title: Vascular dysregulation following SARS-CoV-2 infection involves integrin signaling through a VE-Cadherin mediated pathway

abstract: The vascular barrier is heavily injured following SARS-CoV-2 infection and contributes enormously to life-threatening complications in COVID-19. This endothelial dysfunction is associated with the phlogistic phenomenon of cytokine storms, thrombotic complications, abnormal coagulation, hypoxemia, and multiple organ failure. The mechanisms surrounding COVID-19 associated endotheliitis have been widely attributed to ACE2-mediated pathways. However, integrins have emerged as possible receptor candidates for SARS-CoV-2, and their complex intracellular signalling events are essential for maintaining endothelial homeostasis. Here, we showed that the spike protein of SARS-CoV-2 depends on its RGD motif to drive barrier dysregulation through hijacking integrin V{beta}3. This triggers the redistribution and internalization of major junction protein VE-Cadherin which leads to the barrier disruption phenotype. Both extracellular and intracellular inhibitors of integrin V{beta}3 prevented these effects, similarly to the RGD-cyclic peptide compound Cilengitide, which suggests that the spike protein - through its RGD motif - binds to V{beta}3 and elicits vascular leakage events. These findings support integrins as an additional receptor for SARS-CoV-2, particularly as integrin engagement can elucidate many of the adverse endothelial dysfunction events that stem from COVID-19.


title: Hypoxia inducible factors regulate infectious SARS-CoV-2, epithelial damage and respiratory symptoms in a hamster COVID-19 model.

abstract: Understanding the host pathways that define susceptibility to SARS-CoV-2 infection and disease are essential for the design of new therapies. Oxygen levels in the microenvironment define the transcriptional landscape, however the influence of hypoxia on virus replication and disease in animal models is not well understood. In this study, we identify a role for the hypoxic inducible factor (HIF) signalling axis to inhibit SARS-CoV-2 infection, epithelial damage and respiratory symptoms in Syrian hamsters. Pharmacological activation of HIF with the prolyl-hydroxylase inhibitor FG-4592 significantly reduced the levels of infectious virus in the upper and lower respiratory tract. Nasal and lung epithelia showed a reduction in SARS-CoV-2 RNA and nucleocapsid expression in treated animals. Transcriptomic and pathological analysis showed reduced epithelial damage and increased expression of ciliated cells. Our study provides new insights on the intrinsic antiviral properties of the HIF signalling pathway in SARS-CoV-2 replication that may be applicable to other respiratory pathogens and identifies new therapeutic opportunities.

category: microbiology


title: Omicron variant of SARS-COV-2 gains new mutations in New Zealand and Hong Kong

abstract: Disclaimer statementThe authors have withdrawn this manuscript because an issue was raised about the SARS- COV-2 genome sequence data used in the study. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

category: genomics


title: Structural basis of Omicron immune evasion: A comparative computational study of Spike protein-Antibody interaction.

abstract:
The COVID-19 pandemic has caused more than 424 million infections and 5.9 million deaths so far. The vaccines used against SARS-COV-2 by now have been able to develop some neutralising antibodies in the vaccinated human population and slow down the infection rate. The effectiveness of the vaccines has been challenged by the emergence of the new strains with numerous mutations in the spike (S) protein of SARS-CoV-2. Since S protein is the major immunogenic protein of the virus and also contains Receptor Binding Domain (RBD) that interacts with the human Angiotensin-Converting Enzyme 2 (ACE2) receptors, any mutations in this region should affect the neutralisation potential of the antibodies leading to the immune evasion. Several variants of concern (VOC) of the virus have emerged so far. Among them, the most critical are Delta (B.1.617.2), and recently reported Omicron (B. 1.1.529) which have acquired a lot of mutations in the spike protein. We have mapped those mutations on the modelled RBD and evaluated the binding affinities of various human antibodies with it. Docking and molecular dynamics simulation studies have been used to explore the effect of the mutations on the structure of the RBD and the RBD-antibody interaction. The analysis shows that the mutations mostly at the interface of a nearby region lower the binding affinity of the antibody by ten to forty per cent, with a downfall in the number of interactions formed as a whole and therefore, it implies the generation of immune escape variants. Notable mutations and their effect was characterised by performing various analyses that explain the structural basis of antibody efficacy in Delta and a compromised neutralisation effect for the Omicron variant. Our results pave the way for robust vaccine design that can be effective for many variants.

Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

SynopsisThe research study utilises comparative docking and MD simulations analyses to illustrate how mutations in delta and omicron variants affect the binding of antibodies to the spike receptor binding domain (RBD) of SARS CoV-2.

category: biophysics


title: Neutrophils initiate the destruction of the olfactory epithelium during SARS-CoV-2 infection in hamsters

abstract: The loss of smell related to SARS-CoV-2 infection is one of the most prevalent symptoms of COVID-19. It is now clear that this symptom is related to the massive infection by SARS-CoV-2 of the olfactory epithelium leading to its desquamation. However, the molecular mechanism behind the destabilization of the olfactory epithelium is less clear. Using golden Syrian hamster, we show here that while apoptosis remains at a low level in damaged infected epithelium, the latter is invaded by innate immunity cells. By depleting the neutrophil population or blocking the activity of neutrophil elastase-like proteinases, we reduced the damage induced by the SARS-CoV-2 infection. Surprisingly, the impairment of neutrophil activity led to a decrease of SARS-CoV-2 infection levels in the nasal cavity. Our results indicate a counterproductive role of neutrophils leading to the release of infected cells in the lumen of the nasal cavity and thereby enhanced spreading of the virus.

category: immunology


title: Pulmonary lesions following inoculation with the SARS-CoV-2 Omicron BA.1 (B.1.1.529) variant in Syrian golden hamsters

abstract: The Omicron BA.1 (B.1.1.529) SARS-CoV-2 variant is characterized by a high number of mutations in the viral genome, associated with immune-escape and increased viral spread. It remains unclear whether milder COVID-19 disease progression observed after infection with Omicron BA.1 in humans is due to reduced pathogenicity of the virus or due to pre-existing immunity from vaccination or previous infection. Here, we inoculated hamsters with Omicron BA.1 to evaluate pathogenicity and kinetics of viral shedding, compared to Delta (B.1.617.2) and to animals re-challenged with Omicron BA.1 after previous SARS-CoV-2 614G infection. Omicron BA.1 infected animals showed reduced clinical signs, pathological changes, and viral shedding, compared to Delta-infected animals, but still showed gross- and histopathological evidence of pneumonia. Pre-existing immunity reduced viral shedding and protected against pneumonia. Our data indicate that the observed decrease of disease severity is in part due to intrinsic properties of the Omicron BA.1 variant.


title: Sensorimotor transformation underlying odor-modulated locomotion in walking Drosophila

abstract: Most real-world behaviors are performed with incomplete information. Odor-guided locomotion, an ecologically important behavior essential to an animals survival, is an example of such a behavior. Different odors activate different patterns of olfactory receptor neuron (ORN) classes providing information about which odor is present but does not provide any navigational information. In this study, we investigate the sensorimotor transformation that relates ORN activation to locomotion changes in Drosophila by optogenetically activating different combinations of ORN classes and measuring the resulting changes in locomotion. Three features describe this sensorimotor transformation: First, locomotion depends on both the instantaneous firing frequency (f) and its change (df); the two together serve as short-term memory that allows the fly to automatically adapt its motor program to sensory context. Second, the mapping between f-df and locomotor parameters such as speed or curvature is distinct for each pattern of activated ORNs. Finally, the sensorimotor mapping changes with time after odor exposure allowing integration of information over a longer timescale.

category: neuroscience


title: Motor Cortex Excitability in Chronic Low Back Pain

abstract:
IntroductionChronic pain is associated with dysfunctional cortical excitability. Research has identified altered intracortical motor cortex excitability in Chronic Lower Back Pain (CLBP). However, research identifying the specific intracortical changes underlying CLBP has been met with inconsistent findings. In the present case-control study, we examined intracortical excitability of the primary motor cortex using transcranial magnetic stimulation (TMS) in individuals with CLBP.

MethodsTwenty participants with CLBP (Mage = 54.45 years, SDage = 15.89 years) and 18 age- and gender-matched, pain-free controls (M = 53.83, SD = 16.72) were included in this study. TMS was applied to the hand motor area of the right hemisphere and motor evoked potentials (MEPs) were recorded from the first dorsal interosseous muscle of the contralateral hand. Resting motor threshold (rMT) and MEP amplitude were measured using single-pulse stimulation. Short intracortical inhibition (SICI) and intracortical facilitation (ICF) were assessed using paired-pulse stimulation.

ResultsIndividuals with CLBP had significantly higher rMT (decreased corticospinal excitability) and reduced ICF compared to controls. No significant differences were found in MEP amplitude and SICI.

ConclusionThese findings add to the growing body of evidence that CLBP is associated with deficits in intracortical modulation involving glutamatergic mechanisms.

