title: Chronic clinical signs of upper respiratory tract disease shape gut and respiratory microbiomes in cohabitating domestic felines.

abstract: Feline  upper  respiratory  tract  disease (FURTD), often caused by infections etiologies, is a multifactorial syndrome affecting feline populations worldwide. Because of its highly transmissible nature, infectious FURTD is most prevalent anywhere cats are housed in groups such as animal shelters, and is associated with negative consequences such as decreasing adoption rates, intensifying care costs, and increasing euthanasia rates. Understanding the etiology and pathophysiology of FURTD is thus essential to best mitigate the negative consequences of this disease. Clinical signs of FURTD include acute respiratory disease, with a small fraction of cats developing chronic sequelae. It is thought that nasal mucosal microbiome changes play an active role in the development of acute clinical signs, but it remains unknown if the microbiome may play a role in the development and progression of chronic clinical disease. To address the knowledge gap surrounding how microbiomes link to chronic FURTD, we asked if microbial community structure of upper respiratory and gut microbiomes differed between cats with chronic FURTD signs and clinically normal cats. We selected 8 households with at least one cat exhibiting chronic clinical FURTD, and simultaneously collected samples from cohabitating clinically normal cats. Microbial community structure was assessed via 16S rDNA sequencing of both gut and nasal microbiome communities. Using a previously described ecophylogenetic method, we identified 37 and 27 microbial lineages within gut and nasal microbiomes respectively that significantly associated with presence of active FURTD clinical signs in cats with a history of chronic signs. Overall, we find that nasal and gut microbial communities may contribute to the development of chronic clinical course, but more research is needed to confirm our observations.


title: The Drosophila ZAD zinc finger protein Kipferl guides Rhino to piRNA clusters

abstract: RNA interference systems depend on the synthesis of small RNA precursors whose sequences define the target spectrum of these silencing pathways. The Drosophila Heterochromatin Protein 1 (HP1) variant Rhino permits transcription of PIWI-interacting RNA (piRNA) precursors within transposon-rich heterochromatic loci in germline cells. Current models propose that Rhino's specific chromatin occupancy at piRNA source loci is determined by histone marks and maternally inherited piRNAs, but also imply the existence of other, undiscovered specificity cues. Here, we identify a member of the diverse family of zinc finger associated domain (ZAD)-C2H2 proteins, Kipferl, as critical Rhino cofactor in ovaries. By binding to guanosine-rich DNA motifs and interacting with the Rhino chromodomain, Kipferl recruits Rhino to specific loci and stabilizes it on chromatin. In kipferl mutant flies, Rhino is lost from most of its target chromatin loci and instead accumulates on pericentromeric satellite arrays, resulting in decreased levels of transposon targeting piRNAs and impaired fertility. Our findings reveal that DNA sequence, in addition to the H3K9me3 mark, determines the identity of piRNA source loci and provide insight into how Rhino might be caught in the crossfire of genetic conflicts.

category: genetics


title: Real-time single-molecule observation of chaperone-assisted protein folding

abstract: The ability of Hsp70 molecular chaperones to remodel the conformation of their clients is central to their biological function; however, questions remain regarding the precise molecular mechanisms by which Hsp70 machinery interacts with the client and how this contributes towards efficient protein folding. Here, we used Total Internal Reflection Fluorescence (TIRF) microscopy and single-molecule Fluorescence Resonance Energy Transfer (smFRET) to temporally observe the conformational changes that occur to individual firefly luciferase (Fluc) proteins as they are folded by the bacterial Hsp70 system. For the first time, we observed multiple cycles of chaperone binding-and-release to an individual client during refolding and that high rates of chaperone cycling improves refolding yield. Furthermore, we demonstrate that DnaJ remodels misfolded proteins via a conformational selection mechanism whereas DnaK resolves misfolded states via mechanical unfolding. This study illustrates that the temporal observation of chaperone-assisted folding enables the elucidation of key mechanistic details inaccessible using other approaches.

category: biochemistry


title: Design of an ultrafast pulsed ponderomotive phase plate for cryo-electron tomography

abstract: Ponderomotive phase plates have shown temporally consistent phase contrast is possible within electron microscopes via high fluence static laser modes resonating in Fabry-Perot cavities. Here, we explore using pulsed laser beams as an alternative method of generating high fluences. We find through forward-stepping finite element models that picosecond-or-less interactions are required for meaningful fluences phase shifts, with higher pulse energies and smaller beam waists leading to the predicted higher fluences. An additional model based on quasiclassical assumptions is used to discover the shape of the phase plate by incorporating the oscillatory nature of the electric field. From these results, we find the transient nature of the laser pulses removes the influence of Kapitza-Dirac diffraction patterns that appear in the static resonator cases. The addition of a second laser aligned 90 degrees to the first induces anisotropy to the shape of the phase plate. By incorporating a shifting-electron-beam algorithm, the effects of a finite electron beam crossover are also simulated. A total pulse energy of 8.7 microJoules is enough to induce the required pi/2 phase shift for Zernike-like phase microscopy. As a brief thought experiment, we also explore the usage of high frequency lasers in a standard electron emission scheme to see if a pulsed electron beam is even necessary. Ultimately, frequency requirements limit the laser to nanosecond pulse durations, causing the required pulse energies to reach unreasonable levels before adequate phase shifts are achieved.

category: biophysics


title: Deep Reinforcement Learning for Optimal Experimental Design in Biology

abstract: The field of optimal experimental design uses mathematical techniques to determine experiments that are maximally informative from a given experimental setup. Here we apply a technique from artificial intelligence---reinforcement learning---to the optimal experimental design task of maximizing confidence in estimates of model parameter values. We show that a reinforcement learning approach performs favourably in comparison with a one-step ahead optimisation algorithm and a model predictive controller for the inference of bacterial growth parameters in a simulated chemostat. Further, we demonstrate the ability of reinforcement learning to train over a distribution of parameters, indicating that this approach is robust to parametric uncertainty.

