title: The Alzheimer's disease risk factor APOE4 drives pro-inflammation in human astrocytes via HDAC-dependent repression of TAGLN3

abstract:
The Apolipoprotein E4 (APOE4) is the major allelic risk factor for late-onset Alzheimers disease (AD). APOE4 associates with a pro-inflammatory phenotype increasingly considered as critical in AD initiation and progression. Yet, the mechanisms driving an APOE4-dependent neuroinflammation remain unelucidated. Leveraging patient specific human induced Pluripotent Stem Cells (iPSCs) we demonstrate inflammatory chronicity and hyperactivated responses upon cytokines in human APOE4 astrocytes via a novel mechanism. We uncovered that APOE4 represses Transgelin 3 (TAGLN3), a new interacting partner of I{kappa}B, thus increasing the NF-kB activity. The transcriptional repression of TAGLN3 was shown to result from an APOE4-dependent histone deacetylase (HDAC) activity. The functional relevance of TAGLN3 was demonstrated by the attenuation of APOE4-driven neuroinflammation after TAGLN3 supplementation. Importantly, TAGLN3 downregulation was confirmed in the brain of AD patients. Our findings highlight the APOE4-TAGLN3 axis as a new pathogenic pathway that paves the way for the development of therapeutics to prevent maladaptive inflammatory responses in APOE4 carriers, while placing TAGLN3 downregulation as a potential biomarker of AD.

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title: Neural Sources Underlying Visual Word Form Processing as Revealed by Steady State Visual Evoked Potentials (SSVEP)

abstract: EEG has been central to investigations of the time course of various neural functions underpinning visual word recognition. Recently the steady-state visual evoked potential (SSVEP) paradigm has been increasingly adopted for word recognition studies due to its high signal-to-noise ratio. Such studies, however, have been typically framed around a single source in the left ventral occipitotemporal cortex (vOT). Here, we combine SSVEP recorded from 16 adult native English speakers with a data-driven spatial filtering approach--Reliable Components Analysis (RCA)--to elucidate distinct functional sources with overlapping yet separable time courses and topographies that emerge when contrasting words with pseudofont visual controls. The first component topography was maximal over left vOT regions with an early latency (approximately 180 msec). A second component was maximal over more dorsal parietal regions with a longer latency (approximately 260 msec). Both components consistently emerged across a range of parameter manipulations including changes in the spatial overlap between successive stimuli, and changes in both base and deviation frequency. We then contrasted word-in-nonword and word-in-pseudoword to test the hierarchical processing mechanisms underlying visual word recognition. Results suggest that these hierarchical contrasts fail to evoke a unitary component that might be reasonably associated with lexical access.

category: neuroscience


title: MHC-matched allogeneic bone marrow transplant fails to eliminate SHIV-infected cells from ART-suppressed Mauritian cynomolgus macaques

abstract:
BackgroundAllogeneic hematopoietic stem cell transplants (allo-HSCTs) dramatically reduce HIV reservoirs in antiretroviral therapy (ART) suppressed individuals. However, the mechanism(s) responsible for these post-transplant viral reservoir declines are not fully understood but may include pre-transplant conditioning regimens, ART-mediated protection of donor cells, and graft-versus-host (GvH) responses. Therefore, we modeled allo-HSCT in ART-suppressed simian-human immunodeficiency virus (SHIV)-infected Mauritian cynomolgus macaques (MCMs) to illuminate factors contributing to transplant-induced viral reservoir decay.

ResultsWe infected four MCMs with CCR5-tropic SHIV162P3 and started ART 6-16 weeks post-infection (p.i.) to establish robust viral reservoirs. We maintained the MCMs on continuous ART during myeloablative conditioning with total body irradiation (TBI) and while transplanting allogeneic MHC-matched /{beta} T cell-depleted bone marrow cells. Post-transplant, we prophylactically treated the MCMs with cyclophosphamide and tacrolimus to prevent GvH disease (GvHD). The transplants produced ~85% whole blood donor chimerism without causing high-grade GvHD. Consequently, three MCMs had undetectable SHIV DNA in their peripheral blood mononuclear cells post-transplant. However, SHIV-harboring cells persisted in various tissues. We detected viral DNA in lymph node biopsies and terminal analyses of tissues between 38 and 62 days post-transplant. Further, we removed ART from one MCM at 63 days post-transplant, resulting in viral rebound within seven days and viral loads nearing 1x108 SHIV RNA copies/ml of plasma after treatment interruption.

ConclusionsOur results indicate that myeloablative conditioning and maintaining ART through the peri-transplant period alone are insufficient for eradicating latent viral reservoirs early after allo-HSCTs. Furthermore, our findings suggest that extended ART and GvH responses may be necessary to substantially deplete viral reservoirs after allo-HSCTs.

category: immunology


title: Role of African swine fever virus (ASFV) proteins EP153R and EP402R in reducing viral persistence and virulence from attenuated BeninΔDP148R

abstract: The limited knowledge on the role of many of the approximately 170 proteins encoded by African swine fever virus restricts progress towards vaccine development. In this study we investigated the effect of deleting combinations of different genes from a previously attenuated virus, Benin{Delta}DP148R on: virus replication in macrophages, virus persistence and clinical signs post immunization, and induction of protection against challenge. Deletion of either EP402R or EP153R genes individually or in combination from Benin{Delta}DP148R did not reduce virus replication in vitro. However, deletion of EP402R dramatically reduced viral persistence in vivo, whilst maintaining high levels of protection against challenge. The additional deletion of EP153R (Benin{Delta}DP148R{Delta}EP153R{Delta}EP402R) further attenuated the virus and no viremia or clinical signs were observed post immunization. This was associated with decreased protection and detection of moderate levels of challenge virus in blood. Interestingly, the deletion of EP153R alone from Benin{Delta}DP148R did not result in further virus attenuation and a slight increase in virus genome copies in blood was observed at different times post immunization when compared with Benin{Delta}DP148R. These results show that EP402R and EP153R have a synergistic role in promoting viremia, however EP153R alone does not seem to have a major impact on virus levels in blood.

category: microbiology


title: A competitive activity-based protein profiling platform yields cell wall synthesis inhibitors active against replicating and non-replicating Mycobacterium tuberculosis

abstract: The identification and validation of a small molecules targets is a major bottleneck in the discovery process for tuberculosis antibiotics. Activity-based protein profiling (ABPP) is an efficient tool for determining a small molecules targets within complex proteomes. However, how target inhibition relates to biological activity is often left unexplored. Here we studied the effects of 1,2,3-triazole ureas on Mycobacterium tuberculosis (Mtb). After screening ~200 compounds, we focused on two inhibitors active against both exponentially replicating and hypoxia-induced drug-tolerant Mtb that form part of a four-compound structure-activity series. The compound with negligible activity revealed potential false positive targets not addressed in other ABPP studies. Biochemistry, computational docking, and morphological analysis confirmed that active compounds preferentially inhibit serine hydrolases with cell wall and lipid metabolism functions and that disruption of the cell wall underlies biological activity. Our findings showed that ABPP identifies the targets most likely relevant to a compounds antibacterial activity.


title: Drosophila fabp  is a retinoid-inducible gene required for Rhodopsin-1 homeostasis and photoreceptor survival

abstract:
Retinoids act as chromophore co-factors for light-detecting rhodopsin proteins. In vertebrates, retinoids also actively regulate gene expression. Whether retinoids regulate gene expression in Drosophila for a specific biological function remains unclear. Here, we report that Drosophila fatty acid binding protein (fabp) is a retinoid-inducible gene required for Rhodopsin-1 (Rh1) protein homeostasis and photoreceptor survival. Specifically, we performed a photoreceptor-specific gene expression profiling study in flies bearing a misfolding-prone Rhodopsin-1 (Rh1) mutant, ninaEG69D, which serves as a Drosophila model for Retinitis Pigmentosa. ninaEG69D photoreceptors showed increased expression of genes that control Rh1 protein levels, along with a poorly characterized gene, fabp. We found that in vivo fabp expression was reduced when the retinoids were deprived through independent methods. Conversely, fabp mRNA was induced when we challenged cultured Drosophila cells with retinoic acid. In flies reared under light, loss of fabp caused an accumulation of Rh1 proteins in cytoplasmic vesicles. fabp mutants exhibited light-dependent retinal degeneration, a phenotype also found in other mutants that block light-activated Rh1 degradation. These observations indicate that a retinoid-inducible gene expression program regulates fabp that is required for Rh1 proteostasis and photoreceptor survival.

Author SummaryRhodopsins are light-detecting proteins that use retinoids as chromophore co-factors. In vertebrates, retinoids also actively regulate gene expression. Whether retinoids regulate Rhodopsin function aside from its role as a chromophore remains unclear. Here, we report that Drosophila fatty acid binding protein (fabp) is a retinoid-inducible gene required for Rhodopsin-1 (Rh1) protein homeostasis and photoreceptor survival. Specifically, we found that fabp is among the genes induced by a misfolding-prone Rhodopsin-1 (Rh1) mutant, ninaEG69D, which serves as a Drosophila model for Retinitis Pigmentosa. We further found that fabp induction in ninaEG69D photoreceptors required retinoids. fabp was required in photoreceptors to help degrade light-activated Rh1. In the absence of fabp, Rh1 accumulated in cytoplasmic vesicles in a light-dependent manner, and exhibited light-dependent retinal degeneration. These observations indicate that a retinoid-inducible gene expression program regulates fabp that is required for Rh1 proteostasis and photoreceptor survival.

