title: FAM114A1 Influences Cardiac Fibrosis by Regulating Angiotensin II Signaling in Cardiac Fibroblasts

abstract:
Cardiac fibrosis, a primary contributor to heart failure (HF) and sudden death, is considered as an important target for HF therapy. However, the signaling pathways that govern cardiac fibroblast (CF) function during cardiac fibrosis have not been fully elucidated. Here, we found that a functionally unannotated human myocardial infarction (MI) associated gene, family with sequence similarity 114 member A1 (FAM114A1), is induced in failing human and mouse hearts compared to non-failing hearts. Homozygous knockout of Fam114a1 (Fam114a1-/-) in the mouse genome reduces cardiac hypertrophy and fibrosis while significantly restores cardiac function in angiotensin (Ang) II- and MI-induced HF mouse models. Fam114a1 deletion antagonizes Ang II induced inflammation and oxidative stress. Using isolated mouse primary CFs in wild type and Fam114a1-/- mice, we found that FAM114A1 is a critical autonomous factor for CF proliferation, activation, and migration. We discovered that FAM114A1 interacts with angiotensin receptor associated protein (AGTRAP) and regulates the expression of angiotensin type 1 receptor (AT1R) and downstream Ang II signaling transduction, and subsequently influences pro-fibrotic response. Using RNA-Seq in mouse primary CFs, we identified differentially expressed genes including extracellular matrix proteins such as Adamts15. RNAi-mediated inactivation of Adamts15 attenuates CF activation and collagen deposition. Our results indicate that FAM114A1 regulates Ang II signaling and downstream pro-fibrotic and pro-inflammatory gene expression, thereby activating cardiac fibroblasts and augmenting pathological cardiac remodeling. These findings provide novel insights into regulation of cardiac fibrosis and identify FAM114A1 as a new therapeutic target for treatment of cardiac disease.

SignificanceCardiac fibrosis is a hallmark of heart failure and angiotensin II signaling promotes pro-fibrotic response in the heart. This study is a pioneering investigation of the role of a functionally unknown protein FAM114A1. We show that FAM114A1 expression is induced in human and mouse failing hearts. Genetic ablation of FAM114A1 can effectively reduce cardiac fibrosis and pathological remodeling. Isolated cardiac fibroblasts from Fam114a1 knockout mice show reduced response to Ang II stimulation and compromised myofibroblast activation. Mechanistically, FAM114A1 binds to AGTRAP and influences AT1R protein expression, thereby enhancing angiotensin II signaling and pro-fibrotic response. Thus, FAM114A1 is a novel factor that modulates cardiac fibrosis and pharmacological inhibition of FAM114A1 may be a therapeutic strategy for the treatment of heart disease.

category: pathology


title: De novo homology assessment from landmark data: A workflow to identify and track segmented structures in plant time series images

abstract: Assessing the phenotypes underlying plant growth and development is integral to exploring the development, genetics, and evolution of morphology and plays an essential role in agronomic and basic research studies. Although various automated or semi-automated phenomic approaches have recently been developed, tools assessing differential growth of plant organs remains a key topic of interest, but one which is often difficult to analyze due to the requirements of segmenting and annotating specific structures or positions in the plant body in time-series data. To address this gap, we have developed a generalized workflow linking our previously published function, acute, with a companion function, homology, in the PlantCV environment. The homology function uses a generalized strategy of dimensionality reduction via starscape followed by hierarchical clustering through constella to identify  constellations of segments in eigenspace that represent the same landmark in consecutive images of a time-series. We devised a quality control function, constellaQC, that can test the accuracy of the clustering approach, and we use it to show that the approach accurately clustered the pseudo-landmarks derived from acute, although with several sources of error. We discuss the reasons for and consequences of these errors in automated workflows, and suggest how to develop these functions so that they can easily be repurposed for other phenomics datasets that may vary in dimensional complexity.

category: plant biology


title: Insulin-dependent maturation of newly generated olfactory sensory neurons after injury

abstract:
Loss of olfactory sensory neurons (OSNs) after injury to the olfactory epithelium (OE) triggers the generation of OSNs that are incorporated into olfactory circuits to restore olfactory sensory perception. This study addresses how insulin receptor-mediated signaling affects the functional recovery of OSNs after OE injury. Insulin levels were reduced in mice by ablating the pancreatic beta cells via streptozotocin injections. These streptozotocin-induced diabetic and control mice were then intraperitoneally injected with the olfactotoxic drug methimazole to selectively ablate OSNs. The OE of diabetic and control mice regenerated similarly until day 14 after injury. Thereafter, the OE of diabetic mice contained fewer mature and more apoptotic OSNs than control mice. Functionally, diabetic mice showed reduced electro-olfactogram responses and their olfactory bulbs had fewer c-Fos-active cells following odor stimulation, as well as performed worse in an odor-guided task compared to control mice. Insulin administered intranasally during day 8 to 13 after injury was sufficient to rescue recovery of OSNs in diabetic mice compared to control levels, while insulin administration between days 1 - 6 did not. During this critical time window on day 8 - 13 after injury, insulin receptors are highly expressed and intranasal application of insulin receptor antagonist inhibits regeneration. Furthermore, an insulin-enriched environment could facilitate regeneration even in non-diabetic mice. These results indicate that insulin facilitates the regeneration of OSNs after injury and suggest a critical stage during recovery (8 - 13 days after injury) during which the maturation of newly generated OSNs is highly dependent on and promoted by insulin.

Significance StatementAlthough insulin receptor signaling is known to influence on cellular processes such as proliferation and apoptosis, it is poorly understood whether the insulin influences the regeneration of olfactory sensory neurons (OSNs) after injury. We compared the maturation processes of new OSNs after the methimazole-induced loss of pre-existing OSNs between diabetic and control mice. The results show that the regeneration of new OSNs depend on sufficient insulin levels during a specific temporal window, when insulin receptor expression is highly upregulated. Furthermore, an insulin-enriched environment via nasal insulin application during the critical period facilitates OSNs regeneration even in non-diabetic mice. The present results have implications for intranasal application of insulin as potential clinical therapeutics to facilitate OSNs regeneration after the injury.

category: neuroscience


title: Epidermal Green Autofluorescence is A Novel Biomarker for Local Inflammation of the Skin

abstract: Inflammation of the skin is not only a key pathological factor of multiple major skin diseases, but also a hallmark of the pathological state of the skin. However, there has been significant deficiency in the biomarkers and approaches for non-invasive evaluations of local inflammation of the skin. In our current study, we used a mouse model of skin inflammation to test our hypothesis that the inflammation of the skin can lead to increased epidermal green autofluorescence (AF), which can become a novel biomarker for non-invasive evaluations of the local inflammation of the skin. We found that 12-O-tetradecanoylphorbol-13-acetate (TPA), a widely used inducer of skin inflammation, induced not only inflammation of the skin, but also increased green AF of the skin. The distinct polyhedral structure of the increased AF has indicated that the AF originates from the keratin 1 and/or keratin 10 of the suprabasal keratinocytes. Our Western blot assays showed that TPA produced dose-dependent decreases in the levels of both keratin 1 and keratin 10, suggesting that TPA produced the increased epidermal green AF at least partially by inducing cleavage of keratin 1 and/or keratin 10. Collectively, our study has indicated that epidermal green AF is a novel biomarker for non-invasive evaluations of the local inflammation of the skin. This finding is of profound and extensive significance for non-invasive and efficient diagnosis of multiple inflammation-mediated skin diseases. This biomarker may also be used for non-invasive and rapid evaluations of the health state of the skin.

category: immunology


title: Precise topology of adjacent domain-general and sensory-biased regions in the human brain

abstract: Recent functional MRI studies identified sensory-biased regions across much of the association cortices and cerebellum. However, their anatomical relationship to multiple-demand (MD) regions, characterized as domain-general due to their co-activation during multiple cognitive demands, remains unclear. For a better anatomical delineation, we used multimodal MRI techniques of the Human Connectome Project to scan subjects performing visual and auditory versions of a working memory (WM) task. The contrast between hard and easy WM showed strong domain generality, with essentially identical patterns of cortical, subcortical and cerebellar MD activity for visual and auditory materials. In contrast, modality preferences were shown by contrasting easy WM with baseline; most MD regions showed visual preference while immediately adjacent to cortical MD regions, there were interleaved regions of both visual and auditory preference. The results may exemplify a general motif whereby domain-specific regions feed information into and out of an adjacent, integrative MD core.


title: Behavioural synchronization in a multilevel society of feral horses

abstract: AO_SCPLOWBSTRACTC_SCPLOWBehavioural synchrony among individuals is essential for group-living organisms. It is still largely unknown how synchronization functions in a multilevel society, which is a nested assemblage of multiple social levels between many individuals. Our aim was to build a model that explained the synchronization of activity in a multilevel society of feral horses. We used multi-agent based models based on four hypotheses: A) horses do not synchronize, B) horses synchronize with any individual in any unit, C) horses synchronize only within units and D) horses synchronize across and within units, but internal synchronization is stronger. Our empirical data obtained from drone observations best supported hypothesis D. This result suggests that animals in a multilevel society coordinate with other conspecifics not only within a unit but at an inter-unit level. In this case, inter-individual distances are much longer than those in most previous models which only considered local interaction within a few body lengths.

category: animal behavior and cognition


title: Quantitative profiling of axonal guidance proteins during the differentiation of human neurospheres

abstract: Axon guidance is required for the establishment of brain circuits. Whether much of the molecular basis of axon guidance is known from animal models, the molecular machinery coordinating axon growth and pathfinding in humans remains to be elucidated. The use of induced pluripotent stem cells (iPSC) from human donors has revolutionized in vitro studies of the human brain. iPSC can be differentiated into neuronal stem cells which can be used to generate neural tissue-like cultures, known as neurospheres, that reproduce, in many aspects, the cell types and molecules present in the brain. Here, we analyzed quantitative changes in the proteome of neurospheres during differentiation. Relative quantification was performed at early time points during differentiation using iTRAQ-based labeling and LC-MS/MS analysis. We identified 6,438 proteins, from which 433 were downregulated and 479 were upregulated during differentiation. We show that human neurospheres have a molecular profile that correlates to the fetal brain. During differentiation, upregulated pathways are related to neuronal development and differentiation, cell adhesion, and axonal guidance whereas cell proliferation pathways were downregulated. We developed a functional assay to check for neurite outgrowth in neurospheres and confirmed that neurite outgrowth potential is increased after 10 days of differentiation and is enhanced by increasing cyclic AMP levels. The proteins identified here represent a resource to monitor neurosphere differentiation and coupled to the neurite outgrowth assay can be used to functionally explore neurological disorders using human neurospheres as a model.


