molecular structure data and interactome data outperforms state-of-the-art machine learning methods for protein-compound interaction prediction tasks. The performance improvement is proven to be statistically significant by the Wilcoxon signed-rank test. This reveals that the multi-interactome captures different perspectives than amino acid sequence homology and chemical structure similarity, and both type of data have a synergistic effect in improving prediction accuracy. Furthermore, experiments on three benchmark datasets show that our method is more robust than existing methods in accurately predicting interactions between proteins and compounds that are unseen in the training samples.


title: Clearing spheroids for 3D fluorescent microscopy:combining safe and soft chemicals with deep convolutional neural network

abstract: In life sciences, there are increasing interest in 3D culture models to better reproduce the 3D environment encountered in-vivo. Imaging of such 3D culture models is instrumental for drug discovery, but face several issues before its use becomes widespread. Extensive microscopic investigation of these 3D cell models faces the challenge of light penetration in depth in opaque biological tissues. To overcome this limit, diverse clearing techniques have emerged over the past decades. However, it is not straightforward to choose the best clearing protocols, and assess quantitatively their clearing efficiency. Focusing on spheroids, we propose a combination of fast and cost-effective clearing procedure for such medium-sized samples. A generic method with local contrast metrics and deep convolutional neural network-based segmentation of nuclei is proposed to quantify the efficiency of clearing. We challenged this method by testing the possibility to transfer segmentation knowledge from a clearing protocol to another. The later results support the pertinence of training deep learning algorithms on cleared samples to further use the segmentation pipeline on non-cleared ones. This second step of the protocol gives access to digital clearing possibilities applicable to live and high-throughput optical imaging.


title: Crystal structure determination of the armadillo repeat domain of Drosophila SARM1 using MIRAS phasing

abstract: We describe the crystal structure determination of the ARM domain of Drosophila SARM1 (dSARM1ARM), which required combination of a number of sources of phase information in order to obtain interpretable electron density maps. SARM1 is a central executioner of the process of axon degeneration, a common feature of the early phase of a range of neurodegenerative diseases. SARM1 is held in the inactive state in healthy axons by its N-terminal auto-inhibitory ARM domain, and is activated to cleave NAD+ upon injury, triggering the subsequent axon degeneration. To characterize the molecular mechanism of SARM1 activation, we sought to determine the crystal structure of the SARM1 ARM domain. Here we describe the recombinant production and crystallization of dSARM1ARM, as well as unconventional process used for structure determination. Crystals were obtained in the presence of NMN, a precursor of NAD+ and a potential activator of SARM1, only after in situ proteolysis of the N-terminal 63 residues. After molecular replacement attempts failed, we determined the crystal structure of dSARM1ARM at 1.65 [A] resolution using the MIRAS phasing technique with the program autoSHARP, combining data from the native, SeMet-labelled, and Br-soaked crystals. The structure will further our understanding of the regulation of SARM1.


title: Single cell analysis of population-wide nuclear and cytosolic drug responses using high-content FRET imaging: measuring protein kinase activation in rat primary striatal neurons

abstract: Genetically-encoded biosensors are used to track biochemical activities in living cells by measuring changes in fluorescence emitted by one or more fluorescent proteins. In the present article, we describe the application of genetically-encoded FRET biosensors with high content microscopy to image the signaling responses of thousands of neurons in response to drug treatments. We applied this approach to reveal intercellular variation in signaling responses among cultured striatal neurons stimulated with multiple drugs. The striatum is largely composed of medium-spiny GABAergic neurons which are divided into two broad sub-types based in part on their expression of dopamine D1 vs. D2 receptors. Using high content FRET imaging and immunofluorescence, we identified neuronal sub-populations with unique responses to pharmacological manipulation. Focusing on dopamine- and glutamate-regulated PKA and ERK1/2 signaling in both the cytoplasm and nucleus, we identified pronounced intercellular differences, in both the magnitude and kinetics of signaling responses to drug application. Importantly, we found that a conventional "bulk" analysis that included all cells in culture yielded a different rank order of drug potency than that revealed by our single-cell analysis. The high degree of heterogeneity that we observed at the single cell level would not have been detectable using common population-level analyses, derived for example from western blotting or plate reader-based measurements. In conclusion, our single-cell analytical approach highlights the limitations of population-level analyses, and provides a novel way to study signaling biology.


title: Neuronal morphology and physiology, functional brain networks and memory in temporal lobe epilepsy

abstract: Temporal lobe epilepsy (TLE) patients are at risk of memory deficits, which have been linked to functional network disturbances, particularly of integration of the default mode network (DMN). However, the cellular substrates of functional network integration are unknown. We leverage a unique cross-scale dataset of therapy-resistant TLE patients, who underwent fMRI, MEG and/or neuropsychological testing before neurosurgery. fMRI and MEG underwent atlas-based connectivity analyses. Functional network centrality of the lateral middle temporal gyrus, part of the DMN, was used as a measure of local network integration. Subsequently, non-pathological cortical tissue from this region was used for single cell morphological and electrophysiological patch-clamp analysis, assessing integration in terms of total dendritic length and action potential rise speed. As could be hypothesized, greater network centrality related to better memory performance. Moreover, greater network centrality correlated with more integrative properties at the cellular level across patients. We conclude that individual differences in cognitively relevant functional network integration of a DMN region are mirrored by differences in cellular integrative properties of this region in TLE patients. These findings connect previously separate scales of investigation, increasing translational insight into focal pathology and large-scale network disturbances in TLE.