category: synthetic biology


title: Persistent serum protein signatures define an inflammatory subset of long COVID

abstract: Long COVID or post-acute sequelae of SARS-CoV-2 (PASC) is a clinical syndrome featuring diverse symptoms that can persist for months after acute SARS-CoV-2 infection. The etiologies are unknown but may include persistent inflammation, unresolved tissue damage, or delayed clearance of viral protein or RNA. Attempts to classify subsets of PASC by symptoms alone have been unsuccessful. To molecularly define PASC, we evaluated the serum proteome in longitudinal samples from 55 PASC individuals with symptoms lasting [≥]60 days after onset of acute infection and compared this to symptomatically recovered SARS-CoV-2 infected and uninfected individuals. We identified subsets of PASC with distinct signatures of persistent inflammation. Type II interferon signaling and canonical NF-{kappa}B signaling (particularly associated with TNF), were the most differentially enriched pathways. These findings help to resolve the heterogeneity of PASC, identify patients with molecular evidence of persistent inflammation, and highlight dominant pathways that may have diagnostic or therapeutic relevance.

category: immunology


title: SARS-CoV-2 Spike N-Terminal Domain modulates TMPRSS2-dependent viral entry and fusogenicity

abstract: Over 20 mutations have been identified in the N-Terminal Domain (NTD) of SARS-CoV-2 spike and yet few of them are fully characterised. Here we first examined the contribution of the NTD to infection and cell-cell fusion by constructing different VOC-based chimeric spikes bearing B.1617 lineage (Delta and Kappa variants) NTDs and generating spike pseudotyped lentivirus (PV). We found the Delta NTD on a Kappa or WT background increased spike S1/S2 cleavage efficiency and virus entry, specifically in Calu-3 lung cells and airway organoids, through use of TMPRSS2. Delta was previously shown to have fast cell-cell fusion kinetics and increased fusogenicity that could be conferred to WT and Kappa variant spikes by transfer of the Delta NTD. Moving to contemporary variants, we found that BA.2 had higher entry efficiency in a range of cell types as compared to BA.1. BA.2 showed higher fusogenic activity than BA.1, but the BA.2 NTD could not confer higher fusion to BA.1 spike. There was low efficiency of TMPRSS2 usage by both BA.1 and BA.2, and chimeras of Omicron BA.1 and BA.2 spikes with a Delta NTD did not result in more efficient use of TMRPSS2 or cell-cell fusogenicity. We conclude that the NTD allosterically modulates S1/S2 cleavage and spike-mediated functions such as entry and cell-cell fusion in a spike context dependent manner, and allosteric interactions may be lost when combining regions from more distantly related spike proteins. These data may explain the lack of dominant SARS-CoV-2 inter-variant recombinants bearing breakpoints within spike.

category: microbiology


title: Soil nitrogen impacts the rhizosphere microbial community owing to microbial hitchhiking

abstract: Microbial hitchhiking demonstrates that some nonmotile microbes utilize trans-species motility to traverse their environment; however, whether driving forces, such as plants and nitrogen, affect microbial hitchhiking is not clear. In our study, we explored the effects of plants and nitrogen fertilizer on Bacillus- hitchhiking by setting filter membranes and different nitrogen fertilizer concentration gradients. In the experimental treatment, we added a filter membrane to the soil to prevent hitchhiking. In the absence of plants, nitrogen alone had little influence on motile bacteria and hitchhiking. However, Bacillus contents were significantly impacted by the nitrogen concentration when the plants were rooted, leading to a great variation in cell motility function according to the functional analysis in the soil microbial community. After applying the filter membrane, there were no significant differences in Bacillus contents, microbial community structure or cell motility functional abundance, which illustrated that hitchhiking impacted the microbial community. Our analysis of co-occurrence between bulk soil motile bacteria (Bacillus) and rhizosphere bacteria also confirmed this. The correlation between bulk soil motile bacteria and the rhizosphere microbial community was strong in the groups with suitable nitrogen concentrations without filter membranes and was weak at all nitrogen levels in the no-membrane treatments. Thus, we concluded that plants and different nitrogen doses synergistically altered the soil microbiome by hitchhiking, whose effect depends on nitrogen.


title: A marine probiotic treatment against the bacterial pathogen Vibrio coralliilyticus to improve the performance of Pacific (Crassostrea gigas) and Kumamoto (C. sikamea) oyster larvae

abstract: Oyster larvae reared in hatcheries on the U.S. West coast often experience severe Vibrio coralliilyticus-related mortalities early in their development. Current treatment options for these molluscs are not available or feasible; however, for decades, probiotics have been successfully used in finfish and crustacean shellfish culture. Consequently, the objectives of this work were to 1) isolate marine bacteria from oysters and evaluate their protective activity against Vibrio coralliilyticus infection of Pacific oyster (Crassostrea gigas) larvae, and 2) to determine the long-term effects of probiotic additions on growth and metamorphosis of larval Pacific and Kumamoto oysters (C. sikamea). A combination of three probiotic strains applied once 24 hours post-fertilization was more effective in improving survival of larval C. gigas exposed to lethal concentrations of V. coralliilyticus strain RE22, compared with separate additions of individual probiotics. In addition, a single application of the probiotic combination to one-day-old larvae increased the larval metamorphosis success of C. sikamea and both the Midori and Myiagi stocks of C. gigas. These results suggest that probiotics are effective at preventing disease and can significantly improve performance of oyster larvae, using a single application early in their development.


