title: The cryptic gonadotropin-releasing hormone neuronal system of human basal ganglia

abstract: Human reproduction is controlled by [~]2,000 hypothalamic gonadotropin-releasing hormone (GnRH) neurons. Here we report the discovery and characterization of additional 150-200,000 GnRH-synthesizing cells in the human basal ganglia and basal forebrain. Extrahypothalamic GnRH neurons were cholinergic. Though undetectable in adult rodents, the GnRH-GFP transgene was expressed transiently by caudate-putamen cholinergic interneurons in newborn transgenic mice. In slice electrophysiological studies, GnRH inhibited these interneurons via GnRHR1 autoreceptors. Whole-transcriptome analysis of cholinergic interneurons and medium spiny projection neurons laser-microdissected from the human putamen confirmed selective expression of GNRH1 and GNRHR1 autoreceptors in cholinergic cells and uncovered the detailed transcriptome profile and molecular connectome of these two cell types. Higher- order non-reproductive functions regulated by GnRH under physiological conditions in the human basal ganglia and basal forebrain require clarification. GnRH/GnRHR1 signaling as a potential therapeutic target in the treatment of neurodegenerative disorders affecting cholinergic neurocircuitries, including Parkinsons and Alzheimers diseases, needs to be explored.


title: SARS-CoV-2 surveillance in Norway rats (Rattus norvergicus) from Antwerp sewer system, Belgium

abstract:
BackgroundSARS-CoV-2 human-to-animal transmission can lead to the establishment of novel reservoirs and the evolution of new variants with the potential to start new outbreaks in humans.

AimWe tested Norway rats inhabiting the sewer system of Antwerp, Belgium, for the presence of SARS-CoV-2 following a local COVID-19 epidemic peak. In addition, we discuss the use and interpretation of SARS-CoV-2 serological tests on non-human samples.

MethodsBetween November and December 2020, Norway rat oral swabs, feces and tissues from the sewer system of Antwerp were collected to be tested by RT-qPCR for the presence of SARS-CoV-2. Serum samples were screened for the presence of anti-SARS-CoV-2 IgG antibodies using a Luminex microsphere immunoassay (MIA). Samples considered positive were then checked for neutralizing antibodies using a conventional viral neutralization test (cVNT).

ResultsThe serum of 35 rats was tested by MIA showing 3 potentially positive sera that were later shown to be negative by cVNT. All tissue samples of 39 rats analyzed tested negative for SARS-CoV-2 RNA.

ConclusionThis is the first study that evaluates SARS-CoV-2 infection in urban rats. We can conclude that the sample of 39 rats had never been infected with SARS-CoV-2. We show that diagnostic serology tests can give misleading results when applied on non-human samples. SARS-CoV-2 monitoring activities should continue due to the emergence of new variants prone to infect Muridae rodents.

category: ecology


title: Long-read sequencing of SARS-CoV-2 reveals novel transcripts and a diverse complex transcriptome landscape.

abstract: Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2 (COVID-19), is a positive single-stranded RNA virus with a 30 kb genome that is responsible for the current pandemic. To date, the genomes of global COVID-19 variants have been primarily characterized via short-read sequencing methods. Here, we devised a long-read RNA (IsoSeq) sequencing approach to characterize the COVID-19 transcript landscape and expression of its [~]27 coding regions. Our analysis identified novel COVID-19 transcripts including a) a short [~]65-70 nt 5-UTR fused to various downstream ORFs encoding accessory proteins such as the envelope, ORF 8, and ORF 9 (nucleocapsid) proteins, that are relatively highly expressed, b) novel SNVs that are differentially expressed, whereby a subset are suggestive of partial RNA editing events, and c) SNVs at functional sites, whereby at least one is associated with a differentially expressed spike protein isoform. These previously uncharacterized COVID-19 isoforms, expressed genes, and gene variants were corroborated using ddPCR. Understanding this transcriptional complexity may help provide insight into the biology and pathogenicity of SARS-CoV-2 compared to other coronaviruses.

category: genetics


title: Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics

abstract: Coronavirus (CoV) non-structural proteins (nsps) assemble to form the replication-transcription complex (RTC) responsible for viral RNA synthesis. nsp7 and nsp8 are important cofactors of the RTC, as they interact and regulate the activity of RNA-dependent RNA polymerase (RdRp) and other nsps. To date, no structure of full-length SARS-CoV-2 nsp7:nsp8 complex has been published. Current understanding of this complex is based on structures from truncated constructs or with missing electron densities and complexes from related CoV species with which SARS-CoV-2 nsp7 and nsp8 share upwards of 90% sequence identity. Despite available structures being solved using crystallography and cryo-EM representing detailed snapshots of the nsp7:nsp8 complex, it is evident that the complex has a high degree of structural plasticity. However, relatively little is known about the conformational dynamics of the complex and how it assembles to interact with other nsps. Here, the solution-based structural proteomic techniques, hydrogen-deuterium exchange mass spectrometry (HDX-MS) and crosslinking mass spectrometry (XL-MS), illuminate the structural dynamics of the SARS-CoV-2 full-length nsp7:nsp8 complex. The results presented from the two techniques are complementary and validate the interaction surfaces identified from the published three-dimensional heterotetrameric crystal structure of SARS-CoV-2 truncated nsp7:nsp8 complex. Furthermore, mapping of XL-MS data onto higher order complexes suggests that SARS-CoV-2 nsp7 and nsp8 do not assemble into a hexadecameric structure as implied by the SARS-CoV full-length nsp7:nsp8 crystal structure. Instead our results suggest that the nsp7:nsp8 heterotetramer can dissociate into a stable dimeric unit that might bind to nsp12 in the RTC without altering nsp7-nsp8 interactions.

category: biophysics


title: SARS-CoV-2 501Y.V2 (B.1.351) elicits cross-reactive neutralizing antibodies

abstract: Neutralization escape by SARS-CoV-2 variants, as has been observed in the 501Y.V2 (B.1.351) variant, has impacted the efficacy of first generation COVID-19 vaccines. Here, the antibody response to the 501Y.V2 variant was examined in a cohort of patients hospitalized with COVID-19 in early 2021 - when over 90% of infections in South Africa were attributed to 501Y.V2. Robust binding and neutralizing antibody titers to the 501Y.V2 variant were detected and these binding antibodies showed high levels of cross-reactivity for the original variant, from the first wave. In contrast to an earlier study where sera from individuals infected with the original variant showed dramatically reduced potency against 501Y.V2, sera from 501Y.V2-infected patients maintained good cross-reactivity against viruses from the first wave. Furthermore, sera from 501Y.V2-infected patients also neutralized the 501Y.V3 (P.1) variant first described in Brazil, and now circulating globally. Collectively these data suggest that the antibody response in patients infected with 501Y.V2 has a broad specificity and that vaccines designed with the 501Y.V2 sequence may elicit more cross-reactive responses.

category: immunology


title: A novel Rab11-Rab3a cascade required for lysosome exocytosis

abstract:
Lysosomes are dynamic organelles, capable of undergoing exocytosis. This process is crucial for several cellular functions, namely plasma membrane repair. Nevertheless, the molecular machinery involved in this process is poorly understood.

Here, we identify Rab11a and Rab11b as regulators of calcium-induced lysosome exocytosis. Interestingly, Rab11-positive vesicles transiently interact with lysosomes at the cell periphery, indicating that this interaction is required for the last steps of lysosome exocytosis. Additionally, we found that the silencing of the exocyst subunit Sec15, a Rab11 effector, impairs lysosome exocytosis independently of the exocyst complex, suggesting that Sec15 acts together with Rab11 in the regulation of lysosome exocytosis. Furthermore, we show that Rab11 binds the guanine nucleotide exchange factor for Rab3a (GRAB) and also Rab3a, which we described previously as a regulator of the positioning and exocytosis of lysosomes.

Thus, our studies suggest that Rab11-positive vesicles transport GRAB to activate Rab3a on lysosomes, establishing a Rab11-Rab3 cascade that is essential for lysosome exocytosis.

category: cell biology


title: Cell competition removes segmental aneuploid cells from Drosophila imaginal disc-derived tissues based on ribosomal protein gene dose

abstract: Aneuploidy causes birth defects and miscarriages, occurs in nearly all cancers, and is a hallmark of aging. Individual aneuploid cells can be eliminated from developing tissues by unknown mechanisms. Cells with ribosomal protein (Rp) gene mutations are also eliminated, by cell competition with normal cells. Because Rp genes are spread across the genome, their copy number is a marker for chromosome aberrations. Elimination of imaginal disc cells with irradiation-induced genome damage often required cell competition genes. When defined chromosome regions were deleted, segmentally-aneuploid cells were eliminated by the RpS12-Xrp1 cell competition pathway in an apoptosis- dependent manner when they differed from neighboring cells in Rp gene dose. Cells with normal doses of the Rp (and eIF2{gamma}) genes survived and differentiated adult tissues. Thus, cell competition, triggered by differences in Rp gene dose between cells, is a significant mechanism for the elimination of aneuploid somatic cells, likely to contribute to preventing cancer.


title: Tissue specific targeting of DNA nanodevices in a multicellular living organism

abstract: Nucleic acid nanodevices present great potential as agents for logic-based therapeutic intervention as well as in basic biology. Often, however, the disease targets that need corrective action are localized in specific organs and thus realizing the full potential of DNA nanodevices also requires ways to target them to specific cell-types in vivo. Here we show that by exploiting either endogenous or synthetic receptor-ligand interactions and by leveraging the biological barriers presented by the organism, we can target extraneously introduced DNA nanodevices to specific cell types in C. elegans, with sub-cellular precision. The amenability of DNA nanostructures to tissue-specific targeting in vivo significantly expands their utility in biomedical applications and discovery biology.

category: biochemistry


title: HOXA9 acts as a regulatory switch in acute myeloid leukaemia and myeloproliferative neoplasms

abstract: Blood malignancies arise from the dysregulation of haematopoiesis. The type of blood cell and the specific order of oncogenic events initiating abnormal growth ultimately determine the cancer subtype and subsequent clinical outcome. HOXA9 plays an important role in acute myeloid leukaemia (AML) prognosis by promoting blood cell expansion and altering differentiation; however, the function of HOXA9 in other blood malignancies is still unclear. Here, we demonstrate the importance of this gene in chronic myeloproliferative neoplasms (MPN) and highlight the biological switch and prognosis marker properties of HOXA9 in AML and MPN. This binary switch function can explain the clinical stratification of these two blood disorders. First, we establish the ability of HOXA9 to stratify AML patients with distinct cellular and clinical outcomes. Then, through the use of a computational network model of MPN, we show that the self-activation of HOXA9 and its relationship to JAK2 and TET2 can explain the branching progression of JAK2/TET2 mutant MPN patients towards divergent clinical characteristics. Finally, we predict a connection between the RUNX1 and MYB genes and a suppressive role for the NOTCH pathway in MPN diseases.