title: Half-life of biodegradable plastics in the marine environment depends on material, habitat, and climate zone

abstract: The performance of the biodegradable plastic materials polyhydroxyalkanoate (PHA), polybutylene sebacate (PBSe) and polybutylene sebacate co-terephthalate (PBSeT), and of polyethylene (LDPE) was assessed under marine environmental conditions in a three-tier approach. Biodegradation lab tests (20 {degrees}C) were complemented by mesocosm tests (20 {degrees}C) with natural sand and seawater and by field tests in the warm-temperate Mediterranean Sea (12 - 30 {degrees}C) and in tropical Southeast Asia (29 {degrees}C) in three typical coastal scenarios. Plastic film samples were exposed in the eulittoral beach, the pelagic open water and the benthic seafloor and their disintegration monitored over time. We used statistical modelling to predict the half-life for each of the materials under the different environmental conditions to render the experimental results numerically comparable across all experimental conditions applied. The biodegradation performance of the materials differed by orders of magnitude depending on climate, habitat and material and revealed the inaccuracy to generically term a material  marine biodegradable. The half-life t0.5 of a film of PHA with 85 m thickness ranged from 54 d on the seafloor in SE Asia to 1247 d in mesocosm pelagic tests. t0.5 for PBSe (25 m) ranged from 99 d in benthic SE Asia to 2614 d in mesocosm benthic tests, and for PBSeT t0.5 ranged from 147 d in the mesocosm eulittoral to 797 d in Mediterranean benthic field tests. For LDPE no biodegradation could be observed. These data can now be used to estimate the persistence of plastic objects should they end up in the marine environments considered here and will help to inform the life cycle (impact) assessment of plastics in the open environment.


title: An unusual aspartic acid cluster in the reovirus attachment fiber σ1 mediates stability at low pH

abstract:
The reovirus attachment protein {sigma}1 mediates cell attachment and receptor binding and is thought to undergo conformational changes during viral disassembly. {sigma}1 is a trimeric filamentous protein with an -helical coil-coiled Tail, a triple {beta}-spiral Body, and a globular Head. The Head domain features an unusual and conserved aspartic acid cluster at the trimer interface, which forms the only significant intra-trimer interactions in the Head, and must be protonated to allow trimer formation.

Here we show that all domains of {sigma}1 are remarkably thermostable across a wide range of pH, even at the low pH of the stomach. Interestingly, we determine the optimal pH for stability to be between pH 5-6, a value close to the pH of the endosome and of the jejunum. The {sigma}1 Head is stable at acidic and neutral pH, but detrimerizes at basic pH. When Asp345 in the aspartic acid cluster is mutated to asparagine, the {sigma}1 Head loses stability at low pH and is more prone to detrimerize. Overall, the presence of the Body stabilizes the {sigma}1 Head.

Our results confirm a role of the aspartic acid cluster as a pH-dependent molecular switch, and highlight its role in enhancing {sigma}1 stability at low pH.


title: A topological characterization of DNA sequences based on chaos geometry and persistent homology

abstract: Methods for analyzing similarities among DNA sequences play a fundamental role in computational biology, and have a variety of applications in public health, and in the field of genetics. In this paper, a novel geometric and topological method for analyzing similarities among DNA sequences is developed, based on persistent homology from algebraic topology, in combination with chaos geometry in 4-dimensional space as a graphical representation of DNA sequences. Our topological framework for DNA similarity analysis is general, alignment-free, and can deal with DNA sequences of various lengths, while proving first-of-the-kind visualization features for visual inspection of DNA sequences directly, based on topological features of point clouds that represent DNA sequences. As an application, we test our methods on three datasets including genome sequences of different types of Hantavirus, Influenza A viruses, and Human Papillomavirus.