title: Restoration of BDNF-TrkB signaling rescues deficits in a mouse model of SCA6

abstract: Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disease resulting in motor coordination deficits and cerebellar pathology. Expression of brain-derived neurotrophic factor (BDNF) is reduced in several neurodegenerative diseases, including in post-mortem tissue from SCA6 patients. Here, we show that cerebellar BDNF levels are reduced at an early disease stage in a mouse model of SCA6 (SCA684Q/84Q). One month of voluntary exercise was sufficient to elevate BDNF expression, as well as rescue both motor coordination and cerebellar Purkinje cell firing rate deficits. A BDNF mimetic, 7,8-dihydroxyflavone (7,8-DHF) likewise improved motor coordination and reversed Purkinje cell firing rate deficits, suggesting that exercise acts via BDNF-TrkB signaling. Prolonged chronic 7,8-DHF administration rescued ataxia when treatment commenced near disease onset, but was ineffective when treatment was started late. These data suggest that 7,8-DHF, which is orally bioavailable and crosses the blood-brain barrier, is a promising therapeutic for SCA6 and argue for the importance of early intervention for SCA6.


title: Investigating the chemolithoautotrophic and formate metabolism of Nitrospira moscoviensis by constraint-based metabolic modeling and 13C-tracer analysis

abstract:
Nitrite-oxidizing bacteria belonging to the genus Nitrospira mediate a key step in nitrification and play important roles in the biogeochemical nitrogen cycle and wastewater treatment. While these organisms have recently been shown to exhibit metabolic flexibility beyond their chemolithoautotrophic lifestyle, including the use of simple organic compounds to fuel their energy metabolism, the metabolic networks controlling their autotrophic and mixotrophic growth remain poorly understood. Here, we reconstructed a genome-scale metabolic model for Nitrospira moscoviensis (iNmo686) and used constraint-based analysis to evaluate the metabolic networks controlling autotrophic and formatotrophic growth on nitrite and formate, respectively. Subsequently, proteomic analysis and 13C-tracer experiments with bicarbonate and formate coupled to metabolomic analysis were performed to experimentally validate model predictions. Our findings support that N. moscoviensis uses the reductive tricarboxylic acid cycle for CO2 fixation. We also show that N. moscoviensis can indirectly use formate as a carbon source by oxidizing it first to CO2 followed by reassimilation, rather than direct incorporation via the reductive glycine pathway. Our study offers the first measurements of Nitrospiras in vivo central carbon metabolism and provides a quantitative tool that can be used for understanding and predicting their metabolic processes.

ImportanceNitrospira are globally abundant nitrifying bacteria in soil and aquatic ecosystems and wastewater treatment plants, where they control the oxidation of nitrite to nitrate. Despite their critical contribution to nitrogen cycling across diverse environments, detailed understanding of their metabolic network and prediction of their function under different environmental conditions remains a major challenge. Here, we provide the first constraint-based metabolic model of N. moscoviensis representing the ubiquitous Nitrospira lineage II and subsequently validate this model using proteomics and 13C-tracers combined with intracellular metabolomic analysis. The resulting genome-scale model will serve as a knowledge base of Nitrospira metabolism and lays the foundation for quantitative systems biology studies of these globally important nitrite- oxidizing bacteria.

category: microbiology


title: Multi-omics reveal intricate network of mitochondrial adaptations to training in human skeletal muscle

abstract: Mitochondrial defects are implicated in multiple diseases and aging. Exercise training is an accessible and inexpensive therapeutic intervention improving mitochondrial bioenergetics and quality of life. By combining a multi-omics approach with biochemical and in silico normalization, we removed the bias arising from the training-induced increase in human skeletal muscle mitochondrial content to unearth an intricate and previously undemonstrated network of differentially prioritized mitochondrial adaptations. We show that changes in hundreds of transcripts, proteins, and lipids are not stoichiometrically linked to the increase in mitochondrial content. We demonstrate that enhancing electron flow to oxidative phosphorylation (OXPHOS) is more important to improve ATP generation than increasing the abundance of the OXPHOS machinery, and that training-induced supercomplex formation does not confer enhancements in mitochondrial bioenergetics. Our study provides a new analytical approach allowing unbiased and in-depth investigations of training-induced mitochondrial adaptations, challenging our current understanding and calling for careful reinterpretation of previous findings.

category: cell biology


title: Training-induced bioenergetic improvement in human skeletal muscle is associated with non-stoichiometric changes in the mitochondrial proteome without reorganization of respiratory chain content


title: QTLs for potato tuber resistance to Dickeya solani are located on chromosomes II and IV

abstract: Soft rot is a bacterial disease that causes heavy losses in potato production worldwide. The goal of this study was to identify quantitative trait loci (QTLs) for potato tuber resistance to bacterium Dickeya solani and for tuber starch content to study the relationship between these traits. A highly resistant diploid hybrid of potato was crossed with a susceptible hybrid to generate the F1 mapping population. Tubers that were wound-inoculated with bacteria were evaluated for disease severity expressed as the mean weight of rotted tubers, and disease incidence measured as the proportion of rotten tubers. Diversity array technology (DArTseq) was used for genetic map construction and QTLs analysis. The most prominent QTLs for disease severity and incidence were identified in overlapping regions on potato chromosome IV and explained 22.4% and 22.9% of the phenotypic variance, respectively. The second QTL for disease severity was mapped to chromosome II and explained 16.5% of the variance. QTLs for starch content were detected on chromosomes III, V, VI, VII, VIII, IX, XI, and XII in regions different than the QTLs for soft rot resistance. Two strong and reproducible QTLs for resistance to Dickeya solani on potato chromosomes IV and II might be useful for further study of candidate genes and marker development in potato breeding programs. The relationship between tuber resistance to bacteria and the starch content in potato tubers was not confirmed by QTL mapping, which makes the selection of genotypes highly resistant to soft rot with a desirable starch content feasible.

category: genetics


title: Structural basis of RICs iron donation for iron-sulfur cluster biogenesis

abstract:
Escherichia coli YtfE is a di-iron protein, of the widespread RIC family, with capacity to donate iron, which is a crucial component of the biogenesis of the ubiquitous family of iron-sulfur proteins. Herein we identify in E. coli a previously unrecognized link between the YtfE protein and the major bacterial system for iron-sulfur cluster (ISC) assembly. We show that YtfE establishes protein-protein interactions with the scaffold IscU, where the transient cluster is formed, and the cysteine desulfurase IscS. Moreover, we found that promotion by YtfE of the formation of an Fe-S cluster in IscU requires two glutamates, E125 and E159 in YtfE. Both glutamates form part of the entrance of a protein channel in YtfE that links the di-iron centre to the surface. In particular, E125 is crucial for the exit of iron, as a single mutation to leucine closes the channel rendering YtfE inactive for the build-up of Fe-S clusters. Hence, we provide evidence for the key role of RICs as bacterial iron donor proteins involved in the biogenesis of Fe-S clusters.

ImportanceThe ubiquitous iron-sulfur proteins require specialized cellular machineries to promote the assembly of the cofactor. These systems include proteins that provide sulfur and iron, and scaffold proteins where the cluster is formed. Although largely studied the nature of the iron donor remains to be fully clarified. In this work, we show that Escherichia coli YtfE, which belongs to the RIC protein family, establishes protein-protein interactions with two of the major proteins of the ISC system, and we reveal the structural characteristics necessary for the exit of iron ions from YtfE. Altogether our results prove that RICs can be considered a family of iron donor proteins involved in the biogenesis of iron-sulfur containing proteins.

category: biochemistry


title: Comprehensive Assessment of Somatic Copy Number Variation Calling Using Next-Generation Sequencing Data

abstract: Copy number variation (CNV) is a common type of mutation that often drives cancer progression. With advances in next-generation sequencing (NGS), CNVs can be detected in a detailed manner via newly developed computational tools but quality of such CNV calls has not been carefully evaluated. We analyzed CNV calls reported by 6 cutting-edge callers for 91 samples which were derived from the same cancer cell line, prepared and sequenced by varying the following factors: type of tissue sample (Fresh vs. Formalin Fixed Paraffin Embedded (FFPE)), library DNA amount, tumor purity, sequencing platform (Whole-Genome Sequencing (WGS) versus Whole-Exome Sequencing (WES)), and sequencing coverage. We found that callers greatly determined the pattern of CNV calls. Calling quality was drastically impaired by low purity (<50%) and became variable when WES, FFPE, and medium purity (50%-75%) were applied. Effects of low DNA amount and low coverage were relatively minor. Our analysis demonstrates the limitation of benchmarking somatic CNV callers when the real ground truth is not available. Our comprehensive analysis has further characterized each caller with respect to confounding factors and examined the consistency of CNV calls, thereby providing guidelines for conducting somatic CNV analysis.

category: bioinformatics


title: Time-restricted feeding near dawn entrains long-term behavioral changes through the suprachiasmatic nucleus

abstract: The suprachiasmatic nucleus (SCN) is a master circadian pacemaker known to integrate light intensity and seasonal information with peripheral tissues to coordinate daily rhythms of physiology and behavior. However, the contribution of food information to the regulation of the SCN network remains controversial. Here, we identified the effect induced by time-restricted feeding (TRF) at dawn, but not at another time widow, inducing a robust and long-term shift in locomotor behavior and increased wakefulness. Comparing the oscillations of intracellular Ca2+ signals in the SCN GABAergic neurons of freely moving mice, before and after TRF, revealed significant activation of these neurons in dawn-TRF mice. Moreover, RNA-seq profiling in the dawn TRF-induced behavioral changes identified altered expressed genes involved in regulating extracellular exosome, ion transporters, and ECM-receptor interaction, but not core clock genes. Furthermore, injection in the SCN of insulin-like growth factor (IGF2) inhibitor Chromeceptin, targeting the most upregulated gene in extracellular exosome, abolished the after effect induced by ZT0-4 TRF. Finally, GABAergic-neuron-specific disruption of the potassium-chloride cotransporter Kcc2 intensified the dawn TRF-induced after effect, indicating that Kcc2 encodes food intake derived signals that control SCN clock entrainment. Thus, our study functionally links SCN GABAergic neuron activity and central clock entrainment regulation to both hunger- and food-response-related behaviors in mice.

