title: Exploring a diverse world of effector domains and amyloid signaling motifs in fungal NLR proteins

abstract: NLR proteins are intracellular receptors constituting a conserved component of the innate immune system of multicellular organisms. In fungi, NLRs are characterized by high diversity of architectures and presence of amyloid signaling. Here, we explore the diverse world of effector and signaling domains of fungal NLRs using state-of-the-art bioinformatic methods including MMseqs2 for fast clustering, probabilistic context-free grammars for sequence analysis, and AlphaFold2 deep neural networks for structure prediction. In addition to substantially improving the overall annotation, especially in basidiomycetes, the study identifies novel domains and reveals the structural similarity of MLKL-related HeLo- and Goodbye-like domains forming the most abundant superfamily of fungal NLR effectors. Moreover, compared to previous studies, we found several times more amyloid motifs, including novel families, and validated aggregating and prion-forming properties of the most abundant of them in vitro and in vivo. Also, through an extensive in silico search, the NLR-associated amyloid signaling is for the first time identified in basidiomycetes. The emerging picture highlights similarities and differences in the NLR architectures and amyloid signaling in ascomycetes, basidiomycetes and other branches of life.


title: MiXcan: a Framework for Cell-Type-Specific Transcriptome-Wide Association Studies with an Application to Breast Cancer

abstract: Human bulk tissue samples comprise multiple cell types with diverse roles in disease etiology. Conventional transcriptome-wide association study (TWAS) approaches predict gene expression at the tissue level from genotype data, without considering cell-type heterogeneity, and test associations of the predicted tissue-level gene expression with disease. Here we develop MiXcan, a new TWAS approach that predicts cell-type-specific gene expression levels, identifies disease-associated genes via combination of cell-type-specific association signals for multiple cell types, and provides insight into the disease-critical cell type. We conducted the first cell-type-specific TWAS of breast cancer in 58,648 women and identified 12 transcriptome-wide significant genes using MiXcan compared with only eight genes using conventional approaches. Importantly, MiXcan identified genes with distinct associations in mammary epithelial versus stromal cells, including three new breast cancer susceptibility genes. These findings demonstrate that cell-type-specific TWAS can reveal new insights into the genetic and cellular etiology of breast cancer and other diseases.

category: bioinformatics


title: MSclassifR: an R package for supervised classification of mass spectra with machine learning methods

abstract:
MotivationClassification of mass spectra is essential for identifying microorganisms from matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. However, spectrally close organisms remain difficult to identify. In this context, we developed the MSclassifR R package to improve the classification of mass spectra. Its open code strengthens the reproducibility of analyzes in the community.

ResultsWe applied the functions of our package to raw mass spectra from three different laboratories. The best workflow available in MSclassifR package achieves near 100% accuracy in all three datasets. Thus, MSclassifR constitutes an interesting alternative for reliable MALDI-TOF based diagnosis.

AvailabilityMSclassifR is freely available online from CRAN repository https://cran.r-project.org/web/packages/MSclassifR/index.html. Two vignettes illustrating how to use the functions of this package from real data sets are also available online to help users.

Contactalexandre.godmer@aphp.fr

Supplementary informationSupplementary material is available at Bio-informatics online.


title: Learning deep representations of enzyme thermal adaptation

abstract: Temperature is a fundamental environmental factor that shapes the evolution of organisms. Learning thermal determinants of protein sequences in evolution thus has profound significance for basic biology, drug discovery, and protein engineering. Here, we use a dataset of over 3 million enzymes labeled with optimal growth temperatures (OGT) of their source organisms to train a deep neural network model (DeepET). The protein-temperature representations learned by DeepET provide a temperature-related statistical summary of protein sequences and capture structural properties that affect thermal stability. For prediction of enzyme optimal catalytic temperatures and protein melting temperatures via a transfer learning approach, our DeepET model outperforms classical regression models trained on rationally designed features and other recent deep-learning-based representations. DeepET thus holds promise for understanding enzyme thermal adaptation and guiding the engineering of thermostable enzymes.


title: Systematic evaluation of chromatin immunoprecipitation sequencing to study histone occupancy in dormancy transitions of grapevine buds

abstract: 1.The regulation of DNA accessibility by histone modification has emerged as a paradigm of developmental and environmental programming. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a versatile tool widely used to investigate in vivo protein-DNA interaction. The technique has been successfully demonstrated in several plant species and tissues; however, it has remained challenging in woody tissues. Here we developed a ChIP method specifically for mature dormant grapevine buds (Vitis vinifera cv. Cabernet Sauvignon). Each step of the protocol was systematically optimised, including crosslinking, chromatin extraction, sonication, and antibody validation. Analysis of histone H3-enriched DNA was performed to evaluate the success of the protocol and identify occupancy of histone H3 along grapevine bud chromatin. To our best knowledge, this is the first ChIP experiment protocol optimised for grapevine bud system.

category: plant biology


title: Predictable and stable epimutations induced during clonal propagation with embryonic transcription factors

abstract: Although clonal propagation is frequently used in commercial plant breeding and plant biotechnology programs because it minimizes genetic variation, it is not uncommon to observe clonal plants with stable phenotypic changes, a phenomenon known as somaclonal variation. Several studies have shown that epigenetic modifications induced during regeneration are associated with this newly acquired phenotypic variation. However, the factors that determine the extent of somaclonal variation and the molecular changes associated with it remain poorly understood. To address this gap in our knowledge, we compared clonally propagated Arabidopsis thaliana plants derived from somatic embryogenesis using two different embryonic transcription factors-RWP-RK DOMAIN-CONTAINING 4 (RKD4) or LEAFY COTYLEDON2 (LEC2) and from two epigenetically distinct tissues. We found that both the epi(genetic) status of explant and the regeneration protocol employed play critical roles in shaping the molecular and phenotypic state of clonal plants. Phenotypic variation of regenerated plants can be largely explained by the inheritance of tissue-specific DNA methylation imprints, which are associated with specific transcriptional and metabolic changes in sexual progeny of clonal plants. Moreover, regenerants from roots were particularly affected by the inheritance of epigenetic imprints, which resulted in increased accumulation of salicylic acid in leaves and accelerated plant senescence. Collectively, our data reveal pathways for targeted manipulation of phenotypic variation in clonal plants.


title: Age-dependent lipid droplet-rich microglia worsen stroke outcome in old mice

abstract: Microglial cells of the aged brain manifest signs of dysfunction that could contribute to the worse neurological outcome of stroke in the elderly. Treatment with colony-stimulating factor-1 receptor antagonists enable transient microglia depletion that can be followed by microglia repopulation after interruption of the treatment, causing no known harm to mice. Using this strategy, we aimed to restore microglia function and ameliorate stroke outcome in aged mice. Cerebral ischemia/reperfusion induces strong innate immune responses in microglia highlighted by prominent type I interferon signaling, together with cellular metabolic perturbances and lipid droplet biogenesis in young mice. In aged mice, a subset of microglia accumulates lipid droplets under steady state and displays exacerbated innate immune responses after stroke. Microglia renewal in old mice reduces the lipid droplet content, prevents the ischemia-induced exaggerated type I interferon response, and improves the neurological outcome of stroke. This study shows that age-dependent lipid droplet-enriched microglia contribute to impair stroke outcome in old mice.

category: neuroscience


title: Temporal dynamics of competition between statistical learning and episodic memory in intracranial recordings of human visual cortex

abstract:
The function of long-term memory is not just to reminisce about the past, but also to make predictions that help us behave appropriately and efficiently in the future. This predictive function of memory provides a new perspective on the classic question from memory research of why we remember some things but not others. If prediction is a key outcome of memory, then the extent to which an item generates a prediction signifies that this information already exists in memory and need not be encoded. We tested this principle using human intracranial EEG as a time-resolved method to quantify prediction in visual cortex during a statistical learning task and link the strength of these predictions to subsequent episodic memory behavior. Epilepsy patients of both sexes viewed rapid streams of scenes, some of which contained regularities that allowed the category of the next scene to be predicted. We verified that statistical learning occurred using neural frequency tagging and measured category prediction with multivariate pattern analysis. Although neural prediction was robust overall, this was driven entirely by items that were subsequently forgotten. Such interference provides a mechanism by which prediction can regulate memory formation to prioritize encoding of information that could help learn new predictive relationships.

Significance StatementWhen faced with a new experience, we are rarely at a loss for what to do. Rather, because many aspects of the world are stable over time, we rely upon past experiences to generate expectations that guide behavior. Here we show that these expectations during a new experience come at the expense of memory for that experience. From intracranial recordings of visual cortex, we decoded what humans expected to see next in a series of photographs based on patterns of neural activity. Photographs that generated strong neural expectations were more likely to be forgotten in a later behavioral memory test. Prioritizing the storage of experiences that currently lead to weak expectations could help improve these expectations in future encounters.


title: Dual STDP processes at Purkinje cells contribute to distinct improvements in accuracy and vigor of saccadic eye movements.

abstract:
Saccadic eye-movements play a crucial role in visuo-motor control by allowing rapid foveation onto new targets. However, the neural processes governing saccades adaptation are not fully understood. Saccades, due to the short-time of execution (20-100 ms) and the absence of sensory information for online feedback control, must be controlled in a ballistic manner. Incomplete measurements of the movement trajectory, such as the visual end-point error, are supposedly used to form internal predictions about the movement kinematics resulting in predictive control. In order to characterize the synaptic and neural circuit mechanisms underlying predictive saccadic control, we have reconstructed the saccadic system in a digital controller embedding a spiking neural network of the cerebellum with spike timing-dependent plasticity (STDP) rules driving parallel fiber - Purkinje cell long-term potentiation and depression (LTP and LTD). This model implements a control policy based on a dual plasticity mechanism, resulting in the identification of the roles of LTP and LTD in optimizing saccade movement control: it turns out that LTD regulates the accuracy and LTP the speed (vigor) of the ballistic eye movement. The control policy also required cerebellar PCs to be divided into two subpopulations, characterized by burst or pause responses. To our knowledge, this is the first model that explains in mechanistic terms the accuracy and vigor regulation of ballistic eye movements in forward mode exploiting spike-timing to regulate firing in different populations of the neuronal network. This elementary model of saccades could be extended and applied to other more complex cases in which single jerks are concatenated to compose articulated and coordinated movements.

