Biology subjects

Ramos, A.

Publications and source records attributed to Ramos, A..

At least 19 recordsLinked to original sources

bioRxiv2026

BettaAI: a machine-learning object detection model for the quantification of aggressive displays in the Siamese fighting fish Betta splendens

The Siamese fighting fish (Betta splendens) is well-known for its high levels of aggression and complex, stereotyped displays, making it an ideal model for testing deep learning-based tools for high-throughput, unbiased quantification of aggressive behaviour. A deep-learning object recognition model (BettaAI) was developed to detect the species' aggressive displays, including threat (opercula opening and fin distension), attack (bites) behaviours, as well as surface air breathing, a behaviour correlated with the frequency and intensity of aggression. Videos of males confronting their mirror image were acquired simultaneously with synchronised top and side cameras. Two separate models were trained, with some behaviours identified from top-view frames and others from side-view frames; outputs from both models were combined and post-processed for final quantification. BettaAI performed with high accuracy: F1 scores were 0.99 for the top-view model and 0.93 for the side-view model. Average Precision (AP) at 0.5 IoU exceeded 0.98 for all categories except bites, a rare behaviour in the dataset, which reached 0.865. Comparisons with a manually curated "ground-truth" dataset from videos of 10 males showed strong concordance between automated quantification and manual annotations by expert observers across all behaviours. A case study on 461 males reacting either to a mirror or to a video playback of a conspecific demonstrated the high-throughput and unbiased advantages of BettaAI, with threat displays being more frequent toward the mirror image and attack rates not differing between treatments. This work underscores the potential of BettaAI for automated, bias-free quantification of aggressive displays in B. splendens.

animal behavior and cognition↗

bioRxiv2026

Physiological folate levels constrain nucleotide synthesis and increase dependence on nucleotide salvage

Proliferating cells must acquire nucleotides to support DNA replication, yet how cells meet these nucleotide demands for proliferation under physiological conditions remains understudied. Here, we investigated how physiological nutrient availability shapes nucleotide acquisition strategies in a mouse model of B-cell acute lymphoblastic leukemia (B-ALL). To assess how environmental nutrients impact nucleotide metabolism, we formulated a mouse plasma-like medium (MPM) that reproduces the circulating metabolite composition of plasma from mice with B-ALL and assessed how this influenced nucleotide metabolism relative to standard culture conditions, where nucleotide acquisition has historically been studied. We find that leukemia cells cultured in MPM acquire nucleotides through salvage pathways, and that select nucleotide salvage pathways are required for proliferation under physiological conditions. Of note, this dependency on nucleotide salvage in plasma-like conditions was not caused by precursor metabolite limitation for de novo synthesis. Instead, we found that physiological folate levels are insufficient to support deoxynucleotide triphosphate (dNTP) synthesis for genome replication, leading to DNA replication stress and impaired proliferation when nucleotide salvage is disrupted. Consistently, dietary folate restriction exacerbates the impaired leukemia progression phenotype of nucleotide salvage-deficient B-ALL cells. Together, these findings demonstrate that access to folates is an endogenous limitation for nucleotide synthesis in plasma-like nutrient conditions, increasing the relevance of nucleotide salvage pathways for leukemia progression. More broadly, this work highlights how micronutrient abundance can influence metabolic dependencies and reveals that folate levels shape nucleotide metabolism under physiological conditions.

cancer biology↗

bioRxiv2026

Mesenchymal predominance in olfactory epithelium-derived cultures limits modeling of neurodevelopmental brain disorders

The human olfactory epithelium (OE) represents a lifelong source of neural progenitor cells and has been proposed as an accessible model to investigate molecular alterations associated with neurodevelopmental disorders in postnatal individuals. Globose basal cells are considered the immediate neuronal progenitors within the OE, and several studies have attempted to culture these cells from nasal exfoliates. However, the actual contribution of neurogenic lineages in these cultures remains largely unquantified. Here, we cultured human nasal explants using an established protocol and characterized the resulting cell populations by immunohistochemistry and single-cell RNA sequencing. Integration with primary in vivo OE datasets revealed that these cultures are predominantly composed of mesenchymal-like cells, with limited representation of globose basal cells and neurons, and low expression of canonical neuronal markers. Using curated gene sets associated with neurodevelopmental disorders and malformations of cortical development, we assessed the extent to which disease-relevant transcriptional programs are captured in OE-derived cultures. While disease-associated genes are enriched in neurogenic lineages in vivo, their representation in mesenchymal cells is reduced. Together, our results challenge the assumption that standard OE culture systems faithfully model neurogenic compartments and suggest that current approaches may need refinement to recover neurogenic lineages.