category: physiology


title: The aversive value of pain in human decision-making

abstract: According to basic utilitarian principles, people should try to maximize rewards and minimize pain. Here, participants were put in a situation where monetary rewards were paired to electric shocks spanning between pain detection and tolerance thresholds. Monetary offers ranged linearly from $0 to $5 or $10 in Group1 and 2, respectively, and exponentially from $0 to $5 in Group3. The value of pain increased quadratically as a function of the anticipated pain intensity. While increasing the range of monetary offers increased the price requested to accept pain, skewing the distribution of rewards encouraged profit maximization. Participants scoring higher on harm avoidance and lower on persistence scales requested more money to accept pain. Accepting highly painful offers slowed decisions regardless of the value of the concurrent reward. Altogether, pain-related decisions are highly relative to the local range of available rewards and may be under the control of more automatic avoidance mechanisms.


title: NAMPT-derived NAD+ fuels PARP1 to promote skin inflammation through parthanatos

abstract:
Several studies have revealed a correlation between chronic inflammation and NAD+ metabolism but the precise mechanism involved is unknown. Here we report that the genetic and pharmacological inhibition of nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in the salvage pathway of NAD+ biosynthesis, reduced oxidative stress, inflammation, and keratinocyte DNA damage, hyperproliferation and cell death in zebrafish models of chronic skin inflammation, while all these effects were reversed by NAD+ supplementation. Similarly, genetic and pharmacological inhibition of poly ADP-ribose (PAR) polymerase 1 (Parp1), overexpression of PAR glycohydrolase, inhibition of apoptosis-inducing factor 1, inhibition of NADPH oxidases and reactive oxygen species (ROS) scavenging, all phenocopied the effects of Nampt inhibition. Pharmacological inhibition of NADPH oxidases/NAMPT/PARP/AIFM1 axis decreased expression of pathology-associated genes in human organotypic 3D skin models of psoriasis. Consistently, an aberrant induction of both NAMPT amounts and PARP activity was observed in lesional skin from psoriasis patients. In conclusion, hyperactivation of PARP1 in response to ROS-induced DNA damage, fueled by NAMPT-derived NAD+, mediates skin inflammation through parthanatos cell death.

HighlightsO_LINAMPT inhibition alleviates inflammation in zebrafish and human epidermis organoid models of psoriasis.
C_LIO_LINADPH oxidase-derived ROS mediates keratinocyte DNA damage and Parp1 overactivation.
C_LIO_LIInhibition of parthanatos cell death phenocopies the effects of NAMPT inhibition in zebrafish and human psoriasis models.
C_LIO_LINAMPT and PAR metabolism is altered in psoriasis patients.
C_LI


title: Antiviral effect of a nucleic acid hydrolyzing scFv against oseltamivir resistant influenza A virus

abstract: Influenza viral genome is frequently mutated due to antigenic shift and drift, and therefore the existing antiviral drugs have been suffered from low efficacy against the viruses. Here we report an innovative strategy for treating influenza Type A (IAV) infection by 3D8 single chain variable fragment (scFv) showing intrinsic viral RNA hydrolyzing activity, cell penetration activity and the epidermal cell penetration ability. In this study, we first analyzed antiviral activity directed by 3D8 scFv to three different strains, two oseltamivir-sensitive (A/PuertoRico/8/1934, A/X-31) and oseltamivir-resistant (A/Korea/2785/2009pdm) using cell culture models, suggesting that 3D8 scFv reduces viral genomic RNA. Moreover, we further addressed antiviral effect to analyze clinical outcomes in in vivo mice model. Intranasal administered 3D8 scFv rescued the mice challenged by oseltamivir resistant H1N1. Consistent results are observed through IHC pathology and molecular virological analysis. Taken together, these results demonstrate that 3D8 scFv has antiviral potential against a wide range of IAV.


title: Nanoscale dynamics of cellulase TrCel7A digesting cellulose

abstract: Understanding how cellulases catalyze the digestion of lignocellulose is a major goal of bioenergy research. Cel7A from Trichoderma reesei is a model exoglucanase that degrades cellulose strands from their reducing ends by processively cleaving individual cellobiose units. Despite being one of the most studied cellulases, the binding and hydrolysis mechanisms of Cel7A are still debated. We used single-molecule tracking to analyze the dynamics of 11,116 quantum dot-labeled TrCel7A binding to and moving processively along immobilized Gluconoacetobacter cellulose. Enzyme molecules were localized with a spatial precision of a few nanometers and followed for hundreds of seconds. Most enzymes bound into a static state and dissociated without detectable movement. Processive enzymes moved an average distance of 39 nm at an average speed of 3.2 nm/s. Static binding episodes preceding and following processive runs were of similar duration to static binding events that lacked any processive movement. Transient jumps of >20 nm were observed, but no diffusive behavior indicative of a diffusive search of the enzyme for a free cellulose strand end was observed. These data were integrated into a three-state model in which TrCel7A molecules can bind from solution into either a static or a processive state, and can reversibly switch between static and processive states before dissociating. From these results, we conclude that the rate-limiting step for cellulose degradation by Cel7A is the transition out of the static state either by dissociation from the cellulose surface or initiation of a processive run.


title: CCM2 deficient endothelial cells undergo a mechano-dependent reprogramming into senescence associated secretory phenotype used to recruit endothelial and immune cells.

abstract: Cerebral Cavernous Malformations (CCM) is a cerebrovascular disease in which stacks of dilated haemorrhagic capillaries form focally in the brain. Whether and how defective mechanotransduction, cellular mosaicism and inflammation interplay to sustain the progression of CCM diseases is unknown. Here, we reveal that CCM1- and CCM2-silenced endothelial cells enter into senescence associated with secretory phenotype (SASP) that they use to invade the extracellular matrix and attract surrounding wild-type endothelial and immune cells. Further, we demonstrate that this SASP is driven by the mechanical and molecular disorders provoked by ROCKs dysfunctions. By this, we identify CCM1/2 and ROCKs as parts of a scaffold controlling senescence, bringing new insights into the emerging field of the control of aging by cellular mechanics. This discovery reconciles the dysregulated traits of CCM1/2-deficient endothelial cells into a unique mechano-dependent endothelial fate that links perturbed mechanics to microenvironment remodelling and long-range activation of endothelial and immune cells.


title: Rare genera differentiate urban green space soil bacterial communities in three cities across the world

abstract: Vegetation complexity is potentially important for urban green space designs aimed at fostering microbial biodiversity to benefit human health. Exposure to urban microbial biodiversity may influence human health outcomes via immune training and regulation. In this context, improving human exposure to microbiota via biodiversity-centric urban green space designs is an underused opportunity. There is currently little knowledge on the association between vegetation complexity (i.e., diversity and structure) and soil microbiota of urban green spaces. Here, we investigated the association between vegetation complexity and soil bacteria in urban green spaces in Bournemouth, UK; Haikou, China; and the City of Playford, Australia by sequencing the 16S rRNA V4 gene region of soil samples and assessing bacterial diversity. We characterized these green spaces as having  low or  high vegetation complexity and explored whether these two broad categories contained similar bacterial community compositions and diversity around the world. Within cities, we observed significantly different alpha and beta diversities between vegetation complexities; however, these results varied between cities. Rare genera (< 1 % relative abundance individually, on average 35 % relative abundance when pooled) were most likely to be significantly different in sequence abundance between vegetation complexities and therefore explained much of the differences in microbial communities observed. Overall, general associations exist between soil bacterial communities and vegetation complexity, although these are not consistent between cities. Therefore, more in-depth work is required to be done locally to derive practical actions to assist the conservation and restoration of microbial communities in urban areas.

category: ecology


title: The Antiviral Enzyme, Viperin, Activates Protein Ubiquitination by the E3 Ubiquitin Ligase, TRAF6

abstract: Viperin is a broadly conserved radical SAM enzyme that synthesizes the antiviral nucleotide ddhCTP. In higher animals viperin expression also accelerates the degradation of various cellular and viral proteins necessary for viral replication; however, the details of this process remain largely unknown. Here we show that viperin activates a component of the protein ubiquitination machinery, which plays an important role in both protein degradation and immune signaling pathways. We demonstrate that viperin binds the E3 ubiquitin ligase, TRAF6, which catalyzes K63-linked ubiquitination associated with immune signaling pathways. Viperin activates ubiquitin transfer by TRAF6 ~2.5-fold and causes a significant increase in polyubiquitinated forms of TRAF6 that are important for mediating signal transduction. Our observations both imply a role for viperin as an agonist of immune signaling and suggest that viperin may activate other K48-linked E3-ligases involved in targeting proteins for proteasomal degradation.


title: A Framework for Evaluating Myocardial Stiffness Using 3D-Printed Hearts

abstract: MRI-driven computational modeling is increasingly used to simulate in vivo cardiac mechanical behavior and estimate subject-specific myocardial stiffness. However, in vivo validation of these estimates is exceedingly difficult due to the lack of a known ground-truth in vivo myocardial stiffness. We have developed 3D-printed heart phantoms of known myocardium-mimicking stiffness and MRI relaxation properties and incorporated the heart phantoms within a highly controlled MRI-compatible setup to simulate in vivo diastolic filling. The setup enables the acquisition of experimental data needed to evaluate myocardial stiffness using computational constitutive modeling: phantom geometry, loading pressures, boundary conditions, and filling strains. The pressure-volume relationship obtained from the phantom setup was used to calibrate an in silico model of the heart phantom undergoing simulated diastolic filling. The model estimated stiffness was compared with ground-truth stiffness obtained from uniaxial tensile testing. Ultimately, the setup is designed to enable extensive validation of MRI and FEM-based myocardial stiffness estimation frameworks.

category: bioengineering


title: Illustrating Potential Effects of Alternate Control Populations on Real-World Evidence-based Statistical Analyses

abstract: Case-control study designs are commonly used in retrospective analyses of Real-World Evidence (RWE). Due to the increasingly wide availability of RWE, it can be difficult to determine whether findings are robust or the result of testing multiple hypotheses. We investigate the potential effects of modifying cohort definitions in a case-control association study between depression and Type 2 Diabetes Mellitus (T2D). We found that small permutations to the criteria used to define the control population result in significant shifts in both the demographic structure of the identified cohort as well as the odds ratio of association. These differences remain present when testing against age and sex-matched controls. We believe this offers strong support for the need for robust guidelines, best practices and regulations around the use of observational RWE for clinical or regulatory decision making.