Author summaryIt has been suggested that the cerebellum plays a crucial role in oculomotor adaptation. Computationally, the cerebellum is described as a supervised learner whose activity can be adjusted by synaptic changes proportional to the amount of mismatch between expected and actual movements outcomes (sensorimotor-errors). However, the spike-timing-dependent-plasticity (STDP) underlying adaptation has been so far modeled in behaviors where the error is continuously available as tracking-error. Such models depend on detailed tracking-errors to improve movement quality. We asked if the cerebellum can maintain good motor control even if the error is not completely available? This is important considering that error-dependent-STDP is only a subset among a family of STDP processes in the cerebellum. Moreover, even the physiological signals that were generally thought to carry sensorimotor-errors (called complex-spikes) are shown to encode multiple types of information regarding the movement. In this context, we characterize the role of cerebellar STDPs in saccade control, where the error information is constrained because of sensory suppression. We show that even in the absence of detailed error, the cerebellum can leverage two of its STDPs to increase movement quality. Hence, we emphasize the need to go beyond error-centric view to understand how the cerebellum improves motor behavior.


title: A conserved immune trajectory of recovery in hospitalized COVID-19 patients

abstract: Many studies have provided insights into the immune response to COVID-19; however, little is known about the immunological changes and immune signaling occurring during COVID-19 resolution. Individual heterogeneity and variable disease resolution timelines obscure unifying immune characteristics. Here, we collected and profiled >200 longitudinal peripheral blood samples from patients hospitalized with COVID-19, with other respiratory infections, and healthy individuals, using mass cytometry to measure immune cells and signaling states at single cell resolution. COVID-19 patients showed a unique immune composition and an early, coordinated and elevated immune cell signaling profile, which correlated with early hospital discharge. Intra-patient time course analysis tied to clinically relevant events of recovery revealed a conserved set of immunological processes that accompany, and are unique to, disease resolution and discharge. This immunological process, together with additional changes in CD4 regulatory T cells and basophils, accompanies recovery from respiratory failure and is associated with better clinical outcomes at the time of admission. Our work elucidates the biological timeline of immune recovery from COVID-19 and provides insights into the fundamental processes of COVID-19 resolution in hospitalized patients.

category: immunology


title: Size-selective mortality fosters evolutionary changes in collective learning across ontogeny and decision-making in zebrafish, Danio rerio

abstract: Size-selective harvesting evolutionary alters the life-history, behavioural and physiological traits in exploited fish populations. Changes in these traits may cause alteration in learning and decision-making abilities, either due to energetic trade-offs with brain investment that may vary across development or via correlations with boldness, sociability or other personality traits. Whether size-selective harvesting evolutionarily alters learning and decision-making abilities in fish remains unexplored, despite the global scale of fisheries. We tested the hypothesis that persistent removal of large-bodied individuals typical of many fisheries reduces learning ability in adults but not in juveniles, increases cognitive flexibility but reduces decision-making ability in adults. We examined associative learning through ontogeny, and reversal learning and collective decision-making in adults in three selection lines of zebrafish (Danio rerio) generated through positive, negative and random size-selective harvesting for five generations. Fish groups of each selection line were tested across ontogeny using a colour-discrimination paradigm with a food reward. The associative reversal task was conducted with a social reward, and the propensity to make group decisions was tested in an associative task. All selection lines showed significant learning ability and improved performance across ontogeny. Consistent with our hypothesis, the large-harvested line fish revealed a significantly reduced learning speed as subadults and adults, while the small-harvested line fish showed slower error rate compared to controls as 4-month old adults. We found no evidence of memory decay, and the selection lines did not vary in associative reversal ability. Decision-making speed did not vary across lines, but the large-harvested line made faster decisions during the probe trial. We conclude that size-selective harvesting typical of many fisheries evolutionarily alters learning and decision making. As this is likely to negatively and persistently affect resource acquisition and survival in exploited populations, we suggest that the cognition-related mechanism we identify may increase natural mortality.

category: ecology


title: Targeting an evolutionarily conserved "E-L-L" motif in the spike protein to develop a small molecule fusion inhibitor against SARS-CoV-2

abstract: As newer variants of SARS-CoV-2 continue to pose major threats to global human health and economy, identifying novel druggable antiviral targets is the key towards sustenance. Here, we identify an evolutionary conserved "E-L-L" motif present within the HR2 domain of all human and non-human coronavirus spike (S) proteins that play a crucial role in stabilizing the post-fusion six-helix bundle (6-HB) structure and thus, fusion-mediated viral entry. Mutations within this motif reduce the fusogenicity of the S protein without affecting its stability or membrane localization. We found that posaconazole, an FDA-approved drug, binds to this "E-L-L" motif resulting in effective inhibition of SARS-CoV-2 infection in cells. While posaconazole exhibits high efficacy towards blocking S protein-mediated viral entry, mutations within the "E-L-L" motif rendered the protein completely resistant to the drug, establishing its specificity towards this motif. Our data demonstrate that posaconazole restricts early stages of infection through specific inhibition of membrane fusion and viral genome release into the host cell and is equally effective towards all major variants of concerns of SARS-CoV-2 including beta, kappa, delta, and omicron. Together, we show that this conserved essential "E-L-L" motif is an ideal target for the development of prophylactic and therapeutic interventions against SARS-CoV-2.

category: biochemistry


title: Allosteric binders of ACE2 are promising anti-SARS-CoV-2 agents

abstract:
The COVID-19 pandemic has had enormous health, economic, and social consequences. Vaccines have been successful in reducing rates of infection and hospitalization, but there is still a need for an acute treatment for the disease. We investigate whether compounds that bind the human ACE2 protein can interrupt SARS-CoV-2 replication without damaging ACE2s natural enzymatic function. Initial compounds were screened for binding to ACE2 but little interruption of ACE2 enzymatic activity. This set of compounds was extended by application of quantitative structure-activity analysis, which resulted in 512 virtual hits for further confirmatory screening. A subsequent SARS-CoV-2 replication assay revealed that five of these compounds inhibit SARS-CoV-2 replication in human cells. Further effort is required to completely determine the antiviral mechanism of these compounds, but they serve as a strong starting point for both development of acute treatments for COVID-19 and research into the mechanism of infection.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/484484v1_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@173d7c9org.highwire.dtl.DTLVardef@5c0021org.highwire.dtl.DTLVardef@c9caaorg.highwire.dtl.DTLVardef@18d23_HPS_FORMAT_FIGEXP  M_FIG TOC Graphic: Overall study design.

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title: Nitrogen and phosphorus deficiencies alter primary and secondary metabolites of soybean roots

abstract: Nitrogen (N) and phosphorus (P) are two essential plant macronutrients that can limit plant growth by different mechanisms. We aimed to shed light on how soybean respond to low nitrogen (LN), low phosphorus (LP) and their combined deficiency (LNP). Generally, these conditions triggered changes in gene expression of the same processes, including cell wall organization, defense response, response to oxidative stress, and photosynthesis, however, response was different in each condition. A typical primary response to LN and LP was detected also in soybean, i.e., the enhanced uptake of N and P, respectively, by upregulation of genes for the corresponding transporters. The regulation of genes involved in cell wall organization showed that in LP roots tended to produce more casparian strip, in LN more secondary wall biosynthesis occurred, and in LNP reduction in expression of genes involved in secondary wall production accompanied by cell wall loosening was observed. Flavonoid biosynthesis also showed distinct pattern of regulation in different conditions: more anthocyanin production in LP, and more isoflavonoid production in LN and LNP, which we confirmed also on the metabolite level. Interestingly, in soybean the nutrient deficiencies reduced defense response by lowering expression of genes involved in defense response, suggesting a role of N and P nutrition in plant disease resistance. In conclusion, we provide detailed information on how LN, LP, and LNP affect different processes in soybean roots on the molecular and physiological levels.


title: A free classified advertising website is a high-risk unregulated source of domestic wild boar, pot-bellied pigs, and their hybrids with domestic swine in Canada: Implications for African Swine Fever Risk Preparedness

abstract: Invasive wild pigs are a global problem and are established in large areas across Canada. Domestic European wild boar were first introduced to Canadian farms in the 1980s, and their escape and intentional release have since led to free-ranging populations, which have expanded rapidly in numbers and range. Wild pigs are associated with widespread damage to crops and natural environments, as well as risks of disease transmission. African Swine Fever (ASF) is a highly contagious viral disease of global concern, that can spread between domestic and free-ranging swine and cause catastrophic economic impacts. As ASF preparedness requires a detailed understanding of the distribution and movements of all pig types, this paper aimed to characterize an unregulated  grey swine market in Canada to understand risks to domestic swine production and potential contributions to free-ranging wild pig populations. Kijiji.ca is a free Canadian internet classified advertising service, almost exclusively cash-based with no receipts and few, if any, records kept of transactions. We monitored Kijiji for sales of domestic wild boar, pot-bellied pigs, and their hybrids across Canada over two months from April 28 - June 30, 2021. Data was collected from all advertisements, including how the seller labelled pigs breed, age, sex, number for sale, sexual intactness, presence of tattoos and/or ear tags, as well as the date and location of the posting. Locations were mapped and compared to the spatial distribution of existing free-ranging wild pigs in Canada to identify areas they were likely to facilitate new populations or potentially supplement and genetically diversify existing populations. We identified 151 advertisements on Kijiji, the most coming from Ontario (34.4%; n = 52) and Alberta (29.1%; n = 44), followed by Saskatchewan (11.3%; n = 17), British Columbia (10.6%; n = 16), Nova Scotia (7.3%; n = 11), New Brunswick (4.0%; n = 6), Manitoba (2.0%; n = 3), Quebec (0.7%; n = 1), and Prince Edward Island (0.7%; n = 1). No listings were observed in Newfoundland and Labrador, or Yukon, Nunavut and Northwest Territories. Overall, 62 (41%) of advertisements were within watersheds with existing wild pigs that may contribute to supplementing additional swine, and 89 (59%) were within watersheds where wild pigs have not yet been identified. African swine fever preparedness should include policies and action to address this unmonitored and unregulated sale of swine in Canada, including ear tags, tattoos, genetic analysis, and mandatory reporting.