title: SIRT3 deficiency decreases oxidative-metabolism capacity but increases lifespan under caloric restriction

abstract: Mitochondrial NAD+-dependent protein deacetylase Sirtuin3 (SIRT3) has been proposed to mediate calorie restriction (CR)-dependent metabolic regulation and lifespan extension. Here, we investigated the role of SIRT3 in CR-mediated longevity, mitochondrial function, and aerobic fitness. We report that SIRT3 is required for whole-body aerobic capacity but is dispensable for CR-dependent lifespan extension. Under CR, loss of SIRT3 (Sirt3-/-) yielded a longer overall and maximum lifespan as compared to Sirt3+/+ mice. This unexpected lifespan extension was associated with altered mitochondrial protein acetylation in oxidative metabolic pathways, reduced mitochondrial respiration, and reduced aerobic exercise capacity. Also, Sirt3-/- CR mice exhibit lower physical activity and favor fatty acid oxidation during the postprandial period, leading to a pseudo-fasting condition that extends the fasting period. This study shows the uncoupling of lifespan and healthspan parameters (aerobic fitness and spontaneous activity) and provides new insights into SIRT3 function in CR adaptation, fuel utilization, and aging.

category: molecular biology


title: Developmentally regulated alternate 3' end cleavage of nascent transcripts controls dynamic changes in protein expression in an adult stem cell lineage

abstract: Alternative polyadenylation (APA) generates transcript isoforms that differ in the position of the 3' cleavage site, resulting in the production of mRNA isoforms with different length 3'UTRs. Although widespread, the role of APA in the biology of cells, tissues and organisms has been controversial. We identified over 500 Drosophila genes that express mRNA isoforms with a long 3'UTR in proliferating spermatogonia but a short 3'UTR in differentiating spermatocytes due to APA. We show that the stage- specific choice of the 3' end cleavage site can be regulated by the arrangement of a canonical polyadenylation signal (PAS) near the distal cleavage site but a variant or no recognizable PAS near the proximal cleavage site. The emergence of transcripts with shorter 3'UTRs in differentiating cells correlated with changes in expression of the encoded proteins, either from off in spermatogonia to on in spermatocytes or vice versa. Polysome gradient fractionation revealed over 250 genes where the long 3'UTR versus short 3'UTR mRNA isoforms migrated differently, consistent with dramatic stage-specific changes in translation state. Thus, the developmentally regulated choice of an alternative site at which to make the 3'end cut that terminates nascent transcripts can profoundly affect the suite of proteins expressed as cells advance through sequential steps in a differentiation lineage.

category: developmental biology


title: Opportunistic binding of EcR to open chromatin drives tissue-specific developmental responses

abstract: Steroid hormones perform diverse biological functions in developing and adult animals. However, the mechanistic basis for their tissue specificity remains unclear. In Drosophila, the ecdysone steroid hormone is essential for coordinating developmental timing across physically separated tissues. Ecdysone directly impacts genome function through its nuclear receptor, a heterodimer of the EcR and Usp proteins. Ligand binding to EcR triggers a transcriptional cascade, including activation of a set of primary response transcription factors. The hierarchical organization of this pathway has left the direct role of EcR in mediating ecdysone responses obscured. Here, we investigate the role of EcR in controlling tissue-specific ecdysone responses, focusing on two tissues that diverge in their response to rising ecdysone titers: the larval salivary gland, which undergoes programmed destruction, and the wing imaginal disc, which initiates metamorphosis. We find that EcR functions bimodally, with both gene repressive and activating functions, even at the same developmental stage. EcR DNA binding profiles are highly tissue-specific, and transgenic reporter analyses demonstrate that EcR plays a direct role in controlling enhancer activity. Finally, despite a strong correlation between tissue-specific EcR binding and tissue-specific open chromatin, we find that EcR does not control chromatin accessibility at genomic targets. We conclude that EcR contributes extensively to tissue-specific ecdysone responses. However, control over access to its binding sites is subordinated to other transcription factors.


title: Inferring selection effects in SARS-CoV-2 with Bayesian Viral Allele Selection

abstract: The global effort to sequence millions of SARS CoV-2 genomes has provided an unprecedented view of viral evolution. Characterizing how selection acts on SARS-CoV-2 is critical to developing effective, long-lasting vaccines and other treatments, but the scale and complexity of genomic surveillance data make rigorous analysis distinctly challenging. To meet this challenge, we develop Bayesian Viral Allele Selection (BVAS), a principled and scalable probabilistic method for inferring the genetic determinants of differential viral fitness and the relative growth rates of viral lineages, including newly emergent lineages. After demonstrating the accuracy and efficacy of our method through simulation, we apply BVAS to 6.9 million SARS-CoV-2 genomes. We identify numerous mutations that increase fitness, including previously identified mutations in the SARS-CoV-2 Spike and Nucleocapsid proteins, as well as mutations in non-structural proteins whose contribution to fitness is less well characterized. In addition, we extend our baseline model to identify mutations whose fitness exhibits strong dependence on vaccination status. Our method, which couples Bayesian variable selection with a diffusion approximation in allele frequency space, lays a foundation for identifying fitness-associated mutations under the assumption that most alleles are neutral.

category: genomics


title: Deciphering the Impact of Genetic Variation on Human Polyadenylation

abstract: Genetic variants that disrupt polyadenylation can cause or contribute to genetic disorders. Yet, due to the complex cis-regulation of polyadenylation, variant interpretation remains challenging. Here, we introduce a residual neural network model, APARENT2, that can infer 3'-cleavage and polyadenylation from DNA sequence more accurately than any previous model. This model generalizes to the case of alternative polyadenylation (APA) for a variable number of polyadenylation signals. We demonstrate APARENT2's performance on several variant datasets, including functional reporter data and human 3' aQTLs from GTEx. We apply neural network interpretation methods to gain insights into disrupted or protective higher-order features of polyadenylation. We fine-tune APARENT2 on human tissue-resolved transcriptomic data to elucidate tissue-specific variant effects. Finally, we perform in-silico saturation mutagenesis of all human polyadenylation signals and compare the predicted effects of >44 million variants against gnomAD. While loss-of-function variants were generally selected against, we also find specific clinical conditions linked to gain-of-function mutations. For example, using APARENT2's predictions we detect an association between gain-of-function mutations in the 3'-end and Autism Spectrum Disorder.