category: cell biology


title: SARS-CoV-2 proteins bind heme and hemoglobin

abstract: The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome virus 2 (SARS-CoV-2), has led to a global crisis that included collapsing healthcare systems and shut-down communities, producing considerable economic burden. Despite the number of effective vaccines quickly implemented, the emergence of new variants is a primary concern. The scientific community undertook a rapid response to better study this new virus. However, critical questions about viral protein-protein interactions and mechanisms of its physiopathology are still unclear. Although severe COVID-19 was associated with hematological dysfunctions, scarce experimental data were produced about iron dysmetabolism and the viral proteins possible interaction with hemoglobin (Hb) chains. This work demonstrates the binding of SARS-CoV-2 proteins to hemin and Hb using a multimethodological approach. In silico analysis indicated binding motifs between a cavity in the viral nucleoprotein and hemoglobins porphyrin coordination region. Different hemin binding capacities of mock and SARS-CoV-2-infected culture extracts were noticed using gel electrophoresis and TMB staining. Hemin-binding proteins were isolated from SARS-CoV-2-infected cells by affinity chromatography and identified by shotgun proteomics, indicating that structural (nucleoprotein, spike, and membrane protein) and non-structural (Nsp3 and Nsp7) viral proteins interact with hemin. In vitro analyses of virus adsorption to host cells and viral replication studies in Vero cells demonstrated inhibitory activities - at different levels - by hemin, protoporphyrin IX (PpIX) Hb. Strikingly, free Hb at 1M suppressed viral replication (99 %), and its interaction with SARS-CoV-2 was localized to the RBD region of the Spike protein. The findings showed clear evidence of new avenues to disrupt viral replication and understand virus physiopathology that warrants further investigation.

category: biochemistry


title: Meta-analytic markers reveal a generalizable description of cortical cell types

abstract: Our understanding of cell types has advanced considerably with the publication of single cell atlases. Marker genes play an essential role for experimental validation and computational analyses such as physiological characterization through pathway enrichment, annotation, and deconvolution. However, a framework for quantifying marker replicability and picking replicable markers is currently lacking. Here, using high quality data from the Brain Initiative Cell Census Network (BICCN), we systematically investigate marker replicability for 85 neuronal cell types. We show that, due to dataset-specific noise, we need to combine 5 datasets to obtain robust differentially expressed (DE) genes, particularly for rare populations and lowly expressed genes. We estimate that 10 to 200 meta-analytic markers provide optimal performance in downstream computational tasks. Replicable marker lists condense single cell atlases into interpretable and generalizable information about cell types, opening avenues for downstream applications, including cell type annotation, selection of gene panels and bulk data deconvolution.

category: bioinformatics


title: How many markers are needed to robustly determine a cell's type?


title: 4 SPECIES OF BACTERIA DETERMINISTICALLY FORM A STABLE BIOFILM IN A MILLIFLUIDIC CHANNEL: ASSEMBLY PRINCIPLES

abstract: AO_SCPLOWBSTRACTC_SCPLOWMultispecies microbial adherent communities are widespread in nature and organisms but the principles of their assembly and development remain unclear. Yet, the demand to understand and predict the responses of such living communities to environmental changes is increasing, calling for new approaches. Here, we test the possibility to establish a simplified but relevant model of multispecies biofilm in a laboratory setup enabling in situ real-time monitoring of the community development and control of the environmental parameters in order to decipher the mechanisms underlying the formation of the community. Using video-microscopy and species combinatorial approach, we assess the global and individual species spatiotemporal development in millifluidic channels under constant flow of nutrients. Based on quantitative measurements of expansion kinetics, local dynamics and spatial distribution, we demonstrate that the four chosen species (Bacillus thuringiensis, Pseudomonas fluorescens, Kocuria varians and Rhodocyclus sp.) form a dynamical community that deterministically reaches its equilibrium after about 30 hours of growth. We evidence the emergence of complexity in this simplified community as reported by spatial heterogeneity rise and non-monotonic developmental kinetics. We find interspecies interactions consisting in competition for resources -- in particular oxygen -- and both direct and indirect physical interactions but no positive feedback. Thereby, we introduce a model of multispecies adherent community where effective couplings result from individual species quest for fitness optimization in a moving and heterogenous environment. This control and the understanding of this simplified experimental model shall open new avenues to apprehend adherent bacterial communities behavior in a context of rapid global change.

category: biophysics


title: Mycobacterial infection of precision cut lung slices reveals that the type 1 interferon pathway is locally induced by Mycobacterium bovis but not M. tuberculosis in different cattle breeds

abstract: Tuberculosis exacts a terrible toll on human and animal health. While Mycobacterium tuberculosis (Mtb) is restricted to humans, Mycobacterium bovis (Mb) is present in a large range of mammalian hosts. In cattle, bovine TB (bTB) is a notifiable disease responsible for important economic losses in developed countries and underestimated zoonosis in the developing world. Early interactions that take place between mycobacteria and the lung tissue early after aerosol infection govern the outcome of the disease. In cattle, these early steps remain poorly characterized. The precision-cut lung slice (PCLS) model preserves the structure and cell diversity of the lung. We developed this model in cattle in order to study the early lung response to mycobacterial infection. In situ imaging of PCLS infected with fluorescent Mb revealed bacilli in the alveolar compartment, adjacent or inside alveolar macrophages (AMPs) and in close contact with pneumocytes. We analyzed the global transcriptional lung inflammation signature following infection of PCLS with Mb and Mtb in two French beef breeds: Blonde dAquitaine and Charolaise. Whereas lungs from the Blonde dAquitaine produced high levels of mediators of neutrophil and monocyte recruitment in response to infection, such signatures were not observed in the Charolaise in our study. In the Blonde dAquitaine lung, whereas the inflammatory response was highly induced by two Mb strains, AF2122 isolated from cattle in the UK and Mb3601 circulating in France, the response against two Mtb strains, H37Rv the reference laboratory strain and BTB1558 isolated from zebu in Ethiopia, was very low. Strikingly, the type I interferon pathway was only induced by Mb but not Mtb strains indicating that this pathway may be involved in mycobacterial virulence and host tropism. Hence, the PCLS model in cattle is a valuable tool to deepen our understanding of early interactions between lung host cells and mycobacteria. It revealed striking differences between cattle breeds and mycobacterial strains. This model could help deciphering biomarkers of resistance versus susceptibility to bTB in cattle as such information is still critically needed for bovine genetic selection programs and would greatly help the global effort to eradicate bTB.


title: A polysaccharide deacetylase enhances bacterial adhesion in high ionic strength environments

abstract: The adhesion of organisms to surfaces in aquatic environments provides a diversity of benefits such as better access to nutrients or protection from the elements or from predation. Differences in ionic strength, pH, temperature, shear forces, and other environmental factors impact adhesion and organisms have evolved various strategies to optimize their adhesins for their specific environmental conditions. We know essentially nothing about how bacteria evolved their adhesive mechanisms to attach efficiently in environments with different physico-chemical conditions. Many species of Alphaproteobacteria, including members of the order Caulobacterales, use a polar adhesin, called holdfast, for surface attachment and subsequent biofilm formation in both freshwater and marine environments. Hirschia baltica, a marine member of Caulobacterales, produces a holdfast adhesin that tolerates a drastically higher ionic strength than the holdfast produced by its freshwater relative, Caulobacter crescentus. In this work, we show that the holdfast polysaccharide deacetylase HfsH plays an important role in adherence in high ionic strength environments. We show that deletion of hfsH in H. baltica disrupts holdfast binding properties and structure. Increasing expression of HfsH in C. crescentus improved holdfast binding in high salinity, whereas lowering HfsH expression in H. baltica reduced holdfast binding at high ionic strength. We conclude that HfsH plays a role in modulating holdfast binding at high ionic strength and hypothesize that this modulation occurs through varied deacetylation of holdfast polysaccharides.


title: The UBAP2L ortholog PQN-59 contributes to stress granule assembly and development in C. elegans

abstract:
When exposed to stressful conditions, eukaryotic cells respond by inducing the formation of cytoplasmic ribonucleoprotein complexes called stress granules. Stress granules are thought to have a protective function but their exact role is still unclear. Here we use C. elegans to study two proteins that have been shown to be important for stress granule assembly in human cells: PQN-59, the ortholog of human UBAP2L, and GTBP-1, the ortholog of the human G3BP1 and G3BP2 proteins. Both proteins fall into stress granules in the embryo and in the germline when C. elegans is exposed to stressful conditions. None of the two proteins is essential for the assembly of stress induced granules, but the granules formed in absence of PQN-59 or GTBP-1 are less numerous and dissolve faster than the ones formed in control embryos. Despite these differences, pqn-59 or gtbp-1 mutant embryos do not show a higher sensitivity to stress than control embryos. pqn-59 mutants display reduced progeny and a high percentage of embryonic lethality, phenotypes that are not dependent on stress exposure and that are not shared with gtbp-1 mutants. Our data indicate that both GTBP-1 and PQN-59 contribute to stress granule formation but that PQN-59 is, in addition, required for C. elegans development.

Author summaryThe formation of so-called stress granules is an adaptive response that cells and organisms put into action to cope with changes in internal and environmental conditions and thus to survive to stressful conditions. Although it is generally thought that stress granule formation protects cells from stress-related damage, the exact role of stress granules in cells and organisms is not well understood. Moreover, the mechanisms governing stress granule assembly, and if and how the ability to form stress granules is important for C. elegans development is still unclear.

Our work focuses on two conserved proteins, known to be involved in stress granule assembly in mammalian cells, and investigates their role in C. elegans embryos. We find that these proteins are important but not essential to assemble stress-induced granules in C. elegans. We moreover did not observe a different sensitivity to stress exposure between wild-type and mutant developing embryos, suggesting that at least in these conditions these proteins do not exert a protective role.


title: Developmental regulation of neuronal gene expression by Elongator complex protein 1 dosage.

abstract: Elongator is a highly conserved protein complex required for transcriptional elongation, intracellular transport and translation. Elongator complex protein 1 (ELP1) is the scaffolding protein of Elongator and is essential for its assembly and stability. Familial dysautonomia (FD), a hereditary sensory and autonomic neuropathy, is caused by a mutation in ELP1 that lead to a tissue-specific reduction of ELP1 protein. Our work to generate a phenotypic mouse model for FD led to the discovery that homozygous deletion of the mouse Elp1 gene leads to embryonic lethality prior to mid-gestation. Given that FD is caused by a reduction, not loss, of ELP1, we generated two new mouse models by introducing different copy numbers of the human FD ELP1 transgene into the Elp1 knockout mouse (Elp1-/-) and observed that human ELP1 expression rescues embryonic development in a dose dependent manner. We then conducted a comprehensive transcriptome analysis in mouse embryos to identify genes and pathways whose expression correlates with the amount of ELP1. We found that ELP1 is essential for the expression of genes responsible for the formation and development of the nervous system. Further, gene length analysis of the differentially expressed genes showed that the loss of Elp1 mainly impacts the expression of long genes and that by gradually restoring Elongator their expression is progressively rescued. Finally, through evaluation of co-expression modules, we identified gene sets with unique expression patterns that depended on ELP1 expression. Overall, this study highlights the crucial role of ELP1 during early embryonic neuronal development and reveals gene networks and biological pathways that are regulated by Elongator.

category: developmental biology


title: Decellularized Articular Cartilage Microparticles for Expansion of Mesenchymal Stem Cells and Zonal Regeneration of Articular Cartilage

abstract:
IntroductionThe objective was to create multilayer cellular constructs using fetal or adult, decellularized articular cartilage in particulate form as microcarriers for expansion and fusion of mesenchymal stem cells (MSCs) to regenerate the stratified structure of articular cartilage.