category: scientific communication and education


title: Neuroligin-3 in Dopamine Neurons Promotes Behavioral and Neurobiological Adaptations to Chronic Morphine Exposure

abstract:
Chronic opioid exposure causes structural and functional changes in brain circuits, which may contribute to opioid use disorders. Synaptic cell-adhesion molecules are prime candidates for mediating this opioid-evoked plasticity. Neuroligin-3 (NL3) is a postsynaptic adhesion protein that shapes synaptic function at multiple sites in the mesolimbic dopamine system. We therefore studied how genetic knockout of NL3 alters responses to chronic morphine in male mice. Constitutive NL3 knockout caused a persistent reduction in psychomotor sensitization after chronic morphine exposure, as well as a change in the topography of locomotor stimulation produced by morphine. This latter change was recapitulated by conditional genetic deletion of NL3 from cells expressing the Drd1 dopamine receptor, whereas the reduction in psychomotor sensitization was recapitulated by conditional genetic deletion from dopamine neurons. In the absence of NL3 expression, dopamine neurons in the ventral tegmental area showed diminished activation following chronic morphine exposure, as measured by in vivo calcium imaging with fiber photometry. This altered pattern of dopamine neuron activity may be driven by aberrant forms of opioid-evoked synaptic plasticity in the absence of NL3: dopamine neurons lacking NL3 showed weaker synaptic inhibition at baseline, which was subsequently strengthened after chronic morphine. In total, our study highlights neurobiological adaptations in dopamine neurons of the ventral tegmental area that correspond with increased behavioral sensitivity to opioids, and further suggests that NL3 expression by dopamine neurons provides a molecular substrate for opioid-evoked adaptations in brain function and behavior.

SIGNIFICANCE STATEMENTExposure to opioids and other drugs of abuse can cause long-lasting changes in the brain. Some of the most durable alterations involve functional and structural changes at synapses in brain reward circuits, including dopamine neurons in the ventral tegmental area and their projection to the nucleus accumbens. Synaptic cell-adhesion molecules support structural and functional aspects of synaptic transmission. Dysregulation of these molecules has been implicated in addiction and other neuropsychiatric disorders. We find that a specific synaptic cell-adhesion molecule, neuroligin-3, mediates changes in brain reward circuits caused by morphine exposure. In the absence of neuroligin-3, male mice are less sensitive to the behavioral effects of chronic morphine exposure, and dopamine neurons in the ventral tegmental area do not function normally.


title: Omicron BA.1 and BA.2 neutralizing activity elicited by a comprehensive panel of human vaccines

abstract: The SARS-CoV-2 Omicron variant of concern comprises three sublineages designated BA.1, BA.2, and BA.3, with BA.2 steadily replacing the globally dominant BA.1. We show that the large number of BA.1 and BA.2 spike mutations severely dampen plasma neutralizing activity elicited by infection or seven clinical vaccines, with cross-neutralization of BA.2 being consistently more potent than that of BA.1, independent of the vaccine platform and number of doses. Although mRNA vaccines induced the greatest magnitude of Omicron BA.1 and BA.2 plasma neutralizing activity, administration of a booster based on the Wuhan-Hu-1 spike sequence markedly increased neutralizing antibody titers and breadth against BA.1 and BA.2 across all vaccines evaluated. Our data suggest that although BA.1 and BA.2 evade polyclonal neutralizing antibody responses, current vaccine boosting regimens may provide sufficient protection against Omicron-induced disease.


title: Direct detection of natural selection in Bronze Age Britain

abstract: We developed a novel method for efficiently estimating time-varying selection coefficients from genome-wide ancient DNA data. In simulations, our method accurately recovers selective trajectories, and is robust to mis-specification of population size. We applied it to a large dataset of ancient and present-day human genomes from Britain, and identified seven loci with genome-wide significant evidence of selection in the past 4500 years. Almost all of them are related to increased vitamin D or calcium levels, and we conclude that lack of vitamin D and consequent low calcium was consistently the most important selective pressure in Britain since the Bronze Age. However, the strength of selection on individual loci varied substantially over time, suggesting that cultural or environmental factors moderated the genetic response to this pressure. Of 28 complex anthropometric and metabolic traits, skin pigmentation was the only one with significant evidence of polygenic selection, further underscoring the importance of phenotypes related to vitamin D. Our approach illustrates the power of ancient DNA to characterize selection in human populations and illuminates the recent evolutionary history of Britain.

category: evolutionary biology


title: MePMe-seq: Antibody-free simultaneous m6A and m5C mapping in mRNA by metabolic propargyl labeling and sequencing

abstract: Internal modifications of mRNA have emerged as widespread and versatile regulatory mechanism to control gene expression at the post-transcriptional level. Current insights rely on the ability to make a modified nucleoside amenable to sequencing. Most of the modifications are methylations involving the co-factor S-adenosyl-L-methionine (SAM), however, simultaneous detection of different methylation sites in the same sample has remained elusive. We present metabolic labeling with propargyl-selenohomocysteine (PSH) in combination with click chemistry to detect N6- methyladenosine (m6A) and 5-methylcytidine (m5C) sites in mRNA with single nucleotide precision in the same sequencing run (MePMe-seq). Intracellular formation of the corresponding SAM analogue leads to detectable levels of N6-propargyl-A (prop6A) and 5-propargyl-C (prop5C). MePMe-seq overcomes the problems of antibodies for enrichment and sequence-motifs for evaluation, limiting previous methodologies. The joint evaluation of m6A and m5C sites opens the door to study their interconnectivity and improve our understanding of mechanisms and functions of the RNA methylome.


title: Long range electromagnetic effects drive protein-protein approaches: an egg of Coulomb

abstract:
Living systems cannot rely on random intermolecular approaches inside cell crowding and hidden mechanisms must be present to favor only those molecular interactions which are specifically required by biological functions. Electromagnetic messaging among proteins is here hypothesized upon the observation that charged amino acids are most commonly located in adjacent sequence positions and/or in spatial close proximity. Molecular Dynamics simulations have been used to explore possible effects arising from concerted motions of charged amino acid side chains in two protein model systems. Protein electrodynamics seems to emerge as the framework for understanding long distance protein-ligand interactions.

HighlightsO_LIProtein surfaces are often occupied by nearby side chains bearing opposite charges;
C_LIO_LICoulomb interactions determine hindered reorientations of charged side chains;
C_LIO_LIMD simulations suggest time scales and extents of surface charge interactions;
C_LIO_LIconcerted motions of electric charges can yield electromagnetic protein signaling.
C_LI


title: Molecular Dynamics simulation of TDP-43 RRM in the presence and absence of RNA

abstract: Structural characterization of the prion prone TAR DNA Binding protein (TDP)-43 has been challenging since its intrinsically disordered regions represents 15-30% of the total protein. TDP-43 is a nucleic acid binding protein with an N-terminal domain, two RNA Recognition Motifs (RRM1 and RRM2) and the C-terminal domain. In this study, we seek to define possible new targetable sites on the apo structure of TDP-43 RRM domains. To do so, we used molecular dynamic (MD) simulations on the NMR solved TDP-43RRM1-2 structure bound to RNA to predict the apo structure. Contact analysis of TDP-43 showed that while the integrity of the individual domains was maintained upon RNA removal, a decrease in interdomain contacts was observed. Moreover, we compared apo TDP-43 structures obtained from MD to AlphaFold 2 (AF2) predicted TDP-43 structures and found differences in loop regions. A Sitemap analysis identified five druggable sites for the RNA bound structure solved by NMR, while fewer sites were identified following MD simulations and AF2 predicted apo structures.

category: biophysics


title: Quorum sensing as a mechanism to harness the wisdom of the crowds

abstract: Bacteria release and sense small molecules called autoinducers (AIs) in a process known as  quorum sensing (QS). The prevailing interpretation of QS is that by sensing AI concentrations, bacteria estimate population density to regulate the expression of functions that are only beneficial when carried out by a sufficiently large number of cells. However, a major challenge to this interpretation is that the concentration of AIs strongly depends on the environment, often rendering AI-based estimates of cell density unreliable. Here we propose an alternative interpretation of QS, where bacteria, by releasing and sensing AIs, harness social interactions to sense the environment as a collective. As shown by a model, this functionality can explain the evolution of QS, and results from individuals improving their estimation accuracy by pooling many imperfect estimates - analogous to the  wisdom of the crowds in decision theory. Importantly, our model reconciles the observed dependence of QS on both population density and the environment and explains why several QS systems regulate the production of private goods.

category: microbiology


title: Chlamydia trachomatis suppresses host cell store-operated Ca2+ entry and inhibits NFAT/calcineurin signaling

abstract: The obligate intracellular bacterium, Chlamydia trachomatis, replicates within a parasitophorous vacuole termed an inclusion. During development, host proteins critical for regulating intracellular calcium (Ca2+) homeostasis interact with the inclusion membrane. The inclusion membrane protein, MrcA, interacts with the inositol-trisphosphate receptor (IP3R), an ER cationic channel that conducts Ca2+. Stromal interaction molecule 1 (STIM1), an ER transmembrane protein important for regulating store-operated Ca2+ entry (SOCE), localizes to the inclusion membrane via an uncharacterized interaction. We therefore examined Ca2+ mobilization in C. trachomatis infected cells. Utilizing a variety of Ca2+ indicators to assess changes in cytosolic Ca2+ concentration, we demonstrate that C. trachomatis impairs host cell SOCE. Ca2+ regulates many cellular signaling pathways. We find that the SOCE-dependent NFAT/calcineurin signaling pathway is impaired in C. trachomatis L2 infected HeLa cells and likely has major implications on host cell physiology as it relates to C. trachomatis pathogenesis.


title: Is virulence phenotype evolution driven exclusively by Lr gene deployment in French Puccinia triticina populations?