title: Liquid-like assembly of VASP drives actin polymerization and bundling

abstract: The organization of actin filaments into bundles is required for cellular processes such as motility, morphogenesis, and cell division. Filament bundling is controlled by a network of actin binding proteins. Recently, several proteins that comprise this network have been found to undergo liquid-liquid phase separation. How might liquid-like condensates contribute to filament bundling? Here we show that the processive actin polymerase, VASP, forms liquid-like droplets under physiological conditions. As actin polymerizes within VASP droplets, elongating filaments partition to the edges of the droplet to minimize filament curvature, forming an actin-rich ring within the droplet. The rigidity of this ring is balanced by the surface tension of the droplet, as predicted by a continuum-scale computational model. However, as the ring grows thicker, its rigidity increases and eventually overcomes the surface tension of the droplet, deforming into a linear bundle. The resulting bundles contain parallel actin filaments that grow from their tips. Growing bundles zipper together upon contact with one another, an effect which is mediated by the surface tension of the liquid-like VASP droplets that encapsulate them. Once the parallel arrangement of filaments is created within a VASP droplet, it propagates through the addition of new actin monomers to achieve a length that is many times greater than the initial droplet. This droplet-based mechanism of bundling may be relevant to the assembly of cellular architectures rich in parallel actin filaments, such as filopodia, stress fibers, and focal adhesions.


title: Cryo-EM structure of ex vivo fibrils associated with extreme AA amyloidosis prevalence in a cat shelter

abstract: AA amyloidosis is a systemic disease characterized by deposition of misfolded serum amyloid A protein (SAA) into amyloid in multiple organs in humans and animals. AA amyloidosis occurs at high SAA serum levels during chronic inflammation. The disease can be transmitted horizontally, likely facilitated by prion-like mechanism, in captive animals leading to extreme disease prevalence, e.g. 70% in captive cheetah and 57-73% in domestic short hair (DSH) cats kept in shelters. Herein, we present the 3.3 [A] cryo-EM structure of an AA amyloid extracted post-mortem from the kidney of a DSH cat with renal failure. The structure reveals a cross-{beta} architecture assembled from two 76-residue long proto-filaments. Despite >70% sequence homology to mouse and human SAA, the cat SAA variant adopts a distinct amyloid fold. Based on shared disease profiles and almost identical protein sequences, we propose a similar amyloid fold of deposits identified previously in captive cheetah.


title: A novel ALDH1A1 inhibitor blocks platinum-induced senescence and stemness in ovarian cancer

abstract: Ovarian cancer is a deadly disease attributed to late-stage detection as well as recurrence and development of chemoresistance. Ovarian cancer stem cells (OCSCs) are hypothesized to be largely responsible for emergence of chemoresistant tumors. Although chemotherapy may initially succeed at decreasing the size and number of tumors, it leaves behind residual malignant OCSCs. In this study, we demonstrate that Aldehyde dehydrogenase 1A1 (ALDH1A1) is essential for the survival of OCSCs. We identified a first in class ALDH1A1 inhibitor, compound 974, and used 974 as a tool to decipher the mechanism of stemness regulation by ALDH1A1. Treatment of OCSCs with 974 significantly inhibited ALDH activity, expression of stemness genes, spheroid, and colony formation. In vivo limiting dilution assay demonstrated that 974 significantly inhibited CSC frequency. Transcriptomic sequencing of cells treated with 974 revealed significant downregulation of genes related to stemness and chemoresistance as well as senescence and senescence associated secretory phenotype (SASP). We confirmed that 974 inhibited senescence and stemness induced by platinum-based chemotherapy in functional assays. Overall, these data establish that ALDH1A1 is essential for OCSCs survival and ALDH1A1 inhibition suppresses chemotherapy induced senescence and stemness. Targeting ALDH1A1 using small molecule inhibitors in combination with chemotherapy therefore presents a promising strategy to prevent ovarian cancer recurrence and has potential for clinical translation.

category: cancer biology


title: Integrative multi-omics analysis reveals molecular subtypes and tumor evolution of synovial sarcoma

abstract: Synovial sarcomas (SS) are malignant mesenchymal tumors characterized by the SS18-SSX fusion gene, which drives tumorigenesis by altering the composition of the BAF complex. Secondary genomic alterations that determine variations in tumor phenotype or clinical presentation are largely unknown. Herein, we present transcriptome, targeted DNA-sequencing, and proteomics analysis of 91 synovial sarcomas from 55 patients. We identified three SS clusters (SSCs) characterized by distinct histology, tumor microenvironments, genomic complexities, therapeutic effects, and clinical outcomes. Eight BAF complex components are differentially expressed among SSCs, and their role in mesenchymal-epithelial-transition is supported by single cell sequencing. The epithelial cells of biphasic tumors are more susceptible to developing copy number alterations, including amplification of PDCD1 and TMPRSS2. Our findings explain broad concepts in SS biology and imply that the BAF composition at the start of the tumorigenesis (i.e. the cellular linage) may determine the SS subtype, providing a rationale for individualized treatment strategies.