category: cancer biology


title: Multiplexed VaxArray Immunoassay for Rapid Antigen Quantification in Measles and Rubella Vaccine Manufacturing

abstract: Measles-containing vaccines (MCV), specifically vaccines against measles and rubella (MR), are extremely effective and critical for the eradication of measles and rubella diseases. In developed countries, vaccination rates are high and vaccines are readily available, but continued high prevalence of both diseases in developing countries and surges in measles deaths in recent years have highlighted the need to expand vaccination efforts. To meet demand for additional vaccines at a globally affordable price, it is highly desirable to streamline vaccine production thereby reducing cost and speeding up time to delivery. MR vaccine characterization currently relies on the 50% cell culture infectious dose (CCID50) assay, an endpoint assay with low reproducibility that requires 10-14 days to complete. For streamlining bioprocess analysis and improving measurement precision relative to CCID50, we developed the VaxArray Measles and Rubella assay kit, which is based on a multiplexed microarray immunoassay with a 5-hour time to result. Here we demonstrate vaccine-relevant sensitivity ranging from 345 - 800 IFU/mL up to 100,000 IFU/mL and specificity that allows simultaneous analysis in bivalent vaccine samples. The assay is sensitive to antigen stability and has minimal interference from common vaccine additives. The assay exhibits high reproducibility and repeatability, with 15% CV, much lower than the typical 0.3 log10 error (~65%) observed for the CCID50 assay. The intact protein concentration measured by VaxArray is reasonably correlated to, but not equivalent to, CCID50 infectivity measurements for harvest samples. However, the measured protein concentration exhibits equivalency to CCID50 for more purified samples, including concentrated virus pools and monovalent bulks, making the assay a useful new tool for same-day analysis of vaccine samples for bioprocess development, optimization, and monitoring.


title: An Investigation of the Mechanism for a Novel Influenza Inhibitor Designed to Target the Import of PB1

abstract: A novel inhibitor of PB1 import, referred to here as GM30, previously displayed nuclear retention of viral nucleoprotein (NP), which spurred the question of whether the compound directly or indirectly blocked viral ribonucleoprotein (vRNP) complex export. vRNPs are exported from the nucleus via exportin 1 (XPO1). Using verdinexor (VNXR), a direct inhibitor of vRNP export that binds to XPO1 and also blocks nuclear factor kappa B (NF-{kappa}B) export, we found that GM30 does not block nuclear export of NF-{kappa}B. GM30 likewise demonstrated high nuclear retention of NP but not as much as the direct inhibition of nuclear export by VNXR. When the compound was added hours after infection, the compound lost its ability to block nuclear export but VNXR retained its ability to block nuclear export. GM30 is therefore likely an indirect inhibitor of nuclear export because it disrupts the vRNP complex formation and impedes export from the nucleus by reducing nuclear import of PB1.

category: microbiology


title: Biochemical reconstitution defines new functions for membrane-bound glycosidases in assembly of the bacterial cell wall

abstract: The peptidoglycan cell wall is a macromolecular structure that encases bacteria and is essential for their survival. Proper assembly of the cell wall requires peptidoglycan synthases as well as membrane-bound cleavage enzymes that control where new peptidoglycan is made and inserted. We are only beginning to understand the roles of peptidoglycan cleavage enzymes in cell wall assembly. Previous studies have shown that two membrane-bound proteins in Streptococcus pneumoniae, here named MpgA and MpgB, are important in maintaining cell wall integrity. MpgA was predicted to be a lytic transglycosylase based on its homology to Escherichia coli MltG while the enzymatic activity of MpgB was unclear. Using nascent peptidoglycan substrates synthesized in vitro from the peptidoglycan precursor Lipid II, we report that both MpgA and MpgB are muramidases. We show that replacing a single amino acid in E. coli MltG with the corresponding amino acid from MpgA results in muramidase activity, allowing us to predict from the presence of this amino acid that other putative lytic transglycosylases actually function as muramidases. Strikingly, we report that MpgA and MpgB cut nascent peptidoglycan at different positions along the sugar backbone relative to the reducing end. MpgA produces much longer peptidoglycan oligomers and we show that its cleavage site selectivity is controlled by the LysM-like subdomain, which is also present in MltG. We propose that MltGs ability to complement loss of MpgA in S. pneumoniae despite performing different cleavage chemistry is because it can cleave nascent peptidoglycan at the same distance from the lipid anchor.


title: Multiple classes of bactericidal antibiotics cause DNA double strand breaks in Staphylococcus aureus

abstract: Antibiotics inhibit essential bacterial processes, resulting in arrest of growth and in some cases cell death. Many antibiotics are also reported to trigger endogenous production of reactive oxygen species (ROS), which damage DNA and other macromolecules. However, the type of DNA damage that arises and the mechanisms used by bacteria to repair it are largely unclear. We found that several different classes of antibiotic triggered dose-dependent DNA damage in Staphylococcus aureus, including some bacteriostatic drugs. Damage was heterogenous and varied in magnitude between strains. However, antibiotic-triggered DNA damage led to double strand breaks, the processing of which by the RexAB helicase/nuclease complex triggered the SOS response and reduced staphylococcal susceptibility to most of the antibacterials tested. In most cases, DNA DSBs occurred under aerobic but not anaerobic conditions, suggesting a role for ROS. We conclude that DNA double strand breaks are a common occurrence during bacterial exposure to several different antibiotic classes and that repair of this damage by the RexAB complex promotes bacterial survival.


title: Exploration of polyyne biosynthetic gene cluster diversity in bacteria leads to the discovery of the Pseudomonas polyyne protegencin

abstract: Natural products that possess alkyne or polyyne moieties have been isolated from a variety of biological sources. In bacteria their biosynthesis has been defined, however, the distribution of polyyne biosynthetic gene clusters (BGCs), and their evolutionary relationship to alkyne biosynthesis, have not been addressed. We explored the distribution of alkyne biosynthesis gene cassettes throughout bacteria, revealing evidence of multiple horizontal gene transfer events. Investigating the evolutionary connection between alkyne and polyyne biosynthesis identified a monophyletic clade possessing a conserved seven-gene cassette for polyyne biosynthesis. Mapping the diversity of these conserved genes revealed a phylogenetic clade representing a polyyne BGC in Pseudomonas, pgn, and subsequent pathway mutagenesis and analytical chemistry characterised the associated metabolite, protegencin. In addition to unifying and expanding our knowledge of polyyne diversity, our results show that alkyne and polyyne biosynthetic gene clusters are promiscuous within bacteria. Systematic mapping of conserved biosynthetic genes across bacterial genomic diversity has proven to be a successful method for discovering natural products.


title: Anaerobic microbiota facilitate P. aeruginosa access to airway epithelia in a novel co-culture model of colonization

abstract: The role(s) of anaerobic microbiota in chronic airway disease are poorly understood due to inherent limitations of existing laboratory models. To address this knowledge gap, we use a dual oxic-anoxic co-culture approach that maintains an oxygen-limited apical epithelial microenvironment while host cells are oxygenated basolaterally. Reduced oxygen culture did not alter the physiology or gene expression of Calu-3 cells but supported anaerobe-epithelial interactions for 24h without affecting bacterial or host cell viability. Anaerobe challenge led to increased expression of inflammatory marker genes and compromised integrity of apical mucins, leading to our hypothesis that anaerobe-host interactions prime the airways for chronic infection. Indeed, anaerobe pre-treatment of Calu-3 cells led to an increase in Pseudomonas aeruginosa colonization. This model system offers new insight into anaerobe-host interactions in airway disease pathophysiology and motivates further study of the lung, gut, and oral cavity, where etiological roles of anaerobes have been proposed but specific pathogenic mechanisms remain unclear.


title: Binding of the Andes Virus Nucleocapsid Protein to RhoGDI Induces the Release and Activation of the Permeability Factor RhoA

abstract:
Andes virus (ANDV) nonlytically infects pulmonary microvascular endothelial cells (PMECs) causing acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). In HPS patients virtually every PMEC is infected, however the mechanism by which ANDV induces vascular permeability and edema remains to be resolved. The ANDV nucleocapsid (N) protein activates the GTPase, RhoA, in primary human PMECs causing VE-Cadherin internalization from adherens junctions and PMEC permeability. We found that ANDV N protein failed to bind RhoA, but co-precipitates RhoGDI (Rho GDP-dissociation inhibitor), the primary RhoA repressor that normally sequesters RhoA in an inactive state. ANDV N protein selectively binds the RhoGDI C-terminus (69-204) but fails to form ternary complexes with RhoA or inhibit RhoA binding to the RhoGDI N-terminus (1-69). However, we found that ANDV N protein uniquely inhibits RhoA binding to an S34D phosphomimetic RhoGDI mutant. Hypoxia and VEGF increase RhoA induced PMEC permeability by directing Protein Kinase C (PKC) phosphorylation of S34 on RhoGDI. Collectively, ANDV N protein alone activates RhoA by sequestering and reducing RhoGDI available to suppress RhoA. In response to hypoxia and VEGF activated PKC, ANDV N protein additionally directs the release of RhoA from S34-phosphorylated RhoGDI, synergistically activating RhoA and PMEC permeability. These findings reveal a fundamental edemagenic mechanism that permits ANDV to amplify PMEC permeability in hypoxic HPS patients. Our results rationalize therapeutically targeting PKC and opposing Protein Kinase A (PKA) pathways that control RhoGDI phosphorylation as a means of resolving ANDV induced capillary permeability, edema and HPS.