abstract: Puccinia triticina is a highly damaging wheat pathogen. The efficacy of leaf rust control by genetic resistance is mitigated by the adaptive capacity of the pathogen, expressed as changes in its virulence combinations (pathotypes). An extensive P. triticina population survey has been carried out in France over the last 30 years, describing the evolution dynamics of this pathogen in response to cultivar deployment. We analyzed the dataset for the 2006-2016 period to determine the relationship between the Lr genes in the cultivars and virulence in the pathotypes. Rust populations were dominated by a small number of pathotypes, with variations in most of the virulence frequencies related to the corresponding Lr gene frequencies in the cultivated landscape. Furthermore, the emergence and spread of a new virulence matched the introduction and use of the corresponding Lr gene (Lr28), confirming that the deployment of qualitative resistance genes is an essential driver of evolution in P. triticina populations. However, PCA revealed that certain pathotype-cultivar associations cannot be explained solely by the distribution of Lr genes in the landscape. This conclusion is supported by the predominance of a few pathotypes on some cultivars, with the persistence of several other compatible pathotypes at low frequencies. Specific interactions are not, therefore, sufficient to explain the distribution of virulence in rust populations. Our findings suggest that aggressiveness is a driver of changes in pathotype frequencies. Accordingly, the hypothesis of "dual selection", based on both qualitative and quantitative interactions between P. triticina populations and bread wheat cultivars, is favored.


title: Dynamic model of bovine brucellosis to investigate control strategies in endemic settings

abstract: Brucellosis imposes substantial impacts on livestock production and public health worldwide. A stochastic, age-structured model incorporating herd demographics was developed describing within- and between-herd transmission of Brucella abortus in dairy cattle herds. The model was fitted to data from a cross-sectional study conducted in Punjab State of India and used to evaluate the effectiveness of control strategies under consideration. Based on model results, stakeholder acceptance and constraints regarding vaccine supply, vaccination of replacement calves in large farms should be prioritised. Test and removal applied at early stages of the control programme where seroprevalence is high would not constitute an effective use of resources. Critically, under current model assumptions, significant numbers of animals  removed (culled or not used for breeding) in this strategy would be removed based on false positive results. To achieve sustained reductions in brucellosis, policymakers must commit to maintaining vaccination in the long term, which may eventually reduce frequency of infection in the livestock reservoir to a low enough level for elimination to be a realistic objective. This exercise provided important insights into the control of brucellosis in India, which has the largest cattle population globally, and a general framework for evaluating control strategies in endemic settings.


title: A coevolution experiment reveals parallel mutations in the AcrA-AcrB-TolC efflux pump that contributes to bacterial antibiotic resistance

abstract: One interference mechanism of bacterial competition is the production of antibiotics. Bacteria exposed to antibiotics can resist antibiotic inhibition through intrinsic and/or acquired mechanisms. Here, we performed a coevolution experiment to understand long-term consequences of antibiotic production and antibiotic resistance for two environmental bacterial strains. We grew five independent lines of the antibiotic-producing environmental strain, Burkholderia thailandensis E264, and the antibiotic-inhibited environmental strain, Flavobacterium johnsoniae UW101, together and separately on agar plates for 7.5 months (1.5 month incubations), transferring each line five times to new agar plates. We first observed that the F. johnsoniae ancestor could tolerate the B. thailandensis-produced antibiotic through efflux mechanisms. We then sequenced the genomes of clonal isolates from the coevolved and monoculture F. johnsoniae lines, and uncovered mutational ramifications to the long-term antibiotic exposure. The coevolved genomes from F. johnsoniae revealed four potential mutational signatures of antibiotic resistance that were not observed in the evolved monoculture lines. Two mutations were found in tolC: one corresponding to a 33 bp deletion and the other corresponding to a nonsynonymous mutation. A third mutation was observed as a 1 bp insertion coding for a RagB/SusD nutrient uptake protein. The last mutation was a G83R nonsynonymous mutation in acetyl-coA carboxylayse carboxyltransferase subunit alpha (AccA). Placing the tolC 33 bp deletion back into the F. johnsoniae ancestor conferred some antibiotic resistance, but not to the degree of resistance observed in coevolved lines. Furthermore, the accA mutation matched a previously described mutation conferring resistance to B. thailandensis-produced thailandamide. Analysis of B. thailandensis transposon mutants for thailandamide production revealed that thailandamide was bioactive against F. johnsoniae, but also suggested that additional B. thailandensis-produced antibiotics were involved in the inhibition of F. johnsoniae. This study reveals how long-term interspecies chemical interactions can result in a novel mutation in efflux that contribute to antibiotic resistance.


title: Tales of Two α-Carboxysomes: the Structure and Assembly of Cargo Rubisco

abstract: Carboxysomes are a family of bacterial microcompartments in cyanobacteria and chemoautotrophs. It encapsulates carbonic anhydrase and Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalysing carbon fixation inside a proteinaceous shell. How Rubisco packs into the carboxysomes is unknown. Using cryo-electron tomography and subtomogram averaging, we present 3D organization of Rubisco inside two types of native -carboxysomes from a marine -cyanobacterium Cyanobium sp. PCC 7001 and a chemoautotrophic bacterium Halothiobacillus neapolitanus. We determined the structures of Rubiscos within native Halothiobacillus and Cyanobium carboxysomes at 3.3 [A] and 3.8 [A] resolution respectively and further identified an associated CsoS2 segment. Interestingly, CsoS2 is only associated with a sub-population of Rubiscos that are close to the shell in Halothiobacillus, but with all Rubiscos throughout Cyanobium carboxysome. Moreover, Rubiscos in Cyanobium carboxysomes are organized in three concentric layers whereas Rubiscos in Halothiobacillus carboxysomes are arranged in spiral arrays. Calcium treatment induced a drastic re-organization of Rubiscos, converting these two distinct assemblies into ordered lattice arrays in both -carboxysomes. Our findings provide critical knowledge of the assembly principles of -carboxysomes, which may aid in rational design and repurpose of carboxysome structures for new functions.


title: Rapid detection of Staphylococcus aureus and Streptococcus pneumoniae by real-time analysis of volatile metabolites

abstract:
Rapid detection of pathogenic bacteria is needed for rapid diagnostics allowing adequate and timely treatment. In this study, we aimed to evaluate the technical feasibility of Secondary Electro-Spray Ionization-High Resolution Mass Spectrometry (SESI-HRMS) as a diagnostic tool for rapid detection of bacterial infections and compare its performance with the current standard of diagnostics. We compared the time required to confirm growth of the pathogenic bacteria Staphylococcus aureus and Streptococcus pneumoniae by conventional detection by culture and MAL-DI-TOF vs. detection of specific volatile organic compounds (VOCs) produced by these human pathobionts. SESI-HRMS could consistently detect VOCs produced by S. aureus or S. pneumoniae on blood agar plates within minutes, allowing to positively identify bacteria within hours. Unique S. aureus and S. pneumoniae features were detected already at bacterial densities as low as [~]103 colony forming units. Rich mass spectral fingerprints allowed for the distinction of these two bacteria on a species and even strain level. To give an incentive towards clinical application of this technology, further analyzed 17 clinical samples previously diagnosed by conventional methods. We predominantly obtained a separation of samples which showed growth (i.e. presence of living bacteria) compared to samples with no bacterial growth (i.e. presence of dead bacteria). We conclude that SESI-HRMS allows rapid identification of unique bacterial features. Further development of real-time analysis of clinical samples by SESI-HRMS will shorten the time required for microbiological diagnosis with a high level of confidence and sensitivity and should help to improve patients tailored treatment.

IMPORTANCEA timely identification of a pathogenic bacteria causing the infection is of pivotal importance for the initiation of an adequate antimicrobial therapy. In this regard, different technologies have been developed with the aim to achieve a highly reliable, specific, and overall fast identification of pathogenic bacteria. However, conventional diagnostic techniques still require long preprocessing times (hours to days) to acquire enough biological material for an accurate identification of the pathogen. Therefore, in this work, we aimed to further shorten the detection time of current gold standards for microbiological diagnostics by providing a system capable of a fast, sensitive and specific discrimination of different pathogenic bacteria. This system relies on the real-time mass spectrometric detection of volatile organic compounds (VOCs) produced by a given organism during its growth, potentially leading to a significant shortening of the time required to obtain a positive reliable diagnostic.


title: Phylogenetic analysis of human parainfluenza type 3 virus strains responsible for the outbreak during the COVID-19 pandemic in Seoul, South Korea

abstract:
BackgroundHuman parainfluenza virus 3 (HPIV3) is a major respiratory pathogen that causes acute respiratory infections in infants and children. Since September 2021, an out-of-season HPIV3 rebound has been noted in Korea. The objective of this study was to analyze the molecular characteristics of the HPIV3 strains responsible for the outbreak in Seoul, South Korea.

MethodsA total of 61 HPIV3-positive nasopharyngeal swab specimens were collected between October and November 2021. Using 33 HPIV3-positive specimens, partial nucleotide sequences of the HPIV3 hemagglutinin-neuraminidase (HN) gene were aligned with previously published HN gene sequences for phylogenetic and genetic distance (p-distance) analyses.

ResultsPhylogenetic tree revealed that all Seoul HPIV3 strains grouped within the phylogenetic subcluster C3. However, these strains formed a unique cluster that branched separately from the C3a lineage. This cluster showed 99% bootstrap support with a p-distance < 0.001. Genetic distances within the other C3 lineages ranged from 0.013 (C3a) to 0.023 (C3c). Deduced amino acid sequences of the HN gene revealed four protein substitutions in Seoul HPIV3 strains that have rarely been observed in other reference strains: A22T, K31N, G387S, and E514K.