title: A requirement for Kruppel-Like Factor-4 in the maintenance of endothelial cell quiescence

abstract: Rationale and Goal: Endothelial cells (ECs) are quiescent and critical for maintaining homeostatic functions of the mature vascular system, while disruption of quiescence is at the heart of endothelial to mesenchymal transition (EndMT) and tumor angiogenesis. Here, we addressed the hypothesis that KLF4 maintains the EC quiescence. Methods and Results: In ECs, KLF4 bound to KLF2, and the KLF4-transctivation domain (TAD) interacted directly with KLF2. KLF4-depletion increased KLF2 expression, accompanied by phosphorylation of SMAD3, increased expression of alpha-smooth muscle actin (SMA), VCAM-1, TGF-{beta}1 and ACE2, but decreased VE-cadherin expression. In the absence of Klf4, Klf2 bound to the Klf2-promoter/enhancer region and autoregulated its own expression. Loss of EC-Klf4 in Rosa(mT/mG)::Klf4(fl/fl)::Cdh5(CreERT2) engineered mice, increased Klf2 levels and these cells underwent EndMT. Conclusion: In quiescent ECs, KLF2 and KLF4 partnered to regulate a combinatorial mechanism. The loss of KLF4 disrupted this combinatorial mechanism, thereby upregulating KLF2 as an adaptive response. However, increased KLF2 expression overdrives for the loss of KLF4, giving rise to an EndMT phenotype.

category: physiology


title: The Role of Conjunctive Representations in Prioritizing and Selecting Planned Actions

abstract: For flexible goal-directed behavior, prioritizing and selecting a specific action among multiple candidates is often important. Working memory has long been assumed to play a role in prioritization and planning, while bridging cross-temporal contingencies during action selection. However, studies of working memory have mostly focused on memory for single components of an action plan, such as a rule or a stimulus, rather than management of all of these elements during planning. Therefore, it is not known how post-encoding prioritization and selection operate on the entire profile of representations for prospective actions. Here, we assessed how such control processes unfold over action representations, highlighting the role of conjunctive representations that nonlinearly integrate task-relevant features during maintenance and prioritization of action plans. For each trial, participants prepared two independent rule- based actions simultaneously, then they were retro-cued to select one as their response. Prior to the start of the trial, one rule-based action was randomly assigned to be high priority by cueing that it was more likely to be tested. We found that both full action plans were maintained as conjunctive representations during action preparation, regardless of priority. However, during output selection, the conjunctive representation of the high priority action plan was more enhanced and readily selected as an output. Further, the strength of conjunctive representation was related to behavioral interference when the low priority action was tested. Thus, multiple integrated representations were maintained for upcoming actions and served as the target of post-encoding attentional selection mechanisms to prioritize and select an action from those in working memory.

category: neuroscience


title: Persistence of Cajal-Retzius cells in the postnatal hippocampus is required for development of dendritic spines of CA1 pyramidal cells

abstract: Cajal-Retzius (CR) cells are a transient type of neuron that populate the postnatal hippocampus. The role of transient cell types and circuits have been vastly addressed in neocortical regions, but poorly studied in the hippocampus. To understand how CR cells persistence influences the maturation of hippocampal circuits, we specifically ablated CR cells from the postnatal hippocampus. Our results highlighted layer-specific effects on dendritic spines and synaptic-related genes and revealed a critical role of CR cells in the establishment of the hippocampal network.


title: The evolution of information transmission in mammalian brain networks

abstract: Brain communication, defined as information transmission through white-matter connections, is at the foundation of the brain's computational capacities that virtually subtend all aspects of behavior: from sensory perception shared across mammalian species, to complex cognitive functions in humans. How did communication strategies in macroscale brain networks adapted across evolution to accomplish increasingly complex functions? By applying a novel approach to measure information transmission in mouse, macaque and human brains, we found an evolutionary gradient from selective information processing, where brain regions share information through single polysynaptic pathways, to parallel information processing, where regions communicate through multiple parallel pathways. In humans, parallel processing acts as a major connector between unimodal and transmodal systems. Communication strategies are unique to individuals across different mammalian species, pointing at the individual-level specificity of information routing architecture. Our work provides compelling evidence that different communication strategies are tied to the evolutionary complexity of mammalian brain networks.


title: Neuroinflammation plays a critical role in cerebral cavernous malformation disease

abstract: Background: Cerebral Cavernous Malformations (CCMs) are neurovascular lesions caused by loss-of-function mutations in one of three genes, including KRIT1 (CCM1), CCM2, and PDCD10 (CCM3). CCMs affect ~1/200 children and adults, and no pharmacologic therapy is available. CCM lesion count, size, and aggressiveness vary widely among patients of similar ages with the same mutation or even within members of the same family. However, what determines the transition from quiescent lesions into mature and active (aggressive) CCM lesions is unknown. Methods: We use genetic, RNA-seq, histology, flow cytometry, and imaging techniques to report the interaction between CCM-endothelium, astrocytes, leukocytes, microglia/macrophages, neutrophils (CALMN interaction) during the pathogenesis of CCMs in the brain tissue. Results: Expression profile of astrocytes in adult mouse brains using translated mRNAs obtained from the purification of EGFP-tagged ribosomes (Aldh1l1-EGFP/Rpl10a) in the presence or absence of CCM lesions (Slco1c1-iCreERT2;Pdcd10fl/fl; Pdcd10BECKO) identifies a novel gene signature for neuroinflammatory astrogliosis. CCM reactive astrocytes have a neuroinflammatory capacity by expressing genes involved in angiogenesis, chemotaxis, hypoxia signaling, and inflammation. RNA-seq analysis on RNA isolated from brain endothelial cells (BECs) in chronic Pdcd10BECKO mice (CCM-endothelium), identified crucial genes involved in recruiting inflammatory cells and thrombus formation through chemotaxis and coagulation pathways. In addition, CCM-endothelium was associated with increased expression of Nlrp3 and Il1b. Pharmacological inhibition of NLRP3 significantly decreased inflammasome activity as assessed by quantification of a fluorescent indicator of caspase-1 activity (FAM-FLICA caspase-1) in BECs from Pdcd10BECKO in the chronic stage. Importantly, our results support the hypothesis of the crosstalk between astrocytes and CCM endothelium can trigger the recruitment of inflammatory cells arising from brain parenchyma (microglia) and the peripheral immune system (leukocytes) into mature active CCM lesions that propagate lesion growth, immunothrombosis, and bleedings. Unexpectedly, partial or total loss of brain endothelial NF-kB activity (using Ikkbfl/fl mice) in chronic Pdcd10BECKO mice does not prevent lesion genesis or neuroinflammation. Instead, this resulted in an elevated number of lesions and immunothrombosis, suggesting that therapeutic approaches designed to target inflammation through endothelial NF-kB inhibition may contribute to detrimental side effects. Conclusions: Our study reveals previously unknown links between neuroinflammatory astrocytes and inflamed CCM endothelium as contributors that trigger leukocyte recruitment and precipitate immunothrombosis in CCM lesions. However, therapeutic approaches targeting brain endothelial NF-kB activity may contribute to detrimental side effects.