title: IL-32 is a metabolic regulator promoting survival and proliferation of malignant plasma cells

abstract: IL-32 is a non-classical cytokine expressed in cancers, inflammatory diseases and infections. IL-32 can have both extracellular and intracellular functions, and its receptor is not identified. We here demonstrate that endogenously expressed, intracellular IL-32 binds to components of the mitochondrial respiratory chain and promotes oxidative phosphorylation. Knocking out IL-32 in malignant plasma cells significantly reduced survival and proliferation in vitro and in vivo. High throughput transcriptomic and MS-metabolomic profiling of IL-32 KO cells revealed that loss of IL-32 leads to profound perturbations in metabolic pathways, with accumulation of lipids, pyruvate precursors and citrate, indicative of reduced mitochondrial function. IL-32 is expressed in a subgroup of multiple myeloma patients with an inferior prognosis. Primary myeloma cells expressing IL-32 were characterized by a plasma cell gene signature associated with immune activation, proliferation and oxidative phosphorylation. We propose a novel concept for regulation of metabolism by an intracellular cytokine and identify IL-32 as an endogenous growth and survival factor for malignant plasma cells. IL-32 is a potential prognostic biomarker and a treatment target in multiple myeloma.

category: cancer biology


title: Assembly of chromosome-scale and allele-aware autotetraploid genome of the Chinese alfalfa cultivar Zhongmu-4 and identification of SNP loci associated with 27 agronomic traits

abstract: Alfalfa (Medicago sativa L.), the most valuable perennial legume crop, referred to as "Queen of the Forages" for its high nutritional value and yield production among forage crops. Comprehensive genomic information of germplasm resources from different ecological regions and modern breeding strategies, such as molecular-marker assisted breeding are of great importance to breed new alfalfa varieties with environmental resilience. Here, we report assembly of the genome sequence of Zhongmu-4 (ZM-4), one of the most planted cultivars in China, and identification of SNPs associated with alfalfa agronomic traits by Genome-wide Association Studies (GWAS). Sequence of 32 allelic chromosomes was assembled successfully by single molecule real time sequencing and Hi-C technique with ALLHiC algorithm. About 2.74 Gbp contigs, accounting for 88.39% of the estimated genome, were assembled with 2.56 Gbp contigs anchored to 32 pseudo-chromosomes. In comparison with M. truncatula A17, distinctive inversion and translocation on chromosome 1, and between chromosome 4 and 8, respectively, were detected. Moreover, we conducted resequencing of 220 alfalfa accessions collected globally and performed GWAS analysis based on our assembled genome. Population structure analysis demonstrated that alfalfa has a complex genetic relationship among germplasm with different geographic origins. GWAS identified 101 SNPs associated with 27 out of 93 agronomic traits. The updated chromosome-scale and allele-aware genome sequence, coupled with the resequencing data of most global alfalfa germplasm, provides valuable information for alfalfa genetic research, and further analysis of major SNP loci will accelerate unravelling the molecular basis of important agronomic traits and facilitate genetic improvement of alfalfa.


title: Dynamic Predictive Coding with Hypernetworks

abstract: The original predictive coding model of Rao & Ballard [1] focused on spatial prediction to explain spatial receptive fields and contextual effects in the visual cortex. Here, we introduce a new dynamic predictive coding model that achieves spatiotemporal prediction of complex natural image sequences using time-varying transition matrices. We overcome the limitations of static linear transition models (as in, e.g., Kalman filters) using a hypernetwork to adjust the transition matrix dynamically for every time step, allowing the model to predict using a time-varying mixture of possible transition dynamics. We developed a single level model with recurrent modulation of transition weights by a hypernetwork and a two-level hierarchical model with top-down modulation based on a hypernetwork. At each time step, the model predicts the next input and estimates a sparse neural code by minimizing prediction error. When exposed to natural movies, the model learned localized, oriented spatial filters as well as both separable and inseparable (direction-selective) space-time receptive fields at the first level, similar to those found in the primary visual cortex (V1). Longer timescale responses and stability at the second level also emerged naturally from minimizing prediction errors for the first level dynamics. Our results suggest that the multiscale temporal response properties of cortical neurons could be the result of the cortex learning a hierarchical generative model of the visual world with higher order areas predicting the transition dynamics of lower order areas.


title: Volumetric Calcium Imaging of 1 Million Neurons Across Cortical Regions at Cellular Resolution Using Light Beads Microscopy

abstract: Two-photon microscopy together with genetically encodable calcium indicators has emerged as a standard tool for high-resolution imaging of neuroactivity in scattering brain tissue. However, its various realizations have not overcome the inherent tradeoffs between speed and spatiotemporal sampling in a principled manner which would be necessary to enable, amongst other applications, mesoscale volumetric recording of neuroactivity at cellular resolution and speed compatible with resolving calcium transients. Here, we introduce Light Beads Microscopy (LBM), a scalable and spatiotemporally optimal acquisition approach limited only by fluorescence life-time, where a set of axially-separated and temporally-distinct foci record the entire axial imaging range near-simultaneously, enabling volumetric recording at 1.41 x 108 voxels per second. Using LBM, we demonstrate mesoscopic and volumetric imaging at multiple scales in the mouse cortex, including cellular resolution recordings within ~3x5x0.5 mm3 volumes containing >200,000 neurons at ~5 Hz, recording of populations of ~1 million neurons within ~5.4x6x0.5 mm3 volumes at ~2Hz as well as higher-speed (9.6 Hz) sub-cellular resolution volumetric recordings. LBM provides an unprecedented opportunity for discovering the neurocomputations underlying cortex-wide encoding and processing of information in the mammalian brain.


title: High-Speed, Cortex-Wide Volumetric Recording of Neuroactivity at Cellular Resolution using Light Beads Microscopy


title: A chimeric nuclease substitutes CRISPR-Cas: A phage weaponizes laterally acquired specificity to destroy subviral parasites

abstract: Mobile genetic elements, elements that can move horizontally between genomes, have profound effects on their hosts fitness. The PLE is a mobile element that integrates into the chromosome of Vibrio cholerae and parasitizes the bacteriophage ICP1 to move between cells. This parasitism by PLE is such that it abolishes the production of ICP1 progeny and provides a defensive boon to the host cell population. In response to the severe parasitism imposed by PLE, ICP1 has acquired an adaptive CRISPR-Cas system that targets the PLE genome during infection. However, ICP1 isolates that naturally lack CRISPR-Cas are still able to overcome certain PLE variants, and the mechanism of this immunity against PLE has thus far remained unknown. Here we show that ICP1 isolates that lack CRISPR-Cas encode an endonuclease in the same locus, and that the endonuclease provides ICP1 with immunity to a subset of PLEs. Further analysis shows that this endonuclease is of chimeric origin, incorporating a DNA binding domain that is highly similar to some PLE replication origin binding proteins. This similarity allows the endonuclease to bind and cleave PLE origins of replication. The endonuclease appears to exert considerable selective pressure on PLEs and may drive PLE replication module swapping and origin restructuring as mechanisms of escape. This work demonstrates that new genome defense systems can arise through domain shuffling and provides a greater understanding of the evolutionary forces driving genome modularity and temporal succession in mobile elements.


title: Chemical rescue of mutant proteins in living cells by naturally occurring small molecules

abstract: Intracellular proteins function in a complex milieu wherein small molecules influence protein folding and act as essential cofactors for enzymatic reactions. Thus protein function depends not only on amino acid sequence but also on the concentrations of such molecules, which are subject to wide variation between organisms, metabolic states, and environmental conditions. We previously found evidence that exogenous guanidine reverses the phenotypes of specific budding yeast septin mutants by binding to a WT septin at the former site of an Arg side chain that was lost during fungal evolution. Here we used a combination of targeted and unbiased approaches to look for other cases of "chemical rescue" by naturally occurring small molecules. We report in vivo rescue of hundreds of yeast mutants representing a variety of genes, including likely examples of Arg or Lys side chain replacement by the guanidinium ion. Failed rescue of targeted mutants highlight features required for rescue, as well as key differences between the in vitro and in vivo environments. Some non-Arg mutants rescued by guanidine likely result from "off-target" effects on specific cellular processes in WT cells. Molecules isosteric to guanidine and known to influence protein folding had a range of effects, from essentially none for urea, to rescue of a few mutants by DMSO. Strikingly, the osmolyte trimethylamine-N-oxide rescued [~]20% of the mutants we tested, likely reflecting combinations of direct and indirect effects on mutant protein function. Our findings illustrate the potential of natural small molecules as therapeutic interventions and drivers of evolution.


title: Automatic segmentation and cardiac mechanics analysis of evolving zebrafish using deep-learning

abstract:
ObjectiveIn the study of early cardiac development, it is important to acquire accurate volume changes of the heart chambers. Although advanced imaging techniques, such as light-sheet fluorescent microscopy (LSFM), provide an accurate procedure for analyzing the structure of the heart, rapid and robust segmentation is required to reduce laborious time and accurately quantify developmental cardiac mechanics.

MethodsThe traditional biomedical analysis involving segmentation of the intracardiac volume is usually carried out manually, presenting bottlenecks due to enormous data volume at high axial resolution. Our advanced deep-learning techniques provide a robust method to segment the volume within a few minutes. Our U-net based segmentation adopted manually segmented intracardiac volume changes as training data and produced the other LSFM zebrafish cardiac motion images automatically.

ResultsThree cardiac cycles from 2 days post fertilization (dpf) to 5 dpf were successfully segmented by our U-net based network providing volume changes over time. In addition to understanding the cardiac function for each of the two chambers, the ventricle and atrium were separated by 3D erode morphology methods. Therefore, cardiac mechanical properties were measured rapidly and demonstrated incremental volume changes of both chambers separately. Interestingly, stroke volume (SV) remains similar in the atrium while that of the ventricle increases SV gradually.

ConclusionOur U-net based segmentation provides a delicate method to segment the intricate inner volume of zebrafish heart during development; thus providing an accurate, robust and efficient algorithm to accelerate cardiac research by bypassing the labor-intensive task as well as improving the consistency in the results.


title: Structure-function Specialisation of the Interfascicular Matrix in the Human Achilles Tendon

abstract:
ObjectiveTendon consists of highly aligned collagen-rich fascicles surrounded by interfascicular matrix (IFM). Some tendons act as energy stores to improve locomotion efficiency; these tendons are prone to debilitating injuries, the incidence of which increases with ageing. In equine tendons, energy storage is achieved primarily through specialisation of the IFM. However, no studies have investigated IFM structure-function specialisation in human tendons. Here, we compare the positional anterior tibialis and energy storing Achilles tendons, testing the hypothesis that the Achilles IFM has specialised composition and mechanical properties, which are lost with ageing.