title: Hedgehog pathway inhibitors significantly reduce the formation of heterotopic ossification in a direct trauma/burn mouse model

abstract: Heterotopic ossification (HO), either acquired or hereditary, is featured by ectopic bone formation outside of the normal skeleton. The acquired form of HO is a debilitating and common complication of musculoskeletal trauma, central nervous system injury, burns, combat trauma, hip and elbow fractures, and total joint replacement surgeries. It can be characterized as abnormal bone formation that occurs mostly by endochondral ossification. Recent studies have implicated inflammation and dysregulation of Hedgehog (Hh) signaling as major early contributors to HO formation. Here, we demonstrate that administration of the Hh pathway inhibitor, arsenic trioxide (ATO), prevented acquired HO in a clinically-relevant trauma/burn mouse model. We further evaluated the effects of two additional Hh pathway antagonists: cholecalciferol and pravastatin on mitigating osteoblast differentiation. Finally, we assessed the effect of a combination of Hh pathway inhibitors on reducing systemic proinflammatory responses. A targeted combination approach using Hh pathway inhibitors may offer potential therapeutic benefits though targeting differential components of the Hh pathway. Taken together, our study demonstrates that the administration of single or multiple Hh pathway inhibitors may have the potential to reduce the formation of acquired HO.

category: pharmacology and toxicology


title: Multiplexed activation in mammalian cells using dFnCas12a-VPR

abstract: The adoption of CRISPR systems for the generation of synthetic transcription factors has greatly simplified the process for upregulating endogenous gene expression, with a plethora of applications in cell biology, bioproduction and cell reprogramming. In particular the recently discovered Cas12a systems offer extended potential, as Cas12a is capable of processing its own crRNA array to provide multiple individual crRNAs for subsequent targeting from a single transcript. Here we show the application of dFnCas12a-VPR in mammalian cells, with FnCas12a possessing a shorter PAM sequence than As or Lb variants, enabling denser targeting of genomic loci. We observe that synergistic activation and multiplexing can be achieved using crRNA arrays but also show that crRNAs expressed towards the 5 of 6-crRNA arrays show evidence of enhanced activity. This not only represents a more flexible tool for transcriptional modulation but further expands our understanding of the design capabilities and limitations when considering longer crRNA arrays for multiplexed targeting.

category: synthetic biology


title: In vitro inactivation of SARS-CoV-2 with 0.5% povidone iodine nasal spray (Nasodine) at clinically relevant concentrations and timeframes using tissue culture and PCR based assays

abstract:
BACKGROUNDThere has been considerable speculation regarding the potential of PVP-I nasal disinfection as an adjunct to other countermeasures during the ongoing SARS-CoV-2 pandemic. Nasodine is a commercial formulation of 0.5% PVP-I that has been evaluated for safety and efficacy in human trials as a treatment for the common cold, including a Phase III trial (ANZCTR: ACTRN12619000764134). This study presents the first report of the in vitro efficacy of this formulation against SARS-CoV-2.

METHODSWe conducted in vitro experiments to determine if the PVP-I formulation inactivated SARS-CoV-2 using two independent assays and virus isolates, and incorporating both PCR-based detection and cell culture methods to assess residual virus after exposure to the formulation.

RESULTSBased on cell culture results, the PVP-I formulation was found to rapidly inactivate SARS-CoV-2 isolates in vitro in short timeframes (15 seconds to 15 minutes) consistent with the minimum and maximum potential residence time in the nose. The Nasodine formula was found to be more effective than 0.5% PVP-I in saline. Importantly, it was found that the formulation inactivated culturable virus but had no effect on PCR-detectable viral RNA.

CONCLUSIONSThe PVP-I formulation eliminated the viability of SARS-CoV-2 virus with short exposure times consistent with nasal use. PCR alone may not be adequate for viral quantification in nasal PVP-I studies; future studies should incorporate cell culture to assess viral viability. Nasal disinfection with PVP-I may be a useful intervention for newly-diagnosed COVID-19 patients to reduce transmission risk and disease progression to the lower respiratory tract.


title: β-importins Tnpo-SR and Cadmus and the small GTPase Ran promote ovarian cyst formation in Drosophila

abstract:
Germ cells undergo mitotic expansion via incomplete cytokinesis, forming cysts of undifferentiated cells that remain interconnected prior to meiotic initiation, through mechanisms that are not well-defined. In somatic cells, Ras-related nuclear protein (Ran) spatiotemporally regulates mitotic spindle assembly, cleavage furrow formation and abscission. Here, we identify Ran and {beta}-importins as critical regulators of cyst development in the Drosophila ovary. Depletion of Ran or the {beta}-importins Tnpo-SR and cadmus disrupts oocyte selection and results in egg chambers with variable numbers of germ cells, suggesting abnormal cyst development and cyst fragmentation. We demonstrate that Ran, Tnpo-SR, and Cadmus regulate key cellular processes during cyst formation, including cell cycle dynamics, fusome biogenesis, and ring canal stability, yet do so independently of mitotic spindle assembly. Further, Tnpo-SR and Cadmus control cyclin accumulation and suppress cytokinesis independent of Ran-GTP, suggesting that {beta}-importins sequester protein cargos that normally promote the mitotic-to-meiotic transition. Our data demonstrates that Ran and {beta}-importins are critical for germ cell cyst formation, a role that is likely conserved in other organisms.

SUMMARY STATEMENTRan and two {beta}-importins function coordinately to promote oocyte selection and cyst development in the Drosophila ovary.

category: developmental biology