title: Species sympatry shapes brain size evolution in Primates

abstract: The main hypotheses about the evolution of animal cognition emphasise the role of conspecifics. Yet, space is often simultaneously occupied by multiple species from the same ecological guild. These sympatric species can compete for food, which may thereby stimulate or hamper cognition. Considering brain size as a proxy for cognition, we tested whether species sympatry impacted the evolution of cognition in frugivorous primates. We first retraced the evolutionary history of sympatry between frugivorous primate lineages. We then fitted phylogenetic models of the evolution of the size of several brain areas in frugivorous primates, considering or not species sympatry. We found that the whole brain or brain areas used in immediate information processing were best fitted by models not considering sympatry. By contrast, models considering species sympatry best predicted the evolution of brain areas related to long-term memory of interactions with the social or ecological environment, with a decrease of their size the higher the sympatry. We speculate that species sympatry, by generating intense food depletion, leads to an over-complexification of resource spatio-temporality that counteracts the benefits of high cognitive abilities and thereby induces lower brain area sizes. In addition, we reported that species in sympatry diversify more slowly. This comparative study suggests that species sympatry significantly contributes to shaping primate cognition and diversification.

category: ecology


title: Mutation rates and fitness consequences of mosaic chromosomal alterations in blood

abstract: Mosaic chromosomal alterations (mCAs) are commonly detected in many cancers and have been found to arise decades before diagnosis. A quantitative understanding of the rate at which these events occur and their functional consequences could improve cancer risk prediction and yet they remain poorly characterised. Here we use clone size estimates of mCAs from the blood of 500,000 participants in the UK Biobank to estimate the mutation rates and fitness consequences of acquired gain, loss and copy-neutral loss of heterozygosity (CN-LOH) events at the chromosomal arm level. Most mCAs have moderate to high fitness effects, but occur at a low rate, being over 10-fold less common than equivalently fit SNVs. While the majority of mCAs increase in prevalence with age in a way that is consistent with a constant growth rate, we find specific examples of mCAs whose behaviour deviates from this suggesting fitness effects for these mCAs may depend on inherited variants or be influenced by extrinsic factors. We find an association between mCA fitness effect and future blood cancer risk, highlighting the important role mCAs may play in risk stratification.


title: The effect of sociality on competitive interactions among birds

abstract: Sociality can provide many benefits, including increased foraging success, reproductive opportunities, and defence against predation. How does sociality influence the dominance hierarchies of ecological competitors? Here, we address this question using a large dataset of competitive interactions among birds foraging at backyard feeders across North America, representing a network of over 88,000 interactions among 196 bird species. We quantify sociality as the number of conspecifics observed together, and show that this measure of group size varies across bird species. More social species are less likely to dominate similarly sized opponents, suggesting that the evolution of social clustering is associated with reduced competitive ability overall. At the same time, we find that more social species also gain a greater competitive advantage from the presence of their conspecifics. We show that within-species competition occurs more often than expected in a null model, with the most social species exhibiting the greatest proportion of conflict originating from conspecifics. Overall, these results demonstrate that sociality influences the outcome of competition in ecological networks. Species that evolve greater sociality exhibit decreased competitive ability as individuals, but increased competitive ability in groups.

category: animal behavior and cognition


title: TRPV1 drugs alter core body temperature via central projections of primary afferent sensory neurons

abstract: TRPV1, a capsaicin- and heat-sensitive ion channel, is expressed by peripheral nociceptors and has been implicated in various inflammatory and neuropathic pain conditions. Although pharmacological modulation of TRPV1 has attracted therapeutic interest, their utility is limited because TRPV1 agonists and antagonists thus far examined show thermo-modulatory side effects in animal models and human clinical trials. These on-target effects may result from the perturbation of TRPV1 receptors on nociceptors, which transduce signals to central thermoregulatory circuits and also release pro-inflammatory factors from their peripheral terminals, such as the vasodilative neuropeptide calcitonin gene-related peptide (CGRP). Alternatively, they may originate from the modulation of TRPV1 on vascular smooth muscle cells (vSMCs), where channel activation promotes arteriole constriction. Here, we ask which of these pathways is most responsible for the body temperature perturbations elicited by TRPV1 drugs in vivo. We address this question by selectively eliminating TRPV1 expression in sensory neurons or vSMCs and show that only the former abrogates agonist-induced hypothermia and antagonist-induced hyperthermia. Furthermore, lesioning the central projections of TRPV1-positive sensory nerve fibers also abrogates drug-mediated thermo-modulation, whereas eliminating CGRP has no effect. Thus, TRPV1 drugs alter core body temperature by modulating sensory input to the central nervous system, rather than through peripheral actions on the vasculature. These findings suggest how mechanically distinct TRPV1 antagonists may diminish inflammatory pain without affecting core body temperature.


title: Cellular And Molecular Effects Of Understudied Kinase Pregnancy Upregulated Non-Ubiquitous Calcium-Calmodulin Dependent Kinase (PNCK) In Renal Cell Carcinoma.