MethodsPorous microparticles (CMPs) generated from decellularized fetal or adult bovine articular cartilage were used as microcarriers for expansion of human MSCs. The CMP expanded MSCs (CMP-MSCs) were used to generate injectable hydrogels or preformed multilayer constructs for articular cartilage regeneration. In the injectable approach, CMP-MSCs were suspended in alginate gel, crosslinked with calcium chloride, and incubated in chondrogenic medium to generate an injectable regenerative construct. In the preformed approach, fetal or adult CMP-MSCs were suspended in a culture medium, allowed to settle sequentially by the force of gravity, and fused by incubation in chondrogenic medium to generate multilayer cell sheets. The constructs were characterized with respect to compressive modulus, cellularity, and expression of chondrogenic markers.

ResultsHuman MSCs expanded on fetal or adult CMPs in basal medium maintained the expression of mesenchymal markers. The injectable CMP-MSCs hydrogels had significantly higher expression of chondrogenic markers and compressive modulus after four weeks incubation in chondrogenic medium compared to MSCs directly encapsulated in alginate gel; preformed CMP-MSCs cell sheets had significantly higher compressive modulus and expression of chondrogenic markers compared to MSCs in the pellet culture.

ConclusionThe preformed cell sheet approach is potentially useful for creating multilayer constructs by sequential gravitational settling of CMP-MSCs to mimic the stratified structure of articular cartilage.

Insight, Innovation, IntegrationThis work described a novel approach to recreate the zonal structure of articular cartilage. Human MSCs were expanded on porous microcarrier beads generated from decellularized fetal or adult bovine articular cartilage. The cell-seeded microbeads were fused by gravitational settling to form mono- or bi-layer cell sheets. The cell sheets were cultured in chondrogenic medium to regenerate the articular cartilage tissue. The in vitro regenerated tissue had higher compressive modulus and expression of chondrogenic markers compared to the MSC pellet culture.

category: bioengineering


title: Contrasting processing tomato cultivars unlink yield and pollen viability under heat stress

abstract: The occurring climate change is causing temperature increment in crop production areas worldwide, generating conditions of heat stress that negatively affect crop productivity. Tomato (Solanum lycopersicum), a major vegetable crop, is highly susceptible to conditions of heat stress. When tomato plants are exposed to ambient day/night temperatures that exceed 32{degrees}C/20{degrees}C respectively during the reproductive phase, fruit set and fruit weight are reduced, leading to a significant decrease in yield. Processing tomato cultivars are cultivated in open fields, where environmental conditions are not controlled, therefore plants are exposed to multiple abiotic stresses, including heat stress. Understanding the physiological response of modern processing tomato cultivars to heat stress may facilitate the development of thermotolerant cultivars. Here, we compared two tomato processing cultivars, H4107 and H9780, that we found to be constantly differing in yield performance. Using field and temperature-controlled greenhouse experiments, we show that the observed difference in yield is attributed to the occurrence of heat stress conditions. In addition, fruit-set and seed production were significantly improved in the thermotolerant cultivar H4107, compared with H9780. Despite the general acceptance of pollen viability as a measure of thermotolerance, there was no difference in the percentage of viable pollen between H4107 and H9780 under either of the conditions tested. Therefore, processing tomato cultivars may present a particular case, in which other factors are central for heat stress tolerance. Our results also demonstrate the value of combining controlled with uncontrolled experimental settings, in order to identify heat stress related responses and facilitate the development of thermotolerant processing tomato cultivars.

category: plant biology


title: Extracellular Hsp90α Detoxifies β-Amyloid Fibrils Through an NRF2 and Autophagy Dependent Pathway

abstract: We have investigated the role of extracellular Heat shock protein 90 alpha (eHsp90) in conferring protection of neuronal cells against fibrillary amyloid beta (f-A{beta}1-42) toxicity mediated by microglial cells. Formation of f-A{beta}1-42 plaques leads to neurotoxic inflammation, a critical pathological feature of Alzheimers Disease. We observed increased uptake and clearance of internalized f-A{beta}1-42 by microglial cells treated with eHsp90, an effect associated with activation of NRF2 (NF-E2-related factor 2) - mediated autophagy. eHsp90 thus mitigated the neuronal toxicity of f-A{beta}1-42-activated microglia. In addition, eHsp90 facilitated f-A{beta}1-42 engulfment by microglial cells in vitro. In summary, eHsp90 triggers NRF2-mediated autophagy in microglia and thus protects against the neurotoxic effects of f-A{beta}1-42.


title: Extended frontal networks for visual and auditory working memory

abstract: Working memory (WM) supports the persistent representation of transient sensory information. Visual and auditory stimuli place different demands on WM and recruit different brain networks. Separate auditory- and visual-biased WM networks extend into the frontal lobes, but several challenges confront attempts to parcellate human frontal cortex, including fine-grained organization and between-subject variability. Here, we use differential intrinsic functional connectivity from two visual-biased and two auditory-biased frontal structures to identify additional candidate sensory-biased regions in frontal cortex. We then examine direct contrasts of task fMRI during visual vs. auditory 2-back WM to validate those candidate regions. Three visual-biased and five auditory-biased regions are robustly activated bilaterally in the frontal lobes of individual subjects (N=14, 7 women). These regions exhibit a sensory preference during passive exposure to task stimuli, and that preference is stronger during WM. Hierarchical clustering analysis of intrinsic connectivity among novel and previously identified bilateral sensory-biased regions confirms that they functionally segregate into visual and auditory networks, even though the networks are anatomically interdigitated. We also observe that the fronto-temporal auditory WM network is highly selective and exhibits strong functional connectivity to structures serving non-WM functions, while the fronto-parietal visual WM network hierarchically merges into the multiple-demand cognitive system.


title: Structural brain connectivity in children with developmental dyscalculia

abstract: Developmental dyscalculia (DD) is a neurodevelopmental disorder specific to arithmetic learning even with normal intelligence and age-appropriate education. Difficulties often persist from childhood through adulthood. Underlying neurobiological mechanisms of DD, however, are poorly understood. This study aimed to identify possible structural connectivity alterations in DD. We evaluated 10 children with pure DD (11.3 {+/-} 0.7 years) and 16 typically developing (TD) peers (11.2 {+/-} 0.6 years) using diffusion tensor imaging. We first assessed white matter microstructure with tract-based spatial statistics. Then we used probabilistic tractography to evaluate tract lengths and probabilistic connectivity maps in specific regions. At whole brain level, we found no significant microstructural differences in white matter between children with DD and TD peers. Also, seed-based connectivity probabilities did not differ between groups. However, we did find significant differences in regions-of-interest tracts which had previously been related to math ability in children. The major findings of our study were reduced white matter coherence and shorter tract lengths of the left superior longitudinal/arcuate fasciculus and left anterior thalamic radiation in the DD group. Furthermore, lower white matter coherence and shorter pathways corresponded with the lower math performance as a result of the correlation analyses. These results from regional analyses indicate that learning, memory and language-related pathways in the left hemisphere might underlie DD.


title: A cortico-hippocampal scaffold for representing and recalling lifelike events

abstract: Real-world events are complex, featuring elements that may be unique to, or shared across, multiple situations. In the present study, we used fMRI to identify how different event components are represented in real-time and during memory retrieval. Twenty participants viewed and recalled eight videos depicting real-world events, combining people, contexts, and context types. Multi-voxel pattern similarity analyses revealed specific  person representations, persistent across contexts, in regions of an Anterior-Temporal Network. Conversely, we found specific  context representations, persistent across people, in regions of a Posterior-Medial Network. We also found schema-like generalization across contexts in medial prefrontal cortex, and episodic specificity in the hippocampus. Event patterns were reinstated during recall, and hippocampal reinstatement predicted the number of details retrieved. Finally, we observed distinct representational timescales across the hippocampus and cortical regions. These findings reveal mechanisms for scaffolding different aspects of lifelike event representations in cortico-hippocampal networks as experiences are observed and recalled.


title: Protein visualization and manipulation in Drosophila through the use of epitope tags recognized by nanobodies

abstract: Expansion of the available repertoire of reagents for visualization and manipulation of proteins will help understand their function. Short epitope tags linked to proteins of interest and recognized by existing binders such as nanobodies facilitate protein studies by obviating the need to isolate new antibodies directed against them. Nanobodies have several advantages over conventional antibodies, as they can be expressed and used as tools for visualization and manipulation of proteins in vivo. Here, we characterize two short (<15 aa) NanoTag epitopes, 127D01 and VHH05) and their corresponding high-affinity nanobodies. We demonstrate their use in Drosophila for in vivo protein detection and re-localization, direct and indirect immunofluorescence, immunoblotting, and immunoprecipitation. We further show that CRISPR-mediated gene targeting provides a straightforward approach to tagging endogenous proteins with the NanoTags. Single copies of the NanoTags, regardless of their location, suffice for detection. This versatile and validated toolbox of tags and nanobodies will serve as a resource for a wide array of applications, including functional studies in Drosophila and beyond.

category: genetics


title: Protein content and lipid profiling of isolated native autophagosomes

abstract: Autophagy is a central eukaryotic catabolic pathway responsible for clearance and recycling of an extensive portfolio of cargoes, which are packed in vesicles, called autophagosomes, and are delivered to lysosomes for degradation. Besides basal autophagy, which constantly degrades cellular material, the pathway is highly responsive to several stress conditions. However, the exact protein content and phospholipid composition of autophagosomes under changing autophagy conditions remain elusive so far. Here, we introduce a FACS-based approach for isolation of native unmanipulated autophagosomes and ensure the quality of the preparations. Employing quantitative proteomics and phospholipidomics, we obtained a profound cargo and lipid profile of autophagosomes purified upon basal autophagy conditions, nutrient deprivation, and proteasome inhibition. Indeed, starvation only mildly affected the content profile, while interference with proteasome activity showed stronger effects and specifically altered autophagosome cargoes. Interestingly, the phospholipid composition of autophagosomes was unaffected by the different treatments. Thus, the novel isolation method enables purification of intact autophagosomes in large quantities and allows protein content and phospholipid profiling without the requirement of exhaustive cellular fractionation or genetic manipulation.