MethodsWe used a multidisciplinary combination of mechanical testing, immunolocalisation and proteomics to investigate structure-function specialisations in functionally distinct human tendons and how these are altered with ageing.

ResultsThe IFM in the energy storing Achilles tendon is more elastic and fatigue resistant than the IFM in the positional anterior tibialis tendon, with a trend towards decreased fatigue resistance with age in the Achilles IFM. With ageing, alterations occur predominantly to the proteome of the Achilles IFM.

ConclusionThe Achilles tendon IFM is specialised for energy storage, and changes to its proteome with ageing are likely responsible for the observed trends towards decreased fatigue resistance. Knowledge of key energy storing specialisations and their changes with ageing offers insight towards developing effective treatments for tendinopathy.

Key messagesO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LIEnergy storing tendons in animals and humans are particularly prone to tendinopathy and the incidence increases with increasing age.
C_LIO_LIPrevious work in some animal models has shown that the specialisation of tendon properties for energy storage is achieved primarily through adaptation of the interfascicular matrix, with specialisation lost in ageing. However, the structural specialisations that provide the human Achilles tendon with its energy storing ability, and how these are affected by ageing, remain to be established.
C_LI

What does this study add?O_LIWe demonstrate that the interfascicular matrix in the human Achilles tendon is specialised for energy storing, with increased elastic recoil and fatigue resistance, and that these specialisations are partially lost with ageing, likely due to alterations to the proteome of the interfascicular matrix.
C_LI

How might this impact on clinical practice or future developments?O_LIShort term, the specialist IFM mechanics we have demonstrated can be detected with new developments in ultrasound functional imaging, offering improved opportunities for contextual tendinopathy diagnostics. Personalised rehabilitation programmes can now be explored and designed specifically to target IFM mechanics.
C_LIO_LILonger term, the knowledge of key specialisations in injury prone energy storing tendons and how they are affected by ageing, offers crucial insight towards developing cell or tissue engineering treatments targeted at restoring tendon structure and function post-injury, specifically targeted at the IFM.
C_LI


title: Recovery of small plasmid sequences via Oxford Nanopore sequencing

abstract:
Oxford Nanopore Technologies (ONT) sequencing platforms currently offer two approaches to whole-genome native-DNA library preparation: ligation and rapid. In this study, we compared these two approaches for bacterial whole-genome sequencing, with a specific aim of assessing their ability to recover small plasmid sequences. To do so, we sequenced DNA from seven plasmid-rich bacterial isolates in three different ways: ONT ligation, ONT rapid and Illumina. Using the Illumina read depths to approximate true plasmid abundance, we found that small plasmids (<20 kbp) were underrepresented in ONT ligation read sets (by a mean factor of [~]4) but were not underrepresented in ONT rapid read sets. This effect correlated with plasmid size, with the smallest plasmids being the most underrepresented in ONT ligation read sets. We also found lower rates of chimeric reads in the rapid read sets relative to ligation read sets. These results show that when small plasmid recovery is important, ONT rapid library preparations are preferable to ligation-based protocols.

Impact statementResearchers who use Oxford Nanopore Technologies (ONT) platforms to sequence bacterial genomes can currently choose from two library preparation methods. The first is a ligation-based approach, which uses ligase to attach sequencing adapters to the ends of DNA molecules. The second is a rapid approach, which uses a transposase enzyme to cleave DNA and attach adapters in a single step. There are advantages to each preparation, for example ligation can produce better yields but rapid is a simpler procedure. Our study reveals another advantage of rapid preparations: they are more effective at sequencing small plasmids. We show that sequencing of ligation-based libraries yields fewer reads derived from small plasmids, making such plasmids harder to detect in bacterial genomes. Since small plasmids can contain clinically relevant genes, including antimicrobial resistance (AMR) or virulence determinants, their exclusion could lead to unreliable conclusions that have serious consequences for AMR surveillance and prediction. We therefore recommend that researchers performing ONT-only sequencing of bacterial genomes should consider using rapid preparations whenever small plasmid recovery is important.

Data summarySupplementary figures, tables, data and code can be found at: github.com/rrwick/Small-plasmid-Nanopore


title: B/tMSI-CAST: a systematic approach for microsatellite instability detection in both tumor tissue and plasma specimens from next-generation sequencing data

abstract: Microsatellite instability (MSI) is a well-established prognostic and predictive biomarker in certain types of cancers. MSI detection using tumour tissue is often limited by the availability of specimens. Next generation sequencing (NGS)-based MSI detection in plasma cell-free DNA (cfDNA) is challenged by a much lower signal-to-noise ratio. We developed a highly accurate cfDNA MSI detection method called bMSI-CAST (blood MSI Caller Adjusted with Sequence duplicaTes), with improvement on three features including a set of locus selection principles ensuring loci with high robustness and compatibility across sequencing platforms, an MSI-specific duplicate removal strategy, and a calling algorithm that dynamically matches baselines with a broad range of duplication levels. Analytical validation via MSI-high (MSI-H) cell gDNA showed an LOD of 0.15%. Furthermore, in an analysis of 95 evaluable cfDNA samples from patients with gastrointestinal cancers, bMSI-CAST exhibited a positive predictive agreement (PPA) of 92.9% (39/42) and negative predictive agreement (NPA) of 100% (53/53) with tissue MSI-PCR. In conclusion, bMSI-CAST provides novel and advanced solutions to key aspects fundamental to cfDNA MSI calling but not sufficiently addressed by existing methods, and it is a validated method ready to be applied to aid clinical decisions for cancer patients.


title: bMSI-CAST: a systematic method for next generation sequencing-based microsatellite instability detection in plasma cell-free DNA


title: High-level representations in human occipito-temporal cortex are indexed by distal connectivity

abstract:
Human object recognition is dependent on occipito-temporal cortex, but a complete understanding of the complex functional architecture of this area must account for how it is connected to the wider brain. Converging functional magnetic resonance imaging evidence shows that univariate responses to different categories of information (e.g. faces, bodies, & non-human objects) are strongly related to, and potentially shaped by, functional and structural connectivity to the wider brain. However, to date, there have been no systematic attempts to determine how distal connectivity and complex local high-level responses in occipito-temporal cortex (i.e. multivoxel response patterns) are related. Here, we show that distal functional connectivity is related to, and can reliably index, high-level representations for several visual categories (i.e. tools, faces, & places) within occipito-temporal cortex; that is, voxels sets that are strongly connected to distal brain areas show higher pattern discriminability than less well-connected sets do. We further show that, in several cases, pattern discriminability is higher in sets of well-connected voxels than sets defined by  local activation (e.g. strong amplitude responses to faces in fusiform face area). Together, these findings demonstrate the important relationship between the complex functional organization of occipito-temporal cortex and wider brain connectivity.

Significance statementHuman object recognition relies strongly on occipito-temporal cortex (OTC), yet responses in this broad area are often considered in relative isolation to the rest of the brain. We employ a novel  connectivity-guided voxel selection approach with functional MRI data to show higher sensitivity to information (i.e. higher multivoxel pattern discriminability) in voxel sets that share strong connectivity to distal brain areas, relative to: 1) voxel sets that are less-strongly connected; and in several cases 2) voxel sets that are defined by strong  local response amplitude. These findings underscore the importance of distal contributions to local processing in OTC.


title: Nano Pom-poms Prepared Highly Specific Extracellular Vesicles Expand the Detectable Cancer Biomarkers

abstract: Extracellular vesicles (EVs), particularly exosomes, are emerging biomarker sources. However, due to heterogeneous populations secreted from diverse cell types, mapping EV multi-omic molecular information specifically to their pathogenesis origin for cancer biomarker identification is still extraordinary challenging. Herein, we introduced a novel 3D-structured nanographene immunomagnetic particles (NanoPoms) with unique flower pom-poms morphology and photo-click chemistry for specific marker-defined capture and release of intact small EVs. This specific EV isolation approach leads to the expanded identification of targetable cancer biomarkers with enhanced specificity and sensitivity, as demonstrated by multi-omic EV analysis of bladder cancer patient tissue fluids using the next generation sequencing of somatic DNA mutations, miRNAs, and the global proteome. The NanoPoms prepared sEVs also exhibit distinctive in vivo biodistribution patterns, highlighting the highly viable and integral quality. The developed method is simple and straightforward, and is applicable to nearly all types of biological fluids and amenable for scale up and high-throughput EV isolation.


title: Unbiased choice of global clustering parameters in single-molecule localization microscopy

abstract: Single-molecule localization microscopy resolves objects below the diffraction limit of light via sparse, stochastic detection of target molecules. Single molecules appear as clustered detection events after image reconstruction. However, identification of clusters of localizations is often complicated by the spatial proximity of target molecules and by background noise. Clustering results of existing algorithms often depend on user-generated training data or user-selected parameters, which can lead to unintentional clustering errors. Here we suggest an unbiased algorithm (FINDER) based on adaptive global parameter selection and demonstrate that the algorithm is robust to noise inclusion and target molecule density. We benchmark the most common density based clustering algorithms in test scenarios which we designed based on experimental datasets. We show that FINDER can keep the number of false positive inclusions low while also maintaining a low number of false negative detections in densely populated regions.


title: Statistical testing and annotation of gene transcriptomic-neuroimaging associations

abstract: Multiscale integration of gene transcriptomic and neuroimaging data is becoming a widely used approach for exploring the molecular underpinnings of large-scale brain structure and function. Proper statistical evaluation of computed associations between imaging-based phenotypic and transcriptomic data is key in these explorations, in particular to establish whether observed associations exceed  chance level of random, non-specific effects. Recent approaches have shown the importance of spatial null models to test for spatial specificity of effects to avoid serious inflation of reported statistics. Here, we discuss the need for examination of the second category of specificity in transcriptomic-neuroimaging analyses, namely that of gene specificity, examined using null models built upon effects that occur from sets of random genes. Through simple examples of commonly performed transcriptomic-neuroimaging analyses, we show that providing additional gene specificity on observed transcriptomic-neuroimaging effects is of high importance to avoid non-specific (potentially false-positive) effects. Through simulations we further show that the rate of reported non-specific effects (i.e., effects that are generally observed and cannot be specifically linked to a gene-set of interest) can run as high as 60%, with only less than 5% of transcriptomic-neuroimaging associations observed through ordinary linear regression analyses showing spatial and gene specificity. We explain that using proper null models that test for both spatial specificity and gene specificity is warranted.


title: Statistical testing in gene transcriptomic-neuroimaging associations: an evaluation of methods that assess spatial and gene specificity


title: Cardiac Atrial Compartmentalisation Proteomics: A Modified Density Gradient Method to Analyse Endo-lysosomal Proteins

abstract:
The importance of lysosomes in cardiac physiology and pathology are well established, and evidence for roles in calcium signalling are emerging. We describe a label-free proteomics method suitable for small cardiac tissue biopsies based on density-separated fractionation, which allows study of endo-lysosomal (EL) proteins.