ConclusionsPhylogenetic analysis of Seoul HPIV3 strains revealed that the strain belonged to a separate cluster within subcluster C3. Genetic distances among strains within subcluster C3 suggest the emergence of a new genetic lineage. The emergence of a new genetic lineage could pose a potential risk of a new epidemic. Further monitoring of the circulating HPIV3 strains is needed to understand the importance of newly discovered mutations.


title: Zika virus NS4A hijacks host ANKLE2 to promote viral replication

abstract: Zika virus (ZIKV) is infamous among flaviviruses for its unique association with congenital birth defects, notably microcephaly. We previously mapped ZIKV-host protein interactions and identified the interaction between ZIKV NS4A and host ANKLE2, which itself has established ties to congenital microcephaly. In fruit flies, NS4A induces microcephaly phenotypes in an ANKLE2-dependent manner. This suggests that NS4A interacts with ANKLE2 to dysregulate cell behavior and contributes to abnormal host neurodevelopment. Here, we explore the role of ANKLE2 in ZIKV replication to understand the biological significance of the interaction from the viral perspective. We show that knockdown of ANKLE2 reduces replication of two ZIKV strains, across multiple MOIs. We observe that localization of ANKLE2 is drastically shifted to sites of NS4A and viral replication. We investigate which domains of ANKLE2 mediate this behavior and the interaction with NS4A. Using co-immunoprecipitation, we show that deletion of either the transmembrane or LEM domain has little impact on the interaction, but deletion of both significantly reduces interaction with NS4A. Finally, we show that the C-terminal transmembrane domains of NS4A stabilize the interaction with ANKLE2. Together, these results suggest NS4A interacts with ANKLE2 through a combination of its transmembrane and LEM domains, bringing it to sites of ZIKV replication to promote replication through an unknown mechanism. Taken together with our previous results, our findings indicate that, in the process of hijacking ANKLE2 for replication, ZIKV disrupts its physiological function to cause disease.

category: molecular biology


title: ANTARCTIC FUNGI: A BIO-SOURCE ALTERNATIVE TO PRODUCE POLYUNSATURATED FATTY ACIDS (PUFAs)

abstract:
The Antarctic ecosystem is a combination of conditions including extremely low values of temperature. The environmental temperature is one of the parameters thoroughly affecting the structure and composition of fungal membranes lipids. The psychrophilic fungi generally increase the disorder within macromolecules to maintain membrane fluidity at low temperatures. The strategy adopted by Antarctic fungi is to increase the proportion of unsaturated fatty acid that allows maintaining a semi-fluid state of the membranes. This ecological feature might be exploited for using Antarctic fungi as potential alternative source of polyunsaturated fatty acids (PUFAs) for human diet.

This study provides both the characterization of fungal strains isolated from Antarctica by lipidomic analysis and the laboratory/large-scale production of fungal biomass with high content of beneficial PUFAs. In detail, three fungal species isolated from environmental matrices from Antarctica were tested and identified at genome level. Growth experiments to evaluate the influence of temperature and substrate in the yield in biomass and unsaturated fatty acid (UFA) were conducted. The results showed that the selected fungi have a high percentage of UFA compared to saturated ones; low growth temperatures increase the yield in linolenic fatty acid (C18:3); the biomass yield depends on the composition of the growth substrate and a satisfying qualitative-quantitative yield has also been obtained by using an agri-food chain waste product as growth substrate.

IMPORTANCEThe presence of polyunsaturated fatty acids (PUFAs) in human and animal diet is gaining attention because PUFAs have several recognized functional properties: they modulate immune response, have anti-allergic and anti-inflammatory activity, cardio-protective effect and reduce blood LDL cholesterol levels. Human diets typically do not contain sufficient PUFAs because foods rich in PUFAs are few and it is therefore necessary to supplement this diet. Food supplements with these types of fatty acid currently commercially available come from marine fish oils and this source is no longer sustainable. It is necessary to develop efficient industrial processes capable of producing good quality PUFAs and in quantity, even using as carbon and nitrogen sources agro-industrial chains (in our case spent yeast from brewing and whey waste) waste products. Like microorganisms we used Antarctic fungi because they are adapted at very low temperature increasing the proportion of unsaturated fatty acid that allows maintaining a semi-fluid state of the membranes.


title: Hibernation shows no apparent effect on germline mutation rates in grizzly bears

abstract: A male mutation bias is observed across vertebrates, and, where data are available, this bias is accompanied by increased per-generation mutation rates with parental age. While continuing mitotic cell division in the male germline post-puberty has been proposed as the major cellular mechanism underlying both patterns, little direct evidence for this role has been found. Understanding the evolution of the per-generation mutation rate among species requires that we identify the molecular mechanisms that change between species. Here, we study the per-generation mutation rate in an extended pedigree of the brown (grizzly) bear, Ursus arctos horribilis. Brown bears hibernate for one-third of the year, a period during which spermatogenesis slows or stops altogether. The cessation of spermatogenesis is predicted to lessen the male mutation bias and to lower the per-generation mutation rate in this species. However, using whole-genome sequencing, we find that both male bias and per-generation mutation rates are the same as expected for a non-hibernating species. We also carry out a phylogenetic comparison of substitution rates along the lineage leading to brown bear and panda (a non-hibernating species) and find no slowing of the substitution rate in the hibernator. Our results contribute to accumulating evidence that suggests that male germline cell division is not the major determinant of mutation rates and mutation biases. The results also provide a quantitative basis for improved estimates of the timing of carnivore evolution.


title: Plasmid stability in fluctuating environments:population genetics of multi-copy plasmids

abstract: Plasmids are extra-chromosomal genetic elements that encode a wide variety of phenotypes and can be maintained in bacterial populations through vertical and horizontal transmission, thus increasing bacterial adaptation to hostile environmental conditions like those imposed by antimicrobial substances. To circumvent the segregational instability resulting from randomly distributing plasmids between daughter cells upon division, non-transmissible plasmids tend to be carried in multiple copies per cell, with the added benefit of exhibiting increased gene dosage. But carrying multiple copies also results in a high metabolic burden to the bacterial host, therefore reducing the overall fitness of the population. This trade-off poses an existential question for plasmids: What is the optimal plasmid copy number? In this manuscript, we address this question using a combination of population genetics modeling with microbiology experiments consisting of Escherichia coli K12 bearing a multi-copy plasmid encoding for blaTEM-1, a gene conferring resistance to {beta}-lactam antibiotics. We postulate and analyze a Wright-Fisher model to evaluate the interaction between selective pressure, the number of plasmid copies carried by each cell, and the energetic cost associated with plasmid bearing. By numerically determining the optimal plasmid copy number for constant and fluctuating selection regimes, we show that the stability of multi-copy plasmids is maximized at intermediate plasmid copy numbers. We conclude by arguing that plasmid copy number is a highly optimized evolutionary trait that depends on the rate of environmental fluctuation and balances the benefit between increased stability in the absence of selection with the burden associated with carrying multiple copies of the plasmid.


title: Genome-wide binding of Drosophila Mask reveals its role in maintenance of gene activation by Trithorax group

abstract: The Trithorax group (trxG) proteins counteract repressive effect of Polycomb group (PcG) complexes and maintain transcriptional memory of active states of key developmental genes. Although, chromatin structure and modifications appear to play a fundamental role in this process, it is not clear how trxG prevents PcG-silencing and heritably maintain an active gene expression state. Here, we report a hitherto unknown role of Drosophila Mask which emerged as one of the candidate trxG genes in our screen. The genome-wide binding profile of Mask correlates with known Trithorax binding sites across Drosophila genome. Association of Mask at chromatin overlaps with H3K27ac and depletion of Mask results in strongly diminished H3K27ac levels. Moreover, Mask predominantly associates with actively transcribed genes and its depletion results in downregulation of trxG targets. Importantly, strong suppression of extra sex comb phenotype by mask mutant provides physiological relevance that Mask is required for maintenance of gene activation by trxG.

category: genomics


title: In depth exploration of the alternative proteome of Drosophila melanogaster

abstract: Recent studies have shown that hundreds of small proteins were occulted when protein coding genes were annotated. These proteins, called alternative proteins, have failed to be annotated notably due to the short length of their open reading frame (less than 100 codons) or the enforced rule establishing that messenger RNAs (mRNAs) are monocystronic. Several alternative proteins were shown to be biologically active molecules and seem to be involved in a wide range of biological functions. However, genome wide exploration of the alternative proteome is still limited to a few species. In the present article we describe a deep peptidomics workflow which enabled the identification of 401 alternative proteins in Drosophila melanogaster. Subcellular localization, protein domains and short linear motifs were predicted for 235 of the alternative proteins identified and point towards specific functions of these small proteins. Several alternative proteins had approximated abundances higher than their canonical counterparts, suggesting that these alternative proteins are actually the main products of their corresponding genes. Finally, we observed fourteen alternative proteins with developmentally regulated expression patterns and ten induced upon heat shock treatment of embryos, demonstrating stage or stress specific production of alternative proteins.


title: A single-cell clusters similarity measure for different batches, datasets, and samples

abstract:
SummarySince the inception of single-cell level measuring techniques, identification of distinct cell stages, phenotypes and populations has been a challenge. Cell clustering and dimensionality reduction methods are the most popular approaches to identify heterogeneity of single-cell data. But, as public repositories continue to grow in number, integrative analyses and merging of large pools of samples from different and heterogeneous datasets becomes a difficult challenge, which showcases the impossibility of scalability of some of the existing methods. Here we present ClusterFoldSimilarity, an R package that calculates a measure of similarity between clusters from different datasets/batches, without the need of correcting for batch effect or normalizing and merging the data, thus avoiding artifacts and the loss of information derived from these kinds of techniques. The similarity metric is based on the average vector module and sign of the product of logarithmic fold-changes. ClusterFoldSimilarity compares every single pair of clusters from any number of different samples/datasets, including different number of clusters for each sample. Additionally, the algorithm is able to select the top genes which contribute the most to the similarity of two specific clusters, serving also as a feature selection tool.