title: Antibody Cocktail Exhibits Broad Neutralization against SARS-CoV-2 and SARS-CoV-2 variants

abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has precipitated multiple variants resistant to therapeutic antibodies. In this study, 12 high-affinity antibodies were generated from convalescent donors in early outbreaks using immune antibody phage display libraries. Of them, two RBD-binding antibodies (F61 and H121) showed high affinity neutralization against SARS-CoV-2, whereas three S2-target antibodies failed to neutralize SARS-CoV-2. Following structure analysis, F61 identified a linear epitope located in residues G446 -S494, which overlapped with angiotensin-converting enzyme 2 (ACE2) binding sites, while H121 recognized a conformational epitope located on the side face of RBD, outside from ACE2 binding domain. Hence the cocktail of the two antibodies achieved better performance of neutralization to SARS-CoV-2. Importantly, F61 and H121 exhibited efficient neutralizing activity against variants B.1.1.7 and B.1.351, those showed immune escape. Efficient neutralization of F61 and H121 against multiple mutations within RBD revealed a broad neutralizing activity against SARS-CoV-2 variants, which mitigated the risk of viral escape. Our findings defined the basis of therapeutic cocktails of F61 and H121 with broad neutralization and delivered a guideline for the current and future vaccine design, therapeutic antibody development, and antigen diagnosis of SARS-CoV-2 and its novel variants.


title: Uncovering divergence in gene expression regulation in the adaptation of yeast to nitrogen scarcity

abstract:
Saccharomyces cerevisiae rewires its transcriptional output to survive stressful environments, such as nitrogen scarcity under fermentative conditions. Although divergence in nitrogen metabolism has been described among natural yeast populations, the impact of regulatory genetic variants modulating gene expression and nitrogen consumption remains to be investigated. Here, we employed an F1 hybrid from two contrasting S. cerevisiae strains, providing a controlled genetic environment to map cis factors involved in the divergence of gene expression regulation in response to nitrogen scarcity. We used a dual approach to obtain genome-wide allele-specific profiles of chromatin accessibility, transcription factor binding, and gene expression through ATAC-seq and RNA-seq. We observed large variability in allele-specific expression and accessibility between the two genetic backgrounds, with a third of these differences specific to a deficient nitrogen environment. Furthermore, we discovered events of allelic bias in gene expression correlating with allelic bias in transcription factor binding solely under nitrogen scarcity, where the majority of these transcription factors orchestrates the Nitrogen Catabolite Repression regulatory pathway and demonstrates a cis x environment-specific response. Our approach allowed us to find cis variants modulating gene expression, chromatin accessibility and allelic differences in transcription factor binding in response to low nitrogen culture conditions.

ImportanceHistorically, coding variants were prioritized when searching for causal mechanisms driving adaptation of natural populations to stressful environments. However, the recent focus on non-coding variants demonstrated their ubiquitous role in adaptation. Here we performed genome-wide regulatory variation profiles between two divergent yeast strains when facing nitrogen nutritional stress. The open chromatin availability of several regulatory regions changes in response to nitrogen scarcity. Importantly we describe regulatory events that deviate between strains. Our results demonstrate a widespread variation in gene expression regulation between naturally occurring populations in response to stressful environments.

category: genomics


title: Innate immunity mediator STING modulates nascent DNA metabolism at stalled forks in human cells

abstract: The cGAS/STING pathway, part of the innate immune response to foreign DNA, is known to be activated by cells own DNA arising from the processing of the genome, including the excision of nascent DNA at arrested replication forks. We found STING activation to affect nascent DNA processing, suggesting a novel, unexpected feedback connection between the two events. Depletion of STING suppressed and re-expression of the protein in STING-deficient cells upregulated degradation of nascent DNA. Fork arrest was accompanied by the STING pathway activation, and a STING mutant that does not activate the pathway failed to upregulate nascent strand degradation. Consistent with this, cells expressing the STING mutant had a reduced level of RPA on parental and nascent DNA of arrested forks as well as a reduced CHK1 activation compared to the cells with wild type STING. Together our findings reveal a novel connection between replication stress and innate immunity.

category: molecular biology


title: State and stimulus dependence reconcile motion computation and the Drosophila connectome

abstract: Sensory systems dynamically optimize their processing properties in order to process a wide range of environmental and behavioral conditions. However, attempts to infer the function of these systems via modeling often treat system components as having static processing properties. This is particularly evident in the Drosophila motion detection circuit, where the core algorithm for motion detection is still debated, and where inputs to motion detecting neurons remain underdescribed. Using whole-cell patch clamp electrophysiology, we measured the state- and stimulus-dependent filtering properties of inputs to the OFF motion-detecting T5 cell in Drosophila. Simply summing these inputs within the framework of a connectomic-constrained model of the circuit demonstrates that changes in the shape of input temporal filters are sufficient to explain conflicting theories of T5 function. Therefore, with our measurements and our model, we reconcile motion computation with the anatomy of the circuit.


title: A programmable system to methylate and demethylate m6A on specific mRNAs

abstract: RNA N6-Methyladenosine (m6A) is the most abundant mRNA modification, and forms part of an epitranscriptomic system that modulates RNA function. RNA modifications can be reversibly catalyzed by several specific enzymes, and those modifications can be recognized by RNA binding proteins that in turn regulate biological processes. Although there are many reports demonstrating m6A participation in critical biological functions, this exploration has mainly been conducted through the global knockout or knockdown of the writers, erasers, or readers of m6A. Consequently, there is a lack of information about the role of m6A on single transcripts in biological processes, posing a challenge in understanding the biological functions of m6A. Here, we demonstrate a CRISPR/dCas13a-based RNA m6A-editor which can target mRNAs using single crRNA or multiple crRNAs array to methylate or demethylate m6A. We systematically assay its capabilities to enable the targeted rewriting of m6A dynamics, including modulation of circular RNA translation and transcript half-life. Finally, we demonstrate the utility of the system by specifically modulating XIST m6A levels, which can control X chromosome silencing and activation. Based on our editors, m6A on single and multiple transcripts can be modified to allow the exploration of the role of m6A on in biological processes.


title: Human adipose-derived mesenchymal stromal cells from face and abdomen undergo replicative senescence and loss of genetic integrity after long-term culture

abstract: Body fat depots are heterogeneous concerning their embryonic origin, structure, exposure to environmental stressors, and availability. Thus, investigating adipose-derived mesenchymal stromal cells (ASCs) from different sources is essential to standardization for future therapies. In vitro amplification is also critical because it may predispose cell senescence and mutations, reducing regenerative properties and safety. Here, we evaluated long-term culture of human facial ASCs (fASCs) and abdominal ASCs (aASCs) and showed that both met the criteria for MSCs characterization, but presented differences in their immunophenotypic profile, and differentiation and clonogenic potentials. The abdominal tissue yielded more ASCs, but facial cells displayed fewer mitotic errors at higher passages. However, both cell types reduced clonal efficiency over time and entered replicative senescence around P12, as evaluated by progressive morphological alterations, reduced proliferative capacity, and SA-{beta}-galactosidase expression. Loss of genetic integrity was detected by a higher proportion of cells showing nuclear alterations and {gamma}-H2AX expression. Our findings indicate that the source of ASCs can substantially influence their phenotype and therefore should be carefully considered in future cell therapies, avoiding, however, long-term culture to ensure genetic stability.


title: Time-resolved single-cell sequencing identifies multiple waves of mRNA decay during mitotic exit

abstract: Accurate control of the cell cycle is critical for development and tissue homeostasis and requires precisely-timed expression of many genes. Cell cycle gene expression is regulated through transcriptional and translational control, as well as through regulated protein degradation. Here, we show that widespread and temporally-controlled mRNA decay acts as an additional mechanism for gene expression regulation during the cell cycle. We find that two waves of mRNA decay occur sequentially during the mitosis-to-G1 phase transition, and identify the deadenylase CNOT1 as a factor that contributes to mRNA decay during this cell cycle transition. Collectively, our data show that, akin to protein degradation, scheduled mRNA decay helps to reshape cell cycle gene expression as cells move from mitosis into G1 phase.


title: Image-based phenotypic profiling of a chemogenomic screening library identifies novel druggable targets in the EGFR pathway

abstract: The gene encoding epidermal growth factor receptor (EGFR) is a major driver gene in cancer. Many drugs targeting EGFR-associated molecules have been developed, yet many have failed in clinical trials due to a lack of efficacy and/or unexpected side effects. In this study, I used image-based phenotypic profiling to screen a pharmacologically active compound library with the aim of identifying new druggable targets in the EGFR pathway. As anticipated, the phenotypic screen identified compounds that produce phenotypes resulting from targeting a known specific molecule or pathway. The assay also showed that compounds with diverse known mechanisms of action produced similar, EGFR-related cellular phenotypes. Biochemical assays revealed that those compounds share a previously unappreciated common target/pathway, showing that the image-based assay can identify new target molecules that are independent of the compounds known target. Further experiments showed that ROCK1 and PSMD2 are novel druggable targets within the EGFR pathway.


title: Generation of photocaged nanobodies for in vivo  applications using genetic code expansion and computationally guided protein engineering

abstract: Nanobodies are becoming increasingly popular as tools for manipulating and visualising proteins in vivo. The ability to control nanobody/antigen interactions using light could provide precise spatiotemporal control over protein function. We develop a general approach to engineer photo-activatable nanobodies using photocaged amino acids that are introduced into the target binding interface by genetic code expansion. Guided by computational alanine scanning and molecular-dynamics simulations, we tune nanobody/target binding affinity to eliminate binding before uncaging. Upon photo-activation, binding is restored. We use this approach to generate improved photocaged variants of two anti-GFP nanobodies. These variants exhibit photo-activatable binding triggered by illumination with 365nm light. We demonstrate that the photocaged nanobodies we have created are highly robust and function in a complex cellular environment. We apply them to control subcellular protein localisation in the nematode worm C. elegans. Our approach provides a rare example of computationally designed proteins being directly applied in living animals and demonstrates the importance of accounting for in vivo effects on protein-protein interactions.