Density gradient fractions corresponding to tissue lysate; sarcoplasmic reticulum (SR), mitochondria (Mito) (1.3 g/ml); and EL with negligible contamination from SR or Mito (1.04 g/ml), were analysed using Western Blot, enzyme activity assay and LC-MS/MS analysis (adapted discontinuous Percoll, and sucrose differential density gradient).

Kyoto Encyclopedia of Genes and Genomes, Reactome, Panther and Gene Ontology pathway analysis showed good coverage of RAB proteins and lysosomal cathepsins (including cardiac-specific cathepsin D) in the purified EL fraction. Significant EL proteins recovered included catalytic activity proteins. We thus present a comprehensive protocol and dataset of guinea-pig atrial EL organelle proteomics using techniques also applicable for non-cardiac tissue.

category: molecular biology


title: Old players in new posts: the role of P53, ATM and DNAPK in DNA damage-related ubiquitylation-dependent removal of S2P RNAPII

abstract:
DNA double-strand breaks are the most deleterious lesions for the cells, therefore understanding the macromolecular interactions in the DNA repair-related mechanisms is essential. DNA damage triggers transcription silencing at the damage site, leading to the removal of the elongating RNA polymerase II (S2P RNAPII) from this locus, which provides accessibility for the repair factors to the lesion. Ataxia-telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNAPK) are the two main regulatory kinases of homologous recombination and non-homologous end joining, respectively. Although these kinases are involved in the activation of different repair pathways, they have common target proteins, such as P53. We previously demonstrated that following transcription block, P53 plays a pivotal role in transcription elongation process by interacting with S2P RNAPII. In the current study, we reveal that P53, ATM and DNAPK are involved in the fine-tune regulation of the ubiquitin-proteasome system-related degradation of S2P RNAPII. However, they act differently in this process: P53 delays the removal of S2P RNAPII, while ATM and DNAPK participate in the activation of members of E3 ligase complexes involved in the ubiquitylation of S2P RNAPII. We also demonstrate that WW domain-containing protein 2 (WWP2) and Cullin-3 (CUL3) are interaction partners of S2P RNAPII, thus forming a complex with the transcribing RNAPII complex.

Simple SummaryTo ensure the proper repair following DNA double-strand breaks, the eviction of the arrested elongating RNA polymerase II (S2P RNAPII) is required. Here, we report an emerging role of P53, Ataxia-telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNAPK) in the ubiquitin-proteasome system-dependent removal of S2P RNAPII. We also identified interactions between S2P RNAPII and WW domain-containing protein 2 (WWP2) or Cullin-3 (CUL3) (members of E3 ligase complexes), which are involved in the ubiquitylation of S2P RNAPII following DNA damage. Furthermore, the RNAPII-E3 ligase complex interactions are mediated by P53, ATM and DNAPK, which suggests potential participation of all three proteins in the effective resolution of transcription block at the damage site. Altogether, our results provide a better comprehension of the molecular background of transcription elongation block-related DNA repair processes and highlight an indispensable function of P53, ATM and DNAPK in these mechanisms.


title: Structural Basis for Accommodation of Emerging B.1.351 and B.1.1.7 Variants by Two Potent SARS-CoV-2 Neutralizing Antibodies

abstract: Emerging SARS-CoV-2 strains, B.1.1.7 and B.1.351, from the UK and South Africa, respectively show decreased neutralization by monoclonal antibodies and convalescent or vaccinee sera raised against the original wild-type virus, and are thus of clinical concern. However, the neutralization potency of two antibodies, 1-57 and 2-7, which target the receptor-binding domain (RBD) of spike, was unaffected by these emerging strains. Here, we report cryo-EM structures of 1-57 and 2-7 in complex with spike, revealing each of these antibodies to utilize a distinct mechanism to bypass or accommodate RBD mutations. Notably, each antibody represented a response with recognition distinct from those of frequent antibody classes. Moreover, many epitope residues recognized by 1-57 and 2-7 were outside hotspots of evolutionary pressure for both ACE2 binding and neutralizing antibody escape. We suggest the therapeutic use of antibodies like 1-57 and 2-7, which target less prevalent epitopes, could ameliorate issues of monoclonal antibody escape.


title: Reduced binding and neutralization of infection- and vaccine-induced antibodies to the B.1.351 (South African) SARS-CoV-2 variant

abstract: The emergence of SARS-CoV-2 variants with mutations in the spike protein is raising concerns about the efficacy of infection- or vaccine-induced antibodies to neutralize these variants. We compared antibody binding and live virus neutralization of sera from naturally infected and spike mRNA vaccinated individuals against a circulating SARS-CoV-2 B.1 variant and the emerging B.1.351 variant. In acutely-infected (5-19 days post-symptom onset), convalescent COVID-19 individuals (through 8 months post-symptom onset) and mRNA-1273 vaccinated individuals (day 14 post-second dose), we observed an average 4.3-fold reduction in antibody titers to the B.1.351-derived receptor binding domain of the spike protein and an average 3.5-fold reduction in neutralizing antibody titers to the SARS-CoV-2 B.1.351 variant as compared to the B.1 variant (spike D614G). However, most acute and convalescent sera from infected and all vaccinated individuals neutralize the SARS-CoV-2 B.1.351 variant, suggesting that protective immunity is retained against COVID-19.


title: A novel glucocorticoid and androgen receptor modulator reduces viral entry and innate immune inflammatory responses in the Syrian Hamster model of SARS-CoV-2

abstract: Since its initial discovery in late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of COVID19, has spread worldwide and despite significant research efforts, treatment options remain limited. Replication of SARS-CoV-2 in lung is associated with marked infiltration of macrophages and activation of innate immune inflammatory responses triggered, in part, by heightened production of interleukin-6 (IL-6) that recruits lymphocytes to the site of infection that amplify tissue injury. Antagonists of the glucocorticoid and androgen receptors have shown promise in experimental models of COVID19 and in clinical studies, because cell surface proteins required for viral entry, angiotensin converting enzyme 2 (ACE2) and the transmembrane serine protease 2 (TMPRSS2), are transcriptionally regulated by these receptors. We therefore postulated that the glucocorticoid (GR) and androgen receptor (AR) antagonist, PT150, would reduce infectivity of SARS-CoV-2 and prevent inflammatory lung injury in the Syrian golden hamster model of COVID19. Animals were infected intranasally with 2.5 x 104 TCID50/ml equivalents of SARS-CoV-2 (strain 2019-nCoV/USA-WA1/ 2020) and PT150 was administered by oral gavage at 30 and 100 mg/Kg/day for a total of 7 days. Animals were then examined at days 3, 5 and 7 post-infection (DPI) for lung histopathology, viral load and production of proteins regulating the initiation and progression of SARS-CoV-2 infection. Results of these studies indicated that oral administration of PT150 decreased replication of SARS-CoV-2 in lung, as well as expression of ACE2 and TMPRSS2 protein. Hypercellularity and inflammatory cell infiltration driven by macrophage responses were dramatically decreased in PT150-treated animals, as was tissue damage and expression of IL-6. Molecular modeling suggested that PT150 binds to the co-activator interface of the ligand binding domain of both AR and GR and thereby acts as an allosteric modulator and transcriptional repressor of these receptors. Phylogenetic analysis of AR and GR across multiple species permissive to SARS-CoV-2 infection revealed a high degree of sequence identity maintained across species, including human, suggesting that the mechanism of action and therapeutic efficacy observed in Syrian hamsters would likely be predictive of positive outcomes in patients. PT150 is therefore a strong candidate for further clinical development for the treatment of COVID19 across variants of SARS-CoV-2.


title: Complete map of SARS-CoV-2 RBD mutations that escape the monoclonal antibody LY-CoV555 and its cocktail with LY-CoV016

abstract: Monoclonal antibodies and antibody cocktails are a promising therapeutic and prophylaxis for COVID-19. However, ongoing evolution of SARS-CoV-2 can render monoclonal antibodies ineffective. Here we completely map all mutations to the SARS-CoV-2 spike receptor binding domain (RBD) that escape binding by a leading monoclonal antibody, LY-CoV555, and its cocktail combination with LY-CoV016. Individual mutations that escape binding by each antibody are combined in the circulating B.1.351 and P.1 SARS-CoV-2 lineages (E484K escapes LY-CoV555, K417N/T escape LY-CoV016). Additionally, the L452R mutation in the B.1.429 lineage escapes LY-CoV555. Furthermore, we identify single amino acid changes that escape the combined LY-CoV555+LY-CoV016 cocktail. We suggest that future efforts should diversify the epitopes targeted by antibodies and antibody cocktails to make them more resilient to antigenic evolution of SARS-CoV-2.


title: Fundamental Role Of The H2A.Z C-Terminal Tail In The Formation Of Constitutive Heterochromatin

abstract: Nucleosome stability, a crucial determinant of gene regulation, was measured in a robust in situ assay to assess the molecular determinants of the stability of H2A.Z-containig nucleosomes. Surprisingly, a large fraction of H2A.Z detected by three different antibodies was released from the nucleosomes by salt together with H3, and was associated with H3K9me3 but not with H3K27me3 marked nucleosomes. This unusual behavior relied on the presence of the unstructured C-terminal chain of the histone variant, rather than on isoform specificity, posttranslational modifications or binding of the reader protein PWWPA2, as determined using cell lines expressing only particular forms of the variant. In the absence of this tail, or upon addition of an excess of the tail peptide to the nuclei of control cells, the canonical H2A-like stability features were readily restored and most of the H2A.Z-containing nucleosomes left the periphery and ended up in scattered foci in the nuclei. Concomitantly, the H3K9me3-marked constitutive heterochromatin was also dispersed, what was accompanied by increased overall nuclease sensitivity and significantly enhanced binding of intercalating dyes to the DNA. The DT40 cells expressing the tailless H2A.Z showed marked differences in their gene expression pattern and were distinguished by compromised DNA damage response. Thus, interactions involving a short H2A.Z peptide chain simultaneously determine the stability and accessibility features of chromatin involving the nucleosomes containing this histone variant and the localization of these large chromatin regions in the nucleus. Our data suggest that H2A.Z can function in both heterochromatic and in euchromatic scenarios depending on the molecular interactions involving its C-terminal unstructured tail, shedding light on the enigmatic double-faced character of this histone variant.