SignificanceThis article reports chronic lower back pain is associated with changes in intracortical excitability, specifically reduced intracortical facilitation. Furthermore, the imbalance between facilitation and inhibition may be related to pain intensity. These findings may help clinicians in the treatment of chronic pain with an increased focus on using neuromodulation techniques, targeting maladaptive intracortical facilitation, as a potential therapeutic tool in chronic pain.


title: APOE ϵ2 vs APOE ϵ4 dosage shows sex-specific links to hippocampus-default network subregion co-variation

abstract: Alzheimers disease and related dementias (ADRD) are marked by intracellular tau aggregates in the medial-temporal lobe (MTL) and extracellular amyloid aggregates in the default network (DN). Here, we sought to clarify ADRD-related co-dependencies between the MTLs most vulnerable structure, the hippocampus (HC), and the highly associative DN at a subregion resolution. We confronted the effects of APOE {varepsilon}2 and {varepsilon}4, rarely investigated together, with their impact on HC-DN co-variation regimes at the population level. In a two-pronged decomposition of structural brain scans from [~]40,000 UK Biobank participants, we located co-deviating structural patterns in HC and DN subregions as a function of ADRD family risk. Across the disclosed HC-DN signatures, recurrent deviations in the CA1, CA2/3, molecular layer, fornixs fimbria, and their cortical partners related to ADRD risk. Phenome-wide profiling of HC-DN co- variation expressions from these population signatures revealed male-specific associations with air-pollution, and female-specific associations with cardiovascular traits. We highlighted three main factors associated with brain-APOE associations across the different gene variants: happiness, and satisfaction with friendships, and with family. We further showed that APOE {varepsilon}2/2 interacts preferentially with HC-DN co-variation patterns in estimating social lifestyle in males and physical activity in females. Our findings reinvigorate the often-neglected interplay between APOE {varepsilon}2 dosage and sex, which we have linked to fine-grained structural divergences indicative of ADRD susceptibility.


title: Dynamic and selective engrams emerge with memory consolidation

abstract: Episodic memories are encoded by sparse populations of neurons activated during an experience.1 These neural ensembles constitute memory engrams that are both necessary and sufficient for inducing recall even long after memory acquisition.2 This suggests that following encoding, engrams are stabilized to reliably support memory retrieval. However, little is known about the temporal evolution of engrams over the course of memory consolidation or how it impacts mnemonic properties. Here we employed computational and experimental approaches to examine how the composition and selectivity of engrams change with memory consolidation. We modeled engram cells using a spiking recurrent neural network that yielded three testable predictions: memories transition from unselective to selective as neurons are removed from and added to the engram, inhibitory activity during recall is essential for memory selectivity, and inhibitory synaptic plasticity during memory consolidation is critical for engrams to become selective. Using the Cal-Light system to tag activated neurons in vivo with high spatiotemporal precision3 as well as optogenetic and chemogenetic techniques, we conducted contextual fear conditioning experiments that supported each of our models predictions. Our results reveal that engrams are dynamic even within hours of memory consolidation and that changes in engram composition mediated by inhibitory synaptic plasticity are crucial for the emergence of memory selectivity. These findings challenge classical theories of stable memory traces and point to a close link between engram state and memory expression.


title: A large and rich EEG dataset for modeling human visual object recognition

abstract: The human brain achieves visual object recognition through multiple stages of nonlinear transformations operating at a millisecond scale. To predict and explain these rapid transformations, computational neuroscientists employ machine learning modeling techniques. However, state-of-the-art models require massive amounts of data to properly train, and to the present day there is a lack of vast brain datasets which extensively sample the temporal dynamics of visual object recognition. Here we collected a large and rich dataset of high temporal resolution EEG responses to images of objects on a natural background. This dataset includes 10 participants, each with 82,160 trials spanning 16,740 image conditions. Through computational modeling we established the quality of this dataset in five ways. First, we trained linearizing encoding models that successfully synthesized the EEG responses to arbitrary images. Second, we correctly identified the recorded EEG data image conditions in a zero-shot fashion, using EEG synthesized responses to hundreds of thousands of candidate image conditions. Third, we show that both the high number of conditions as well as the trial repetitions of the EEG dataset contribute to the trained models prediction accuracy. Fourth, we built encoding models whose predictions well generalize to novel participants. Fifth, we demonstrate full end-to-end training of randomly initialized DNNs that output M/EEG responses for arbitrary input images. We release this dataset as a tool to foster research in visual neuroscience and computer vision.


title: Place cells are non-randomly clustered by field location in CA1 hippocampus

abstract: A challenge in both modern and historic neuroscience has been achieving an understanding of neuron circuits, and determining the computational and organizational principles that underlie these circuits. Deeper understanding of the organization of brain circuits and cell types, including in the hippocampus, is required for advances in behavioral and cognitive neuroscience, as well as for understanding principles governing brain development and evolution. In this manuscript, we pioneer a new method to analyze the spatial clustering of active neurons in the hippocampus. We use calcium imaging and a rewarded navigation task to record from 100s of place cells in the CA1 of freely moving rats. We then use statistical techniques developed for and in widespread use in geographic mapping studies, global Morans I and local Morans I to demonstrate that cells that code for similar spatial locations tend to form small spatial clusters. We present evidence that this clustering is not the result of artifacts from calcium imaging, and show that these clusters are primarily formed by cells that have place field around previously rewarded locations. We go on to show that, although cells with similar place fields tend to form clusters, there is no obvious topographic mapping of environmental location onto the hippocampus, such as seen in the visual cortex. Insights into hippocampal organization, as in this study, can elucidate mechanisms underlying motivational behaviors, spatial navigation, and memory formation.


title: Dynamic single-cell RNA sequencing reveals BCG vaccination curtails SARS-CoV-2 induced disease severity and lung inflammation

abstract: COVID-19 continues to exact a toll on human health despite the availability of several vaccines. Bacillus Calmette Guerin (BCG) has been shown to confer heterologous immune protection against viral infections including COVID-19 and has been proposed as vaccine against SARS-CoV-2 (SCV2). Here we tested intravenous BCG vaccination against COVID-19 using the golden Syrian hamster model together with immune profiling and single cell RNA sequencing (scRNAseq). We observed that BCG reduced both lung SCV2 viral load and bronchopneumonia. This was accompanied by an increase in lung alveolar macrophages, a reversal of SCV2-mediated T cell lymphopenia, and reduced lung granulocytes. Single cell transcriptome profiling showed that BCG uniquely recruits immunoglobulin-producing plasma cells to the lung suggesting accelerated antibody production. BCG vaccination also recruited elevated levels of Th1, Th17, Treg, CTLs, and Tmem cells, and differentially expressed gene (DEG) analysis showed a transcriptional shift away from exhaustion markers and towards antigen presentation and repair. Similarly, BCG enhanced lung recruitment of alveolar macrophages and reduced key interstitial macrophage subsets, with both cell-types also showing reduced IFN-associated gene expression. Our observations indicate that BCG vaccination protects against SCV2 immunopathology by promoting early lung immunoglobulin production and immunotolerizing transcriptional patterns among key myeloid and lymphoid populations.


title: Dual spatially resolved transcriptomics for SARS-CoV-2 host-pathogen colocalization studies in humans

abstract: To advance our understanding of cellular host-pathogen interactions, technologies that facilitate the co-capture of both host and pathogen spatial transcriptome information are needed. Here, we present an approach to simultaneously capture host and pathogen spatial gene expression information from the same formalin-fixed paraffin embedded (FFPE) tissue section using the spatial transcriptomics technology. We applied the method to COVID-19 patient lung samples and enabled the dual detection of human and SARS-CoV-2 transcriptomes at 55 m resolution. We validated our spatial detection of SARS-CoV-2 and identified an average specificity of 94.92% in comparison to RNAScope and 82.20% in comparison to in situ sequencing (ISS). COVID-19 tissues showed an upregulation of host immune response, such as increased expression of inflammatory cytokines, lymphocyte and fibroblast markers. Our colocalization analysis revealed that SARS-CoV-2+ spots presented shifts in host RNA metabolism, autophagy, NF{kappa}B, and interferon response pathways. Future applications of our approach will enable new insights into host response to pathogen infection through the simultaneous, unbiased detection of two transcriptomes.


title: A pathogenicity locus of Streptococcus gallolyticus subspecies gallolyticus

abstract: Streptococcus gallolyticus subspecies gallolyticus (Sgg) is known to be strongly associated with colorectal cancer (CRC). Recent functional studies further demonstrated that Sgg actively stimulates CRC cell proliferation and promotes the development of colon tumors. However, the Sgg factors important for the pro-proliferative and pro-tumor activities of Sgg remain unclear. Here, we identified a chromosomal locus in Sgg strain TX20005. Deletion of this locus significantly reduced Sgg adherence to CRC cells and abrogated the ability of Sgg to stimulate CRC cell proliferation. Thus, we designate this locus as the Sgg pathogenicity-associated region (SPAR). More importantly, we found that SPAR is important for Sgg pathogenicity in vivo. In a gut colonization model, mice exposed to the SPAR deletion mutant showed significantly reduced Sgg load in the colonic tissues and fecal materials, suggesting that SPAR contributes to the colonization capacity of Sgg. In a mouse model of CRC, deletion of SPAR abolished the ability of Sgg to promote the development of colon tumors growth. Taken together, these results highlight SPAR as a critical pathogenicity determinant of Sgg.


title: Halophilic bacteria Bacillus altitudinis MIM2 producing melanin isolated from Mundra port, Kutch, Gujarat, India

abstract: Melanins are ubiquitous black or brown color pigments exhibiting a wide variety of bioactivities. They are stable and insoluble in nature. Melanin are industrially important pigment currently used in cosmetics, medicine and pharmaceuticals, industries. Bacteria mainly produce three types of melanin namely, Eumelanin, pheomelanin and pyomelanin which is usually extracellularly secreted. This makes the downstream processing of bacterial melanin easier. Stress conditions like salt stress, radiation stress etc. triggers the bacteria to produce melanin and several bacterial species were found to produce melanin under different stress induced conditions. In this present study we had isolated melanin producing bacteria from saline sediment sample of Mundra port, Kutch region of Gujarat state of India. The melanin producing bacteria was characterized using staining, biochemical and molecular methods. The melanin produced was extracted and analyzed using physicochemical techniques. To our knowledge this is the first report on Bacillus altitudinis producing melanin.


title: Broad-spectrum extracellular antiviral properties of Cucurbiturils

abstract: Viruses are microscopic pathogens capable of causing disease and are responsible for a range of human mortality and morbidity worldwide. They can be rendered harmless or destroyed with a range of antiviral chemical compounds. Cucurbit[n]urils (CB[n]s) are a macrocycle chemical compound existing as a range of homologues; due to their structure they can bind to biological materials, acting as supramolecular "hosts" to "guests", such as amino acids. Due to the increasing need for a non-toxic antiviral compound, we investigated whether cucurbit[n]urils could act in an antiviral manner. We have found that certain cucurbit[n]uril homologues do indeed have an antiviral effect against a range of viruses, including RSV and SARS-CoV-2. In particular, we demonstrate that CB[7] is the active homologue of CB[n] mixtures, having an antiviral effect against enveloped and non-enveloped species. High levels of efficacy were observed with five-minute contact times across different viruses. We also demonstrate that CB[7] acts with an extracellular virucidal mode of action via host-guest supramolecular interactions between viral surface proteins and the CB[n] cavity, rather than via cell internalisation or a virustatic mechanism. This finding demonstrates that CB[7] acts as a supramolecular virucidal antiviral (a mechanism distinct from other current extracellular antivirals) demonstrating the potential of supramolecular interactions for future antiviral disinfectants.