category: synthetic biology


title: Cytoplasmic zinc regulates IL-1β production by monocytes/macrophages via mTORC1-induced glycolysis in rheumatoid arthritis (RA)

abstract:
The essential micronutrient zinc plays regulatory roles in immune responses through its ability to affect signaling pathways. In activated monocytes/macrophages, signaling networks mediate metabolic reprogramming in order to meet the demands of participating in immune responses. Despite its known immunoregulatory roles, the effect of zinc on metabolic reprogramming in monocytes/macrophages remains unclear. Here, we demonstrate that cytoplasmic bioavailable zinc is essential for regulating IL-1{beta} production in activated human monocytes/macrophages downstream of mTORC1-induced glycolysis. The cytoplasmic zinc level was influenced by extracellular zinc concentration through a zinc-specific importer, Zip8, which was markedly increased in monocytes of patients with rheumatoid arthritis (RA), a chronic inflammatory disease, and even in LPS-stimulated monocytes/macrophages of healthy individuals. Mechanically, phosphorylation of S6 kinase, a substrate of mTORC1, was significantly enhanced by zinc-mediated inhibition of PP2A, an S6 kinase phosphatase. As a result, IL-1{beta} production was increased due to the activation of mTORC1-induced glycolysis. The expression of Zip8 and MT2A, a zinc-inducible gene, and the phosphorylation of S6 kinase by monocytes of RA patients was significantly enhanced compared with those of HCs and Zip8 levels positively correlated with RA clinical parameters, suggesting that Zip8-mediated zinc influx is related to inflammatory conditions. These results provide insight into the role of cytoplasmic bioavailable zinc in the metabolic reprogramming of human monocytes/macrophages which is an essential process for inflammatory responses.

One Sentence SummaryCytoplasmic zinc regulates IL-1{beta} production in monocytes/macrophages downstream of mTORC1-S6K-induced glycolysis via zinc-mediated inhibition of PP2A.


title: Rhinovirus C replication is associated with the endoplasmic reticulum and triggers cytopathic effects in an in vitro model of human airway epithelium

abstract:
The clinical impact of rhinovirus C (RV-C) is well-documented; yet the viral life cycle remains poorly defined. Thus, we characterized RV-C15 replication at the single-cell level and its impact on the human airway epithelium (HAE) using a physiologically-relevant in vitro model. RV-C15 replication was restricted to ciliated cells where viral RNA levels peaked at 12 hours post-infection (hpi), correlating with elevated titers in the apical compartment at 24 hpi. Notably, infection was associated with a loss of polarized expression of the RV-C receptor, cadherin-related family member 3. Visualization of double-stranded RNA (dsRNA) during RV-C15 replication revealed two distinct replication complex arrangements within the cell, likely corresponding to different time points in infection and correlating with the formation of large intracellular vesicles. To further define RV-C15 replication sites, we analyzed the expression of giantin, phosphatidylinositol-4- phosphate, and calnexin, as well as the colocalization of these markers with dsRNA. Fluorescence levels of all three cellular markers were elevated during infection and altered giantin distribution further indicated Golgi fragmentation. However, unlike previously characterized RVs, the high ratio of calnexin-dsRNA colocalization implicated the endoplasmic reticulum as the primary site for RV-C15 replication in HAE. RV-C15 infection was also associated with elevated stimulator of interferon genes (STING) expression, facilitating replication, and the induction of incomplete autophagy, a mechanism used by other RVs to promote non-lytic release of progeny virions. Finally, RV-C15 infection resulted in a temporary loss in epithelial barrier integrity and the translocation of tight junction proteins while a reduction in mucociliary clearance indicated cytopathic effects on epithelial function. Together, our findings identify both shared and unique features of RV-C replication compared to related rhinoviruses and define the impact of RV-C on both epithelial cell organization and tissue functionality - aspects of infection that may contribute to pathogenesis in vivo.

Author summaryRhinovirus C has a global distribution and significant clinical impact - especially in those with underlying lung disease. Although RV-C is genetically, structurally, and biologically distinct from RV-A and -B viruses, our understanding of the RV-C life cycle has been largely inferred from these and other related viruses. Here, we performed a detailed analysis of RV-C15 replication in a physiologically-relevant model of human airway epithelium. Our single-cell, microscopy-based approach revealed that - unlike other RVs - the endoplasmic reticulum is the primary site for RV- C15 replication. RV-C15 replication also stimulated STING expression, which was proviral, and triggered dramatic changes in cellular organization, including altered virus receptor distribution, fragmented Golgi stacks, and the induction of incomplete autophagy. Additionally, we observed a loss of epithelial barrier function and a decrease in mucociliary clearance, a major defense mechanism in the lung, during RV-C15 infection. Together, these data reveal novel insight into RV-C15 replication dynamics and resulting cytopathic effects in the primary target cells for infection, thereby furthering our understanding of the pathogenesis of RV-C. Our work highlights similar, as well as unique, aspects of RV-C15 replication compared to related pathogens, which will help guide future studies on the molecular mechanisms of RV-C infection.


title: Mucin-mimetic glycan arrays integrating machine learning for analyzing receptor pattern recognition by influenza A viruses

abstract: Influenza A viruses (IAVs) exploit host glycans in airway epithelial mucosa to gain entry and initiate infection. Rapid detection of changes in IAV specificity towards host glycan classes can provide early indication of virus transmissibility and infection potential. IAVs use hemagglutinins (HA) to bind sialic acids linked to larger glycan structures and a switch in HA specificity from 2,3-to 2,6-linked sialoglycans is considered a prerequisite for viral transmission from birds to humans. While the changes in HA structure associated with the evolution of binding phenotype have been mapped, the influence of glycan receptor presentation on IAV specificity remains obscured. Here, we describe a glycan array platform which uses synthetic mimetics of mucin glycoproteins to model how receptor presentation in the mucinous glycocalyx, including glycan type and valency of the glycoconjugates and their surface density, impact IAV binding. We found that H1N1 virus produced in embryonated chicken eggs, which recognizes both receptor types, exclusively engaged mucin-mimetics carrying 2,3-linked sialic acids in their soluble form. The virus was able utilize both receptor structures when the probes were immobilized on the array; however, increasing density in the mucin-mimetic brush diminished viral adhesion. Propagation in mammalian cells produced a change in receptor pattern recognition by the virus, without altering its HA affinity, toward improved binding of 2,6-sialylated mucin mimetics and reduced sensitivity to surface crowding of the probes. Application of a support vector machine (SVM) learning algorithm as part of the glycan array binding analysis efficiently characterized this shift in binding preference and may prove useful to study the evolution of viral responses to a new host.


title: A genome-scale antibiotic screen in Serratia marcescens identifies YdgH as a conserved modifier of cephalosporin and detergent susceptibility

abstract: Serratia marcescens, a member of the order Enterobacterales, is adept at colonizing healthcare environments and an important cause of invasive infections. Antibiotic resistance is a daunting problem in S. marcescens because in addition to plasmid-mediated mechanisms, most isolates have considerable intrinsic resistance to multiple antibiotic classes. To discover endogenous modifiers of antibiotic susceptibility in S. marcescens, a high-density transposon insertion library was subjected to sub-minimal inhibitory concentrations of two cephalosporins, cefoxitin and cefepime, as well as the fluoroquinolone ciprofloxacin. Comparisons of transposon insertion abundance before and after antibiotic exposure identified hundreds of potential modifiers of susceptibility to these agents. Using single gene deletions, we validated several candidate modifiers of cefoxitin susceptibility and chose ydgH, a gene of unknown function, for further characterization. In addition to cefoxitin, deletion of ydgH in S. marcescens resulted in decreased susceptibility to multiple 3rd generation cephalosporins, and in contrast, to increased susceptibility to both cationic and anionic detergents. YdgH is highly conserved throughout the Enterobacterales, and we observed similar phenotypes in Escherichia coli O157:H7 and Enterobacter cloacae mutants. YdgH is predicted to localize to the periplasm and we speculate that it may be involved there in cell envelope homeostasis. Collectively, our findings provide insight into chromosomal mediators of antibiotic resistance in S. marcescens and will serve as a resource for further investigations of this important pathogen.


title: Phytophthora capsici sterol reductase PcDHCR7 has a role in mycelium development and pathogenicity

abstract: The de novo biosynthesis of sterols is critical for eukaryotes, however, some organisms lack this pathway including most oomycetes. Phytophthora spp. are sterol auxotroph but remarkably, have retained a few genes encoding enzymes in the sterol biosynthesis pathway. Here we investigated the function of PcDHCR7, a gene in Phytophthora capsici predicted to encode the {bigtriangleup}7-sterol reductase. When expressed in Saccharomyces cerevisiae, PcDHCR7 showed a {bigtriangleup}7-sterol reductase activity. Knocking out PcDHCR7 in P. capsici resulted in loss of the capacity to transform ergosterol into brassicasterol, which means PcDHCR7 has a {bigtriangleup}7-sterol reductase activity in P. capsici itself. This enables P. capsici to transform sterols recruited from the environment for better use. Biological characteristics were compared between wild-type isolate and PcDHCR7 knock-out transformants. The results indicated that PcDHCR7 plays a key role in mycelium development and pathogenicity of zoospores in P. capsici.


title: An infectious Rous Sarcoma Virus Gag mutant that is defective in nuclear cycling

abstract:
During retroviral replication, unspliced viral genomic RNA (gRNA) must escape the nucleus for translation into viral proteins and packaging into virions. "Complex" retroviruses such as Human Immunodeficiency Virus (HIV) use cis-acting elements on the unspliced gRNA in conjunction with trans-acting viral proteins to facilitate this escape. "Simple" retroviruses such as Mason-Pfizer Monkey Virus (MPMV) and Murine Leukemia Virus (MLV) exclusively use cis-acting elements on the gRNA in conjunction with host nuclear export proteins for nuclear escape. Uniquely, the simple retrovirus Rous Sarcoma Virus (RSV) has a Gag structural protein that cycles through the nucleus prior to plasma membrane binding. This trafficking has been implicated in facilitating gRNA nuclear export and is thought to be a required mechanism. Previously described mutants that abolish nuclear cycling displayed enhanced plasma membrane binding, enhanced virion release, and a significant loss in genome incorporation resulting in loss of infectivity. Here, we describe a nuclear cycling deficient RSV Gag mutant that has similar plasma membrane binding and genome incorporation to WT virus and surprisingly, is replication competent albeit with a slower rate of spread compared to WT. This mutant suggests that RSV Gag nuclear cycling is not strictly required for RSV replication.