title: GABARAP membrane conjugation sequesters the FLCN-FNIP tumor suppressor complex to activate TFEB and lysosomal biogenesis

abstract: Adaptive changes in lysosomal capacity are driven by the transcription factors TFEB and TFE3 in response to increased autophagic flux and endolysosomal stress, yet the molecular details of their activation are unclear. LC3 and GABARAP members of the ATG8 protein family are required for selective autophagy and sensing perturbation within the endolysosomal system. Here we show that during single membrane ATG8 conjugation (SMAC), Parkin-dependent mitophagy, and Salmonella-induced xenophagy, the membrane conjugation of GABARAP, but not LC3, is required for activation of TFEB/TFE3 to control lysosomal homeostasis and capacity. GABARAP directly binds to a novel LC3-interacting motif (LIR) in the FLCN/FNIP tumor suppressor complex with picomolar affinity and regulates its relocalization to these GABARAP-conjugated membrane compartments. This disrupts the regulation of RagC/D by the FLCN/FNIP GAP complex, resulting in impaired mTOR-dependent phosphorylation of TFEB without changing mTOR activity towards other substrates. Thus, the GABARAP-FLCN/FNIP-TFEB axis serves as a universal molecular sensor that coordinates lysosomal homeostasis with perturbations and cargo flux within the autophagy-lysosomal network.


title: Physically interacting beta-delta pairs in the regenerating pancreas revealed by single cell sequencing

abstract:
The endocrine pancreas is able to regenerate in response to insult, including by driving beta-cells into the cell division cycle. Until recently, communication between neighboring cells in islets of Langerhans was overlooked by single-cell genomic technologies, which require rigorous tissue dissociation into single cells. Here, we utilize sorting of physically interacting cells (PICs) with single-cell RNA-sequencing to systematically map cellular interactions in the regenerating endocrine pancreas. The cellular landscape of the regenerated pancreas features regeneration-associated endocrine populations.

We explore the unexpected heterogeneity of beta-cells in regeneration, including an interaction-specific program between paired beta and delta-cells. Our analysis suggests that the particular cluster of beta-cells that pair with delta-cells benefits from stress protection, implying that the interaction between beta and delta-cells safeguards against regeneration-associated challenges.


title: Comparative Perturbation-Based Modeling of the SARS-CoV-2 Spike Protein Binding with Host Receptor and Neutralizing Antibodies : Structurally Adaptable Allosteric Communication Hotspots Define Spike Sites Targeted by Global Circulating Mutations

abstract: In this study, we used an integrative computational approach focused on comparative perturbation-based modeling to examine molecular mechanisms and determine functional signatures underlying role of functional residues in the SARS-CoV-2 spike protein that are targeted by novel mutational variants and antibody-escaping mutations. Atomistic simulations and functional dynamics analysis are combined with alanine scanning and mutational sensitivity profiling for the SARS-CoV-2 spike protein complexes with the ACE2 host receptor are REGN-COV2 antibody cocktail (REG10987+REG10933). Using alanine scanning and mutational sensitivity analysis, we have shown that K417, E484 and N501 residues correspond to key interacting centers with a significant degree of structural and energetic plasticity that allow mutants in these positions to afford the improved binding affinity with ACE2. Through perturbation-based network modeling and community analysis of the SARS-CoV-2 spike protein complexes with ACE2 we demonstrate that E406, N439, K417 and N501 residues serve as effector centers of allosteric interactions and anchor major inter-molecular communities that mediate long-range communication in the complexes. The results provide support to a model according to which mutational variants and antibody-escaping mutations constrained by the requirements for host receptor binding and preservation of stability may preferentially select structurally plastic and energetically adaptable allosteric centers to differentially modulate collective motions and allosteric interactions in the complexes with the ACE2 enzyme and REGN-COV2 antibody combination. This study suggests that SARS-CoV-2 spike protein may function as a versatile and functionally adaptable allosteric machine that exploits plasticity of allosteric regulatory centers to fine-tune response to antibody binding without compromising activity of the spike protein.

category: biophysics


title: Engineering protease-resistant peptides to inhibit human parainfluenza viral respiratory infection

abstract: The lower respiratory tract infections affecting children worldwide are in large part caused by the parainfluenza viruses (HPIVs), particularly HPIV3, along with human metapneumovirus and respiratory syncytial virus, enveloped negative-strand RNA viruses. There are no vaccines for these important human pathogens, and existing treatments have limited or no efficacy. Infection by HPIV is initiated by viral glycoprotein-mediated fusion between viral and host cell membranes. A viral fusion protein (F), once activated in proximity to a target cell, undergoes a series of conformational changes that first extend the trimer subunits to allow insertion of the hydrophobic domains into the target cell membrane, and then refold the trimer into a stable postfusion state, driving the merger of the viral and host cell membranes. Lipopeptides derived from the C-terminal heptad repeat (HRC) domain of HPIV3 F inhibit infection by interfering with the structural transitions of the trimeric F assembly. Clinical application of this strategy, however, requires improving the in vivo stability of antiviral peptides. We show that the HRC peptide backbone can be modified via partial replacement of -amino acid residues with {beta}-amino acid residues to generate /{beta}-peptides that retain antiviral activity but are poor protease substrates. Relative to a conventional -lipopeptide, our best /{beta}-lipopeptide exhibits improved persistence in vivo and improved anti-HPIV3 antiviral activity in animals.


title: Dual proteomics of Drosophila melanogaster hemolymph infected with the heritable endosymbiont Spiroplasma poulsonii

abstract:
Insects are frequently infected with heritable bacterial endosymbionts. Endosymbionts have a dramatic impact on their host physiology and evolution. Their tissue distribution is variable with some species being housed intracellularly, some extracellularly and some having a mixed lifestyle. The impact of extracellular endosymbionts on the biofluids they colonize (e.g. insect hemolymph) is however difficult to appreciate because biofluids composition can depend on the contribution of numerous tissues.

Here we address the question of Drosophila hemolymph proteome response to the infection with the endosymbiont Spiroplasma poulsonii. S. poulsonii inhabits the fly hemolymph and gets vertically transmitted over generations by hijacking the oogenesis in females. Using dual proteomics on infected hemolymph, we uncovered a low level and chronic activation of the Toll immune pathway by S. poulsonii that was previously undetected by transcriptomics-based approaches. Using Drosophila genetics, we also identified candidate proteins putatively involved in controlling S. poulsonii growth. Last, we also provide a deep proteome of S. poulsonii, which, in combination with previously published transcriptomics data, improves our understanding of the post-transcriptional regulations operating in this bacterium.

Summary statementWe report the changes in Drosophila melanogaster hemolymph proteome upon infection with the heritable bacterial endosymbiont Spiroplasma poulsonii.


title: Small-RNA sequencing reveals altered skeletal muscle microRNAs and snoRNAs signatures in weanling male offspring from mouse dams fed a low protein diet during lactation

abstract: Nutrition plays a key role in pre- and postnatal growth of the musculoskeletal system. Maternal diet during gestation and lactation affects the development of skeletal muscles in the offspring and determines muscle health in later life, however, the molecular mechanisms that govern these effects are largely unknown. In this study, we aim to describe the association between maternal low protein diet-induced changes in offspring skeletal muscle and the differential expression (DE) of small non-coding RNAs (sncRNAs). We used a mouse model of maternal protein restriction to characterise the impact of early-life undernutrition on skeletal muscle morphology in male offspring at weaning. Mouse dams were fed either a normal (N, 20%) or a low protein (L, 8%) diet during gestation and newborn pups were cross-fostered to N or L lactating dams, resulting in the generation of NN, NL and LN offspring groups. Total body and tibialis anterior (TA) weights were decreased in NL males but not different in the LN group, as compared to NN, although neonates from low protein fed dams were smaller at birth than those born to dams fed a normal protein. However, histological evaluation of TA muscle revealed reduced muscle fibre size in both groups at the end of lactation. Small RNA-seq analysis demonstrated DE of multiple classes of sncRNAs, including miRs, snoRNAs and snRNAs. Bioinformatic analyses of miRs-15a, -34a, -122 and -199a, in combination with known myomiRs, confirmed their implication in key muscle-specific biological processes and cellular functions and suggest a promising set of miRs in muscle physiology studies. To our knowledge, this is the first comprehensive report for the DE of sncRNAs in nutrition-associated programming of skeletal muscle development, highlighting the need for further research.


title: A spindle and thread-mechanism unblocks translation of N-terminally disordered proteins

abstract: Protein disorder is a major hurdle for structural biology. A prominent example is the tumour suppressor p53, whose low expression levels and poor conformational stability due to a high degree of disorder pose major challenges to the development of cancer therapeutics. Here, we address these issues by fusing p53 to an engineered spider silk domain termed NT*. The chimeric protein displays highly efficient translation in vitro and in E. coli and is fully active in human cancer cells. The transmission electron microscopy structure and native mass spectrometry reveal that the full-length p53 fusion protein adopts a compact conformation. Molecular dynamics simulations show that the disordered transactivation domain of p53 is wound around the NT* domain via a series of folding events, resulting in a globular structure. We find that expression of B-Raf, another partially disordered cancer target, is similarly enhanced by fusion to NT*. In summary, we demonstrate how inducing co-translational folding via a molecular "spindle and thread" mechanism can overcome poor translation efficiency of partially disordered proteins.