title: High resolution reconstruction of a Jumbo bacteriophage infecting capsulated bacteria using hyperbranched tail fibers

abstract: The Klebsiella jumbo myophage {phi}Kp24 displays an unusually complex arrangement of tail fibers interacting with a host cell. In this study, we combined cryo-electron microscopy methods, protein structure prediction methods, molecular simulations, and machine learning approaches to explore the capsid, tail, and tail fibers of this phage at high resolution. We determined the structure of the capsid and tail at 4.3[A] and 4.1[A] resolution. We observed that the tail fibers were highly branched and rearranged dramatically upon cell surface attachment. This complex configuration involves fourteen putative tail fibers with depolymerase activity that provide {phi}Kp24 with the ability to infect a broad panel of capsular polysaccharide (CPS) types of Klebsiella pneumoniae. Taken together, our study provides structural and functional insight into how {phi}Kp24 adapts to the highly variable surfaces of capsulated bacterial pathogens, which will be useful for the development of phage therapy approaches against pan-drug resistant K. pneumoniae strains.


title: Defining the genes required for survival of Mycobacterium bovis in the bovine host offers novel insights into the genetic basis of survival of pathogenic mycobacteria

abstract:
Tuberculosis has severe impacts in both humans and animals. Understanding the genetic basis of survival of both Mycobacterium tuberculosis, the human adapted species, and Mycobacterium bovis, the animal adapted species is crucial to deciphering the biology of both pathogens. There are several studies that identify the genes required for survival of M. tuberculosis in vivo using mouse models, however, there are currently no studies probing the genetic basis of survival of M. bovis in vivo. In this study we utilise transposon insertion sequencing in M. bovis to determine the genes required for survival in cattle. We identify genes encoding established mycobacterial virulence functions such as the ESX-1 secretion system, PDIM synthesis, mycobactin synthesis and cholesterol catabolism that are required in vivo. We show that, as in M. tuberculosis, phoPR is required by M. bovis in vivo despite the known defect in signalling through this system. Comparison to studies performed in glycerol adapted species such as M. bovis BCG and M. tuberculosis suggests that there are differences in the requirement for genes involved in cholesterol import (mce4 operon), oxidation (hsd) and detoxification (cyp125). We report good correlation with existing mycobacterial virulence functions, but also find several novel virulence factors, including genes involved in protein mannosylation, aspartate metabolism and glycerol-phosphate metabolism. These findings further extend our knowledge of the genetic basis of survival in vivo in bacteria that cause tuberculosis and provide insight for the development of novel diagnostics and therapeutics.

ImportanceThis is the first report of the genetic requirements of an animal adapted member of the MTBC in a natural host. M. bovis has devastating impacts in cattle and bovine tuberculosis is a considerable economic, animal welfare and public health concern. The data highlight the importance of mycobacterial cholesterol catabolism and identifies several new virulence factors. Additionally, the work informs the development of novel differential diagnostics and therapeutics for TB in both human and animal populations.


title: Growth inhibition of Akkermansia muciniphila by a secreted pathobiont sialidase

abstract: Akkermansia muciniphila is considered a key constituent of a healthy gut microbiota. In inflammatory bowel disease (IBD), A. muciniphila has a reduced abundance while other, putative pathogenic, mucus colonizers bloom. We hypothesized that interbacterial competition may contribute to this observation. By screening the supernatants of a panel of enteric bacteria, we discovered that a previously uncharacterized Allobaculum species potently inhibits the growth of A. muciniphila. Mass spectrometry analysis identified a secreted Allobaculum sialidase as inhibitor of A. muciniphila growth. The sialidase targets sialic acids on casein O-glycans, thereby altering the accessibility of nutrients critical for A. muciniphila. The altered glycometabolic niche results in distorted A. muciniphila cell division and efficiently arrests its growth. The identification of a novel mechanism of A. muciniphila growth inhibition by a competing bacterial pathobiont may provide a rationale for interventions aimed at restoring and maintaining a healthy microbiota symbiosis in patients with intestinal disease.


title: On-Ramp: a tool for rapid, multiplexed validation of plasmids using nanopore sequencing

abstract: The ability to generate vectors made of recombinant DNA has facilitated many achievements in molecular biology. As these vectors are assembled from many different parts and the enzymatic and bacterial processes used to create them can introduce errors, validation of the final product is an essential part of plasmid assembly. Sanger sequencing is the primary validation method available at a base-pair level, however it has many limitations. These include its inability to sequence through complex secondary structure, read length, and cost when validating the full sequence of plasmids or large numbers of plasmids. There is a need for a sequence validation method that can rapidly and affordably sequence the entirety of many plasmids simultaneously, using tools that are accessible to molecular cloning labs, without the need for the time and cost associated with next-generation sequencing. On-Ramp (Oxford-nanopore based Rapid Analysis of Multiplexed Plasmids) is a nanopore-based sequencing based approach that addresses this need by leveraging novel long-read technology. On-Ramp combines preparation protocols designed specifically for multi-plasmid pools and an analysis pipeline modified for accurate alignment of nanopore sequencing reads resulting from these plasmid pool methods. We demonstrate that through On-Ramp, large numbers of dissimilar or highly similar plasmids can be sequenced simultaneously and consensus sequences generated that capture single base-pair mutations, insertions or deletions, and highly repetitive region sequences can be resolved. Our tool allows for sequencing of multiple plasmids in their entirety at one-third the cost of Sanger sequencing, and through the use of our On-Ramp webapp, labs can obtain sequence results rapidly without the need for bioinformatic expertise. On-Ramp is available at http://OnRamp.BoyleLab.org.

category: molecular biology


title: Endogenous IL-10 Contributes to Wound Healing and Regulates Tissue Repair

abstract:
BackgroundInterleukin-10 (IL-10) is essential in fetal regenerative wound healing and likewise promotes a regenerative phenotype in adult dermal wounds. However, the role of endogenous IL-10 in postnatal dermal wound healing is not well established. We sought to determine the role of IL-10 in murine full thickness, excisional wounds that are splinted to prevent contracture and mimic human patterns of wound closure.

MethodsFull thickness, excisional wounds were made in wildtype (WT) and IL-10-/- mice on a C57BL/6J background (F/M, 8wks old). In a subset of wounds, contraction was prevented by splinting with silicone stents (stenting) and maintaining a moist wound microenvironment using a semi-occlusive dressing. Wounds were examined for re-epithelialization, granulation tissue deposition, and inflammatory cell infiltrate at day 7 and fibrosis and scarring at day 30 post-wounding.

ResultsWe observed no difference in wound healing rate between WT and IL-10-/- mice in either the stented or unstented group. At day 7, unstented IL-10-/- wounds had a larger granulation tissue area and more inflammatory infiltrate than their WT counterparts. However, we did observe more F4/80+ cell infiltrate in stented IL-10-/- wounds at day 7. At day 30, stented wounds had increased scar area and epithelial thickness compared to unstented wounds.

ConclusionsThese data suggest that endogenous IL-10 expression does not alter closure of full thickness excisional wounds when wound hydration and excessive contraction are controlled. However, the loss of IL-10 leads to increased inflammatory cell infiltration and scarring. These data suggest that previous reports of increased rates of healing in IL-10-/- mice ought to be revisited considering recent advances in wound healing models. Moreover, these new findings suggest that IL-10 contributes to regulation of inflammation without compromising the healing response.


title: A subpopulation of Talin 1 resides in the nucleus and regulates gene expression

abstract: Talin 1 (TLN1) is best known for its role at focal adhesions, where it activates {beta}-integrin receptors and transmits mechanical stimuli to the actin cytoskeleton. Interestingly, the localization of TLN1 is not restricted to the focal adhesions, but its function in other cellular compartments remains poorly understood. By utilizing both biochemical and confocal microscopy analyses, we show that TLN1 localizes to the nucleus and that it strongly interacts with the chromatin. Importantly, depletion of endogenous TLN1 results in extensive changes in the gene expression profile of human breast epithelial cells. To determine the impact of nuclear TLN1 on gene regulation, we expressed a TLN1 fusion protein containing a nuclear localization signal. Our results revealed that nuclear TLN1 regulates a specific subset of the TLN1-dependent genes. Taken together, we show that apart from localizing at the plasma membrane and cytoplasm, TLN1 also resides in the nucleus where it functions in the regulation of gene expression.


title: Graph deep learning reveals multiple signal pathways activated in anti-citrullinated protein antibodies stimulated synoviocytes

abstract: AO_SCPLOWBSTRACTC_SCPLOWWe present a study of anti-citrullinated protein antibodies stimulated fibroblast-like synoviocytes from patients with rheumatoid arthritis, which is powered by a novel graph deep learning framework applied to single-cell mRNA expression data. This new analytical framework discovered dominant pathways that suggest IL1-IL1R mediated signaling, a novel signal transduction response to ACPA-stimulation, and comfirmed our previous finding of PI3K/AKT activation in response of ACPA-stimulation as well. The study demonstrated the capability of graph deep learning framework on signaling pathway analysis. The findings suggest that ACPAs contribute to distinct pathogenic events through activating multiple signaling pathways.


title: Dual targeting of Saccharomyces cerevisiae Pso2 to mitochondria and the nucleus, and its functional relevance in the repair of DNA interstrand crosslinks

abstract: Repair of DNA interstrand crosslinks (ICLs) involves a functional interplay among different DNA surveillance and repair pathways. Previous work has shown that ICL- inducing agents cause damage to Saccharomyces cerevisiae nuclear and mitochondrial DNA (mtDNA), and its pso2/snm1 mutants exhibit a petite phenotype followed by loss of mtDNA integrity and copy number. Complex as it is, the cause and underlying molecular mechanisms remains elusive. Here, by combining a wide range of approaches with in vitro and in vivo analyses, we assessed the subcellular localization and function of Pso2. We found evidence that the nuclear-encoded Pso2 contains one mitochondrial targeting sequence (MTS) and two nuclear localization signals (NLS1 and NLS2), although NLS1 resides within the MTS. Further analysis revealed that Pso2 is a dual-localized ICL repair protein; it can be imported into both nucleus and mitochondria, and that genotoxic agents enhance its abundance in the latter. While MTS is essential for mitochondrial Pso2 import, either NLS1 or NLS2 is sufficient for its nuclear import; this implies that the two NLS motifs are functionally redundant. Ablation of MTS abrogated mitochondrial Pso2 import, and concomitantly, raised its levels in the nucleus. Strikingly, mutational disruption of both NLS motifs blocked the nuclear Pso2 import; at the same time, they enhanced its translocation into the mitochondria, consistent with the notion that the relationship between MTS and NLS motifs is competitive. However, the nuclease activity of import-deficient species of Pso2 was not impaired. The potential relevance of dual-targeting of Pso2 into two DNA-bearing organelles is discussed.