ImportanceHPS causing hantaviruses infect pulmonary endothelial cells causing vascular leakage, pulmonary edema and a 35% fatal acute respiratory distress syndrome (ARDS). Hantaviruses dont lyse or disrupt the endothelium but dysregulate normal EC barrier functions and increase hypoxia directed permeability. Our findings reveal a novel underlying mechanism of EC permeability resulting from ANDV N protein binding to RhoGDI, a regulatory protein that normally maintains edemagenic RhoA in an inactive state and inhibits EC permeability. ANDV N sequesters RhoGDI and enhances the release of RhoA from S34 phosphorylated RhoGDI. These findings indicate that ANDV N induces the release of RhoA from PKC phosphorylated RhoGDI, synergistically enhancing hypoxia directed RhoA activation and PMEC permeability. Our data suggests inhibiting PKC and activating PKA phosphorylation of RhoGDI as mechanisms of inhibiting ANDV directed EC permeability and therapeutically restricting edema in HPS patients. These findings may be broadly applicable to other causes of ARDS.


title: Fungal taste for minerals: the ectomycorrhizal fungus Paxillus involutus triggers specific genes when extracting potassium from different silicates

abstract: Silicates make up about 90% of the Earths crust and constitute the main source of mineral nutrients for microorganisms and plants. Fungi can actively weather silicates to extract nutrients. However, it is unclear whether they are able to obtain the same amounts of nutrients and use the same mechanisms when tapping into different mineral sources. We performed a microcosm experiment using the ectomycorrhizal basidiomycetes Paxillus involutus and the silicates K-vermiculite, muscovite and phlogopite as only potassium sources, as they show a different resistance for the removal of K cations from the mineral structure. A combination of transcriptomic, elemental and SEM analyses showed that different minerals stimulated specific weathering mechanisms and led to a change in fungal genes expression. The differential expression of the fungal genes generated alternative chemical attacks on the minerals, resulting in a tailored dissolution and selective uptake of chemical elements according to the leachability of K from the silicate mineral. The K uptake capacity of the fungus was highest with vermiculite in comparison to growth on phlogopite and muscovite. The findings provide new insights into fungal-mineral interactions that will help to interpret key processes for the homeostasis of soil environments.


title: Mycobacterium abscessus biofilms produce an ECM and have a distinct mycolic acid profile

abstract: A non-tuberculous mycobacterium (NTM), Mycobacterium abscessus is an emerging opportunistic pathogen associated with difficult to treat pulmonary infections, particularly in patients suffering from cystic fibrosis. It is capable of forming biofilms in vitro that result in an increase of already high levels of antibiotic resistance in this bacterium. Evidence that M. abscessus forms biofilm-like microcolonies in patient lungs and on medical devices further implicated the need to investigate this biofilm in detail. Therefore, in this study we characterized the M. abscessus pellicular biofilm, formed on a liquid-air interface, by studying its molecular composition, and its transcriptional profile in comparison to planktonic cells. Using scanning electron micrographs and fluorescence microscopy, we showed that M. abscessus biofilms produce an extracellular matrix composed of lipids, proteins, carbohydrates and eDNA. Transcriptomic analysis of biofilms revealed an upregulation of pathways involved in the glyoxylate shunt, redox metabolism and mycolic acid biosynthesis. Genes involved in elongation and desaturation of mycolic acids were highly upregulated in biofilms and, mirroring those findings, biochemical analysis of mycolates revealed molecular changes and an increase in mycolic acid chain length. Together these results give us an insight into the complex structure of M. abscessus biofilms, the understanding of which may be adapted for clinical use in treatment of biofilm infections, including strategies for dispersing the ECM, allowing antibiotics to gain access to bacteria within the biofilm.


title: The Mycobacterium tuberculosis transposon sequencing database (MtbTnDB): a large-scale guide to genetic conditional essentiality

abstract: Characterization of gene essentiality across different conditions is a useful approach for predicting gene function. Transposon sequencing (TnSeq) is a powerful means of generating genome-wide profiles of essentiality and has been used extensively in Mycobacterium tuberculosis (Mtb) genetic research. Over the past two decades, dozens of TnSeq screens have been published, yielding valuable insights into the biology of Mtb in vitro, inside macrophages, and in model host organisms. However, these Mtb TnSeq profiles are distributed across dozens of research papers within supplementary materials, which makes querying them cumbersome and assembling a complete and consistent synthesis of existing data challenging. Here, we address this problem by building a central repository of publicly available TnSeq screens performed in M. tuberculosis, which we call the Mtb transposon sequencing database (MtbTnDB). The MtbTnDB encompasses 64 published and unpublished TnSeq screens, and is standardized, open-access, and allows users easy access to data, visualizations, and functional predictions through an interactive web-app (www.mtbtndb.app). We also present evidence that (i) genes in the same genomic neighborhood tend to have similar TnSeq profiles, and (ii) clusters of genes with similar TnSeq profiles tend to be enriched for genes belonging to the same functional categories. Finally, we test and evaluate machine learning models trained on TnSeq profiles to guide functional annotation of orphan genes in Mtb. In addition to facilitating the exploration of conditional genetic essentiality in this important human pathogen via a centralized TnSeq data repository, the MtbTnDB will enable hypothesis generation and the extraction of meaningful patterns by facilitating the comparison of datasets across conditions. This will provide a basis for insights into the functional organization of Mtb genes as well as gene function prediction.


title: Critical Assessment of Metaproteome Investigation (CAMPI): a Multi-Lab Comparison of Established Workflows

abstract:
Metaproteomics has matured into a powerful tool to assess functional interactions in microbial communities. While many metaproteomic workflows are available, the impact of method choice on results remains unclear.

Here, we carried out the first community-driven, multi-laboratory comparison in metaproteomics: the critical assessment of metaproteome investigation study (CAMPI). Based on well-established workflows, we evaluated the effect of sample preparation, mass spectrometry, and bioinformatic analysis using two samples: a simplified, laboratory-assembled human intestinal model and a human fecal sample.

We observed that variability at the peptide level was predominantly due to sample processing workflows, with a smaller contribution of bioinformatic pipelines. These peptide-level differences largely disappeared at the protein group level. While differences were observed for predicted community composition, similar functional profiles were obtained across workflows.

CAMPI demonstrates the robustness of present-day metaproteomics research, serves as a template for multi-laboratory studies in metaproteomics, and provides publicly available data sets for benchmarking future developments.


title: Dysbiotic oral and gut viromes in untreated and treated rheumatoid arthritis patients

abstract:
BackgroundRheumatoid arthritis (RA) has been considered to be influenced by bacteria from the oral cavity and gut for many years. Despite potential impact of viruses in RA was mentioned in some studies, specific roles of oral and gut viromes in RA is still unclear.

ResultsIn this study, we observed the viral community variation in the oral and gut samples, performed a comparative analysis of oral and gut viromes in health controls, untreated and treated RA patients, and constructed interaction networks among viruses, bacteria, and RA-associated clinical indexes to address the potential associations between viral community and RA. The results showed that the viromes could be isolated from dental plaque, saliva, and feces samples, among which the saliva having the highest with in-sample diversity. Meanwhile, remarkable variations of viral diversity and composition in the oral (i.e., dental plaque and saliva) virome could be observed in RA patients and healthy controls yet in untreated and treated RA patients, with a relatively low variability in the gut virome. Distraction of viruses-bacteria interaction network was discovered in three sites of RA patients. In addition, some RA-associated oral taxa, including Lactococcus phage vOTU70, Bacteroides vulgatus, Lactococcus lactis, Escherichia coli, Neisseria elongate, were correlated to the RA-related clinical indexes.

ConclusionWhole-virome analysis illustrated the potential role of oral and gut viral communities in the development of RA.


title: Defining proximity proteomics of post-translationally modified proteins by antibody-mediated protein A-APEX2 labeling

abstract: Proximity labeling catalyzed by promiscuous enzymes, such as APEX2, has emerged as a powerful approach to characterize multiprotein complexes and protein-protein interactions. However, current methods depend on the expression of exogenous fusion proteins and cannot be applied to post-translational modifications. To address this limitation, we developed a new method to label proximal proteins of interest by antibody-mediated protein A-APEX2 labeling (AMAPEX). In this method, a modified protein is bound in situ by a specific antibody, which then tethers a protein A-APEX2 (pA-APEX2) fusion protein. Activation of APEX2 labels the nearby proteins with biotin; these proteins are then purified using streptavidin beads and are identified by mass spectrometry. We demonstrate the utility of this approach by profiling the binding proteins of histone modifications including H3K27me3, H3K9me3, H3K4me3, H4K5ac and H4K12ac, and we verified the genome-wide colocalization of these identified proteins with bait proteins by published ChIP-seq analysis. Overall, AMAPEX is an efficient tool to identify proteins that interact with modified proteins.

category: molecular biology


title: A genetically encoded fluorescent biosensor for extracellular L-lactate

abstract: To enable investigations of the emerging roles of cell-to-cell shuttling of L-lactate, we have developed an intensiometric green fluorescent genetically encoded biosensor for extracellular L-lactate. We demonstrate that this biosensor, designated eLACCO1.1, enables minimally invasive cellular resolution imaging of extracellular L-lactate in cultured mammalian cells and brain tissue.


title: Engineered Peptide-Functionalized Hydrogels Modulate the RNA Transcriptome of Human Nucleus Pulposus Cells In Vitro

abstract: Degeneration and aging of the nucleus pulposus (NP) of the intervertebral disc (IVD) is accompanied by alterations in NP cell phenotype marked by a shift towards a fibroblast-like, catabolic state. We have recently demonstrated an ability to manipulate the phenotype of human adult degenerative NP cells through 2D culture upon poly(ethylene glycol) (PEG) based hydrogels dually functionalized with integrin- and syndecan-binding laminin-mimetic peptides (LMPs). In the present study, we sought to understand the transcriptomic changes elicited through NP cell interactions with the LMP-functionalized hydrogel system (LMP gel) by examining targets of interest a priori and by conducting unbiased analysis to identify novel mechanosensitive targets. The results of gene specific analysis demonstrated that the LMP gel promoted adult degenerative NP cells to upregulate 148 genes including several NP markers (e.g. NOG and ITGA6) and downregulate 277 genes, namely several known fibroblastic markers. Additionally, 13 genes associated with G protein-coupled receptors, many of which are known drug targets, were identified as differentially regulated following culture upon the gel. Furthermore, through gene set enrichment analysis we identified over 700 pathways enriched amongst the up- and downregulated genes including pathways related to cell differentiation, notochord morphogenesis, and intracellular signaling. Together these findings demonstrate the global mechanobiological effects induced by the LMP gel and confirm the ability of this substrate to modulate NP cell phenotype.


title: Recombinant haplotypes reflect sexual reproduction in symbiotic arbuscular mycorrhizal fungi

abstract:
Arbuscular mycorrhizal fungi (AMF) are part of the most widespread fungal-plant symbiosis. They colonize at least 80% of plant species, promote plant growth and plant diversity. These fungi are multinucleated and contain either one or two haploid nuclear genotypes (monokaryon and dikaryon) identified by the alleles at a putative mating-type locus. This taxon has been considered as an ancient asexual scandal because of the lack of observable sexual structures. Despite identification of a putative mating-type locus and functional activation of genes related to mating when two isolates co-exist, it remains unknown if AMF take part in a mainly sexual life cycle.