abstract: Renal Cell Carcinoma (RCC) is a uniformly fatal disease when advanced. While immunotherapy and tyrosine kinase inhibitor-based combinations are associated with improved progression-free and overall survival, the majority of patients eventually develop treatment resistance and succumb to progressive, refractory disease. This underscores the urgent need to identify novel, non-canonical RCC targets for drug development. Through a comprehensive pan-cancer, pan-kinome analysis of the Cancer Genome Atlas (TCGA), the understudied kinase, pregnancy upregulated non-ubiquitous calcium-calmodulin dependent kinase (PNCK) was identified as the most differentially overexpressed kinase in RCC. PNCK mRNA was significantly overexpressed in RCC tissues compared to adjacent normal tissue, and its overexpression correlated with tumor T-stage grade and poor disease specific survival in both clear cell and papillary RCCs. PNCK overexpression in VHL mutant and VHL wild type RCC cell lines was associated with increased CREB phosphorylation, as well as increased cell proliferation and cell cycle progression. PNCK down-regulation, conversely, was associated with inhibition of CREB phosphorylation, decreased cell proliferation, cell cycle arrest and increased apoptosis, with differential effects observed between VHL mutant and VHL wild type cell lines. Pathway analyses in PNCK knockdown cells showed significant down regulation of hypoxia and angiogenesis pathways, as well as modulation of pathways promoting cell cycle arrest and apoptosis. The above results demonstrate for the first time the biological role of PNCK, an understudied kinase, in renal cell carcinoma and validate PNCK as a potential novel target for drug development in this fatal disease.


title: Molecular architecture of the C. elegans centriole

abstract: Uncovering organizing principles of organelle assembly is a fundamental pursuit in the life sciences. C. elegans was key in identifying evolutionary conserved components governing assembly of the centriole organelle. However, localizing these components with high precision has been hampered by the minute size of the worm centriole, thus impeding understanding of underlying assembly mechanisms. Here, we used Ultrastructure Expansion coupled with STimulated Emission Depletion microscopy (U-Ex-STED), as well as electron microscopy (EM) and tomography (ET), to decipher the molecular architecture of the worm centriole. Achieving an effective lateral resolution of ~14 nm, we localize centriolar and PeriCentriolar Material (PCM) components in a comprehensive manner with utmost spatial precision. We uncovered that the procentriole assembles from a location on the centriole margin characterized by SPD-2 and ZYG-1 accumulation. Moreover, we found that SAS-6 and SAS-5 are present in the nascent procentriole, with SAS-4 and microtubules recruited thereafter. We registered U-Ex-STED and EM data using the radial array of microtubules, thus allowing us to map each centriolar and PCM protein to a specific ultrastructural compartment. Importantly, we discovered that SAS-6 and SAS-4 exhibit a radial symmetry that is offset relative to microtubules, leading to a chiral centriole ensemble. Furthermore, we establish that the centriole is surrounded by a region from which ribosomes are excluded and to which SAS-7 localizes. Overall, our work uncovers the molecular architecture of the C. elegans centriole in unprecedented detail and establishes a comprehensive framework for understanding mechanisms of organelle biogenesis and function.

category: cell biology


title: Structure of maize BZR1-type β-amylase BAM8 provides new insights into its noncatalytic adaptation

abstract: Plant {beta}-Amylase (BAM) proteins play an essential role in growth, development, stress response, and hormone regulation. Despite their typical ({beta}/)8 barrel structure as active catalysts in starch breakdown, catalytically inactive BAMs are implicated in diverse yet elusive functions in plants. The noncatalytic BAM7/8 contain N-terminal BZR1 domains and were shown to be involved in the regulation of brassinosteroid signaling and possibly serve as sensors of yet an uncharacterized metabolic signal. While the structures of several catalytically active BAMs have been reported, structural characterization of the catalytically inactive BZR1-type BAMs remain unknown. Here, we determine the crystal structure of Zea mays BZR1-type BAM8 and provide comprehensive insights into its noncatalytic adaptation. Using structural-guided comparison combined with biochemical analysis and molecular dynamics simulations, we revealed conformational changes in multiple distinct highly conserved regions resulting in rearrangement of the binding pocket. Altogether, this study adds a new layer of understanding to starch breakdown mechanism and elucidates the acquired adjustments of noncatalytic BZR1-type BAMs as putative regulatory domains and/or metabolic sensors in plants.


title: MPZ-T124M mouse model replicates human axonopathy and suggest alteration in axo-glia communication

abstract: Myelin is essential for rapid nerve impulse propagation and axon protection. Accordingly, defects in myelination or myelin maintenance lead to secondary axonal damage and subsequent degeneration. Studies utilizing genetic (CNPase-, MAG-, and PLP-null mice) and naturally occurring neuropathy models suggest that myelinating glia also support axons independently from myelin. Myelin protein zero (MPZ or P0), which is expressed only by Schwann cells, is critical for myelin formation and maintenance in the peripheral nervous system. Many mutations in MPZ are associated with demyelinating neuropathies (Charcot-Marie-Tooth disease type 1B [CMT1B]). Surprisingly, the substitution of threonine by methionine at position 124 of P0 (P0T124M) causes axonal neuropathy (CMT2J) with little to no myelin damage. This disease provides an excellent paradigm to understand how myelinating glia support axons independently from myelin. To study this, we generated targeted knock-in P0T124M mutant mice, a genetically authentic model of T124M-CMT2J neuropathy. Similar to patients, these mice develop axonopathy between 2 and 12 months of age, characterized by impaired motor performance, normal nerve conduction velocities but reduced compound motor action potential amplitudes, and axonal damage with only minor compact myelin modifications. Mechanistically, we detected metabolic changes that could lead to axonal degeneration, and prominent alterations in non-compact myelin domains such as paranodes, Schmidt-Lanterman incisures, and gap junctions, implicated in Schwann cell-axon communication and axonal metabolic support. Finally, we document perturbed mitochondrial size and distribution along P0T124M axons suggesting altered axonal transport. Our data suggest that Schwann cells in P0T124M mutant mice cannot provide axons with sufficient trophic support, leading to reduced ATP biosynthesis and axonopathy. In conclusion, the P0T124M mouse model faithfully reproduces the human neuropathy and represents a unique tool for identifying the molecular basis for glial support of axons.