Availability and implementationThe algorithm is freely available as an R package at: https://github.com/OscarGVelasco/ClusterFoldSimilarity

Contactoscargvelasco@gmail.com


title: Single cells tell their own story: An updated framework for understanding stochastic variations in cell cycle progression in bacteria

abstract: Illuminating the molecular mechanisms underlying biological phenomena requires an accurate understanding of the context in which these phenomena occur. Currently, population based experiments frame our understanding of the bacterial cell cycle. Most famously, in 1968 Stephen Cooper and Charles Helmstetter took advantage of their ability to synchronize E. coli cells with regard to birth and division using then new technology, the baby machine. Based on baby-machine data, they proposed the existence of two nutrient-dependent growth regimes--slow in which all steps in cell cycle progression are proportional to growth rate and fast in which progression of DNA polymerase plateaus, while new rounds of replication and division remain pegged to growth rate. In the fast regime, the relationship between initiation and division beocome determinant with the latter following the former by a specific amount of time. Still the prevailing model, recent work in single cells reveals a disconnect between the behavior of an idealized average cells (the outcome of population-based analysis) and cell cycle progression in individual cells. To resolve this disconnect, we take an agnostic view of the forces modulating bacterial cell cycle progression and, leveraging extant single cell data, propose a new framework from which to understand this essential process. Significantly, our framework accounts for the stochasticity inherent in biological processes. It is also flexible, providing the ability to assess cell cycle progression independent of medium or genetic background. Using this framework, we test potential explanations for the divergence between single cell growth rate and replication fork elongation rate, identifying limiting reagents as the primary cause of this widespread phenomenon.


title: Integrative DNA methylation analysis of pediatric brain tumors reveals tumor type-specific developmental trajectories and epigenetic signatures of malignancy

abstract: Understanding oncogenic epigenetic mechanisms in brain tumors is crucial for improved diagnosis and treatment. To evaluate tumor type-specific features, we compared atypical teratoid/rhabdoid tumors (AT/RTs), medulloblastomas, and choroid plexus tumors with each other by integrating DNA methylation (507 samples), gene expression (120 samples), and transcription factor (TF) -binding data. Our results suggest that aberrant DNA methylation plays a vital role in the oncogenesis of AT/RTs, which are known for their aggressiveness and exceptionally low mutation rates. In AT/RT, elevated DNA methylation masks the binding sites of TFs driving neural development and is associated with reduced gene expression for specific neural regulators, whereas DNA methylation patterns predict a more advanced differentiation state for MB. By analyzing the data together with pluripotent stem cells, we found two regulatory programs in AT/RT: pluripotent stem cell -like DNA methylation patterns and AT/RT-specific DNA methylation uniquely associated with polycomb repressive complex 2 (PRC2) members. Both methylation patterns cover neural TF binding sites in a TF-specific manner, suggesting their relevance for oncogenic regulation.

category: cancer biology


title: Targeting Cblb-Notch1 axis as a novel immunotherapeutic strategy to boost CD8+ T-cells responses

abstract: A critical feature of cancer is the ability to induce immunosuppression and evade immune responses. Tumor-induced immunosuppression diminishes the efficacy of endogenous immune responses and decreases the efficacy of cancer immunotherapy. In this study, we describe a new immunosuppressive pathway in which adenosine promotes Cbl-b-mediated Notch1 degradation, causing suppression of CD8+ T-cells effector functions. Genetic KO and pharmacological inhibition of Cbl-b prevents Notch1 degradation in response to adenosine and reactivates its signaling. Reactivation of Notch1 results in enhanced CD8+ T-cell effector functions, anti-cancer response and resistance to immunosuppression. Our work demonstrates that targeting Cbl-b-Notch1 axis is a novel promising strategy for cancer immunotherapy.


title: PeroxiHUB: a modular cell-free biosensing platform using H2O2 as signal integrator

abstract: Cell-free systems have great potential for delivering robust, cheap, and field-deployable biosensors. Many cell-free biosensors rely on transcription factors responding to small molecules, but their discovery and implementation still remain challenging. Here we report the engineering of PeroxiHUB, an optimized H2O2-centered sensing platform supporting cell-free detection of different metabolites. H2O2 is a central metabolite and a by-product of numerous enzymatic reactions. PeroxiHUB uses enzymatic transducers to convert metabolites of interest into H2O2, enabling rapid reprogramming of sensor specificity using alternative transducers. We first screen several transcription factors and optimize OxyR for the transcriptional response to H2O2 in cell-free, highlighting the need for pre-incubation steps to obtain suitable signal-to-noise ratios. We then demonstrate modular detection of metabolites of clinical interest - lactate, sarcosine, and choline - using different transducers mined via a custom retro-synthesis workflow publicly available on the SynBioCAD Galaxy portal. We find that expressing the transducer during the pre-incubation step is crucial for optimal sensor operation. Finally, we show that different reporters can be connected to PeroxiHUB, providing high adaptability for various applications. Given the wide range of enzymatic reactions producing H2O2, the PeroxiHUB platform will support cell-free detection of a large number of metabolites in a modular and scalable fashion.

category: synthetic biology


title: Strain-specific alterations in gut microbiome and host immune responses elicited by Bifidobacterium pseudolongum

abstract: The beneficial effects attributed to Bifidobacterium are thought to arise from their host immunomodulatory capabilities, which are likely to be species- and even strain-specific. However, their strain-specificity in direct and indirect immune modulation remain largely uncharacterized. We have shown that B. pseudolongum UMB-MBP-01, a murine isolate, is capable of suppressing inflammation and reducing fibrosis in vivo. To ascertain the mechanism driving this activity and to determine if it is specific to UMB-MBP-01, we compared it to B. pseudolongum type strain ATCC25526 of porcine origin using a combination of in vitro and in vivo experimentation and comparative genomics approaches. Despite many shared features, we demonstrate that these two strains possess distinct genetic repertoires in carbohydrate assimilation, differential activation signatures and cytokine responses in innate immune cells, and differential effects on lymph node morphology with unique local and systemic leukocyte distribution. Importantly, the administration of each B. pseudolongum strain resulted in major divergence in the structure, composition, and function of gut microbiota. This was accompanied by markedly different changes in intestinal transcriptional activities, suggesting strain-specific modulation of the endogenous gut microbiota as a key to host responses of immune modulation and changes in intestinal B. pseudolongum strains. These observations highlight the importance of strain-specificity characteristics of Bifidobacterium for prophylactic supplementation for immune modulation and advance our understanding of the mechanisms which drive the association between Bifidobacterium and health benefit.


title: A potential interacting network identified by quantitative proteomics for the sole adenylyl cyclase Cyr1 of Candida albicans

abstract: Cyr1, the sole adenylyl cyclase of the fungal pathogen Candida albicans, is a central component of the cAMP/PKA signaling pathway that controls the yeast-to-hyphal morphogenesis. Cyr1 functions as a multivalent senor and integrator of various external and internal signals. To better understand how these signals are relayed to Cyr1 and how Cyr1 activity is regulated, we sought to identify global interacting partners of Cyr1 using stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics. From the proteins co-immunoprecipitated with Myc-tagged Cyr1, 38 were assigned as authentic Cyr1-interacting partners, including two previously characterized Cyr1-binding proteins, Cap1 and Act1. Many of the identified proteins remain uncharacterized, but some can be classified into several functional groups, such as actin regulatory proteins, cell wall components, and mitochondrial proteins. We used biochemical and genetic methods to further characterize the interaction of Cyr1 with Mp65, a cell surface mannoprotein previously reported to have a role in cell wall integrity and possess several virulence-related traits. Taken together, our comprehensive analysis of the Cyr1-interacting proteins provides important information for establishing the Cyr1 interactome and uncovers a potential role for cell wall proteins in Cyr1-mediated cAMP signaling.


title: A novel conserved protein in Streptococcus agalactiae, BvaP, is important for vaginal colonization and biofilm formation

abstract:
Streptococcus agalactiae (Group B Strep, GBS) infections in neonates are often fatal and strongly associated with maternal GBS vaginal colonization. Here, we investigated the role of a previously uncharacterized protein, BvaP, in GBS vaginal colonization. BvaP was previously identified as the most highly upregulated gene in the GBS A909 transcriptome when comparing vaginal colonization to growth in liquid culture. We found that expression of BvaP affects GBS adherence to extracellular matrix components and human vaginal epithelial cells and a{Delta} bvaP mutant was significantly decreased in its ability to colonize the murine vaginal tract. Cellular morphological alterations such as changes in cell shape, chain length, and clumping were also observed in a knockout mutant strain. Given its high expression in vivo, high degree of conservation among GBS strains, and role in vaginal colonization, BvaP may be an eligible target for GBS vaccination and/or drug therapy.

IMPORTANCENeonatal GBS disease is a major cause of morbidity and mortality and maternal vaginal colonization is the leading risk factor for disease. Colonization prevention would greatly impact rates of disease transmission, but vaccine development has stalled as capsular polysaccharide vaccines have low immunogenicity in vivo. While these vaccines are still in development, addition of a protein conjugate may prove fruitful in increasing immunogenicity and strain coverage across GBS serotypes. Previous research identified sak_1753 as a gene very highly upregulated gene during murine vaginal colonization. This study reveals that Sak_1753 is required to maintain proper GBS cellular morphology and colonization phenotypes and is required for in vivo vaginal colonization in a murine model. We have renamed Sak_1753 Group B Strep vaginal adherence protein (BvaP). The findings of this study indicate that BvaP is important in GBS colonization of the vaginal tract and may be a candidate for vaccine development.


title: Chemical and genetic screens identify new regulators of tetracycline-inducible gene expression system in mammalian cells

abstract: The tetracycline repressor (tetR)-regulated system is a widely used tool to specifically control gene expression in mammalian cells. Based on this system, we generated a human osteosarcoma cell line which allows for inducible expression of an EGFP-fusion of the TAR DNA-binding protein 43 (TDP-43), which has been linked to neurodegenerative diseases. Consistent with previous findings, TDP-43 overexpression led to the accumulation of aggregates and limited the viability of U2OS. Using this inducible system, we conducted a chemical screen with a library that included FDA-approved drugs. While the primary screen identified several compounds that prevented TDP-43 toxicity, further experiments revealed that these chemicals abrogated doxycyclinedependent TDP-43 expression. This antagonistic effect was observed with both doxycycline and tetracycline, and in several Tet-On cell lines expressing different genes, confirming the general effect of these compounds as inhibitors of the tetR system. Using the same cell line, a genome-wide CRISPR/Cas9 screen identified epigenetic regulators such as the G9a methyltransferase or TRIM28 as potential modifiers of TDP-43 toxicity. Yet again, further experiments revealed that G9a inhibition or TRIM28 loss prevented doxycycline-dependent expression of TDP-43. Together, these results suggest that none of the medically approved drugs significantly mitigates TDP-43 toxicity, identify new chemical and genetic regulators of the tetR system, and raise awareness on the limitations of this approach to conduct chemical or genetic screenings in mammalian cells.