neuroscience↗

bioRxiv2025

Expanding the toolbox for hypervirulent Klebsiella pneumoniae using the social amoeba Dictyostelium discoideum as a virulence biosensor

Hypervirulent Klebsiella pneumoniae (hvKp) represents a critical global threat due to its capacity to cause severe, community-acquired infections in healthy individuals and its convergence with multidrug resistance. However, current methods to evaluate hvKp virulence are limited by their reliance on mammalian models, which are costly, ethically constrained, and unsuitable for high-throughput applications. Here, we introduce a biosensing approach using the social amoeba Dictyostelium discoideum as a living phenotypic sensor for hvKp virulence. Through a suite of quantitative assays integrating predation resistance, social development, and real-time single-cell motility tracking, we show that D. discoideum can discriminate avirulent, virulent, and hypervirulent K. pneumoniae strains. Using the model hvKp strain SGH10 and its capsule-null mutant {Delta}wcaJ, we demonstrate that capsule production is strongly associated with phagocytosis resistance, developmental arrest, and amoebae paralysis, all quantifiable through imaging and motility-based readouts. Multivariate data integration revealed a clear segregation of virulence phenotypes, validating D. discoideum as a sensitive, scalable, and genetically tractable biosensor for functional virulence detection. This work establishes a conceptual and methodological framework for developing cell-based biosensors that report virulence levels through measurable host behavioral responses and outlines a calibration path to vertebrate endpoints. These systems provide a low-cost alternative to mammalian systems, especially for early-stage evaluation, and a potential foundation for automated high-throughput screening and surveillance of virulence in critical priority bacterial pathogens.

microbiology↗

bioRxiv2025

Oxytocin neurons in the anterior and posterior paraventricular nucleus have distinct behavioral functions and electrophysiological profiles

Oxytocin is a neuropeptide that can promote or inhibit affiliative social behaviors. Recent evidence suggests that these diverse effects are mediated by distinct oxytocin receptor-expressing neurons. An outstanding question is whether these behavioral effects are also driven by distinct or overlapping populations of oxytocin-producing neurons. The paraventricular nucleus (PVN) of the hypothalamus is a major source of oxytocin and sends projections to the mesolimbic dopamine system and extended amygdala. Previous work found that social defeat increased oxytocin neuron activity in the anterior PVN (aPVN) but not posterior PVN (pPVN). We reduced oxytocin synthesis with antisense morpholino oligonucleotides in either anterior or posterior PVN in California mice (Peromyscus californicus), a strong model system for studying effects of social stress on brain function and behavior. Antisense morpholinos in aPVN had no effect on behavior in unstressed females but increased social approach and reduced social vigilance in females exposed to social defeat stress. In pPVN, antisense morpholinos reduced social approach in unstressed male and female California mice. We then used OxtCre mice to compare electrophysiological profiles of oxytocin in aPVN and pPVN with a population of oxytocin neurons in the bed nucleus of the stria terminalis (BNST). Oxytocin neurons in aPVN and BNST had higher post-synaptic events and responded more strongly to current injections than oxytocin neurons in pPVN, though they had similar excitatory and inhibitory input balance at the observed resting membrane potential. These findings shed light onto functional and physiological heterogeneity of PVN oxytocin neurons. Our results suggest that context dependent effects of oxytocin are mediated by different populations of oxytocin neurons.

neuroscience↗

bioRxiv2025

Cellular characterization of the peritumoral brain zone of glioblastomas: heterogenous nature and clinical relevance.