category: genetics


title: Expressed therapeutic protein yields are predicted by transiently transfected mammalian cell population

abstract: Therapeutic proteins are currently at the hotspot of innovation in the pharmaceutical medicine. However, their industrial production is technically challenging and improved methods for transcriptional manipulation of mammalian industrial cell cultures are needed. In this work we show that some of the most frequently used lab scale transfection efficacy assays fail to predict performance in the protein production settings. We compare the efficacies of a number of transfection reagents using adherent and suspension mammalian cell cultures and assessment based on several assays that utilize reporter protein quantitation, transfected cell population and post-transfection viability of cells. We validate reporter assays for assessing transfection methods in the lab that predict protein production in industrial settings. We also demonstrate that cell penetrating peptide-based transfection achieve significantly higher protein yields compared to PEI and lipoplex methods in both CHO and HEK293 producer cell lines. Availability of fast lab scale screening methods allows future development of improved transfection methods for protein production. One such potentially effective transient transfection method is the CPP-based approach presented currently.

category: biochemistry


title: Integrated platform for multi-scale molecular imaging andphenotyping of the human brain

abstract: A detailed understanding of the anatomical and molecular architectures of brain cells and their brain-wide organization is essential for interrogating human brain function and dysfunction. Extensive efforts have been made toward mapping brain cells through various lenses, and have established invaluable databases yielding new insights. However, we still lack technologies that allow us to capture multi-scale multi-omic properties of individual brain cells in a holistic and scalable manner. To address this challenge, we developed a fully integrated technology platform for scalable three-dimensional (3D) mapping of human brain cells at subcellular resolution by simultaneously extracting brain-wide structure and high-dimensional features (e.g., spatial, molecular, morphological, and connectivity information) of individual cells from the same brain. We accomplished this by seamlessly integrating new chemical, mechanical, and computational tools to enable highly multiplexed multi-scale 3D proteomic imaging of human brain tissues with minimal information loss. We developed a novel microtome, termed MEGAtome, that enables ultra-precision slicing of a whole-mount intact human brain hemisphere and large arrays of animal organs with minimal loss of intra- and inter-slice information. To preserve structural and molecular information within intact human brain slabs and to enable highly multiplexed multiscale imaging, we developed a tissue-gel technology, termed mELAST, that transforms human brain tissues into an elastic, thermochemically stable, and reversibly expandable tissue-hydrogel. To reconstruct the intact brain to preserve the 3D connectivity information of neural fibers, we developed a semi-automatic tissue reconstruction computational pipeline termed HuBRIS. We demonstrated the utility and scalability of the technology platform by processing whole human brain hemispheres, investigating Alzheimer Disease (AD) pathology, and demonstrating the feasibility of projectome mapping.

category: bioengineering


title: Genetic engineering of Acidithiobacillus ferridurans with CRISPR-Cas9/dCas9 systems

abstract:
Genus Acidithiobacillus includes a group of Gram-negative Fe/S-oxidizing acidophilic chemolithotrophic bacteria that are extensively studied and used for biomining processes. Synthetic biology approaches are key means to study and improve their biomining performance. However, efficient genetic manipulations in Acidithiobacillus are still major bottlenecks. In this study, we report a simple and efficient pAFi system (CRISPR-dCas9) and a scarless pAF system (CRISPR-Cas9) for genetic manipulations in A. ferridurans JAGS. The pAFi system harboring both dCas9 and sgRNA was constructed based on pBBR1MCS-2 to knockdown HdrA and TusA genes, separately, of which the transcription levels were significantly downregulated by 48% and 93%, separately. The pAF system carrying pCas9-sgRNA-homology arms was constructed based on pJRD215 to delete HdrB3 gene and overexpress Rus gene. Our results demonstrated that the pAF system is a fast and efficient genome editing method with an average rate of 15-20% per transconjugant in one recombination event, compared to 10-3 and then 10-2 in two recombination events by traditional markerless engineering strategy. Moreover, with these two systems, we successfully regulated iron and sulfur metabolisms in A. ferridurans JAGS: the deletion of HdrB3 reduced 48% of sulfate production, and substitution overexpression of Rus promoter showed 8.82-fold of mRNA level and enhanced iron oxidation rate. With these high-efficient genetic tools for A. ferridurans, we will be able to study gene functions and create useful recombinants for biomining applications. Moreover, these systems could be extended to other Acidithiobacillus strains and promote the development of synthetic biology-assisted biomining.

HighlightsO_LITwo shuttle vectors were constructed for Acidithiobacillus ferridurans
C_LIO_LIAll-in-one pAFi (CRISPR-dCas9) and pAF (CRISPR-Cas9) systems were built up for gene knockdown and genome editing, separately
C_LIO_LIThe transcription levels of HdrA and TusA were reduced 48% and 93% using pAFi system and thus suppressed sulfur oxidation
C_LIO_LIHdrB3 deletion and Rus overexpression were achieved using pAF system and showed significant effects on sulfur and iron oxidation respectively
C_LIO_LIOur pAF system facilitated genome editing in Acidithiobacillus ferridurans with high efficiency (15-20%) in less than 4 weeks
C_LI


title: Exogenous application of dsRNA for the control of viruses in cucurbits

abstract: The recurrent emergence of virus diseases in intensive horticultural crops requires alternative control strategies. Topical application of dsRNA molecules homologous to pathogens has been proposed as a tool for plant virus control. These dsRNAs induce the silencing mechanism (RNAi) that degrades homologous dsRNAs. Cucumber green mottle mosaic virus (CGMMV) represents a serious threat to cucurbit crops. Since genetic resistance to the virus is not yet available in commercial varieties, we aimed to control this virus by RNAi. For this purpose, we obtained constructions both for expressing dsRNA in bacteria to treat cucumber plants by topical application and for agroinoculation in experiments done in the growth chamber. Besides, greenhouse tests were performed in spring and in summer when plants were challenged with the virus and differences in several parameters were investigated, including severity of symptoms, dry weight, total height, virus accumulation and virus-derived small interfering RNAs (vsiRNAs). Spraying of plants with dsRNA reduced significatively CGMMV symptoms in the plants in growth chamber tests. Agroinfiltration experiments done under identical conditions were also effective in limiting the progress of CGMMV disease. In the greenhouse assay performed in spring, symptoms were significatively reduced in dsRNA-sprayed plants and the development of the plants improved with respect to non-treated plants. Virus titters and vsiRNAs were clearly reduced in dsRNA-treated plants. The effect of protection of the dsRNA was less evident in the greenhouse assay carried out in summer. Besides, we investigated the mobility of long (ds)RNA derived from spraying or agroinfiltrated dsRNA and found that it could be detected in local, close distal and far distal points from the site of application. VsiRNAs were also detected in local and distal points and the differences in accumulation were compared. In parallel, we investigated the capacity of dsRNAs derived from genes of tomato leaf curl New Delhi virus (ToLCNDV), another economically important virus in cucurbits, to limit the disease in zucchini, both by agroinfiltration or direct spraying, but found no protection effect. In view of the results, topical application of dsRNAs is postulated as a promising strategy for CGMMV control in cucumber.

category: plant biology


title: Metabolite interactions in the bacterial Calvin cycle and implications for flux regulation

abstract: Metabolite-level regulation of enzyme activity is important for coping with environmental shifts in bacteria. Improved understanding of such regulation could guide attempts to engineer more efficient strains for biochemical production. Recently developed proteomics methods allow for mapping of post-translational interactions, including metabolite-protein interactions, that may be relevant for quickly regulating pathway activity. While feed-back and feed-forward regulation in glycolysis has been investigated, there is relatively little study of metabolite-level regulation in the Calvin cycle, particularly in bacteria. Here, we applied limited proteolysis small molecule mapping (LiP-SMap) to identify and compare metabolite-protein interactions in four potential metabolic engineering host that fix CO2 using the Calvin cycle, including two photoautotrophs (cyanobacteria) and two chemoautotrophs. Species-specific interactions were observed, such as interactions with glucose-6-phosphate in the chemoautotroph Cupriavidus necator and interactions with glyoxylate in the cyanobacteria Synechocystis sp. PCC 6803, which suggests that metabolite-level regulation could be adapted to a certain metabolic capacity or lifestyle of these bacteria. Identified metabolite interactions with Calvin cycle enzymes fructose-1,6/sedoheptulose-1,7-bisphosphatase (F/SBPase) and transketolase were tested for effects on catalytic activity using kinetic assays. GAP increased the activity of both Synechocystis and Cupriavidus F/SBPase, which may act as a feed-forward activation mechanism in the Calvin cycle. A kinetic model incorporating regulations on F/SBPase generally enhanced flux control of ATP and NADPH supply over the cycle. We show that LiP-SMap is a promising technique to explore and uncover novel post-translational metabolic regulation, although the method could benefit from improved sensitivity and specificity.