title: Phase separated ribosome nascent chain complexes paused in translation are capable to continue expression of proteins playing role in genotoxic stress response upon DNA damage

abstract: EDTA- and RNase-resistant ribonucleoprotein complexes of arrested ribosomes with protruding nascent polypeptide chains have recently been described in yeast and human cells. These complexes have been termed assemblysomes, a type of soluble condensates distinct from other known granules. Here, we use bioinformatics to identify additional proteins that likely form assemblysomes during translation. We characterize soluble condensates of the DNA helicase Sgs1, one such identified protein and a key player in the repair of DNA double-strand breaks in yeast. We show that paused ribosome-associated nascent chains of Sgs1 in condensates are able to resume translation upon UV irradiation, consistent with the return of mRNA to the ribosome pool. By extending our studies to human cell lines, we found that EDTA-resistant pellets of ribosomes from the human prostate cancer cell line DU145 are sensitive to treatment with 1,6-hexanediol, which is known to dissolve liquid-liquid phase-separated condensates. In addition, transmission electron microscopy shows that 1,6-hexanediol dissolves ring ribosomal structures from the cytoplasm of radioresistant A549 cells while making the cells more sensitive to X-rays. These results suggest that the stress response is based on a conserved mechanism involving the regulated return of phase-separated paused ribosome-nascent chain complexes to translating ribosomes.


title: Uterine injury during diestrus leads to embryo spacing defects and perturbations in the COX pathway in subsequent pregnancies

abstract: Uterine injury from procedures such as Cesarean sections (C-sections) often have severe consequences on subsequent pregnancy outcomes, leading to disorders such as placenta previa, placenta accreta, and infertility. With rates of C-section at approximately 30% of deliveries in the US and that are projected to continue to climb, a deeper understanding of the mechanisms by which these pregnancy disorders arise and opportunities for intervention are needed. However, there are no animal models to date that comprehensively assess the consequences of uterine injury. Here we describe a rodent model of uterine injury on subsequent in utero outcomes. We observed three distinct phenotypes: increased rates of resorption and death, embryo spacing defects, and placenta accreta-like features of reduced decidua and expansion of invasive trophoblasts. We show that the appearance of embryo spacing defects depends entirely on the phase of estrous cycle at the time of injury. Using RNA-seq, we identified perturbations in the expression of components of the COX/prostaglandin pathway after recovery from injury, a pathway that has previously been demonstrated to play an important role in embryo spacing. Therefore, we demonstrate that uterine damage in this mouse model causes morphological and molecular changes, most notably perturbed expression of COX/prostaglandin pathway-related genes, that ultimately lead to placental and embryonic developmental defects.

category: developmental biology


title: Leaf bacterial community structure and variation in wild ruderal plants are shaped by the interaction of host species and defense chemistry with environment

abstract: Variable phenotypes help plants ensure fitness and survival in the face of unpredictable environmental stresses. Leaf bacteria (bacteriomes) can extend plant phenotypes and are well-known to vary from one plant to the next, but little is known about controls on this variation. Here, we find in 9 populations of Arabidopsis thaliana that core leaf bacteriomes are largely, but not completely, shared with other ground-dwelling ruderal plant species. Strength of differentiation between plant species and between A. thaliana populations shifts from year to year, becoming stronger when plants within populations have more similar leaf bacteriomes (less plant-to-plant variation or stochasticity). Interestingly, across different populations, plants with shared leaf aliphatic glucosinolate chemotypes exhibited similar strong year-to-year stochasticity shifts. Therefore, stochasticity of leaf bacteriomes in plant populations changes in specific ways and might be controlled by plant traits, with important implications for how plants adapt to complex and shifting environments.


title: Solute exchange through gap junctions lessens the adverse effects of inactivating mutations in metabolite-handling genes

abstract: Experimental inactivation of certain genes involved in metabolism attenuates cancer cell growth in vitro. However, loss-of-function mutations in metabolic pathways are not negatively selected in human cancers, indicating that these genes are not essential in vivo. We hypothesize that spontaneous mutations affecting metabolic pathways do not necessarily result in a functional defect because affected cells may be rescued by exchanging metabolites with neighboring wild-type cells via gap junctions. Using fluorescent substances to probe inter-cellular diffusion, we show that colorectal cancer (CRC) cells are coupled by gap junctions assembled from connexins, particularly the constitutively expressed Cx26. In co-cultures of wild-type cells with cells that had inactivated components of pH regulation (SLC9A1), glycolysis (ALDOA), or mitochondrial metabolism (NDUFS1), we show that diffusive coupling was able to rescue the functional defect associated with the inactivation of metabolite-handling genes. Function rescue was dependent on Cx26 channels and reduced phenotypic heterogeneity among cells. Since the phenotypic landscape did not map onto genotype, an individual cell should not be considered as the unit under selection, at least in the case of metabolite-handling processes. Our findings can explain why certain loss-of-function mutations in genes, previously ascribed as being  essential, do not influence the growth of human cancers.


title: The DNA methylation landscape of the human oxytocin receptor gene (OXTR): Recommendations for future research

abstract: The oxytocin receptor gene (OXTR) is of interest when investigating the effects of early adversity on DNA methylation. However, there is heterogeneity regarding the selection of the most promising CpG sites to target for analyses. The goal of this study was to determine functionally relevant clusters of CpG sites within the OXTR CpG island in 113 mother-infant dyads, with 58 of the mothers having experienced childhood maltreatment (CM). OXTR DNA methylation and gene expression was analyzed in peripheral/umbilical blood mononuclear cells. Different complexity reduction approaches were used to reduce the 188 CpG sites into clusters of co-methylated sites. Furthermore, associations between OXTR DNA methylation (cluster- and site-specific level) and OXTR gene expression and CM were investigated. Results showed that, first, CpG sections differed strongly regarding their statistical utility for research of individual differences in DNA methylation. Second, cluster analyses and Partial Least Squares (PLS) suggested two clusters consisting of intron1/exon2 and the protein-coding region of exon 3, respectively, as most strongly associated with outcome measures. Third, cross-validated PLS regression explained 7% of variance in CM, with low cross-validated variance explained for the prediction of gene expression. Fourth, very high mother-child correspondence was observed in correlation patterns within the identified clusters, but only modest correspondence outside these clusters. This study characterized the DNA methylation landscape of the OXTR CpG island by highlighting clusters of CpG sites that show desirable statistical properties and predictive value. We provide a Companion Web Application to guide future studies in their choice of CpG sites.


title: AHR agonist ITE boosted PD1 antibodys effects by inhibiting myeloid-derived cells suppressive cells in an orthotopic mouse glioma model

abstract: Glioblastoma is a "cold" tumor lacking T cell infiltration and tryptophan metabolites such as kynurenine function as an immune suppressor by binding to aryl hydrocarbon receptor(AHR). Hence AHR antagonists have been developed and some shown to activate the immune response in IDO over-expressing cancer models. Paradoxically, AHR has been reported to block glioma cell invasion like a tumor suppressor, and how to target AHR in cancer remains an open question. We previously discovered that an AHR agonist ITE can effectively inhibit glioma invasion. Here we report that ITE combined with PD1 antibody significantly increased the infiltration of CD8+ T cells while reducing that of the myeloid-derived suppressive cells (MDSCs), extending mouse survival. To identify factors that possibly mediated ITEs effects, we analyzed RNA-seq data and discovered that ITE significantly down-regulated IL11, a known MDSC regulator. In contrast, kynurenine upregulated IL11, further supporting IL11 as an AHR target. Moreover, ITE inhibited IL11s induction of MDSC from mouse PBMC in vitro, supporting that IL11 might mediate ITEs MDSC regulation effects. The discovery of ITEs immune-activating effects highlighted the complexity of AHRs signaling in cancers. The unexpected increase in STAT3, downstream of IL11 provided clues for combination therapy development.


title: Validating methods to eradicate Select Agent and phylotype I Ralstonia solanacearum strains reveals that growth in planta increases bacterial stress tolerance

abstract:
Ralstonia solanacearum is a destructive pathogen that causes bacterial wilt disease of diverse crops. Wilt disease prevention and management is difficult because R. solanacearum persists in soil, water, and plant material. Growers need practical methods to kill R. solanacearum in irrigation water, a common source of disease outbreaks. Additionally, the Race 3 biovar 2 (R3bv2) subgroup is a quarantine pest in many countries and a highly regulated U.S. Select Agent. Plant protection officials and researchers need validated protocols to eradicate R. solanacearum for regulatory compliance. To meet these needs, we measured survival of four R3bv2 and three phylotype I R. solanacearum strains following treatment with hydrogen peroxide, stabilized hydrogen peroxide (HuwaSan), active chlorine, heat, ultraviolet radiation, and desiccation. No surviving R. solanacearum cells were detected after cultured bacteria were exposed for ten minutes to 400 ppm hydrogen peroxide, 50 ppm HuwaSan, 50 ppm active chlorine, or temperatures above 50{degrees}C. R. solanacearum cells on agar plates were eradicated by 30s UV irradiation and killed by desiccation on most biotic and all abiotic surfaces tested. R. solanacearum did not survive the cell lysis steps of four nucleic acid extraction protocols. However, bacteria in planta were more difficult to kill. Stems of infected tomato plants contained a subpopulation of bacteria with increased tolerance of heat and UV light, but not oxidative stress. This result has significant management implications. We demonstrate the utility of these protocols for compliance with Select Agent research regulations and for management of a bacterial wilt outbreak in the field.