We used publicly available genome sequences to test if dikaryon genomes display signatures of sexual reproduction in the form of reciprocal recombination patterns, or if they display signatures of parasexual reproduction involving gene conversion.

We used short-read and long-read sequence data to identify nucleus genotype-specific haplotypes within dikaryons and then compared them to orthologous gene sequences from related monokaryon isolates displaying the same putative MAT-types. We observed that these genotype-specific haplotypes display reciprocal recombination and not gene conversion.

These results are consistent with a sexual origin of the dikaryon rather than a parasexual origin and provides an important step to understand the life cycle of these globally important symbiotic fungi.

category: evolutionary biology


title: Dual RNA modulation of protein mobility and stability within phase-separated condensates

abstract: One of the key mechanisms employed by cells to control their spatiotemporal organization is the formation and dissolution of phase-separated condensates. The balance between condensate assembly and disassembly can be critically regulated by the presence of RNA. In this work, we use a novel sequence-dependent coarse-grained model for proteins and RNA to unravel the impact of RNA in modulating the transport properties and stability of biomolecular condensates. We explore the phase behavior of several RNA-binding proteins such as FUS, hnRNPA1 and TDP-43 proteins along with that of their corresponding prion-like domains and RNA-recognition motifs, from absence to moderately high RNA concentration. By characterising the phase diagram, key molecular interactions, surface tension and transport properties of the condensates, we report a dual RNA-induced behavior: On the one hand, RNA enhances phase separation at low concentration as long as the RNA radius of gyration is comparable to that of the proteins, whilst at high concentration it inhibits the ability of proteins to self-assemble independently of its length. On the other hand, along with the stability modulation, the viscosity of the condensates can be considerably reduced at high RNA concentration as long as the length of the RNA chains is shorter than that of the proteins. Conversely, long RNA strands increase viscosity, even at high concentration, but barely modify protein self-diffusion, which mainly depends on RNA concentration and on its own effect on droplet density. On the whole, our work rationalizes the different routes by which RNA can regulate phase separation and condensate dynamics, as well as the subsequent aberrant rigidification implicated in the emergence of various neuropathologies and age-related diseases.


title: Preferential allelic expression of PIK3CA mutations is frequent in breast cancer and is prognostically significant

abstract:
PIK3CA mutations are the most common in breast cancer, particularly in the estrogen receptor positive cohort, but the benefit of PI3K inhibitors has had limited success compared with approaches targeting other less common mutations. We found allelic imbalances in the expression of PIK3CA in normal breast tissue and mapped a germline candidate regulatory variant. An imbalance was also frequently observed in the expression of the missense mutant and wild-type PIK3CA alleles in breast tumors from METABRIC and TCGA projects. Moreover, although 60% of tumors preferentially expressed the mutant allele, 10% did preferentially express the wild-type allele. Mechanistically, we show that these imbalances are more frequently due to regulatory variants in cis than altered copy-number and predict that somatic variants have a more significant role than germline ones. We further found that imbalanced allelic expression between mutant and wild-type alleles due to cis-regulatory variants associated with poor prognosis (p=0.0081).

Interestingly, ER+, PR+, and Her2- tumors expressing very low levels of the mutant allele had the poorest prognosis (DSS <7.5yrs for ER+ and PR+ tumors and <5yrs for Her2- tumors). Hence, our work provides compelling evidence to support the clinical utility of PIK3CA allelic expression in breast cancer in identifying this cohort of low mutant allele expressing patients of poorer prognosis, who will unlikely benefit from PI3K inhibitors. Furthermore, our work establishes a new model of differential regulation of critical cancer-promoting genes.


title: Allelic expression imbalance of PIK3CA mutations is frequent in breast cancer and prognostically significant


title: Nrf2 Regulates β-cell Mass by Suppressing Cell Death and Promoting Proliferation

abstract: Finding therapies that can protect and expand functional {beta}-cell mass is a major goal of diabetes research. Here we generated {beta}-cell-specific conditional knockout and gain-of-function mouse models and used human islet transplant experiments to examine how manipulating Nrf2 levels affects {beta}-cell survival, proliferation and mass. Depletion of Nrf2 in {beta}-cells resulted in decreased glucose-stimulated {beta}-cell proliferation ex vivo and decreased adaptive {beta}-cell proliferation and {beta}-cell mass expansion after a high fat diet in vivo. Nrf2 protects {beta}-cells from apoptosis after a high fat diet. Nrf2 loss-of-function decreases Pdx1 abundance and insulin content. Activating Nrf2 in a {beta}-cell-specific manner increases {beta}-cell proliferation and {beta}-cell mass. Human islets transplanted under the kidney capsule of immunocompromised mice and treated systemically with CDDO-Me, an Nrf2 activator, display increased {beta}-cell proliferation. Thus, Nrf2 regulates {beta}-cell mass and is an exciting therapeutic target for expanding {beta}-cell mass in diabetes.

category: physiology


title: A Biosensor for Detection of Indole Metabolites

abstract: The lack of diverse health-related biosensors limits the progress towards our understanding of how the microbiome metabolism impacts health. Microbially produced indole-3-aldehyde (I3A) has been associated with reducing inflammation in diseases such as ulcerative colitis by stimulating the aryl hydrocarbon receptor (AhR) pathway. We mined the protein database for gut microbiome metabolites sensors and developed a biosensor for I3A. We engineered E. coli embedded with a single plasmid carrying a chimeric two-component system that detects I3A. Our I3A receptor characterization identified residues that contribute to the sensors high specificity in a range of 0.1-10 {micro}M. The I3A biosensor opens the door to sensing indole metabolites produced at the host-microbe interface and provides new parts for synthetic biology applications.

category: synthetic biology


title: Benzofuran sulfonates and small self-lipid antigens activate type II NKT cells via CD1d

abstract:
Natural Killer T (NKT) cells detect lipids presented by CD1d. Most studies focus on type I NKT cells that express semi-invariant {beta} T cell receptors (TCR) and recognise -galactosylceramides. However, CD1d also presents structurally distinct lipids to NKT cells expressing diverse TCRs (type II NKT cells) but our knowledge of the antigens for type II NKT cells is limited. An early study identified an NKT cell agonist, phenyl pentamethyldihydrobenzofuransulfonate (PPBF), which is notable for its similarity to common sulfa-drugs, but its mechanism of NKT-cell activation remained unknown. Here we demonstrate that a range of pentamethylbenzofuransulfonate (PBFs), including PPBF, activate polyclonal type II NKT cells from human donors. Whereas these sulfa drug-like molecules might have acted pharmacologically on cells, here we demonstrate direct contact between TCRs and PBF-treated CD1d complexes. Further, PBF-treated CD1d-tetramers identified type II NKT cell populations cells expressing {beta} and {gamma}{delta}TCRs, including those with variable and joining region gene usage (TRAV12-1-TRAJ6) that was conserved across donors. By trapping a CD1d-type II NKT TCR complex for direct mass spectrometric analysis, we detected molecules that allow binding of CD1d to TCRs, finding that both PBF and short-chain sphingomyelin lipids are present in these complexes. Furthermore, the combination of PPBF and short-chain sphingomyelin enhances CD1d tetramer staining of PPBF-reactive T cell lines over either molecule alone. This study demonstrates that non-lipidic small molecules, that resemble sulfa-drugs implicated in systemic hypersensitivity and drug allergy reactions, activate a polyclonal population of type II NKT cells in a CD1d-restricted manner.

Significance StatementWhereas T cells are known to recognize peptide, vitamin B metabolite or lipid antigens, we identify several non-lipidic small molecules, pentamethylbenzofuransulfonates (PBFs), that activate a population of CD1d-restricted NKT cells. This represents a breakthrough in the field of NKT cell biology. This study also reveals a previously unknown population of PBF-reactive NKT cells in healthy individuals with stereotyped receptors that paves the way for future studies of the role of these cells in immunity, including sulfa-drug hypersensitivity.


title: Host protease activity on bacterial pathogens promotes complement- and antibiotic-directed killing

abstract: Our understanding of how the host immune system thwarts bacterial evasive mechanisms remains incomplete. Here, we show that host protease neutrophil elastase acts on Acinetobacter baumannii and Pseudomonas aeruginosa to destroy factors that prevent serum-associated, complement-directed killing. The protease activity also enhances bacterial susceptibility to antibiotics in sera. These findings implicate a new paradigm where host protease activity on bacteria acts synergistically with the host complement system and antibiotics to defeat bacterial pathogens.


title: General and cell-type-specific aspects of the motor neuron maturation transcriptional program

abstract: Building a nervous system is a protracted process that starts with the specification of individual neuron types and ends with the formation of mature neural circuits. The molecular mechanisms that regulate the temporal progression of maturation in individual cell types remain poorly understood. In this work, we have mapped the gene expression and chromatin accessibility changes in mouse spinal motor neurons throughout their lifetimes. We found that both motor neuron gene expression and putative regulatory elements are dynamic during the first three weeks of postnatal life, when motor circuits are maturing. Genes that are up-regulated during this time contribute to adult motor neuron diversity and function. Almost all of the chromatin regions that gain accessibility during maturation are motor neuron specific, yet a majority of the transcription factor binding motifs enriched in these regions are shared with other mature neurons. Collectively, these findings suggest that a core transcriptional program operates in a context-dependent manner to access cell-type-specific cis-regulatory systems associated with maturation genes. Discovery of general principles governing neuronal maturation might inform methods for transcriptional reprogramming of neuronal age and for improved modelling of age-related neurodegenerative diseases.