ImportanceWhile mechanisms for retroviral Gag assembly at the plasma membrane are beginning to be characterized, characterization of intermediate trafficking locales remain elusive. This is in part due to the difficulty of tracking individual proteins from translation to plasma membrane binding. RSV Gag nuclear cycling is a unique phenotype that may provide comparative insight to viral trafficking evolution and may present a model intermediate to cis- and trans-acting mechanisms for gRNA export.


title: Cellulomonas zeae sp. nov., Lelliottia zeae sp. nov., Paraburkholderia zeae sp. nov. and Sphingomonas zeigerminis sp. nov., four new bacterial species isolated from Zea mays

abstract: Four novel bacterial species collected from healthy tissues of corn, rice and soybean plants in the United States are described. These include Cellulomonas zeae sp. nov. isolated from corn in Indiana; Lelliottia zeae sp. nov. from corn in Indiana and rice in Arkansas; Paraburkholderia zeae sp. nov. isolated from corn in Iowa; and Sphingomonas zeigerminis sp. nov. from corn in Mississippi and soy in Arkansas. No pathogenic strains are known for any of the novel species based on genome comparisons to assemblies in GenBank.


title: Cellulomonas zeae sp. nov., Lelliottia zeae sp. nov., Paraburkholderia zeae sp. nov. and Sphingomonas zeigerminis sp. nov., four new bacterial species isolated from Zea mays and other hosts


title: Molecular approaches reveal speciation between red and blue flowered plants in the Mediterranean Lysimachia arvensis and Lysimachia monelli (Primulaceae)

abstract: Flower colour constitutes a pivotal evolutionary force in speciation. The Mediterranean Lysimachia arvensis and L. monelli are morphologically variable species having both blue or red flowered plants. Previous studies suggested that L. arvensis plants differing in colour are diverging lineages, but this variation has not been considered in a phylogenetic context. We reconstruct the phylogenetic signal and the ancestral states of flower colour of Mediterranean Lysimachia species by using nuclear (ITS) and three plastid markers. Blue and red specimens are nested in two independent clades in the ITS tree, thus supporting that L. arvensis and L. monelli are polyphyletic, whereas low phylogenetic resolution was found in plastid markers. Blue-flowered L. arvensis is reconstructed sister to L. talaverae in a monophyletic clade sister to the remaining Lysimachia. Red-flowered L. arvensis is reconstructed sister to red-flowered L. monelli in a monophyletic clade sister to blue-flowered L. monelli and L. foemina. Our results suggest that colour lineages in L. arvensis and L. monelli constitute different species, but flower colour did not promote the separation of these lineages. We propose a new name for blue-flowered L. arvensis (L. loeflingii) and a new combination for red-flowered L. monelli (L. collina).


title: Personality-specific pathways from peer victimization to adolescent alcohol misuse: A multilevel longitudinal moderated mediation analysis

abstract: Peer victimization is common in adolescence and have been associated with a broad variety of psychopathology and alcohol use. The present study assessed whether peer victimization has a time-varying effect on alcohol use through internalizing and externalizing symptoms and whether this indirect association throughout time is moderated by personality. This 5-year longitudinal study (3,800 grade 7 adolescents) used Bayesian multilevel moderated mediation models: independent variable was peer victimization; moderators were four personality dimensions (anxiety sensitivity, hopelessness, impulsivity, and sensation seeking); internalizing symptoms (anxiety, depressive symptoms) and externalizing symptoms (conduct, hyperactivity problems) were the mediators; and alcohol use, the outcome. Results indicated significant between, within, and lagged effects on alcohol use through internalizing and externalizing symptoms. There was significant between and within effects on alcohol use through internalizing symptoms for adolescents with high anxiety sensitivity and hopelessness, and significant between, within, and lagged effects on alcohol use through externalizing for adolescents with high impulsivity and sensation seeking. These findings implicate two risk pathways that account for how peer victimization enhances alcohol use risk and emphasize the importance of personality profiles that can shape the immediate and long-term consequences of victimization.


title: Axis formation in annual killifish: Nodal coordinates morphogenesis in absence of Huluwa prepatterning

abstract:
Axis formation in fish and amphibians is initiated by a prepattern of maternal gene products in the blastula. The embryogenesis of annual killifish challenges prepatterning models because blastomeres disperse and then re-aggregate to form the germ layers and body axes. This dispersion-aggregation process prompts the question how axis determinants such as Huluwa and germ layer inducers such as Nodal function in annual killifish. Here we show in Nothobranchius furzeri that huluwa, the factor thought to break symmetry by stabilizing {beta}-catenin, is a non-functional pseudogene. Nuclear {beta}-catenin is not selectively stabilized on one side of the blastula but accumulates in cells forming the incipient aggregate. Inhibition of Nodal signaling blocks aggregation and disrupts coordinated cell migration, establishing a novel role for this signaling pathway. These results reveal a surprising departure from classic mechanisms of axis formation: canonical Huluwa-mediated prepatterning is dispensable and Nodal coordinates morphogenesis.

One Sentence SummaryAxis formation in annual killifish relies on Nodal to coordinate cell migration and is independent of Huluwa-mediated prepatterning.


title: Predicting the Genomic Resolution of Bulk Segregant Analysis

abstract: Bulk segregant analysis (BSA) is a technique for identifying the genetic loci that underlie phenotypic trait differences. The basic approach of this method is to compare two pools of individuals from the opposing tails of the phenotypic distribution, sampled from an interbred population. Each pool is sequenced and scanned for alleles that show divergent frequencies between the pools, indicating potential association with the observed trait differences. BSA has already been successfully applied to the mapping of various quantitative trait loci in organisms ranging from yeast to maize. However, these studies have typically suffered from rather low mapping resolution, and we still lack a detailed understanding of how this resolution is affected by experimental parameters. Here, we use coalescence theory to calculate the expected genomic resolution of BSA. We first show that in an idealized interbreeding population of infinite size, the expected length of the mapped region is inversely proportional to the recombination rate, the number of generations of interbreeding, and the number of genomes sampled, as intuitively expected. In a finite population, coalescence events in the genealogy of the sample reduce the number of potentially informative recombination events during interbreeding, thereby increasing the length of the mapped region. This is incorporated into our theory by an effective population size parameter that specifies the pairwise coalescence rate of the interbreeding population. The mapping resolution predicted by our theory closely matches numerical simulations. Furthermore, we show that the approach can easily be extended to modifications of the crossing scheme. Our framework enables researchers to predict the expected power of their mapping experiments, and to evaluate how their experimental design could be tuned to optimize mapping resolution.


title: Unraveling the influences of sequence and position on yeast uORF activity using massively parallel reporter systems and machine learning.

abstract: Upstream open reading frames (uORFs) are potent cis-acting regulators of mRNA translation and nonsense-mediated decay (NMD). While both AUG- and non-AUG initiated uORFs are ubiquitous in ribosome profiling studies, few uORFs have been experimentally tested. Consequently, the relative influences of sequence, structural, and positional features on uORF activity have not been determined. We quantified thousands of yeast uORFs using massively parallel reporter assays in wildtype and {Delta}upf1 yeast. While nearly all AUG uORFs were robust repressors, most non-AUG uORFs had relatively weak impacts on expression. Machine learning regression modeling revealed that uORF functions are strongly impacted by both their sequences and locations within transcript leaders. Indeed, alternative transcription start sites highly influenced uORF activity. These results define the scope of natural uORF activity, identify features associated with translational repression and NMD, and suggest that the locations of uORFs in transcript leaders are nearly as important as uORF sequences.


title: Telomere-to-telomere gapless chromosomes of banana using nanopore sequencing

abstract: Long-read technologies hold the promise to obtain more complete genome assemblies and to make them easier. Coupled with long-range technologies, they can reveal the architecture of complex regions, like centromeres or rDNA clusters. These technologies also make it possible to know the complete organization of chromosomes, which remained complicated before even when using genetic maps. However, generating a gapless and telomere-to-telomere assembly is still not trivial, and requires a combination of several technologies and the choice of suitable software. Here, we report a chromosome-scale assembly of a banana genome (Musa acuminata) generated using Oxford Nanopore long-reads. We generated a genome coverage of 177X from a single PromethION flowcell with near 17X with reads longer than 75Kb. From the 11 chromosomes, 5 were entirely reconstructed in a single contig from telomere to telomere, revealing for the first time the content of complex regions like centromeres or clusters of paralogous genes.


title: MDH2 is a metabolic switch rewiring the fuelling of respiratory chain and TCA cycle

abstract: The mitochondrial respiratory chain (RC) enables many metabolic processes by regenerating both mitochondrial and cytosolic NAD+ and ATP. The oxidation by the RC of the NADH metabolically produced in the cytosol involves redox shuttles as the malate-aspartate shuttle (MAS) and is of paramount importance for cell fate. However, the specific metabolic regulations allowing mitochondrial respiration to prioritize NADH oxidation in response to high NADH/NAD+ redox stress have not been elucidated. Our work demonstrates the crucial role played by MDH2 in orchestrating the electron fuelling of the RC. On one hand, MDH2 mediated oxaloacetate (OAA) production allows mitochondria to metabolize pyruvate and glutamate and to feed complex I with NADH, whereas on the other hand, OAA inhibits complex II. This regulatory mechanism synergistically increases RCs NADH oxidative capacity and rewires MDH2 driven anaplerosis of the TCA favouring the oxidation of malate produced by the MAS.