category: systems biology


title: Overlap across psychotic disorders: A functional network connectivity analysis

abstract: Functional network connectivity has previously been shown to distinguish patient groups from healthy controls (HC). However, the overlap across schizophrenia (SZ), bipolar (BP), and schizoaffective disorder (SAD) is not clear yet. This study focuses on finding overlap across these three psychotic disorders using dynamic FNC (dFNC) and compares it with static FNC (sFNC). We used resting-state fMRI, demographics, and clinical information from the Bipolar- Schizophrenia Network on Intermediate Phenotypes cohort. The data includes three groups of patients with schizophrenia (SZP, N=102), bipolar (BPP, N=102), and schizoaffective (SADP, N=102), their relatives SZR (N=102), BPR (N=102), SADR (N=102), and HC (N=118) groups. After estimating each individuals dFNC, we put them into three identical states. We estimated five different features, including occupancy rate (OCR), number of transitions, the total number of transitions, and the total distance traveled. Finally, the extracted features are tested statistically across patients and HC groups. In addition, we explored the link between the clinical scores and the extracted features. We found that the OCR difference between SZP and SZR in state2, between BPP and HC in state1, and between SADP and HC in state2. Also, state2 OCR separates SZP from BPP, state 3 OCR separates BPP from SZP and SADP. Moreover, the OCR and traveled distance feature extracted from SZ and BP could significantly predict PANSS Total and PANSS General scores. Finally, combined distance features of all disorders showed a significant relationship to PANSS Total and PANSS General scores.


title: Atypical small GTPase RABL3 interacts with RAB11 to regulate early ciliogenesis in human cells

abstract: Primary cilia are near-ubiquitously assembled on cells in the human body and are broadly associated with genetic diseases and cancers. In the early stage of ciliogenesis, the ciliary vesicle (CV) is formed on the mother centriole, which nucleates the primary cilium. However, the regulatory mechanisms underlying CV formation have not yet been fully elucidated. Here, we found that the atypical small GTPase RAB-Like 3 (RABL3) is necessary to assemble primary cilia in human cells. RABL3 directly interacts with RAB11, which is involved in CV formation. RABL3 localizes around the centrosome during early ciliogenesis, reminiscent of RAB11 dynamics. Furthermore, RABL3 positively controls the CV formation like RAB11. These findings suggest that RABL3 plays an important role, in cooperation with RAB11, in CV formation during early ciliogenesis.

category: cell biology


title: Endocytosis is required for access of surface-bound cargo to the flagellar pocket of trypanosomes

abstract:
All endo- and exocytosis in the African trypanosome Trypanosoma brucei occurs at a single subdomain of the plasma membrane. This subdomain, the flagellar pocket, is a small vase-shaped invagination containing the root of the cells single flagellum. Several cytoskeleton-associated multiprotein complexes are coiled around the neck of the flagellar pocket on its cytoplasmic face. One of these, the hook complex, may affect macromolecule entry into the flagellar pocket lumen. In previous work, knockdown of the hook complex component TbMORN1 resulted in larger cargo being unable to enter the flagellar pocket. In this study, the hook complex component TbSmee1 was characterised in bloodstream form Trypanosoma brucei and was found to be essential for cell viability. TbSmee1 knockdown resulted in flagellar pocket enlargement, and impaired access to the pocket membrane by surface-bound cargo. Inhibition of endocytosis by knockdown of clathrin phenocopied TbSmee1 knockdown, suggesting that endocytic activity itself is a prerequisite for the entry of surface-bound cargo into the flagellar pocket.

SummaryCharacterisation of the essential trypanosome protein TbSmee1 suggests that endocytosis is required for flagellar pocket access of surface-bound cargo


title: Crumbs complex-directed apical membrane dynamics in epithelial cell ingression

abstract: Epithelial cells often leave their tissue context and ingress to form new cell types or acquire migratory ability to move to distant sites during development and tumor progression. Cell lose their apical membrane and epithelial adherens junctions during ingression. However, how factors that organize apical-basal polarity contribute to ingression is unknown. Here, we show that the dynamic regulation of the apical Crumbs polarity complex is crucial for normal neural stem cell ingression. Crumbs endocytosis and recycling allow ingression to occur in a normal timeframe. During early ingression, Crumbs and its complex partner the RhoGEF Cysts support myosin and apical constriction to ensure robust ingression dynamics. During late ingression, the E3-ubiquitin ligase Neuralized facilitates the disassembly of the Crumbs complex and the rapid endocytic removal of the apical cell domain. Our findings reveal a mechanism integrating cell fate, apical polarity, endocytosis, vesicle trafficking, and actomyosin contractility to promote cell ingression, a fundamental morphogenetic process observed in animal development and cancer.

category: developmental biology


title: Honeycomb crystallography: Experiment-theory approach for bee comb formation under geometric frustrations

abstract: 1As honeybees build their nests in pre-existing tree cavities, they must deal with dealing with the presence of geometric constraints, resulting in non-regular hexagons and topological defects in the comb. In this work, we study how bees adapt to their environment in order to regulate the comb structure. Specifically, we identify the irregularities in honeycomb structure in the presence of various geometric frustrations. We 3D-print experimental frames with a variety of constraints imposed on the imprinted foundations. The combs constructed by the bees show clear evidence of reoccurring patterns built by bees in response to specific geometric frustrations on these starter frames. Furthermore, using an experimental-modeling framework, we demonstrate that these patterns can be successfully modeled and replicated through a simulated annealing process, in which the minimized potential is a variation of the Lennard-Jones potential that only considers first-neighbor interactions according to a Delaunay triangulation. Our simulation results not only confirm the connection between honeycomb structures and other crystal systems such as graphene, but also show that irregularities in the honeycomb structure can be explained as the result of local interactions between honeybees and their immediate surroundings, leading to emergent global order. Additionally, our computational model can be used to describe specific strategies that bees use to effectively solve each geometric mismatch problem while minimizing cost of comb building.

category: ecology


title: Honeycomb crystallography: comb formation under geometric frustrations


title: Sex chromosome differentiation via changes in the Y chromosome repeat landscape in African annual killifishes Nothobranchius furzeri and N. kadleci

abstract:
Repetitive DNA represents an important driver of sex chromosome differentiation. Yet, repetitive sequences tend to be misrepresented or overlooked in genomic studies. We analysed repetitive DNA landscape of sex chromosomes in several populations of a turquoise killifish Nothobranchius furzeri and its sister species N. kadleci (Teleostei: Nothobranchiidae), representatives of African annual killifishes with high rate of karyotype and sex chromosome evolution. We combined bioinformatic analyses of repeatome with molecular cytogenetic techniques such as comparative genomic hybridization, fluorescence in situ hybridization with satellite sequences, genes for ribosomal RNAs (rDNA) and bacterial artificial chromosomes (BACs) and immunostaining of SYCP3 and MLH1 proteins, which marked lateral elements of synaptonemal complexes and recombination sites, respectively. We revealed that N. furzeri and N. kadleci share the XY sex chromosome system, which is thus much older than previously assumed. Sex chromosomes are mostly heteromorphic as evidenced by distinct distribution of satellite DNAs and major rDNA. Yet, the heteromorphic XY sex chromosomes pair almost exclusively regularly in meiosis, which implies synaptic adjustment. Physical mapping of BACs identified inversions on Y chromosomes of the N. kadleci populations, akin to the pattern previously reported in N. furzeri. Yet, repetitive DNA landscape of X and Y sex chromosomes either diverged in parallel in populations of both species or it evolved in their common ancestor and thus predates the inversions. The observed differentiation via repeat repatterning thus cannot be explained by the classical sexually antagonistic model. Rather, we hypothesized that relaxed meiotic drive and recombination reduced by neutral processes could drive changes in repeatome and secondary inversions could be maintained by sexually antagonistic regulatory effects resulting from early evolution of dosage compensation.

Author summaryEarly differentiation of sex chromosomes is not yet satisfactorily understood despite intensive research effort. Homomorphic sex chromosomes and their rapid turnover are common in teleost fishes, which makes them excellent models for studying evolution of nascent sex chromosomes. We investigated sex chromosomes in several populations of two sister species of African annual killifishes, Nothobranchius furzeri and N. kadleci, particularly their repetitive landscape, which was misrepresented in previous genomic studies. Combination of cytogenetic and genomic approaches revealed that both species share heteromorphic XY sex chromosome system. The N. furzeri XY sex chromosomes thus evolved earlier than previously expected. In N. kadleci, Y-linked inversions analogous to those reported in N. furzeri were detected. Changes in repetitive DNA distribution on sex chromosomes are either convergent or occurred in a common ancestor of both species, prior to the inversion events. The observed sex chromosome differentiation on repetitive DNA level thus cannot be reconciled with the classical theoretical model of sex chromosome evolution driven by sexually antagonistic selection. We invoke alternative explanations such as relaxed meiotic drive and recombination reduced by neutral processes, and we hypothesize that secondary inversions could be maintained by early evolution of dosage compensation resulting in sexually antagonistic regulatory effects.

category: evolutionary biology


title: Detecting patterns of accessory genome coevolution in bacterial species using data from thousands of bacterial genomes

abstract: Bacterial genomes exhibit widespread horizontal gene transfer, resulting in highly variable genome content that complicates the inference of genetic interactions. In this study, we develop a method for detecting coevolving genes from large datasets of bacterial genomes that we call a "coevolution score". The method is based on pairwise comparisons of closely related individuals, analogous to a pedigree study in eukaryotic populations. This approach avoids the need for an accurate phylogenetic tree and allows very large datasets to be analyzed for signatures of recent coevolution. We apply our method to all of the more than 3 million pairs of genes from the entire annotated Staphylococcus aureus accessory genome of 2,756 annotated genes using a database of over 40,000 whole genomes. We find many pairs of genes that that appear to be gained or lost in a coordinated manner, as well as pairs where the gain of one gene is associated with the loss of the other. These pairs form networks of dozens of rapidly coevolving genes, primarily consisting of genes involved in metal resistance, virulence, mechanisms of horizontal gene transfer, and antibiotic resistance, particularly the SCCmec complex. Our results reflect the fact that the evolution of many bacterial pathogens since the middle of the twentieth century has largely been driven by antibiotic resistance gene gain, and in the case of S. aureus the SCCmec complex is the most prominent of these elements driving the evolution of resistance. The frequent coincidence of these gene gain or loss events suggests that S. aureus switch between antibiotic-resistant niches and antibiotic-susceptible ones. While we focus on gene gain and loss, our method can also detect genes which tend to acquire substitutions in tandem or, in datasets that include phenotypic information, genotype-phenotype or phenotype-phenotype coevolution.