category: developmental biology


title: Genomic structure and diversity of oak populations in British Parklands

abstract: The two predominant oak species in Britain are Quercus robur (English or pedunculate oak) and Q. petraea (sessile oak). We sequenced the whole genomes of 386 oak trees from four British parkland sites and found over 50 million nuclear single nucleotide polymorphisms (SNPs), allowing us to identify 360 Q. robur, ten Q. petraea and 16 hybrid individuals using clustering methods. Comparing Q. robur and Q. petraea trees from Attingham Park, we found that the nuclear genomes of the two species are largely undifferentiated but identified 81 coding regions exhibiting strong interspecific differentiation. The nuclear genomes of our 360 Q. robur individuals showed no clear differentiation among the four parkland sites. Scans for selective sweeps in Q. robur highlighted regions containing genes with putative involvement in stress tolerance, one of which was moderately differentiated from Q. petraea. Reconstructions of past effective population sizes suggested a long population size decline in both Q. robur and Q. petraea over the Pleistocene, but population growth after the last glacial maximum. We assembled the whole chloroplast genomes of 287 Q. robur, 8 Q. petraea and 14 hybrid trees. In a phylogenetic network, these fell into five major haplotypes, which were shared among species but differed in frequency among parkland sites. We matched our chloroplast genome haplotypes to restriction enzyme fragment haplotypes identified in older studies that had surveyed ancient woodlands in Britain and much of Europe. This suggested that the parkland populations in our study derive from local seed sources.

category: genomics


title: Trp53 ablation fails to prevent microcephaly in mouse pallium with impaired minor intron splicing

abstract: Mutations in minor spliceosome component RNU4ATAC, a small nuclear RNA (snRNA), are linked to primary microcephaly. We have reported that in the conditional knockout (cKO) mice for Rnu11, another minor spliceosome snRNA, minor intron splicing defect in minor intron-containing genes (MIGs) regulating cell cycle resulted in cell cycle defects, with a concomitant increase in {gamma}H2aX+ cells and p53-mediated apoptosis. Trp53 ablation in the Rnu11 cKO mice did not prevent microcephaly. However, RNAseq analysis of the double knockout (dKO) pallium reflected transcriptomic shift towards the control from the Rnu11 cKO. We found elevated minor intron retention and alternative splicing across minor introns in the dKO. Disruption of these MIGs resulted in cell cycle defects that were more severe and detected earlier in the dKO, but with delayed detection of {gamma}H2aX+ DNA damage. Thus, p53 might also play a role in causing DNA damage in the developing pallium. In all, our findings further refine our understanding of the role of the minor spliceosome in cortical development and identify MIGs underpinning microcephaly in minor spliceosome-related diseases.


title: CA1 pyramidal cell diversity is rooted in the time of neurogenesis.

abstract: Cellular diversity supports the computational capacity and flexibility of cortical circuits. Accordingly, principal neurons at the CA1 output node of the hippocampus are increasingly recognized as a heterogeneous population. Their genes, molecular content, intrinsic morpho-physiology, connectivity, and function seem to segregate along the main anatomical axes of the hippocampus. Since these axes reflect the temporal order of principal cell neurogenesis, we directly examined the relationship between birthdate and CA1 pyramidal neuron diversity, focusing on the ventral hippocampus. We used a genetic fate-mapping approach that allowed tagging three groups of agematched principal neurons: pioneer, early- and late-born. Using a combination of neuroanatomy, slice physiology, connectivity tracing and cFos staining, we show that birthdate is a strong predictor of CA1 principal cell diversity. We unravel a subpopulation of pioneer neurons recruited in familiar environments with remarkable positioning, morpho-physiological features, and connectivity. Therefore, despite the expected plasticity of hippocampal circuits, given their role in learning and memory, the diversity of their main components is significantly predetermined at the earliest steps of development.

category: neuroscience


title: Mesenchymal Stromal Cell Aging Impairs the Self-Organizing Capacity of Lung Alveolar Epithelial Stem Cells

abstract: Multicellular organisms maintain structure and function of tissues/organs through emergent, self-organizing behavior. In this report, we demonstrate a critical role for lung mesenchymal stromal cell (L-MSC) aging in determining the capacity to form 3-dimentional organoids or "alveolospheres " with type 2 alveolar epithelial cells (AEC2s). In contrast to L-MSCs from aged mice, young L-MSCs support the efficient formation of alveolospheres when co-cultured with young or aged AEC2s. Aged L-MSCs demonstrated features of cellular senescence, altered bioenergetics, and a senescence-associated secretory profile (SASP). The reactive oxygen species generating enzyme, NADPH oxidase 4 (Nox4), was highly activated in aged L-MSCs and Nox4 downregulation was sufficient to, at least partially, reverse this age-related energy deficit, while restoring the self-organizing capacity of alveolospheres. Together, these data indicate a critical role for cellular bioenergetics and redox homeostasis in an organoid model of self-organization, and supports the concept of thermodynamic entropy in aging biology.


title: Active Enterohepatic Cycling is Not Required for the Choleretic Actions of 24-norUrsodeoxycholic Acid in Mice

abstract: The superior ability of norursodeoxycholic acid (norUDCA) to induce a bicarbonate-rich hypercholeresis has been attributed to its ability to undergo cholehepatic shunting and norUDCA is currently being evaluated as a therapeutic for forms of liver disease. The goal of this study was to use mouse models to investigate contributions of bile acid transporters to the choleretic actions of norUDCA. Here, we show that the apical sodium-dependent bile acid transporter (ASBT) and Organic solute transporter-alpha (OST) are dispensable for norUDCA-stimulation of bile flow and biliary bicarbonate secretion in mice. Analysis of the liver transcriptome revealed that norUDCA induced hepatic expression of a limited number of transporter genes, particularly organic anion transporting polypeptide 1a4 (Oatp1a4). However, norUDCA potently stimulated a bicarbonate-rich hypercholeresis in Oatp1a/1b-deficient mice. Blocking intestinal bile acid reabsorption by co-administration of an ASBT inhibitor or bile acid sequestrant did not impact the ability of norUDCA to induce bile flow in wildtype mice. The results support the concept that these major bile acid transporters are not directly involved in the absorption, cholehepatic shunting, or choleretic actions of norUDCA. Additionally, the findings support further investigation of the therapeutic synergy between norUDCA and ASBT inhibitors or bile acid sequestrants for cholestatic liver disease.


title: 3D Cell Neighbour Dynamics in Growing Pseudostratified Epithelia

abstract: During morphogenesis, epithelial sheets remodel into complex geometries. How cells dynamically organize their contact with neighbouring cells in these tightly packed tissues is poorly understood. We have used light-sheet microscopy of growing mouse embryonic lung explants, three-dimensional cell segmentation, and physical theory to unravel the principles behind 3D cell organization in growing pseudostratified epithelia. We find that cells have highly irregular 3D shapes and exhibit numerous neighbour intercalations along the apical-basal axis as well as over time. Despite the fluidic nature, the cell packing configurations follow fundamental relationships previously described for apical epithelial layers, i.e., Eulers formula, Lewis law, and Aboav-Weaires law, at all times and across the entire tissue thickness. This arrangement minimizes the lateral cell-cell surface energy for a given cross-sectional area variability, generated primarily by the distribution and movement of nuclei. We conclude that the complex 3D cell organization in growing epithelia emerges from simple physical principles.


title: Pharmacological targeting of a PWWP domain demonstrates cooperative control of NSD2 localization

abstract: NSD2 is the primary enzyme responsible for the dimethylation of lysine 36 of histone 3 (H3K36me2), a mark associated with active gene transcription and intergenic DNA methylation. In addition to a methyltransferase domain, NSD2 harbors two PWWP and five PHD domains believed to serve as chromatin reading modules, but their exact function in the regulation of NSD2 activity remains underexplored. Here we report a first-in-class chemical probe targeting the N-terminal PWWP (PWWP1) domain of NSD2. UNC6934 binds potently (Kd of 91 {+/-} 8 nM) to PWWP1, antagonizes its interaction with nucleosomal H3K36me2, and selectively engages endogenous NSD2 in cells. Crystal structures show that UNC6934 occupies the canonical H3K36me2-binding pocket of PWWP1 which is juxtaposed to the DNA-binding surface. In cells, UNC6934 induces accumulation of endogenous NSD2 in the nucleolus, phenocopying the localization defects of NSD2 protein isoforms lacking PWWP1 as a result of translocations prevalent in multiple myeloma. Mutation of other NSD2 chromatin reader domains also increases NSD2 nucleolar localization, and enhances the effect of UNC6934. Finally we identified two C-terminal nucleolar localization sequences in NSD2 that appear to drive nucleolar accumulation when one or more chromatin reader domains are disabled. These data support a model in which NSD2 chromatin engagement is achieved in a cooperative manner and subcellular localization is controlled by multiple competitive structural determinants. This chemical probe and the accompanying negative control, UNC7145, will be useful tools in defining NSD2 biology.


title: A specialized bone marrow microenvironment for fetal haematopoiesis

abstract: Local signals provided by cells in specialized niche microenvironments regulate stem cell behaviour in many different organs and species. In adult mammalian bone marrow (BM), leptin receptor-positive (LepR+) reticular cells express secreted factors that control the function of haematopoietic stem and progenitor cells (HSPCs). During fetal development, the developing skeletal system is colonized by c-Kit+ haematopoietic cells de novo after a transient phase of liver haematopoiesis. The cellular and molecular mechanisms regulating de novo haematopoietic cell colonization and expansion remain largely unknown. Here, we report that fetal and adult BM exhibit fundamental differences both in terms of cellular composition and molecular interactions by single cell RNA sequencing (scRNA-seq) analysis. While LepR+ reticular cells are almost completely absent in fetal femur, arterial endothelial cells (AECs) are a source of signals controlling the initial HSPC expansion during BM development. Long-term haematopoietic stem cells (HSCs) and other c-Kit+ HSPCs are reduced when Wnt ligand secretion by AECs is genetically blocked. We identify Wnt2 as an AEC-derived signal that directly activates {beta}-catenin dependent proliferation of fetal HSPCs. Treatment of HSPCs ex vivo with Wnt2 promotes their proliferation and improves engraftment in vivo after transplantation. Our work reveals a fundamental switch in the cellular organization and molecular regulation of BM niches in the embryonic and adult organism.