title: Estimating DNA methylation potential energy landscapes from nanopore sequencing data

abstract: High-throughput third-generation sequencing devices, such as the Oxford Nanopore Technologies (ONT) MinION sequencer, can generate long reads that span thousands of bases. This new technology opens the possibility of considering a wide range of epigenetic modifications and provides the capability of interrogating previously inaccessible regions of the genome, such as highly repetitive regions, as well as of performing comprehensive allele-specific methylation analysis, among other applications. It is well-known, however, that detection of DNA methylation from nanopore data results in a substantially reduced per-read accuracy when comparing to WGBS, due to noise introduced by the sequencer and its underlying chemistry. It is therefore imperative that methods are developed for the reliable modeling and analysis of the DNA methylation landscape using nanopore data. Here we introduce such method that takes into account the presence of noise introduced by the ONT sequencer and, by using simulations, we provide evidence of its potential. The proposed approach establishes a solid foundation for the development of a comprehensive framework for the statistical analysis of DNA methylation, and possibly of other epigenetic marks, using third-generation sequencing.


title: Nanoceutical Fabric Prevents COVID-19 Spread through Expelled Respiratory Droplets: A Combined Computational, Spectroscopic and Anti-microbial Study

abstract:
Centers for Disease Control and Prevention (CDC) warns the use of one-way valves or vents in free masks for potential threat of spreading COVID-19 through expelled respiratory droplets. Here, we have developed a nanoceutical cotton fabric duly sensitized with non-toxic zinc oxide nanomaterial for potential use as membrane filter in the one way valve for the ease of breathing without the threat of COVID-19 spreading. A detailed computational study revealed that zinc oxide nanoflowers (ZnO NF) with almost two-dimensional petals trap SARS-CoV-2 spike proteins, responsible to attach to ACE-2 receptors in human lung epithelial cells. The study also confirm significant denaturation of the spike proteins on the ZnO surface, revealing removal of virus upon efficient trapping. Following the computational study, we have synthesized ZnO NF on cotton matrix using hydrothermal assisted strategy. Electron microscopic, steady-state and picosecond resolved spectroscopic studies confirm attachment of ZnO NF to the cotton (i.e., cellulose) matrix at atomic level to develop the nanoceutical fabric. A detailed antimicrobial assay using Pseudomonas aeruginosa bacteria (model SARS-CoV-2 mimic) reveals excellent anti-microbial efficiency of the developed nanoceutical fabric. To our understanding the novel nanoceutical fabric used in one-way valve of a face mask would be the choice to assure breathing comfort along with source control of COVID-19 infection. The developed nanosensitized cloth can also be used as antibacterial/anti CoV-2 washable dress material in general.

GRAPHICAL ABSTRACT O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

A novel nanoceutical cotton fabric duly sensitized with non-toxic zinc oxide nanoflower can potentially be used as membrane filter in the one way valve of face mask to assure breathing comfort along with source control of COVID-19 infection. The nanoceutical fabric denatures the SARS-CoV-2 spike protein and makes the microorganism ineffective.


title: E. coli chemotaxis is information-limited

abstract: Organisms must acquire and use environmental information to guide their behaviors. However, it is unclear whether and how information quantitatively limits behavioral performance. Here, we relate information to behavioral performance in Escherichia coli chemotaxis. First, we derive a theoretical limit for the maximum achievable gradient-climbing speed given a cells information acquisition rate. Next, we measure cells gradient-climbing speeds and the rate of information acquisition by the chemotaxis pathway. We find that E. coli make behavioral decisions with much less than the 1 bit required to determine whether they are swimming up-gradient. However, they use this information efficiently, performing near the theoretical limit. Thus, information can limit organisms performance, and sensory-motor pathways may have evolved to efficiently use information from the environment.


title: SARS-CoV-2 B.1.1.7 and B.1.351 Spike variants bind human ACE2 with increased affinity

abstract: SARS-CoV2 being highly infectious has been particularly effective in causing widespread infection globally and more variants of SARS-CoV2 are constantly being reported with increased genomic surveillance. In particular, the focus is on mutations of Spike protein, which binds human ACE2 protein enabling SARS-CoV2 entry and infection. Here we present a rapid experimental method leveraging the speed and flexibility of Mircoscale Thermopheresis (MST) to characterize the interaction between Spike Receptor Binding Domain (RBD) and human ACE2 protein. The B.1.351 variant harboring three mutations, (E484K, N501Y, and K417N) binds the ACE2 at nearly five-fold greater affinity than the original SARS-COV-2 RBD. We also find that the B.1.1.7 variant, binds two-fold more tightly to ACE2 than the SARS-COV-2 RBD.


title: Gradients of receptor expression in the macaque cortex

abstract: Dynamics and functions of neural circuits depend on synaptic interactions mediated by receptors. Therefore, a comprehensive map of receptor organization is needed to understand how different functions may emerge across distinct cortical regions. Here we use in-vitro receptor autoradiography to measure the density of 14 neurotransmitter receptor types in 109 areas of macaque cortex. We integrate the receptor data with other anatomical, genetic and functional connectivity data into a common cortical space. We uncovered a principal gradient of increasing receptor expression per neuron aligned with cortical hierarchy from early sensory cortex to higher cognitive areas. A second gradient, primarily driven by 5-HT1A receptors, peaks in the anterior and subcallosal cingulate, suggesting that the macaque may be a promising animal model for major depressive disorder. The receptor gradients may enable rapid, reliable information processing in sensory cortical areas and slow, flexible integration of information in higher cognitive areas.


title: Kynurenine pathway in post-mortem prefrontal cortex and cerebellum in schizophrenia: relationship with monoamines and symptomatology.

abstract:
Backgroundthe cortico-cerebellar-thalamic-cortical circuit has been implicated in the emergence of psychotic symptoms in schizophrenia (SZ). The kynurenine pathway (KP) has been linked to alterations in glutamatergic and monoaminergic neurotransmission and to SZ symptomatology through the production of the metabolites quinolinic acid (QA) and kynurenic acid (KYNA).

Methodsthis work describes alterations in KP in the post-mortem prefrontal cortex (PFC) and cerebellum (CB) of 15 chronic SZ patients and 14 control subjects in PFC and 13 control subjects in CB using immunoblot for protein levels and ELISA for interleukins and QA and KYNA determinations. Monoamine metabolites were analysed by High Performance Liquid Chromatography and SZ symptomatology was assessed by Positive and Negative Syndrome Scale (PANSS). The association of KP with inflammatory mediators, monoamine metabolism and SZ symptomatology was explored.

Resultsin the PFC, the presence of the anti-inflammatory cytokine IL-10 together with IDO2 and KATII enzymes decreased in SZ, while TDO and KMO enzymes expression increased. A network interaction analysis showed that in the PFC IL-10 was coupled to the QA branch of the kynurenine pathway (TDO-KMO-QA), whereas IL-10 associated with KMO in CB. KYNA in the CB inversely correlated with negative and general PANSS psychopathology. Although there were no changes in monoamine metabolites content in the PFC in SZ, a network interaction analysis showed associations between dopamine and methoxyhydroxyphenylglycol degradation metabolite. Direct correlations were found between general PANSS psychopathology and the serotonin degradation metabolite, 5-hydroxyindoleacetic acid. Interestingly, KYNA in the CB inversely correlated with 5-hydroxyindoleacetic acid in the PFC.

Conclusionsthus, this work found alterations in KP in two brain areas belonging to the cortico-cerebellar-thalamic-cortical circuit associated with SZ symptomatology, with a possible impact across areas in 5-HT degradation.


title: Ribosomes in RNA granules are stalled on mRNA sequences that are consensus sites for FMRP association.

abstract: Local translation in neurons is mediated in part by the reactivation of stalled polysomes. Stalled polysomes may be enriched within the pellet of sucrose gradients used to separate polysomes from monosomes. We find that this fraction, isolated from P5 rat brains of both sexes, is enriched in proteins implicated in stalled polysome function, such as the fragile X mental retardation protein (FMRP) and Up-frameshift mutation 1 homolog (UPF1). Cryo-EM analysis of ribosomes in this fraction indicates they are stalled, mainly in the hybrid state. Ribosome profiling of this fraction showed an abundance of footprint reads derived from mRNAs of cytoskeletal proteins implicated in neuronal development and an enrichment of footprint reads on RNA binding proteins. Compared to those usually found in ribosome profiling studies, the footprint reads were more extended on their 3end and were found in reproducible peaks in the mRNAs. These peaks were enriched in motifs previously associated with mRNAs cross-linked to FMRP in vivo, independently linking the ribosomes in the sedimented pellet to the ribosomes associated with FMRP in the cell. The data supports a model in which specific sequences in mRNAs act to stall translation elongation in neurons.


title: Levetiracetam treatment normalizes levels of the presynaptic endocytosis machinery and restores non-amyloidogenic APP processing in App knock-in mice.

abstract: Increasing evidence indicates that toxic amyloid-beta (A{beta}) peptides, produced by sequential proteolytic cleavage of the Amyloid Precursor Protein (APP), induce neuronal circuit hyperexcitability in the early stages of Alzheimers disease (AD). As a result, treatments that modulate this early excitatory/inhibitory imbalance could act as potential AD therapies. Levetiracetam, an atypical antiepileptic drug, has garnered recent interest, despite the mechanism(s) of action remaining elusive. In this study, we set out to identify the pathways and mechanisms primarily affected by levetiracetam in diseased brains of amyloid pathology. Using the App knock-in mouse models and multiplexed TMT-quantitative mass spectrometry-based proteomic analysis to determine how levetiracetam affects the proteome, our findings demonstrate that levetiracetam treatment selectively normalizes levels of presynaptic endocytosis proteins and is capable of lowering A{beta}42 levels by altering APP processing. These novel findings demonstrate a mechanism of action for how levetiracetam lowers A{beta}42 production.


title: Laser-facilitated epicutaneous immunization with SARS-CoV-2 spike protein induces ACE2 blocking antibodies in mice

abstract: The skin represents an attractive target tissue for vaccination against respiratory viruses such as SARS-CoV-2. Laser-facilitated epicutaneous immunization (EPI) has been established as a novel technology to overcome the skin barrier, which combines efficient delivery via micropores with an inherent adjuvant effect due to the release of danger-associated molecular patterns. Here we delivered the S1 subunit of the Spike protein of SARS-CoV-2 to the skin of BALB/c mice via laser-generated micropores with or without CpG-ODN1826 or the B subunit of heat-labile enterotoxin of E.coli (LT-B). EPI induced serum IgG titers of 1:3200 that could be boosted 5 to 10-fold by co-administration of LT-B and CpG, respectively. Sera were able to inhibit binding of the spike protein to its receptor ACE2. Our data indicate that delivery of recombinant spike protein via the skin may represent an alternative route for vaccines against Covid-19.