title: R-BIND 2.0: An Updated Database of Bioactive RNA-Targeting Small Molecules and Associated RNA Secondary Structures

abstract: Discoveries of RNA roles in cellular physiology and pathology are raising the need for new tools that modulate the structure and function of these biomolecules, and small molecules are proving useful. In 2017, we curated the RNA-targeted BIoactive ligaNd Database (R-BIND) and discovered distinguishing physicochemical properties of RNA-targeting ligands, leading us to propose the existence of an "RNA-privileged" chemical space. Biennial updates of the database and the establishment of a website platform (rbind.chem.duke.edu) have provided new insights and tools to design small molecules based on the analyzed physicochemical and spatial properties. In this report and R-BIND 2.0 update, we refined the curation approach and ligand classification system as well as conducted analyses of RNA structure elements for the first time to identify new targeting strategies. Specifically, we curated and analyzed RNA target structural motifs to determine properties of small molecules that may confer selectivity for distinct RNA secondary and tertiary structures. Additionally, we collected sequences of target structures and incorporated an RNA Structure Search algorithm into the website that outputs small molecules targeting similar motifs without a priori secondary structure knowledge. Cheminformatic analyses revealed that, despite the 50% increase in small molecule library size, the distinguishing properties of R-BIND ligands remained significantly different to that of proteins and are therefore still relevant to RNA-targeted probe discovery. Combined, we expect these novel insights and website features to enable rational design of RNA-targeted ligands and to serve as a resource and inspiration for a variety of scientists interested in RNA targeting.


title: Mechanistic insight into the ATP hydrolysis cycle of tick-borne encephalitis virus helicase

abstract: Helicase domain of nonstructural protein 3 (NS3H) unwinds double-stranded RNA replication intermediate during flavivirus life cycle in ATP-dependent manner. While the helicases mechanism of Dengue and Zika viruses has been extensively studied, little is known about helicase activity of the tick-borne encephalitis virus NS3. In the current study, we demonstrated that ATP hydrolysis cycle of NS3H is strongly stimulated by ssRNA but not ssDNA, suggesting that NS3H is an RNA-specific helicase. However, ssDNA binding inhibits ATPase activity in a non-competitive manner. We also captured several structural snapshots of key ATP hydrolysis stages. An intermediate, in which the inorganic phosphate (Pi) and ADP resulted from ATP hydrolysis and remain trapped inside the ATPase site, suggests that Pi release is the rate-limiting step and is accelerated by RNA binding and/or translocation. Based on these structures, we modeled NS3H-ssRNA and -ssDNA binding and performed MD simulations. Our model suggests that NS3H-ssRNA binding triggers conformational changes, revealing the coupling between helicase and ATPase activities. The structural models revealed that ssDNA inhibition may occur via non-specific ssDNA binding to several positively charged surface patches which in turn causes repositioning of ATP molecule within the ATPase site.


title: Multi-level cellular and functional annotation of single-cell transcriptomes

abstract: Single-cell RNA-sequencing (scRNA-seq) offers unprecedented insight into heterogenous biology, allowing for the interrogation of cellular populations and gene expression programs at single-cell resolution. Here, we introduce scPipeline, a single-cell analytic toolbox that offers modular workflows for multi-level cellular annotation and user-friendly analysis reports. Novel methods that are introduced to facilitate scRNA-seq annotation include: (i) co-dependency index (CDI)-based differential expression; (ii) cluster resolution optimization using a marker-specificity criterion; (iii) marker-based cell-type annotation with Miko scoring; and (iv) gene program discovery using scale-free shared nearest neighbor network (SSN) analysis. Our unsupervised and supervised procedures were validated using a diverse collection of scRNA-seq datasets and we provide illustrative examples of cellular and transcriptomic annotation of developmental and immunological scRNA-seq atlases. Overall, scPipeline provides a flexible computational framework for in-depth scRNA-seq analysis.

category: bioinformatics


title: QTLViewer: An interactive webtool for genetic analysis in the Collaborative Cross and Diversity Outbred mouse populations

abstract: The Collaborative Cross (CC) and the Diversity Outbred (DO) mouse populations are related multiparental populations (MPPs), derived from the same eight isogenic founder strains. They carry >50M known genetic variants, which makes them ideal tools for mapping genetic loci that regulate phenotypes, including physiological and molecular traits. Mapping quantitative trait loci (QTLs) requires statistical and computational training, which can present a barrier to access for some researchers. The QTLViewer software is graphical user interface webtool for CC and DO studies that performs QTL mapping through the R/qtl2 package. Additionally, the QTLViewer website serves as a repository for published CC and DO studies, allowing users to explore QTL mapping results interactively and increasing the accessibility of these genetic resources to the broader scientific community.


title: Self-association features of NS1 proteins from different flaviviruses

abstract:
Flaviviruses comprise a large group of arboviral species that are distributed in several countries of the tropics, neotropics, and some temperate zones. Since they can produce neurological pathologies or vascular damage, there has been intense research seeking better diagnosis and treatments for their infections in the last decades. The flavivirus NS1 protein is a relevant clinical target because it is involved in viral replication, immune evasion, and virulence. Being a key factor in endothelial and tissue-specific modulation, NS1 has been largely studied to understand the molecular mechanisms exploited by the virus to reprogram host cells. A central part of the viral maturation processes is the NS1 oligomerization because many stages rely on these protein-protein assemblies. In the present study, the self-associations of NS1 proteins from Zika, Dengue, and West Nile viruses are examined through constant-pH coarse-grained biophysical simulations. Free energies of interactions were estimated for different oligomeric states and pH conditions. Our results show that these proteins can form both dimers and tetramers under conditions near physiological pH even without the presence of lipids. Moreover, pH plays an important role mainly controlling the regimes where van der Waals interactions govern their association. Finally, despite the similarity at the sequence level, we found that each flavivirus has a well-characteristic protein-protein interaction profile. These specific features can provide new hints for the development of binders both for better diagnostic tools and the formulation of new therapeutic drugs.

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title: Replacing bar graphs of continuous data with more informative graphics: Are we making progress?

abstract: Recent work has raised awareness about the need to replace bar graphs of continuous data with informative graphs showing the data distribution. The impact of these efforts is not known. This observational meta-research study examined how often scientists in different fields use various graph types, and assessed whether visualization practices have changed between 2010 and 2020. We developed and validated an automated screening tool, designed to identify bar graphs of counts or proportions, bar graphs of continuous data, bar graphs with dot plots, dot plots, box plots, violin plots, histograms, pie charts, and flow charts. Papers from 23 fields (approximately 1,000 papers/field/year) were randomly selected from PubMed Central and screened (n=227,998). F1 scores for different graphs ranged between 0.83 and 0.95 in the internal validation set. While the tool also performed well in external validation sets, F1 scores were lower for uncommon graphs. Bar graphs are more often used incorrectly to display continuous data than they are used correctly to display counts or proportions. The proportion of papers that use bar graphs of continuous data varies markedly across fields (range in 2020: 4%-58%), with high rates in biochemistry and cell biology, complementary and alternative medicine, physiology, genetics, oncology and carcinogenesis, pharmacology, microbiology and immunology. Visualization practices have improved in some fields in recent years. Fewer than 25% of papers use flow charts, which provide information about attrition and the risk of bias. This study highlights the need for continued interventions to improve visualization and identifies fields that would benefit most.

category: scientific communication and education


title: Cas9-Mediated Knockout of Ndrg2 Enhances the Regenerative Potential of Dendritic Cells for Wound Healing

abstract: Chronic wounds impose a significant healthcare burden to a broad patient population. Cell based therapies, while having shown benefits for the treatment of chronic wounds, have not achieved widespread adoption into clinical practice. Here, we developed a novel CRISPR/Cas9 approach to precisely edit dendritic cells (DCs) to enhance their therapeutic potential for healing chronic wounds. Using single-cell RNA sequencing (scRNA-seq) of tolerogenic DCs, we discover N-myc downregulated gene 2 (Ndrg2), which marks a specific population of DC progenitors, as a promising target for CRISPR knockout (KO). Ndrg2-KO alters the transcriptomic profile of DCs and preserves an immature cell state with a strong, pro-angiogenic and regenerative capacity. We then incorporated our CRISPR-based cell engineering within a hydrogel technology for in vivo cell delivery and developed a highly effective translational approach for DC based immunotherapy that accelerated healing of full-thickness wounds in both non-diabetic and diabetic mouse models. These findings could open the door to future clinical trials using safe gene editing in DCs for treating various types of chronic wounds.


title: The Use and Misuse of Composite Environmental Indices

abstract: Composite indices have been widely used to rank the environmental performance of nations. Such environmental indices can be useful in communicating complex information as a single value and have the potential to generate political and media awareness of environmental issues. However, indices that are poorly constructed can hinder efforts to identify environmental failings. Here, we provide a critical review of the theoretical and methodological foundations of environmental indices to enhance our understanding of the accuracy and applicability of such indices. In the present study we classify existing indices according to ranking goal, measurement components, and weighting methods. Using New Zealand and Niger as case studies, we examine correlations between ranks in ten national level indices to outline how measurement components and the goal of ranking itself may provide a more, or less, optimistic view of the state of a countrys environment. Our results suggest that environmental indices that include human health, socioeconomic, and policy indicators (such as human access to sanitation and clean drinking water) are positively correlated with each other, and that those excluding human health, socioeconomic, and policy indicators are also positively correlated with each other, while these two types of environmental indices are negatively correlated to each other. Our results demonstrate that the inclusion of indicators that do not relate to the actual state of natural environments can confound results. When choosing an existing environmental index - or developing a new one - it is important to assess whether the ranking goal and the included indicators are appropriate. This is important because the inclusion of confounding indicators in environmental indices may misrepresent the actual state of natural environments.


title: Reproducibility problem with a proposed standard method to measure disinfection efficacy

abstract: A collaborative study was carried out in four laboratories to determine the reproducibility of a proposed ASTM International standard method for quantitatively evaluating the efficacy of disinfectants on hard, non-porous surfaces against bacteria. The method, known as the Quantitative Method, has also been suggested as a future regulatory standard for the United States and internationally. The multi-lab study was carried out using Pseudomonas aeruginosa and an alkyl dimethyl benzyl ammonium chloride antimicrobial product diluted in hard water. Results of the study showed acceptable repeatability in log10 reductions within each laboratory, but unacceptable reproducibility across laboratories despite careful analyst training and standardization of test conditions. A follow-up study ruled out analyst-to-analyst differences as the cause of the poor reproducibility. As it currently exists, the Quantitative Method is not sufficiently reproducible. Ruggedness testing to assess the sensitivity of the method to small changes in operational factors is recommended.