title: Substrate mediated elastic coupling between motile cells modulates inter-cell interactions and enhances cell-cell contact

abstract: The mechanical micro-environment of cells and tissues influences key aspects of cell structure and function including cell motility. For proper tissue development, cells need to migrate, interact with other neighbouring cells and form contacts, each of which require the cell to exert physical forces. Cells are known to exert contractile forces on underlying soft substrates. These stresses result in substrate deformation that can affect migratory behavior of cells as well as provide an avenue for cells to sense each other and coordinate their motion. The role of substrate mechanics, particularly its stiffness, in such biological processesis therefore a subject of active investigation. Recent progress in experimental techniques have enabled key insights into pairwise mechanical interactions that control cell motility when they move on compliant soft substrates. Analysis and modeling of such systemsis however still in its nascent stages. Motivated by the role modeling is expected to play in interpreting, informing and guiding experiments, we build a biophysical model for cell migration and cell-cell interactions. Our focus is on situations highly relevant to tissue engineering and regenerative medicine -when substrate traction stresses induced by motile cells enable substrate deformation and serve as a medium of communication. Using a generalizable agent-basedmodel, we compute key metrics of cell motile behavior such as the number of cell-cell contacts over a given time, dispersion of cell trajectories, and probability of permanent cell contact, and analyze how these depend on a cell motility parameter and on substrate stiffness. Our results provide a framework towards modeling the manner in which cells may sense each other mechanically via the substrate and use this information to generate coordinated movements across much longer length scales. Our results also provide a foundation to analyze experiments on the phenomenon known as durotaxis where single cells move preferentially towards regions of high stiffness on patterned substrates.


title: Molecular basis for SPINDOC-Spindlin1 engagement and its role in transcriptional inhibition

abstract: Spindlin1 is a transcriptional coactivator with three Tudor-like domains, of which the first and second Tudors are engaged in histone methylation readout, while the function of the third Tudor is largely unknown. Recent studies revealed that the transcriptional co-activator activity of Spindlin1 could be attenuated by SPIN*DOC. Here we solved the crystal structure of SPIN*DOC-Spindlin1 complex, revealing that a hydrophobic motif, DOCpep3 (256-281), of SPIN*DOC interacts with Tudor 3 of Spindlin1 and completes its {beta}-barrel fold. Massive hydrophobic contacts and hydrogen bonding interactions ensure a high affinity DOCpep3-Spindlin1 engagement with a binding Kd of 30 nM. Interestingly, we characterized two more K/R-rich motifs of SPIN*DOC, DOCpep1 (187-195) and DOCpep2 (228-239), which bind to Spindlin1 at lower affinities with Kd values of 78 M and 31 M, respectively. Structural and binding studies revealed that DOCpep1 and DOCpep2 competitively bind to the aromatic cage of Spindlin1 Tudor 2 that is responsible for H3K4me3 readout. Although DOCpep3-Spindlin1 engagement is compatible with histone readout, an extended SPIN*DOC fragment containing DOCpep1 and DOCpep2 inhibits histone or TCF4 binding by Spindin1 due to introduced competition. This inhibitory effect is more pronounced for weaker binding targets but not for strong ones such as H3 "K4me3-K9me3" bivalent mark. Our RT-qPCR experiment showed that the removal of the hydrophobic motif or the K/R-rich region compromised the inhibitory effects of SPIN*DOC on Spindlin1-mediated transcriptional activation. In sum, here we revealed multivalent engagement between SPIN*DOC and Spindlin1, in which a hydrophobic motif acts as the primary binding site for stable SPIN*DOC-Spindlin1 association, while two more neighboring K/R-rich motifs further modulate the target selectivity of Spindlin1 via competitive inhibition, therefore attenuating the transcriptional co-activator activities of Spindlin1 through affecting its chromatin association.


title: Motor neuron boutons remodel through membrane blebbing

abstract: Wired neurons form new presynaptic boutons in response to increased synaptic activity, but the mechanism by which this occurs remains uncertain. The neuromuscular junction (NMJ) is a synapse formed between motor neurons (MNs) and skeletal muscle fibers and is critical for control of muscle contraction. Because Drosophila MNs have clearly discernible boutons that display robust structural plasticity, it is the ideal system in which to study bouton genesis. Here we show using ex-vivo and by live imaging that in response to depolarization, MNs form new boutons by membrane blebbing, a pressure-driven mechanism used in 3-D migration, but never described as a neuronal remodeling strategy. In accordance, F-actin is decreased during bouton growth (a hallmark of blebs) and we show that non-muscle myosin-II (a master regulator of blebbing) is recruited to newly formed boutons. Furthermore, we discovered that muscle contraction plays a mechanical role in activity-dependent plasticity, promoting bouton addition by increasing MNs confinement. Overall, we provide a novel mechanism by which established circuits create new boutons allowing their structural expansion and plasticity, using trans-synaptic physical forces as the main driving force. Understanding MN-muscle interplay during activity-dependent plasticity can help clarify the mechanisms leading to MN degeneracy observed in neuromuscular diseases.


title: Impact of isolation method on cellular activation and presence of specific tendon cell subpopulations during in vitro culture

abstract:
Tendon injuries are common and heal poorly, due in part to a lack of understanding of fundamental tendon cell biology. A major impediment to the study of tendon cells is the absence of robust, well-characterized in vitro models. Unlike other tissue systems, current tendon cell models do not account for how differences in isolation methodology may affect the activation state of tendon cells or the presence of various tendon cell sub-populations. The objective of this study was to characterize how common isolation methods affect the behavior, fate, and lineage composition of tendon cell cultures. Tendon cells isolated by explant exhibited reduced proliferative capacity, decreased expression of tendon marker genes, and increased expression of genes associated with fibroblast activation compared to digested cells. Consistently, explanted cells also displayed an increased propensity to differentiate to myofibroblasts compared to digested cells. Explanted cultures from multiple different tendons were substantially enriched for the presence of scleraxis-lineage (Scx-lin+) cells compared to digested cultures, while the overall percentage of S100a4-lineage (S100a4-lin+) cells was dependent on both isolation method and tendon of origin. Neither isolation method preserved the ratios of Scx-lin+ or S100a4-lin+ to non-lineage cells seen in tendons in vivo. Combined, these data indicate that further refinement of in vitro cultures models is required in order to more accurately understand the effects of various stimuli on tendon cell behavior.

Statement of clinical significanceThe development of informed in vitro tendon cell models will facilitate enhanced screening of potential therapeutic candidates to improve tendon healing.


title: Computationally reconstructed interactome of Bradyrhizobium diazoefficiens USDA110 reveals novel functional modules and protein hubs for symbiotic nitrogen fixation

abstract: Symbiotic nitrogen fixation is an important part of the nitrogen biogeochemical cycles and the main nitrogen source of the biosphere. As a classical model system for symbiotic nitrogen fixation, rhizobium-legume systems have been studied elaborately for decades. Detailed panorama about the molecular mechanism of the communication and coordination between rhizobia and host plants is becoming clearer. For more systematic insights, there is an increasing demand on new studies integrating multi-omics information. Here we present a comprehensive computational framework, integrating the reconstructed protein interactome of B. diazoefficiens USDA110 with its transcriptome and proteome data, to study the complex protein-protein interaction (PPI) network involved in the symbiosis system. We reconstructed the interactome of B. diazoefficiens USDA110 by computational approaches. Based on the comparison of interactomes between B. diazoefficiens USDA110 and other rhizobia, we inferred that the slow growth of B. diazoefficiens USDA110 may owe to the requirement of more protein modifications and further identified 36 conserved functional PPI modules. Integrated with transcriptome and proteome data, interactomes representing free-living cell and symbiotic nitrogen-fixing (SNF) bacteroid were obtained. Based on the SNF interactome, a core-sub-PPI-network for symbiotic nitrogen fixation was determined and 9 novel functional modules and 11 key protein hubs playing key roles for symbiosis were identified. The reconstructed interactome of B. diazoefficiens USDA110 may serve as a valuable reference for studying the mechanism underlying the SNF system of rhizobia and legumes.

category: systems biology


title: Translational activation by an alternative sigma factor in Bacillus subtilis

abstract: Sigma factors are an important class of bacterial transcription factors that lend specificity to RNA polymerases by binding to distinct promoter elements for genes in their regulons. Here we show that activation of the general stress sigma factor, {sigma}B, in Bacillus subtilis paradoxically leads to dramatic induction of translation for a subset of its regulon genes. These genes are translationally repressed when transcribed by the housekeeping sigma factor, {sigma}A, owing to extended RNA secondary structures as determined in vivo using DMS-MaPseq. Transcription from {sigma}B-dependent promoters liberates the secondary structures and activates translation, leading to dual induction. Translation efficiencies between {sigma}B- and {sigma}A-dependent RNA isoforms can vary by up to 100-fold, which in multiple cases exceeds the magnitude of transcriptional induction. These results highlight the role of long-range RNA folding in modulating translation and demonstrate that a transcription factor can regulate protein synthesis beyond its effects on transcript levels.


title: Lung organoids and microplastic fibers: a new exposure model for emerging contaminants

abstract:
BackgroundThree-dimensional (3D) structured organoids are the most advanced in vitro models for studying human health effects, but they have been applied only once to evaluate the biological effects associated with microplastic exposure. Fibers from synthetic clothes and fabrics are a major source of airborne microplastics, and their release from dryer machines is still poorly understood.

ObjectivesIn this study, we aimed to establish an in vitro organoid model of human lung epithelial cells to evaluate its suitability for studying the effects of airborne microplastic contamination on humans. Furthermore, we aimed to characterize the microplastic fibers (MPFs) released in the exhaust filter of a household dryer and to test their interactions and inflammatory effects on the established lung organoids.

MethodsThe polyester fibers emitted from the drying of synthetic fabrics were collected. Morphological characterization of the fibers released into the air filter was performed by optical microscopy and scanning electron microscopy (SEM)/energy dispersive x-ray spectroscopy (EDS). The organoids were exposed to various MPF concentrations (1, 10, and 50 mg L-1) and analyzed by optical microscopy, SEM, and confocal microscopy. Gene expression analysis of lung-specific genes, inflammatory cytokines, and oxidative stress-related genes was achieved by quantitative reverse transcription-polymerase chain reaction (qRT-PCR).

ResultsWe successfully cultured organoids with lung-specific genes. The presence of MPFs did not inhibit organoid growth, but polarized cell growth was observed along the fibers. Moreover, the MPFs did not cause inflammation or oxidative stress. Interestingly, the MPFs were coated with a cellular layer, resulting in the inclusion of fibers in the organoid.