title: An inter-channel cooperative mechanism mediates PIEZO1's exquisite mechanosensitivity

abstract: The bowl-shaped structure of PIEZO channels is predicted to flatten in response to mechanical stimuli, gating their pore open. However, how this unique structure allows them to detect exquisitely small changes in membrane tension remains unclear. Here, using pressure clamp electrophysiology, modeling, and molecular dynamics simulations, we show that the single channel open probability of PIEZO1 increases weakly with respect to pressure-induced tension. In contrast, when multiple channels are present in a membrane patch, channel open probability increases steeply as a function of the number of open channels. These cooperative effects are consistent with an inter-channel energetic repulsion due to the local membrane deformation created by the non-planar PIEZO structure. When channels open, this deformation shrinks, allowing open channels to diffuse closer to each other, thus delaying closure. This study reveals how PIEZO1 channels acquire their exceptional mechanosensitivity and suggests a possible mechanism by which cells could rapidly tune mechanosensitivity.


title: Sensitive high-throughput assays for tumour burden reveal the response of a Drosophila melanogaster model of colorectal cancer to standard chemotherapies

abstract: Drosophila melanogaster (Drosophila) models of cancer are emerging as powerful tools to investigate the basic mechanisms underlying tumour progression and identify novel therapeutics. Rapid and inexpensive, it is possible to carry out genetic and drug screens at a far larger scale than in vertebrate organisms. Such whole-organism-based drug screens permits assessment of drug absorption and toxicity, reducing the possibility of false positives. Activating mutations in the Wnt and Ras signalling pathways are common in many epithelial cancers, and when driven in the adult Drosophila midgut, it induces aggressive intestinal tumour-like outgrowths that recapitulate many aspects of human colorectal cancer (CRC). Here we have taken a Drosophila CRC model in which tumourous cells are marked with both GFP and luciferase reporter genes, and developed novel high-throughput assays for quantifying tumour burden. Leveraging these assays, we find that the Drosophila CRC model responds rapidly to treatment with standard CRC-drugs, opening the door to future rapid genetic and drug screens.

category: cancer biology


title: Single-cell analysis of localized low- and high-grade prostate cancers

abstract: Approximately, 30% of early-stage localized prostate cancer cases reoccur within 5 to 10 years [1, 2]. However, identifying precise molecular subtypes attributable to specific stages of prostate cancer has proven difficult due to high heterogeneity within localized tumors [3-5]. Bulk assays represent a population average, which is a result of the heterogeneity that exists at the individual prostate cancer cell level [6]. Here, we sequenced the accessible chromatin regions of 14,424 single-cells collected from 18 fresh-frozen prostate tumors using sci-ATAC-seq [7, 8]. We observed that shared chromatin features among low-grade prostate cancer epithelial cells were lost in high-grade tumors. Despite this loss, all high-grade tumors exhibited an enrichment for FOXA1, HOXB13 and CDX2 transcription factor binding sites within their accessible chromatin regions, indicating a shared trans-regulatory program. Single-cell analysis of the differentially accessible regions in high- versus low-grade prostate tumors identified two unique genes encoding neuronal adhesion molecules, NRXN1 and NLGN1. We found that NRXN1 and NLGN1 are expressed in the epithelial luminal, basal and neuroendocrine cells, as well as the immune, endothelial and neuronal cell types in all prostate tumors. Overall, these results provide a deeper understanding of the active gene regulatory networks in low- and high-grade prostate tumors at a striking resolution and provide critical insights for molecular stratification of the disease.


title: Engineering single pan-specific ubiquibodies for targeted degradation of all forms of endogenous ERK protein kinase

abstract: Ubiquibodies (uAbs) are a customizable proteome editing technology that utilizes E3 ubiquitin ligases genetically fused to synthetic binding proteins to steer otherwise stable proteins of interest (POIs) to the proteasome for degradation. The ability of engineered uAbs to accelerate the turnover of exogenous or endogenous POIs in a posttranslational manner offers a simple yet robust tool for dissecting diverse functional properties of cellular proteins as well as for expanding the druggable proteome to include tumorigenic protein families that have yet-to-be successfully drugged by conventional inhibitors. Here, we describe the engineering of uAbs comprised of a highly modular human E3 ubiquitin ligase, human carboxyl terminus of Hsc70-interacting protein (CHIP), tethered to different designed ankyrin repeat proteins (DARPins) that bind to nonphosphorylated (inactive) and/or doubly phosphorylated (active) forms of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Two of the resulting uAbs were found to be global ERK degraders, pan-specifically capturing all endogenous ERK1/2 protein forms and redirecting them to the proteasome for degradation in different cell lines, including MCF7 breast cancer cells. Taken together, these results demonstrate how the substrate specificity of an E3 ubiquitin ligase can be reprogrammed to generate designer uAbs against difficult-to-drug targets, enabling a modular platform for remodeling the mammalian proteome.


title: The ChvG-ChvI and NtrY-NtrX two-component systems coordinately regulate growth of Caulobacter crescentus

abstract:
Two-component signaling systems (TCSs) are comprised of a sensory histidine kinase and a response regulator protein. In response to environmental changes, sensor kinases directly phosphorylate their cognate response regulator to affect gene expression. Bacteria typically express multiple TCSs that are insulated from one another and regulate distinct physiological processes. There are certainly examples of cross-regulation between TCSs, but this phenomenon remains relatively unexplored. We have identified regulatory links between the ChvG-ChvI (ChvGI) and NtrY-NtrX (NtrYX) TCSs, which control important and often overlapping processes in -proteobacteria, including maintenance of the cell envelope. Deletion of chvG and chvI in Caulobacter crescentus limited growth in defined medium and a selection for genetic suppressors of this growth phenotype uncovered interactions among chvGI, ntrYX, and ntrZ, which encodes a previously uncharacterized periplasmic protein. Significant overlap in the experimentally-defined ChvI and NtrX transcriptional regulons provided support for the observed genetic connections between ntrYX and chvGI. Moreover, we present evidence that the growth defect of strains lacking chvGI is influenced by the phosphorylation state of NtrX and, to some extent, by levels of the TonB-dependent receptor ChvT. Measurements of NtrX phosphorylation in vivo indicated that NtrZ is an upstream regulator of NtrY, and that NtrY primarily functions as an NtrX phosphatase. We propose a model in which NtrZ functions in the periplasm to inhibit NtrY phosphatase activity; regulation of phosphorylated NtrX levels by NtrZ and NtrY provides a mechanism to modulate and balance expression of the NtrX and ChvI regulons under different growth conditions.

ImportanceTwo-component signaling systems (TCSs) enable bacteria to regulate gene expression in response to physiochemical changes in their environment. The ChvGI and NtrYX TCSs regulate diverse pathways associated with pathogenesis, growth, and cell envelope function in many -proteobacteria. We used Caulobacter crescentus as a model to investigate regulatory connections between ChvGI and NtrYX. Our work defined the ChvI transcriptional regulon in C. crescentus and revealed a genetic interaction between ChvGI and NtrYX, whereby modulation of NtrYX signaling affects the survival of cells lacking ChvGI. In addition, we identified NtrZ as a periplasmic inhibitor of NtrY phosphatase activity in vivo. Our work establishes C. crescentus as an excellent model to investigate multi-level regulatory connections between ChvGI and NtrYX in -proteobacteria.


title: Topoisomerase I Essentiality, DnaA-independent Chromosomal Replication, and Transcription-Replication Conflict in Escherichia coli

abstract:
Topoisomerase I (Topo I) of Escherichia coli, encoded by topA, acts to relax negative supercoils in DNA. Topo I deficiency results in hypernegative supercoiling, formation of transcription-associated RNA-DNA hybrids (R-loops), and DnaA- and oriC-independent constitutive stable DNA replication (cSDR), but some uncertainty persists as to whether topA is essential for viability in E. coli and related enterobacteria. Here we show that several topA alleles, including {Delta}topA, confer lethality in derivatives of wild-type E. coli strain MG1655. Viability in absence of Topo I was restored with two perturbations, neither of which reversed the hypernegative supercoiling phenotype: (i) in a reduced-genome strain MDS42, or (ii) by an RNA polymerase (RNAP) mutation rpoB*35 that has been reported to alleviate the deleterious consequences of RNAP backtracking and transcription-replication conflicts. Four phenotypes related to cSDR were identified for topA mutants: (i) One of the topA alleles rescued {Delta}dnaA lethality; (ii) in dnaA+ derivatives, Topo I deficiency generated a characteristic copy number peak in the terminus region of the chromosome; (iii) topA was synthetically lethal with rnhA (encoding RNase HI, whose deficiency also confers cSDR); and (iv) topA rnhA synthetic lethality was itself rescued by {Delta}dnaA. We propose that the terminal lethal consequence of hypernegative DNA supercoiling in E. coli topA mutants is RNAP backtracking during transcription elongation and associated R-loop formation, which in turn lead to transcription-replication conflicts and to cSDR.

ImportanceIn all life forms, double helical DNA exists in a topologically supercoiled state. The enzymes DNA gyrase and topoisomerase I act, respectively, to introduce and to relax negative DNA supercoils in Escherichia coli. That gyrase deficiency leads to bacterial death is well established, but the essentiality of topoisomerase I for viability has been less certain. This study confirms that topoisomerase I is essential for E. coli viability, and suggests that in its absence aberrant chromosomal DNA replication and excessive transcription-replication conflicts occur that are responsible for lethality.


title: Deletion of the AMPylase mFICD alters cytokine secretion and affects cognitive plasticity in vivo

abstract: Fic domain-containing AMP transferases (fic AMPylases) are conserved enzymes that catalyze the covalent transfer of AMP to proteins. This post-translational modification regulates the function of several proteins, including the ER-resident chaperone Grp78/BiP. Here we introduce a mFICD AMPylase knock-out mouse model to study fic AMPylase function in vertebrates. We find that mFICD deficiency is well-tolerated in unstressed mice. We show that mFICD-deficient mouse embryonic fibroblasts are depleted of AMPylated proteins. mFICD deletion alters protein synthesis and secretion in splenocytes, including that of IgM and IL-1{beta}, without affecting the unfolded protein response. Finally, we demonstrate that older mFICD-/- mice show improved cognitive plasticity. Together, our results suggest a role for mFICD in adaptive immunity and neuronal plasticity in vivo.