title: The endothelial-specific LINC00607 mediates endothelial angiogenic function

abstract: Long non-coding RNAs (lncRNAs) can act as regulatory RNAs which, by altering the expression of target genes, impact on the cellular phenotype and cardiovascular disease development. Endothelial lncRNAs and their vascular functions are largely undefined. Deep RNA-Seq and FANTOM5 CAGE analysis revealed the lncRNA LINC00607 to be highly enriched in human endothelial cells. LINC00607 was induced in response to hypoxia, arteriosclerosis regression in non-human primates and also in response to propranolol used to induce regression of human arteriovenous malformations. siRNA knockdown or CRISPR/Cas9 knockout of LINC00607 attenuated VEGF-A-induced angiogenic sprouting. LINC00607 knockout in endothelial cells also integrated less into newly formed vascular networks in an in vivo assay in SCID mice. Overexpression of LINC00607 in CRISPR knockout cells restored normal endothelial function. RNA- and ATAC-Seq after LINC00607 knockout revealed changes in the transcription of endothelial gene sets linked to the endothelial phenotype and in chromatin accessibility around ERG-binding sites. Mechanistically, LINC00607 interacted with the SWI/SNF chromatin remodeling protein BRG1. CRISPR/Cas9-mediated knockout of BRG1 in HUVEC followed by CUT&RUN revealed that BRG1 is required to secure a stable chromatin state, mainly on ERG-binding sites. In conclusion, LINC00607 is an endothelial-enriched lncRNA that maintains ERG target gene transcription by interacting with the chromatin remodeler BRG1.


title: The IPDGC/GP2 Hackathon - an open science event for training in data science, genomics, and collaboration using Parkinson's disease data

abstract: Background: Open science and collaboration are necessary to facilitate the advancement of Parkinson's disease (PD) research. Hackathons are collaborative events that bring together people with different skill sets and backgrounds to generate resources and creative solutions to problems. These events can be used as training and networking opportunities. Objective: To coordinate a virtual hackathon to develop novel PD research tools. Methods: 49 early career scientists from 12 countries collaborated in a virtual 3-day hackathon event in May 2021, during which they built tools and pipelines with a focus on PD. Resources were created with the goal of helping scientists accelerate their own research by having access to the necessary code and tools. Results: Each team was allocated one of nine different projects, each with a different goal. These included developing post-genome-wide association studies (GWAS) analysis pipelines, downstream analysis of genetic variation pipelines, and various visualization tools. Conclusion: Hackathons are a valuable approach to inspire creative thinking, supplement training in data science, and foster collaborative scientific relationships, which are foundational practices for early career researchers. The resources generated can be used to accelerate research on the genetics of PD.


title: Chromosome-scale assembly of the lablab genome - A model for inclusive orphan crop genomics

abstract: Orphan crops (also described as underutilised and neglected crops) hold the key to diversified and climate-resilient food systems. After decades of neglect, the genome sequencing of orphan crops is gathering pace, providing the foundations for their accelerated domestication and improvement. Recent attention has however turned to the gross under-representation of researchers in Africa in the genome sequencing efforts of their indigenous orphan crops. Here we report a radically inclusive approach to orphan crop genomics using the case of Lablab purpureus (L.) Sweet (syn. Dolichos lablab, or hyacinth bean) - a legume native to Africa and cultivated throughout the tropics for food and forage. Our Africa-led South-North plant genome collaboration produced a high-quality chromosome-scale assembly of the lablab genome - the first chromosome-scale plant genome assembly locally produced in Africa. We also re-sequenced cultivated and wild accessions of lablab from Africa confirming two domestication events and examined the genetic diversity in lablab germplasm conserved in Africa. Our approach provides a valuable resource for lablab improvement and also presents a model that could be explored by other researchers carrying out sequencing projects of indigenous crops particularly from Low and middle income countries (LMIC).


title: spSeudoMap: Cell type mapping of spatial transcriptomics using unmatched single-cell RNA-seq data

abstract: With advances in computational models, the cellular landscape can be tracked in various tissues using spatial transcriptomics. Since many single-cell RNA-seq (scRNA-seq) data have been obtained after cell sorting, such as when investigating immune cells, integrating these single-cell data with spatial data is limited due to a mismatch of cell types composing the two datasets. Here, we present a method, spSeudoMap, which utilizes sorted scRNA-seq data to train a model for predicting cell types of spatial spots by creating virtual cell mixtures that closely mimic the gene expression profile of spatial transcriptomic data. To overcome the mismatch issue, the cell type exclusively present in the spatial data, pseudotype, was defined. The proportion of pseudotype cells and virtual expression profiles in the cell mixture was determined by pseudobulk transcriptomes. The simulated cell mixture was considered a reference dataset, and the model that predicts the cell composition of the mixture was trained to predict the cell fraction of the spatial data using domain adaptation. First, spSeudoMap was evaluated in human and mouse brain tissues, and the main region-specific neuron types extracted from single-cell data could be precisely mapped to the expected anatomical locations. Moreover, the method was applied to human breast cancer data and described the spatial distribution of immune cell subtypes and their interactions in heterogeneous tissue. Taken together, spSeudoMap is a platform that predicts the spatial composition of cell subpopulations using sorted scRNA-seq data, and it may help to clarify the roles of a few but crucial cell types.