DiscussionThis work could have potential long-term implications regarding lung epithelial cells undergoing repair. This preliminary exposure study using human lung organoids could form the basis for further research regarding the toxicological assessment of emerging contaminants such as micro- or nanoplastics.


title: Novel regulators of growth identified in the evolution of fin proportion in flying fish

abstract: Identifying the genetic foundations of trait variation and evolution is challenging as it is often difficult to parse meaningful signals from confounding signatures such as drift and epistasis. However, identification of the genetic loci underlying morphological and physiological traits can be honed through the use of comparative and complementary genetic approaches, whereby shared sets of genes that are repeatedly implicated across large evolutionary time periods as under selection can illuminate important pathways and epistatic relationships that function as novel regulators of trait development. Here we intersect comparative genomic analyses with unbiased mutagenesis screens in distantly related species to define the control of proportional growth, as changes in the size and relative proportions of tissues underlie a large degree of the variant forms seen in nature. Through a phylogenomic analysis of genome-wide variation in 35 species of flying fishes and relatives, we identify genetic signatures in both coding and regulatory regions underlying the convergent evolution of increased paired fin size and aerial gliding behaviors, key innovations for flying fishes and flying halfbeaks. To refine our analysis, we intersected convergent phylogenomic signatures with mutants identified in distantly related zebrafish with altered fin size. Through these paired approaches, we identify a surprising role for an L-type amino acid transporter, lat4a, and the potassium channel, kcnh2a, in the regulation of fin proportion. We show that specific epistatic interaction between these genetic loci in zebrafish closely phenocopies the observed fin proportions of flying fishes. The congruence of experimental and phylogenomic findings point to a conserved, non-canonical signaling interaction that integrates bioelectric cues and amino acid transport in the establishment of relative size in development and evolution.


title: Proteogenomic characterization of hepatocellular carcinoma

abstract: We performed a proteogenomic analysis of hepatocellular carcinomas (HCCs) across clinical stages and etiologies. We identified pathways differentially regulated on the genomic, transcriptomic, proteomic and phosphoproteomic levels. These pathways are involved in the organization of cellular components, cell cycle control, signaling pathways, transcriptional and translational control and metabolism. Analyses of CNA-mRNA and mRNA-protein correlations identified candidate driver genes involved in epithelial-to-mesenchymal transition, the Wnt-{beta}-catenin pathway, transcriptional control, cholesterol biosynthesis and sphingolipid metabolism. The activity of targetable kinases aurora kinase A and CDKs was upregulated. We found that CTNNB1 mutations are associated with altered phosphorylation of proteins involved in actin filament organization, whereas TP53 mutations are associated with elevated CDK1/2/5 activity and altered phosphorylation of proteins involved in lipid and mRNA metabolism. Integrative clustering identified HCC subgroups with distinct regulation of biological processes, metabolic reprogramming and kinase activation. Our analysis provides insights into the molecular processes underlying HCCs.


title: Choice of alignment pipeline strongly influences clustering quality of scRNA-seq datasets

abstract:
BackgroundSingle-cell RNA sequencing (scRNA-seq) has quickly become one of the most dominant techniques in modern transcriptome assessment. In particular, 10X Genomics Chromium system, with its high throughput approach, turn key and thorough user guide made this cutting-edge technique accessible to many laboratories using diverse animal models. However, standard pre-processing, including the alignment and cell filtering pipelines might not be ideal for every organism or tissue. Here we applied an alternative strategy, based on the pseudoaligner kallisto, on twenty-two publicly available single cell sequencing datasets from a wide range of tissues of eight organisms and compared the results with the standard 10X Genomics Cell Ranger pipeline.

ResultsIn most of the tested samples, kallisto produced higher sequencing read alignment rates and total gene detection rates in comparison to Cell Ranger. Although datasets processed with Cell Ranger had higher cell counts, outside of human and mouse datasets, these additional cells were routinely of low quality, containing low gene detection rates. Thorough downstream analysis of one kallisto processed dataset, obtained from the zebrafish pineal gland, revealed clearer clustering, allowing the identification of an additional photoreceptor cell type that previously went undetected. The finding of the new cluster suggests that the photoreceptive pineal gland is essentially a bi-chromatic tissue containing both green and red cone-like photoreceptors and implies that the alignment and pre-processing pipeline can affect the discovery of biologically-relevant cell types.

ConclusionWhile Cell Ranger favors higher cell numbers, using kallisto results in datasets with higher median gene detection per cell. We could demonstrate that cell type identification was not hampered by the lower cell count, but in fact improved as a result of the high gene detection rate and the more stringent filtering. Depending on the acquired dataset, it can be beneficial to favor high quality cells and accept a lower cell count, leading to an improved classification of cell types.


title: Choice of pre-processing pipeline influences clustering quality of scRNA-seq datasets


title: αB-crystallin affects the morphology of Aβ(1-40) aggregates

abstract:
B-crystallin (ABC) is a human small heat shock protein that is strongly linked to Alzheimers disease (AD). In vitro, it can inhibit the aggregation and amyloid formation of a range of proteins including A{beta}(1-40), a primary component of AD amyloid plaques. Despite the strong links, the mechanism by which ABC inhibits amyloid formation has remained elusive, in part due to the notorious irreproducibility of aggregation assays involving preparations of A{beta}-peptides of native sequence. Here, we present a recombinant expression protocol to produce native A{beta}(1-40), devoid of any modifications or exogenous residues, with yields up to 4 mg/L E. coli. This material provides highly reproducible aggregation kinetics and, by varying the solution conditions, we obtain either highly ordered amyloid fibrils or more disordered aggregates. Addition of ABC slows the aggregation of A{beta}(1-40), and interferes specifically with the formation of ordered amyloid fibrils, favouring instead the more disordered aggregates. Solution-state NMR spectroscopy reveals that the interaction of ABC with A{beta}(1-40) depends on the specific aggregate morphology. These results provide mechanistic insight into how ABC inhibits the formation of amyloid fibrils.

HighlightsO_LIProtocol for production of native recombinant A{beta}(1-40)
C_LIO_LIAmyloid formation under physiological conditions is highly reproducible
C_LIO_LIBoth ordered fibrils and disordered aggregates can be reliably formed
C_LIO_LIB-crystallin specifically inhibits amyloid fibril assembling, favouring disordered aggregates
C_LI

eTOC blurbMuller et al. introduce a protocol for the highly reproducible production of amyloid from native A{beta}(1-40) and determine that the human chaperone ABC specifically destabilises them in favour of disordered aggregates. NMR shows that ABC can distinguish between aggregate morphologies.

Graphical Abstract

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title: A Patient-Specific Model of Transcatheter Valve Replacement in aBicuspid Heart Valve

abstract: Bicuspid Aortic Valves (BAVs) are a common congenital heart disease where two cusps of the aortic heart valve become fused together, this leads to two unequally sized leaflets compared to the normal trileaflet valve. Transcatheter Aortic Valves are currently used in off-label treatmet of stenosed BAVs, however, due to the abnormal valve anatomy, debate surrounds the sizing of transcatheter valves. In this study, finite element models were developed to simulate the deployment of two different valves sizes (a 25 mm and a 27 mm) of the Lotus valve into the patient-specific aortic root geometry of a clinical stenosed BAV case. These models were used to investigate and compare the eccentricity, stress and mal-apposition of the two valve sizes. The results demonstrated that the 25 mm valve was the most suitable in terms of eccentricity and stress reduction. It was also shown that the smaller 25 mm valve size did not increase the likelihood of mal-apposition. As the 25 mm valve was deemed suitable based on current sizing algorithms, on the basis of these results traditional annulus measurement and device sizing may be suitable in the case of the Lotus valve.

category: bioengineering


title: Hepatic Leukemia Factor supports the propagation of leukemia and hematopoietic stem cell function during stress-induced regeneration.

abstract:
The processes regulating hematopoietic stem cells (HSC) during aging are not fully understood1, but it is clear that the incidence of hematological malignancies increases with age, highlighting the importance of unravelling the cellular and molecular networks involved. Recently, we identified Hepatic Leukemia Factor (HLF) as an essential transcription factor in maintaining the HSC pool during regeneration2 and showed that failure to downregulate HLF leads to disrupted differentiation3.

Here, we found that HLF is dispensable for hematopoiesis during systemic aging, but needed during stress-induced hematopoietic recovery of aged HSC after transplantation. Additionally, HLF was dispensable for leukemic initiation but required for disease propagation. Taken together, our findings demonstrate the existence of a HLF-dependent mechanism that uncouples stress-induced regeneration from hematopoietic homeostasis during aging, that can be used by malignant cells to gain stem cell properties to propagate the disease.

Key pointsO_LIHLF is dispensable for HSC function and hematopoietic homeostasis during physiological aging, but crucial during stress induced regeneration.
C_LIO_LIHLF supports the propagation of leukemia-initiating cells
C_LI


title: Intra-epithelial non-canonical Activin A signalling safeguards prostate progenitor quiescence

abstract:
The healthy prostate is a relatively quiescent tissue. Yet, prostate epithelium overgrowth is a common condition during ageing, associated with urinary dysfunction and tumorigenesis. For over thirty years, TGF-{beta} ligands have been known to induce cytostasis in a large variety of epithelia, but the intracellular pathway mediating this signal in the prostate, as well as its relevance for quiescence, have remained elusive.

Here, using mouse prostate organoids to model epithelial progenitors, we found that intra-epithelial non-canonical Activin A signalling inhibited cell proliferation in a Smad-independent manner. Mechanistically, Activin A triggered Tak1 and p38 MAPK activity, leading to p16 and p21 nuclear import. Spontaneous evasion from this quiescent state occurred upon prolonged culture, due to reduced Activin A secretion, a condition associated with DNA replication stress and aneuploidy. Organoids capable to escape quiescence in vitro were also able to implant with increased frequency into immunocompetent mice.

Our study demonstrates that non-canonical Activin A signalling safeguards epithelial quiescence in the healthy prostate, with potential implications for the understanding of cancer initiation, and the development of therapies targeting quiescent tumour progenitors.


title: Procollagen C-Proteinase Enhancer 1 (PCPE-1) is a marker of myocardial fibrosis and impaired cardiac function in a murine model of pressure overload

abstract:
(1) AimsProcollagen C-proteinase enhancer 1 (PCPE-1) is an extracellular matrix protein and a major regulator of fibrillar collagen biosynthesis. Previous work has shown that its abundance is often increased in the context of tissue repair and fibrosis. The present study was designed to evaluate its potential as a biomarker of myocardial interstitial fibrosis (MIF), a well-established pathogenic pathway leading to heart failure.