title: Genomic Skimming and Nanopore Sequencing Uncover Cryptic Hybridization in One of World's Most Threatened Primates

abstract: The Brazilian buffy-tufted-ear marmoset (Callithrix aurita), one of the worlds most endangered primates, is threatened by anthropogenic hybridization with exotic, invasive marmoset species. As there are few genetic data available for C. aurita, we developed a PCR-free protocol with minimal technical requirements to rapidly generate genomic data with genomic skimming and portable nanopore sequencing. With this direct DNA sequencing approach, we successfully determined the complete mitogenome of a marmoset that we initially identified as C. aurita. The obtained nanopore-assembled sequence was highly concordant with a Sanger sequenced version of the same mitogenome. Phylogenetic analyses unexpectedly revealed that our specimen was a cryptic hybrid, with a C. aurita phenotype and C. penicillata mitogenome lineage. We also used publicly available mitogenome data to determine diversity estimates for C. aurita and three other marmoset species. Mitogenomics holds great potential to address deficiencies in genomic data for endangered, non-model species such as C. aurita. However, we discuss why mitogenomic approaches should be used in conjunction with other data for marmoset species identification. Finally, we discuss the utility and implications of our results and genomic skimming/nanopore approach for conservation and evolutionary studies of C. aurita and other marmosets.

category: evolutionary biology


title: Genetic diversity of the LILRB1 and LILRB2 coding regions in an admixed Brazilian population sample

abstract: Leukocyte Immunoglobulin (Ig)-like Receptors (LILR) LILRB1 and LILRB2 play a pivotal role in maintaining self-tolerance and modulating the immune response through interaction with classical and non-classical Human Leukocyte Antigen (HLA) molecules. Although both diversity and natural selection patterns over HLA genes have been extensively evaluated, little information is available concerning the genetic diversity and selection signatures on the LIRB1/2 regions. Therefore, we identified the LILRB1/2 genetic diversity using next-generation sequencing in a population sample comprising 528 healthy control individuals from Sao Paulo State, Brazil. We identified 58 LILRB1 Single Nucleotide Variants (SNVs), which gave rise to 13 haplotypes with at least 1% of frequency. For LILRB2, we identified 41 SNVs arranged into 11 haplotypes with frequencies above 1%. We found evidence of either positive or purifying selection on LILRB1/2 coding regions. Some residues in both proteins showed to be under the effect of positive selection, suggesting that amino acid replacements in these proteins resulted in beneficial functional changes. Finally, we have shown that allelic variation (six and five amino acid exchanges in LILRB1 and LILRB2, respectively) affects the structure and/or stability of both molecules. Nonetheless, LILRB2 has shown higher average stability, with no D1/D2 residue affecting protein structure. Taken together, our findings demonstrate that LILRB1 and LILRB2 are highly polymorphic and provide strong evidence supporting the directional selection regime hypothesis.


title: Improving statistical power in severe malaria genetic association studies by augmenting phenotypic precision

abstract: Severe falciparum malaria has substantially affected human evolution. Genetic association studies of patients with clinically defined severe malaria and matched population controls have helped characterise human genetic susceptibility to severe malaria, but phenotypic imprecision compromises discovered associations. In areas of high malaria transmission the diagnosis of severe malaria in young children and, in particular, the distinction from bacterial sepsis, is imprecise. We developed a probabilistic diagnostic model of severe malaria using platelet and white count data. Under this model we re-analysed clinical and genetic data from 2,220 Kenyan children with clinically defined severe malaria and 3,940 population controls, adjusting for phenotype mis-labelling. Our model, validated by the distribution of sickle trait, estimated that approximately one third of cases did not have severe malaria. We propose a data-tilting approach for case-control studies with phenotype mis-labelling and show that this reduces false discovery rates and improves statistical power in genome-wide association studies.


title: Direct profiling of genome-wide dCas9 and Cas9 specificity using ssDNA mapping (CasKAS)

abstract: Detecting and mitigating off-target activity is critical to the practical application of CRISPR-mediated genome and epigenome editing. While numerous methods have been developed to map Cas9 binding specificity genome-wide, they are generally time-consuming and/or expensive, and not applicable to catalytically dead CRISPR enzymes. We have developed a rapid, inexpensive, and facile assay for identifying off-target CRISPR enzyme binding and cleavage by chemically mapping the unwound single-stranded DNA structures formed upon binding of a sgRNA-loaded Cas9 protein ("CasKAS"). We demonstrate this method in both in vitro and in vivo contexts.


title: Copy-number intratumor heterogeneity contributes to predict relapse in chemotherapy-naive stage II colon cancer

abstract: Optimal selection of high-risk patients with stage II colon cancer is crucial to ensure clinical benefit of adjuvant chemotherapy after surgery. Here, we investigate the prognostic and predictive value of genomic intratumor heterogeneity and aneuploidy for disease recurrence. We combined SNP arrays, targeted next-generation sequencing, fluorescence in situ hybridization and inmunohistochemistry on a retrospective cohort of 84 untreated stage II colon cancer patients. We assessed the subclonality of copy-number alterations (CNAs) and mutations, CD8+ lymphocyte infiltration and their association with time to recurrence (TTR). Prognostic factors were included in machine learning analysis to evaluate their ability to predict individual relapse risk. Tumors from recurrent patients (N = 38) exhibited greater proportion of CNAs compared with non-recurrent (N = 46) (mean 31.3% vs. 23%, respectively; P = 0.014), which was confirmed in an independent cohort. Candidate chromosome-specific aberrations included the gain of the chromosome arm 13q (P = 0.02; HR, 2.67) and loss of heterozygosity at 17q22-q24.3 (P = 0.05; HR, 2.69), both associated with shorter TTR. CNA load positively correlated with intratumor heterogeneity (R = 0.52; P < 0.0001), indicating ongoing chromosomal instability. Consistently, subclonal copy-number heterogeneity was associated with elevated risk of relapse (P = 0.028; HR, 2.20), which we did not observe for subclonal mutations. The clinico-genomic model rated an area under the curve of 0.83, achieving a 10% incremental gain compared to clinicopathological markers. In conclusion, tumor aneuploidy and copy-number heterogeneity were predictive of a poor outcome in early-stage colon cancer, and improved discriminative performance in comparison to clinicopathological data.


title: Precision Genome and Base Editing by Regulating mRNA Nuclear Export Using Selective Inhibitors of Nuclear Export

abstract: CRISPR-based genome engineering tools are associated with off-target effects that constitutively active Cas9 protein may instigate. In the present study, we screened for irreversible small molecule off-switches of CRISPR-Cas9 and discovered that selective inhibitors of nuclear export (SINEs) could inhibit the cellular activity of CRISPR-Cas9 by interfering with the nuclear export of Cas9 mRNA. We subsequently found that SINEs, including an FDA-approved anticancer drug KPT330, could improve the specificities of CRISPR-Cas9-based genome and base editing tools in human cells.


title: GPR171 Agonist Reduces Chronic Neuropathic and Inflammatory Pain in Male, but not in Female Mice

abstract: Chronic pain is a growing public health crisis that requires exigent and efficacious therapeutics. GPR171 is a promising therapeutic target that is widely expressed through the brain, including within the descending pain modulatory regions. Here, we explore the therapeutic potential of the GPR171 agonist, MS15203, in its ability to alleviate chronic pain in male and female mice using a once-daily systemic dose (10mg/kg, i.p.) of MS15203 over the course of 5 days. We found that in our models of Complete Freunds Adjuvant (CFA)-induced inflammatory pain and chemotherapy-induced peripheral neuropathy (CIPN), MS15203 did not reduce thermal hypersensitivity and allodynia, respectively, in female mice. On the other hand, MS15203 treatment decreased the duration of thermal hyper-sensitivity in CFA-treated male mice following 3 days of once-daily administration. MS15203 treatment also produced an improvement in allodynia in male mice, but not female mice, in neuropathic pain after 5 days of treatment. Gene expression of GPR171 and that of its endogenous ligand BigLEN, encoded by the gene PCSK1N, were unaltered within the periaqueductal gray in both male and female mice following inflammatory and neuropathic pain. However, following neuropathic pain in male mice, the protein levels of GPR171 were decreased in the periaqueductal gray. Treatment with MS15203 then rescued the protein levels of GPR171 in the periaqueductal gray of these mice. Taken together, our results identify GPR171 as a GPCR that displays sexual dimorphism in alleviation of chronic pain. Further, our results suggest that GPR171 and MS15203 have demonstrable therapeutic potential in the treatment of chronic pain.

category: pharmacology and toxicology


title: Generative AAV capsid diversification by latent interpolation

abstract: Adeno-associated virus (AAV) capsids have shown clinical promise as delivery vectors for gene therapy. However, the high prevalence of pre-existing immunity against natural capsids poses a challenge for widespread treatment. The generation of diverse capsids that are potentially more capable of immune evasion is challenging because introducing multiple mutations often breaks capsid assembly. Here we target a representative, immunologically relevant 28-amino-acid segment of the AAV2 capsid and show that a low-complexity Variational Auto-encoder (VAE) can interpolate in sequence space to produce diverse and novel capsids capable of packaging their own genomes. We first train the VAE on a 564-sample Multiple-Sequence Alignment (MSA) of dependo-parvoviruses, and then further augment this dataset by adding 22,704 samples from a deep mutational exploration (DME) on the target region. In both cases the VAE generated viable variants with many mutations, which we validated experimentally. We propose that this simple approach can be used to optimize and diversify other proteins, as well as other capsid traits of interest for gene delivery.


title: Profiling the molecular signature of Satellite Glial Cells at the single cell level reveals high similarities between rodent and human

abstract: Peripheral sensory neurons located in dorsal root ganglia relay sensory information from the peripheral tissue to the brain. Satellite glial cells (SGC) are unique glial cells that form an envelope completely surrounding each sensory neuron soma. This organization allows for close bi-directional communication between the neuron and it surrounding glial coat. Morphological and molecular changes in SGC have been observed in multiple pathological conditions such as inflammation, chemotherapy-induced neuropathy, viral infection and nerve injuries. There is evidence that changes in SGC contribute to chronic pain by augmenting neuronal activity in various rodent pain models. SGC also play a critical role in axon regeneration. Whether findings made in rodent model systems are relevant to human physiology have not been investigated. Here we present a detailed characterization of the transcriptional profile of SGC in mouse, rat and human at the single cell level. Our findings suggest that key features of SGC in rodent models are conserved in human. Our study provides the potential to leverage rodent SGC properties and identify potential targets in humans for the treatment of nerve injuries and alleviation of painful conditions.

category: neuroscience