ImportanceRalstonia solanacearum, a globally distributed wilt pathogen of many high-value crops, is spread via diseased plant material and contaminated soil, tools, and irrigation water. The Race 3 biovar 2 Select Agent subgroup of R. solanacearum is subject to stringent and constantly evolving regulations intended to prevent pathogen introduction or release. We validated eradication and inactivation methods that can be used by: 1) growers seeking to disinfest water and manage bacterial wilt disease outbreaks; 2) researchers who must remain in compliance with regulations; and 3) regulators who are expected to define containment practices. Relevant to all these stakeholders, we show that while cultured R. solanacearum cells are sensitive to relatively low levels of oxidative chemicals, dessication, and heat, more aggressive treatment such as autoclaving or incineration is required to eradicate R. solanacearum cells growing inside plant material.


title: Genetic interaction analysis reveals that Cryptococcus neoformans utilizes multiple acetyl-CoA-generating pathways during infection

abstract:
Cryptococcus neoformans is an important human fungal pathogen for which the external environment is its primary niche. To cause infection and disease, C. neoformans must adapt to a plethora of conditions and stresses inherent to the host environment. Of these stresses, the role of central carbon metabolism has been of interest not only at the level of fundamental pathobiology but also as a potential target for new antifungal drug therapy. Previous work has shown that two non-essential acetyl-CoA metabolism enzymes, ATP-citrate lyase (ACL1) and acetyl-CoA synthetase (ACS1), play important roles in C. neoformans infection. Here, we took a genetic interaction approach to studying the interplay between these two enzymes along with an enzyme initially called ACS2 but which we have found is an acetoacetyl-CoA synthetase; we have renamed the gene 2-ketobutyryl CoA synthetase 1 (KBC1) based on its biochemical activity and the systematic name of its substrate. ACL1 and ACS1 represent a synthetic lethal pair of genes based on our genetic interaction studies. Double mutants of KBC1 with either ACS1 or ACL1 do not have significant synthetic phenotypes in vitro, although we find that deletion of any one of these enzymes reduces fitness within macrophages. Importantly, the acs1{Delta} kbc1{Delta} double mutant has significantly reduced fitness in the CNS relative to either single mutant as well as WT (~2 log10 CFU reduction in fungal burden), indicating the important role these enzymes play during infection. The expression of both ACS1 and KBC1 is increased in vivo relative to in vitro conditions. The acs1{Delta} mutant is hypersusceptible to fluconazole in vivo despite its minimal in vitro phenotypes. These data not only provide insights into the in vivo mechanism of action for a new class of antifungal Acs inhibitors but also into metabolic adaptations of C. neoformans to the host environment.

Author SummaryThe adaptation of environmental fungal pathogens to the mammalian host is critical to pathogenesis. Of these adaptations, the remodeling of carbon metabolism is particularly important. Here, we generated a focused set of double mutants of non-essential genes (ACL1, ACS1, KBC1) involved in acetyl-CoA metabolism and examined their fitness in vitro and during CNS infection. From these studies, we found that all three enzymes play important roles during infection but that the role of ACS1/KBC1 was minimal in vitro. Consistent with these observations, the expression of ACS1 and KBC1 was increased in vivo relative to standard in vitro conditions. Furthermore, strains lacking both ACL1 and ACS1 were not viable. These data clearly show that C. neoformans employs multiple carbon metabolism pathways to adapt to the host environment. They also provide insights into the potential mechanism of action for anti-cryptococcal Acs inhibitors.


title: Multigrate: single-cell multi-omic data integration

abstract: Single-cell multimodal omics technologies provide a holistic approach to study cellular decision making. Yet, learning from multimodal data is complicated because of missing and incomplete reference samples, non-overlapping features and batch effects between datasets. To integrate and provide a unified view of multi-modal datasets, we propose Multigrate. Multigrate is a generative multi-view neural network to build multimodal reference atlases. In contrast to existing methods, Multigrate is not limited to specific paired assays, and it compares favorably to existing data-specific methods on both integration and imputation tasks. We further show that Multigrate equipped with transfer learning enables mapping a query multimodal dataset into an existing reference atlas.


title: Multimodal spatiotemporal phenotyping of human organoid development

abstract: Organoids generated from human pluripotent stem cells (PSCs) provide experimental systems to study development and disease. However, we lack quantitative spatiotemporal descriptions of organoid development that incorporate measurements across different molecular modalities. Here we focus on the retina and use a single-cell multimodal approach to reconstruct human retinal organoid development. We establish an experimental and computational pipeline to generate multiplexed spatial protein maps over a retinal organoid time course and primary adult human retina, registering protein expression features at the population, cellular, and subcellular levels. We develop an analytical toolkit to segment nuclei, identify local and global tissue units, infer morphology trajectories, and analyze cell neighborhoods from multiplexed imaging data. We use this toolkit to visualize progenitor and neuron location, the spatial arrangements of extracellular and subcellular components, and global patterning in each organoid and primary tissue. In addition, we generate a single-cell transcriptome and chromatin accessibility time course dataset and infer a gene regulatory network underlying organoid development. We then integrate genomic data with spatially segmented nuclei into a multi-modal atlas enabling virtual exploration of retinal organoid development. We visualize molecular, cellular, and regulatory dynamics during organoid lamination, and identify regulons associated with neuronal differentiation and maintenance. We use the integrated atlas to explore retinal ganglion cell (RGC) spatial neighborhoods, highlighting pathways involved in RGC cell death. Finally, we show that mosaic CRISPR/Cas genetic perturbations in retinal organoids provide insight into cell fate regulation. Altogether, our work is a major advance toward a virtual human retinal organoid, and provides new directions for how to approach disorders of the visual system. More broadly, our approaches can be adapted to many organoid systems.

category: systems biology


title: Hypothalamic-pituitary-adrenal axis dysfunction worsens epilepsy outcomes and increases SUDEP risk

abstract: Epilepsy is often comorbid with psychiatric illnesses, including anxiety and depression. Despite the high incidence of psychiatric comorbidities in people with epilepsy, few studies address the underlying mechanisms. Stress can trigger both epilepsy and depression and significant accumulating evidence, from both human and animal studies, support that hypothalamic-pituitary-adrenal (HPA) axis dysfunction may contribute to the comorbidity of these two disorders1. Here we directly investigate the contribution of HPA axis dysfunction to epilepsy outcomes and psychiatric comorbidities. We generated a novel mouse model (KCC2/Crh mice) that lacks the K+/Cl- co-transporter, KCC2, in corticotropin-releasing hormone (CRH) neurons, which exhibit stress- and seizure-induced hyperactivation of the HPA axis.2,3 Here we demonstrate that HPA axis dysfunction in KCC2/Crh mice is associated with increased vulnerability to behavioral deficits in chronically epileptic mice and more severe neuropathological features associated with chronic epilepsy (e.g. increased mossy fiber sprouting). Despite equivalent seizure burden, HPA axis dysfunction in these chronically epileptic mice increased the incidence of sudden unexpected death in epilepsy (SUDEP). Suppressing HPA axis hyperexcitability in this model using a pharmacological or chemogenetic approach decreased SUDEP incidence, supporting HPA axis dysfunction in SUDEP vulnerability. We also observed changes in neuroendocrine markers in individuals that died of SUDEP compared to people with epilepsy or individuals without a history of epilepsy. Together, these findings describe a novel, nongenetic mouse model of SUDEP which will benefit preclinical SUDEP research to gain a better understanding of the underlying pathological mechanisms contributing to SUDEP. HPA axis dysfunction increases vulnerability to psychiatric comorbidities in epilepsy and may be a risk factor for SUDEP.


title: Proportional intracranial volume correction differentially biases behavioral predictions across neuroanatomical features and populations

abstract: Individual differences in brain anatomy can be used to predict variability in cognitive function. Most studies to date have focused on broad population-level trends, but the extent to which the observed predictive features are shared across sexes and age groups remains to be established. While it is standard practice to account for intracranial volume (ICV) using proportion correction in both regional and whole-brain morphometric analyses, in the context of brain-behavior predictions the possible differential impact of ICV correction on anatomical features and subgroups within the population has yet to be systematically investigated. In this work, we evaluate the effect of proportional ICV correction on sex-independent and sex-specific predictive models of individual cognitive abilities across multiple anatomical properties (surface area, gray matter volume, and cortical thickness) in healthy young adults (Human Connectome Project; n=1013, 548 females) and typically developing children (Adolescent Brain Cognitive Development study; n=1823, 979 females). We demonstrate that ICV correction generally reduces predictive accuracies derived from surface area and gray matter volume, while increasing predictive accuracies based on cortical thickness in both adults and children. Furthermore, the extent to which predictive models generalize across sexes and age groups depends on ICV correction: models based on surface area and gray matter volume are more generalizable without ICV correction, while models based on cortical thickness are more generalizable with ICV correction. Finally, the observed neuroanatomical features predictive of cognitive abilities are unique across age groups regardless of ICV correction, but whether they are shared or unique across sexes (within age groups) depends on ICV correction. These findings highlight the importance of considering individual differences in ICV, and show that proportional ICV correction does not remove the effects of cranium volumes from anatomical measurements and can introduce ICV bias where previously there was none. ICV correction choices affect not just the strength of the relationships captured, but also the conclusions drawn regarding the neuroanatomical features that underlie those relationships.


title: Chimpanzees organize their social relationships like humans

abstract: Human relationships are structured in a set of layers, ordered from higher (intimate relationships) to lower (acquaintances) emotional and cognitive intensity. This structure arises from the limits of our cognitive capacity and the different amounts of resources required by different relationships. However, it is unknown whether nonhuman primate species organize their affiliative relationships following the same pattern. We here show that the time chimpanzees devote to grooming other individuals is well described by the same model used for human relationships, supporting the existence of similar social signatures for both humans and chimpanzees. Furthermore, the relationship structure depends on group size as predicted by the model, the proportion of high intensity connections being larger for smaller groups.

category: animal behavior and cognition


title: Medial septum glutamate neurons are essential for spatial goal-directed memory

abstract: The medial septum (MS), a brain region containing acetylcholine, GABA and glutamate neurons, is essential for learning and memory. However, whether MS glutamate neurons contribute to memory is unknown. Here, we use calcium imaging and optogenetic silencing to determine the function of MS glutamate neurons in mice performing a spatial allocentric memory navigation task. While MS glutamate neurons appeared randomly active during free exploration, two groups of glutamate neurons emerged during training in a five-arm star maze, distinguished by their peak activity. The largest group was predominantly activated before locomotion whereas the other was primarily active when mice reached the reward site. Both populations were preferentially activated for correct over incorrect trajectories. Interestingly, optogenetic silencing of MS glutamate neurons immediately before mice start navigating, induced significant spatial memory impairments. Together these results demonstrate that MS glutamate neurons heterogeneously encode navigationally relevant information and are essential for spatial learning.