(2) Methods and ResultsCardiac fibrosis was induced in rats using an optimized model of chronic pressure overload triggered by angiotensin II and N{omega}-nitro-L-arginine methyl ester (L-NAME). All treated animals suffered from heart hypertrophy and the increase in heart collagen volume fraction (CVF), evidenced by histology and 68Ga-Collagelin uptake, confirmed the development of cardiac fibrosis. Functional analysis by simultaneous PET-MRI further showed that our model closely reflected the pathological features seen in human MIF, including left ventricle thickening and diastolic dysfunction associated with decreased ejection fraction. PCPE-1 mRNA and protein levels were augmented by factors of 3.4 and 6.1 respectively in the heart tissue of treated rats. Moreover, protein abundance was well-correlated with CVF (r=0.92, p<0.0001) and PCPE-1 immuno-detection mainly localized the protein to fibrotic areas. Finally, PCPE-1 plasma levels measured by ELISA were increased in fibrotic rats compared to controls.

(3) ConclusionTogether, our findings demonstrate that PCPE-1 levels in the heart and circulation tightly reflect the cardiac fibrosis status and heart function impairment in rats and suggest that it could be a very useful marker to monitor human heart diseases leading to fibrosis.


title: BoolSim, a Graphical Interface for Open Access Boolean Network Simulations and Use in Guard Cell CO2 Signaling

abstract:
Signaling networks are at the heart of almost all biological processes. Most of these networks contain a large number of components and often the connections between these components are either not known, or the rate equations that govern the dynamics of soluble signaling components are not quantified. This uncertainty in network topology and parameters can make it challenging to formulate detailed mathematical models. Boolean networks, in which all components are either on or off, have emerged as viable alternatives to more detailed mathematical models but can be difficult to implement. Therefore, open source format of such models for community use is desirable. Here we present BoolSim, a freely available graphical user interface (GUI) that allows users to easily construct and analyze Boolean networks. BoolSim can be applied to any Boolean network. We demonstrate BoolSims application using a previously published network for abscisic acid-driven stomatal closure in Arabidopsis. We also show how BoolSim can be used to generate testable predictions by extending the network to include CO2 regulation of stomatal movements. Predictions of the model were experimentally tested and the model was iteratively modified based on experiments showing that ABA closes stomata even at near zero CO2 concentrations (1.5 ppm CO2).

One Sentence SummaryThis study presents an open-source, graphical interface for the simulation of Boolean networks and applies it to an abscisic acid signaling network in guard cells, extended to include input from CO2.

category: plant biology


title: Neurodegeneration in human brain organoids infected with herpes simplex virus type 1

abstract: Herpes simplex virus type 1 (HSV-1) infection of the nervous system may lead to brain damage, including neurodegeneration. However, lack of suitable experimental models hinders understanding molecular mechanisms and cell-type-specific responses triggered by HSV-1. Here, we infected human brain organoids with HSV-1. Known features of HSV-1 infection such as alteration of neuronal electrophysiology and induction of antisense transcription were confirmed. Full-length mRNA-sequencing revealed aberrant 3 end formation and poly(A)-tail lengthening. Single-cell RNA-seq and spatial transcriptomics uncovered changes in the cellular composition of the infected organoids caused by viral replication and dysregulation of molecular pathways in cell-type specific manner. Furthermore, hallmarks of early neurodegeneration were observed, namely extracellular matrix disruption, STMN2 and TARDBP/TDP43 downregulation, and upregulation of the AD-related non-coding RNA BC200/BCYRN1. These hallmarks were weaker/absent when infecting with a mutant HSV-1 control. Together, our data indicate that brain organoids serve as a powerful model to study mechanisms of HSV-1-driven neurodegeneration.


title: Revealing Spatiotemporal Circuit Information of Olfactory Bulb in Large-scale Neural Recordings

abstract: Large-scale multi-site biosensors are essential to probe the olfactory bulb (OB) circuitry for understanding the spatiotemporal dynamics of simultaneous discharge patterns. Current ex-vivo electrophysiological techniques are limited to recording a small set of neurons and cannot provide an inadequate resolution, which hinders revealing the fast dynamic underlying the information coding mechanisms in the OB circuit. Here, we demonstrate a novel biohybrid OB-CMOS platform to decipher the cross-scale dynamics of OB electrogenesis and quantify the distinct neuronal coding properties. The approach with 4096-microelectrodes offers a non-invasive, label-free, bioelectrical imaging to decode simultaneous firing patterns from thousands of connected neuronal ensembles in acute OB slices. The platform can measure spontaneous and drug-induced extracellular field potential activity. We employ our OB-CMOS recordings to perform multidimensional analysis to instantiate specific neurophysiological metrics underlying the olfactory spatiotemporal coding that emerged from the OB interconnected layers. Our results delineate the computational implications of large-scale activity patterns in functional olfactory processing. The high-content characterization of the olfactory circuit could benefit better functional interrogations of the olfactory spatiotemporal coding, connectivity mapping, and, further, the designing of reliable and advanced olfactory cell-based biosensors for diagnostic biomarkers and drug discovery.


title: Inhibition of peripheral VEGF signaling rapidly reduces leucocyte obstructions in brain capillaries and increases cortical blood flow in an Alzheimers disease mouse model

abstract: Increased incidence of stalled capillary blood flow caused by adhesion of leucocytes to the brain microvascular endothelium leads to a 17% reduction of cerebral blood flow (CBF) and exacerbates short-term memory loss in multiple mouse models of Alzheimers disease (AD). Here, we report that Vascular Endothelial Growth Factor (VEGF) signaling at the luminal side of the brain microvasculature plays an integral role in the capillary stalling phenomenon of the APP/PS1 mouse model. Administration of the anti-mouse VEGF-A164 antibody, an isoform that inhibits blood brain barrier (BBB) hyperpermeability, reduced the number of stalled capillaries within an hour of injection, leading to an immediate increase in average capillary blood flow but not capillary diameter. VEGF-A inhibition also reduced the overall eNOS protein concentrations, increased occludin levels, and decreased the penetration of circulating Evans Blue dye across the BBB into the brain parenchyma, suggesting increased BBB integrity. Capillaries prone to neutrophil adhesion after anti-VEGF-A treatment also had lower occludin concentrations than flowing capillaries. Taken together, our findings demonstrate that VEGF-A signaling in APP/PS1 mice contributes to aberrant eNOS/occludin- associated BBB permeability, increases the incidence of capillary stalls, and leads to reductions in CBF. Reducing leucocyte adhesion by inhibiting luminal VEGF signaling may provide a novel and well-tolerated strategy for improving brain microvascular blood flow in AD patients.


title: Gait-phase modulates alpha and beta oscillations in the pedunculopontine nucleus

abstract:
BackgroudThe pedunculopontine nucleus (PPN) is a reticular collection of neurons at the junction of the midbrain and pons, playing an important role in modulating posture and locomotion. Deep brain stimulation of the PPN has been proposed as an emerging treatment for patients with Parkinsons disease (PD) or multiple system atrophy (MSA) suffering gait-related atypical parkinsonian syndromes.

ObjectiveIn this study, we investigated PPN activities during gait to better understand its functional role in locomotion. Specifically, we investigated whether PPN activity is rhythmically modulated during locomotion.

MethodsPPN local field potential (LFP) activities were recorded from PD or MSA patients suffering from gait difficulties during stepping in place or free walking. Simultaneous measurements from force plates or accelerometers were used to determine the phase within each gait cycle at each time point.

ResultsOur results showed that activities in the alpha and beta frequency bands in the PPN LFPs were rhythmically modulated by the gait phase within gait cycles, with a higher modulation index when the stepping rhythm was more regular. Meanwhile, the PPN-cortical coherence was most prominent in the alpha band. Both gait-phase related modulation in the alpha/beta power and the PPN-cortical coherence in the alpha frequency band were spatially specific to the PPN and did not extend to surrounding regions.

ConclusionsThese results raise the possibility that alternating PPN stimulation in tandem with the gait rhythm may be more beneficial for gait control than continuous stimulation, although this remains to be established in future studies.


title: Differential impact of transdiagnostic, dimensional psychopathology on multiple scales of functional connectivity

abstract:
IntroductionDimensional psychopathology strives to associate different domains of cognitive dysfunction with brain circuitry. Connectivity patterns as measured by functional magnetic resonance imaging (fMRI) exist at multiple scales, with global networks of connectivity composed of microscale interactions between individual nodes. It remains unclear how separate dimensions of psychopathology might differentially impact these different scales of organization.

MethodsPatients experiencing anxious misery symptomology (depression, anxiety and trauma; n = 192) were assessed for symptomology and received resting-state fMRI scans. Three modeling approaches (seed-based correlation analysis [SCA], support vector regression [SVR] and Brain Basis Set Modeling [BSS]), each relying on increasingly dense representations of functional connectivity patterns, were used to associate connectivity patterns with six different dimensions of psychopathology: anxiety sensitivity, anxious arousal, rumination, anhedonia, insomnia and negative affect. Importantly, a full 50 patients were held-out in a testing dataset, leaving 142 patients as training data.

ResultsDifferent symptom dimensions were best modeled by different scales of brain connectivity: anhedonia and anxiety sensitivity were best modeled with single connections (SCA), insomnia and anxious arousal by mesoscale patterns (SVR) and negative affect and ruminative thought by broad, cortex-spanning patterns (BBS). Dysfunction within the default mode network was implicated in all symptom dimensions that were best modeled by multivariate models.

ConclusionThese results suggest that symptom dimensions differ in the degree to which they impact different scales of brain organization. In addition to advancing our basic understanding of transdiagnostic psychopathology, this has implications for the translation of basic research paradigms to human disorders.


title: Pathogenic tau disrupts the cellular program that maintains neuronal identity

abstract: Neurons in human Alzheimers disease acquire phenotypes that are also present in various cancers, including over-stabilization of the cytoskeleton, nuclear pleomorphism, decondensation of constitutive heterochromatin, and aberrant activation of the cell cycle. Unlike in cancer, in which cell cycle activation drives tumor formation, activation of the cell cycle in post-mitotic neurons is sufficient to induce neuronal death. Multiple lines of evidence suggest that abortive cell cycle activation is a consequence of pathogenic forms of tau, a protein that drives neurodegeneration in Alzheimers disease and related "tauopathies." We have combined network analysis of human Alzheimers disease and mouse tauopathy with mechanistic studies in Drosophila to discover that pathogenic forms of tau drive abortive cell cycle activation by disrupting the cellular program that maintains neuronal identity. Mechanistically, we identify Moesin, a prognostic biomarker for cancer and mediator of the epithelial-mesenchymal transition (EMT), as a major effector of tau-induced neurotoxicity. We find that aberrant activation of Moesin in neurons acts through the actin cytoskeleton to dysregulate the cellular program that maintains neuronal identity. Our study identifies mechanistic parallels between tauopathy and cancer and sets the stage for novel therapeutic approaches.