category: microbiology


title: Production of novel biosurfactant by a new yeast species isolated from Prunus mume Sieb. et Zucc.

abstract: Biosurfactants reduce surface and interfacial tension due to their amphiphilic properties, and are an eco-friendly alternative for chemical surfactants. In this study, a novel yeast strain JAF-11 that produces biosurfactant was selected using drop collapse method, and the properties of the material were investigated. The nucleotide sequences of the strain were compared with closely related strains and identified based on the D1/D2 domain of the large-subunit rDNA (LSU) and internal transcribed spacer (ITS) regions. Neodothiora populina CPC 39399T, the closest species with strain JAF-11 in the phylogenetic tree, showed a sequence similarity of 97.75% for LSU and 94.27% for ITS, respectively. The result suggests that the strain JAF-11 represent a distinct species which cannot be assigned to any existing genus or species in the family Dothideaceae. Strain JAF-11 was able to produce biosurfactant reducing the surface tension of medium to 34.5 mN/m on the 6th day of culture and the result of measuring the critical micelle concentration (CMC) by extracting the crude biosurfactant was found to be 24 mg l-1. The molecular weight 502 of the purified biosurfactant was confirmed by measuring the fast atom bombardment mass spectrum (FAB-MS). The chemical structure was analyzed by measuring 1H nuclear magnetic resonance (NMR), 13C NMR, two-dimensional NMRs of the compound. The molecular formula was C26H46O9, and it was composed of one octanoyl group and two hexanoyl group to myo-inositol moiety. The new biosurfactant is the first report of a compound produced by a novel yeast strain JAF-11. This new biosurfactant is proposed as potential candidate for use in a variety field.


title: Deep learning based feature extraction for prediction and interpretation of sharp- wave ripples

abstract: Local field potential (LFP) deflections and oscillations define hippocampal sharp-wave ripples (SWR), one of the most synchronous events of the brain. SWR reflect firing and synaptic current sequences emerging from cognitively relevant neuronal ensembles. Current spectral methods fail to capture their mechanistic complexity, thus limiting progress. Here, we show how one-dimensional convolutional networks operating over high-density LFP hippocampal recordings allowed for automatic identification of SWR. When applied to ultra-dense hippocampus-wide recordings, we discovered physiologically relevant processes associated to the emergence of SWR, prompting for novel classification criteria. To gain interpretability, we developed a method to interrogate the operation of the artificial network. We found it relied in feature-based specialization, which permit identification of spatially segregated oscillations and deflections, as well as synchronous population firing. Thus, using deep learning based approaches may change the current heuristic for a better mechanistic interpretation of these relevant neurophysiological events.


title: Claudin5 protects the peripheral endothelial barrier in an organ and vessel type-specific manner

abstract: The pathogenesis of numerous diseases is characterised by disruption of the junctions that form the endothelial cell (EC) barrier, the composition of which may differ greatly between organs. However, the expression level variability and precise contribution of different junctional proteins is poorly understood. Here, we focus on organs with continuous endothelium to identify structural and functional in vivo characteristics of the EC barrier. Assembly of multiple single-cell RNAseq datasets into a single integrated database revealed the variability in EC barrier patterning. Across tissues Claudin5 exhibited diminishing expression along the arteriovenous axis, which correlates with EC barrier integrity. Functional analysis identified tissue-specific differences in leakage patterning and response to agonist-induced leakage. We uncover that Claudin5 loss enhances agonist-induced leakage in an organotypic, vessel type-specific and size-selective manner in an inducible, EC-specific, knock-out mouse. Mechanistically, Claudin5 loss induces no change in junction ultrastructure but alters composition, with concomitant loss of zonula occludens-1 (ZO-1) expression and upregulation of VE-Cadherin. These findings uncover the organ-specific organisation of the EC barrier and distinct importance of Claudin5 in different vascular beds and will aid our ability to modify EC barrier stability in a targeted, organ-specific manner.


title: Human alveolar macrophage response to Mycobacterium tuberculosis: immune characteristics underlying large inter-individual variability

abstract: Mycobacterium tuberculosis (M.tb) establishes residence and growth in human alveolar macrophages (AMs). Large inter-individual variation in M.tb-AM interactions is a potential early indicator of TB risk and efficacy of therapies and vaccines. Herein, we systematically analyze interactions of a virulent M.tb strain with freshly isolated human AMs from 28 healthy adult donors, measuring host RNA expression and secreted candidate proteins associated with TB pathogenesis over 72h. We observe large inter-individual differences in bacterial uptake and growth, with tenfold variation in M.tb load at 72h, reflected by large variation of gene expression programs. Systems analysis of differential and variable RNA and protein expression identifies TB-associated genes and networks (e.g., IL1B and IDO1). RNA time profiles document early stimulation of M1-type macrophage gene expression followed by emergence of an M2-type profile. The fine-scale resolution of this work enables the separation of genes and networks regulating early M.tb growth dynamics, and development of potential markers of individual susceptibility to M.tb infection and response to therapies.


title: Identifying the genes impacted by cell proliferation in proteomics and transcriptomics studies

abstract: Today, hypothesis-free high-throughput profiling allows relative quantification of thousands of proteins or transcripts across samples and thereby identification of differentially expressed genes. It is used in many biological contexts to characterize differences between cell lines and tissues, identify drug mode of action or drivers of drug resistance, among others. Genes can also be differentially regulated because of confounding factors that were not accounted for in the experimental plan, such as change in cell proliferation. Here, we identified genes for which expression consistently correlates with cell proliferation rates in proteomics and transcriptomics high-throughput data sets to determine the overall impact of cell growth rate on these data. We combined the analysis of 449 cell lines and 1,040 cell lines in five proteomics and three transcriptomics data sets to generate a refined list of 223 confounding genes that correlate with cell proliferation rates. These include many actors in DNA replication and mitosis, and genes periodically expressed during the cell cycle. It constitutes a valuable resource when analyzing high-throughput datasets showing changes in proliferation across conditions. We show how to use this resource to analyze in vitro drug screens and tumor samples. By disregarding the proliferation confounders, one can instead focus on the experiment-specific regulation events otherwise buried in the statistical analysis.


title: Integration of multimodal data in the developing tooth reveals candidate dental disease genes

abstract: Dental malformations range from rare syndromes to common nonsyndromic phenotypes. These malformations can predispose individuals to dental disease, which can in turn affect systemic health. While many dental phenotypes are heritable, most cases have not been linked to deleterious mutations in single genes. We demonstrate that human and conserved mouse craniofacial enhancers show enrichment of dental phenotype-associated variants. Given these findings in bulk craniofacial tissues, we looked to determine the role of tooth enhancers in this phenomenon. We used ChIP-seq and machine learning to identify enhancers of E13.5 mouse incisors. Multi-tissue comparisons of human and mouse enhancers revealed that putative tooth enhancers had the strongest enrichment of dental phenotype-associated variants, suggesting a role for dysregulation of tooth development in dental phenotypes. To uncover novel dental phenotype-driving genes in the developing tooth we performed coexpression analysis and annotated the contributing cell types of gene modules using scRNAseq. Through integration of chromatin state, bulk gene coexpression, and cell type resolved gene expression we prioritized a list of candidate novel dental disease genes for future investigations in mouse models and human studies.


title: Decorating chromatin for enhanced genome editing using CRISPR-Cas9

abstract:
CRISPR-associated (Cas) enzymes have revolutionized biology by enabling RNA-guided genome editing. Homology-directed repair (HDR) in the presence of donor templates is currently the most versatile way to introduce precise edits following CRISPR-Cas-induced double-stranded DNA cuts, but HDR efficiency is generally low relative to end-joining pathways that lead to insertions and deletions (indels). We tested the hypothesis that HDR could be increased using a Cas9 construct fused to PRDM9, a chromatin remodeling factor that deposits histone methylations H3K4me3 and H3K36me3 shown to mediate homologous recombination in human cells. Our results show that the fusion protein contacts chromatin specifically at the Cas9 cut site in DNA to double the observed HDR efficiency and increase the HDR:indel ratio by 3-fold compared to that induced by Cas9 alone. HDR enhancement occurred in multiple cell lines with no increase in off-target genome editing. These findings underscore the importance of chromatin structure for the choice of DNA repair pathway during CRISPR-Cas genome editing and provide a new strategy to increase the efficiency of HDR.

Significance StatementCRISPR-Cas-mediated homology-directed repair (HDR) enables precision genome editing for diverse research and clinical applications, but HDR efficiency is often low due to competing end-joining pathways. Here, we describe a simple strategy to influence DNA repair pathway choice and improve HDR efficiency by engineering CRISPR-Cas9-methyltransferase fusion proteins. This strategy highlights the impact of histone modifications on DNA repair following CRISPR-Cas-induced double-stranded breaks and adds to the CRISPR genome editing toolbox.


title: A modality independent proto-organization of human multisensory areas

abstract: The processing of multisensory information is based upon the capacity of brain regions, such as the superior temporal cortex, to combine information across modalities. However, it is still unclear whether the representation of coherent auditory and visual events does require any prior audiovisual experience to develop and function. In three fMRI experiments, intersubject correlation analysis measured brain synchronization during the presentation of an audiovisual, audio-only or video-only versions of the same narrative in distinct groups of sensory-deprived (congenitally blind and deaf) and typically-developed individuals. The superior temporal cortex synchronized across auditory and visual conditions, even in sensory-deprived individuals who lack any audiovisual experience. This synchronization was primarily mediated by low-level perceptual features and relied on a similar modality-independent topographical organization of temporal dynamics. The human superior temporal cortex is naturally endowed with a functional scaffolding to yield a common representation across multisensory events.


title: Functional architecture of cerebral cortex during naturalistic movie-watching

abstract: Characterizing the functional organization of cerebral cortex is a fundamental step in understanding how different kinds of information are processed in the brain. Neuroimaging studies over the past decades have uncovered the function of many cortical areas in response to selected stimulus categories. However, it is still unclear how cortical areas are organized during naturalistic visual and auditory stimulation. Here we used high-resolution functional MRI data from 176 human subjects to map the macro-architecture of the entire cerebral cortex based on responses to a 60-minute audiovisual movie stimulus. A data-driven clustering approach revealed a map of 24 functional areas/networks, each related to a specific aspect of sensory or cognitive processing. The map included three distinct executive control (domain-general) networks which showed a strong push-pull interaction with domain-specific regions in visual, auditory, and language cortex. The cortical parcellation scheme presented here provides a comprehensive and unified map of functionally defined areas, which could replace a large set of functional localizer maps.


title: Reciprocal representation of encoded features by cortical excitatory and inhibitory neuronal populations.

abstract: In the cortex, the interplay between excitation and inhibition determines the fidelity of neuronal representations 1-8. However, while the receptive fields of excitatory neurons are often fine-tuned to the encoded features 9-12, the principles governing the tuning of inhibitory neurons are still elusive 13-21. We addressed this problem by recording populations of neurons in the postsubiculum (PoSub), a cortical area where the receptive fields of most excitatory neurons correspond to a specific head-direction (HD) 12,22-25. In contrast to PoSub-HD cells, the tuning of fast-spiking (FS) cells, the largest class of cortical inhibitory neurons 26-28, was broad and heterogeneous. However, we found that PoSub-FS cell tuning curves were often fine-tuned in the spatial frequency domain, which resulted in various radial symmetries in their HD tuning. In addition, the average frequency spectrum of PoSub-FS cell populations was virtually indistinguishable from that of PoSub-HD cells but different from that of the upstream thalamic HD cells, suggesting that this population co-tuning in the frequency domain has a local origin. Two observations corroborated this hypothesis. First, PoSub-FS cell tuning was independent of upstream thalamic inputs. Second, PoSub-FS cell tuning was tightly coupled to PoSub-HD cell activity even during sleep. Together, these findings provide evidence that the resolution of neuronal tuning is an intrinsic property of local cortical networks, shared by both excitatory and inhibitory cell populations. We hypothesize that this reciprocal feature encoding supports two parallel streams of information processing in thalamocortical networks.


title: Reciprocal feature encoding by corticalexcitatory and inhibitory neurons.


title: The development of efficient communication in the human connectome.

abstract: The structure of the human connectome develops from childhood throughout adolescence to middle age, but how this affects the speed of neuronal signaling is not well described. To understand the maturation process of transmission speed across the connectome, we stimulated electrocorticographic (ECoG) electrode pairs in 75 human subjects of 4 to 51 years old. We then measured the latency of cortico-cortical evoked responses in other electrodes. Each year, responses got faster, until transmission speed reached a minimum latency at the age of 29-44 years old. Responses during middle age were about two times faster compared to childhood. These increases in speed were observed in both long- and short-range connections between and within frontal, central, parietal, and temporal areas. These findings indicate that long-range and short-range communication between brain regions increase in efficiency well throughout adolescence.


title: Corticotropin-releasing hormone signaling from prefrontal cortex to lateral septum supports social novelty preference

abstract: Social preference, the decision to interact with one member of the same species over another, is a key feature of optimizing social interactions. In rodents, social preference relies on both extrinsic factors, such as sex, strain and kinship, and intrinsic ones, such as the memory of previous encounters, which favors interactions with novel compared to familiar animals (social novelty preference). At present, it is unclear which neuronal circuits guide social preferences and whether such circuits promote social interactions with the preferred individuals or suppress interactions with the non-preferred ones. Although both the infra-limbic area of the pre-frontal cortex (ILA) and the lateral septum (LS) have been shown to support social novelty preference, the neuronal circuits and molecular mechanisms by which these brain regions interact to regulate social interactions are unknown. Here, we identify a population of inhibitory neurons in ILA that express the neuropeptide corticotropin releasing hormone (CRH) and project to the rostro-dorsal region of LS (rdLS). Release of CRH from ILA in rdLS during interactions with familiar mice disinhibits rdLS neurons, thereby suppressing interactions with familiar mice and contributing to social novelty preference. We further demonstrate how the maturation of CRH expression during the first two post-natal weeks enables the developmental shift from a preference for littermates in juveniles to a preference for novel mice in adults.


title: Tripartite extended amygdala - basal ganglia CRH circuit drives arousal and avoidance behaviour

abstract:
An adaptive stress response involves various mediators and circuits orchestrating a complex interplay of physiological, emotional and behavioural adjustments. We identified a population of corticotropin-releasing hormone (CRH) neurons in the lateral part of the interstitial nucleus of the anterior commissure (IPACL) - a subdivision of the extended amygdala, which exclusively innervate the substantia nigra (SN). Specific stimulation of this circuit elicits arousal and avoidance behaviour contingent on CRH receptor 1 (CRHR1) located at axon terminals in the SN, which originate from external globus pallidus (GPe) neurons. The neuronal activity prompting the observed behaviour is shaped by IPACLCRH and GPeCRHR1 neurons coalescing in the SN. These results delineate a novel tripartite CRH circuit functionally connecting extended amygdala and basal ganglia nuclei to drive arousal and avoidance behaviour.

One-Sentence SummaryBrain centres involved in emotional and motor control are connected through a stress peptide promoting arousal and avoidance behaviour


title: Neonate personality affects early-life resource acquisition in a large social mammal

abstract: Current debate in the field of animal personality revolves around whether personality is reflecting individual differences in resource allocation or acquisition. Despite the large body of literature, the proximate relationships between personality, resource allocation, and acquisition are still unclear, especially during early stages of development. Here we studied how among-individual differences in behaviour develop over the first 6 months of life, and their potential association with resource acquisition in a free-ranging population of fallow deer (Dama dama). We related proxies of neonate personality - i.e. neonate physiological (heart rate) and behavioural (latency to leave at release) anti-predator responses to human handling - to the proportion of time fawns allocated to scanning during their first summer and autumn of life. We then investigated whether there was a trade-off between scanning time and foraging time in these juveniles, and how it developed over their first 6 months of life. We found that neonates with longer latencies at capture (i.e. risk-takers) allocated less time scanning their environment, but that this relationship was only present when fawns were 3-6 months old during autumn, but not when fawns were only 1-2 months old during summer. We also found that time spent scanning was negatively related to time spent foraging - a relationship rarely tested in juveniles of large mammals - and that this relationship becomes stronger over time, as fawns gradually switch from a nutrition rich (milk) to a nutrition poor (grass) diet. Our results highlight a potential mechanistic pathway in which neonate personality may drive differences in early-life resource acquisition, through allocation, of a large social mammal.


title: On the use of relative brain size

abstract:
O_LIThere is a long standing interest in the effect of relative brain size on other life history variables in a comparative context. Historically, residuals have been used to calculate these effects, but more recently it has been recognised that regression on residuals is not good practice. Instead, absolute brain size and body size are included in a multiple regression, with the idea that this controls for allometry.
C_LIO_LII use a simple simulation to illustrate how a case with brain size as response variable differs from a case with relative brain size as predictor variable. I use the simulated data to test which modelling approach can estimate the underlying causal effects for each case.
C_LIO_LIThe results show that a multiple regression models with both body size and another variable as predictor variable and brain size as response variable work well. However, if relative brain size is a predictor variable, a multiple regression fails to correctly estimate the effects.
C_LIO_LII propose the use of structural equation models to simultaneously estimate relative brain size and its effect on the third variable and discuss other potential methods.
C_LI


title: miR319-targeted TCP4/LANCEOLATE directly regulates OVATE and auxin responses to modulate tomato gynoecium patterning and fruit morphology

abstract: Diversity in fruit morphology is one of the hallmarks of varietal differences among modern cultivars of fruit-bearing crops. As evolutionarily related organs, fruits and leaves share developmental processes, but there are surprisingly few connections between regulatory pathways for fruit and leaf development. Here, we show the regulation of the leaf development-associated TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) TCP4/LANCEOLATE (TCP4/LA) transcription factor by the microRNA319 (miR319) is crucial for gynoecium patterning and establishment of fruit morphology. Loss of miR319 regulation leads to a premature, ectopic TCP4/LA expression during gynoecium patterning, which results in elongated fruits, resembling ovate mutants. TCP4/LA modulates tomato fruit development and morphology partially by directly repressing OVATE expression as early as 5-8 days post-inflorescence (dpi) flower buds. Furthermore, miR319-targeted CINCINNATA-like TCP4/LANCEOLATE controls auxin responses in developing flower buds by directly binding to the SlYUCCA4 promoter. Modulation of auxin biosynthesis by TCP4/LA is shared with other CINCINNATA-like TCPs during Arabidopsis gynoecium patterning. Our study defines a novel miRNA-based molecular link between OVATE, a fundamental gene associated with tomato domestication, and auxin responses in the control of fruit development and morphology. Given the striking variation in fruit shape among members of the Solanaceae family, fine-tuning regulation of gene expression by miRNA coupled with modulation of hormone dynamics may be a common driver in the evolution of fruit-shape diversity.


title: EXTERNAL CONTROL OF CANCER CELLULAR DYNAMICS USING ADAPTIVE MODEL PREDICTIVE CONTROL

abstract: Recent advancements in Cybergenetics have led to the development of new computational and experimental platforms that enable to robustly steer cellular dynamics by applying external feedback control. Such technologies have never been applied to regulate intracellular dynamics of cancer cells. Here, we show in silico that adaptive model predictive control (MPC) can effectively be used to steer signalling dynamics in Non-Small Cell Lung Cancer (NSCLC) cells to resemble those of wild-type cells, and to support the design of combination therapies. Our optimisation-based control algorithm enables tailoring the cost function to force the controller to alternate different drugs and/or reduce drug exposure, minimising both drug-induced toxicity and resistance to treatment. Our results pave the way for new cybergenetics experiments in cancer cells for drug combination therapy design.


title: MODEL PREDICTIVE CONTROL OF CANCER CELLULAR DYNAMICS: A NEW STRATEGY FOR THERAPY DESIGN