BackgroundGlioblastoma (GBM) is a highly aggressive cancer with near-universal recurrence, often due to residual tumor cells that persist after aggressive standard of care treatment. This study aimed to characterize tumor infiltration and microenvironment in the GBM periphery. MethodsWe prospectively collected 161 radiologically guided biopsies from 45 GBM patients and conducted an immunohistochemical analysis. We also integrated single-cell RNA sequencing data to select specific markers for immune, glial, and vascular cells. We measured the expression of these genes in samples from contrast-enhancing (CE) and non-enhancing (nCE) tumor areas, vasogenic edema, and radiologically normal tissue. Correlations with resection extent and clinical outcomes were evaluated. ResultsnCE biopsies exhibited neoplastic features similar to those of the tumor core. However, tumor infiltration was also found in regions classified radiologically as edema, particularly in elderly patients. We found important differences in the composition of the peripheral microenvironment between male and female GBM patients. Prognostic associations with specific cell types, such as myeloid cells, showed intertumor heterogeneity, with variations depending on patient sex, age and extent of resection. Furthermore, in our cohort, minimal residual CE tumor following surgery was associated with significantly poorer patient survival. ConclusionsThe GBM periphery includes regions of active tumor growth that are visible on MRI, as well as infiltrated areas that resemble edema radiologically. Tumor infiltration and microenvironmental features are influenced by patient sex and age, which has major implications for recurrence rates, highlighting the need to tailor surgical and therapeutic strategies based on tumor biology and patient subgroup. Key pointsnCE areas show similar neoplastic traits to the CE tumor. GBM infiltrates edema tissue, predominantly in older adult patients. Prognostic value of the peritumoral phenotype depends on resection extent and patient age/sex. Importance of the studyThis study shows that the impact of peripheral and core cellular features on prognosis differs between patients who undergo complete versus incomplete CE tumor resection. Our results suggest a paradigm shift in the classification and management of these patients, encouraging the inclusion of detailed post-surgical MRI analyses to guide the design of future clinical trials according to the nature and extent of the residual disease. Furthermore, our data confirm the presence of tumorigenic features in non-enhancing areas, supporting the benefits of supratotal resections according to the new RANO classification. Our findings also underscore the need to refine surgical and therapeutic strategies based on a more detailed understanding of the tumor microenvironment beyond the GBM core. This understanding may help identify novel targets for more effective and personalized GBM therapies.

cancer biology↗

bioRxiv2025

A Generalized Platform for Artificial Intelligence-powered Autonomous Protein Engineering

Proteins are the molecular machines of life with numerous applications in energy, health, and sustainability. However, engineering proteins with desired functions for practical applications remains slow, expensive, and specialist-dependent1-3. Here we report a generally applicable platform for autonomous protein engineering that integrates machine learning and large language models with biofoundry automation to eliminate the need for human intervention, judgement, and domain expertise. Requiring only an input protein sequence and a quantifiable way to measure fitness, this autonomous platform can be applied to engineer virtually any protein. As a proof of concept, we engineered Arabidopsis thaliana halide methyltransferase (AtHMT)4 for a 90-fold improvement in substrate preference and 16-fold improvement in ethyltransferase activity, along with developing a Yersinia mollaretii phytase (YmPhytase)5,6 variant with 26-fold improvement in activity at neutral pH. This was accomplished in four rounds over four weeks, while requiring construction and characterization of fewer than a total of 500 variants for each enzyme. This platform for autonomous experimentation paves the way for rapid advancements across diverse industries, from medicine and biotechnology to renewable energy and sustainable chemistry.

synthetic biology↗

bioRxiv2024

Exploring tropical freshwater soundscapes in Southeast Asia: insights into auditory sensory adaptation of wild Siamese fighting fish Betta splendens

While soundscapes shape the structure and function of auditory systems over evolutionary timescales, there is limited information regarding the adaptation of wild fish populations to their natural acoustic environments. This is particularly relevant for freshwater ecosystems, which are extremely diverse and face escalating pressures from human activities and associated noise pollution. The Siamese fighting fish Betta splendens is one of the most important cultured species in the global ornamental fish market and is increasingly recognized as a model organism for genetics and behavioural studies. This air-breathing species (Anabantoidei), characterized by the presence of a suprabranchial labyrinth organ, is native to Southeast Asia and inhabits low flow freshwater ecosystems that are increasingly threatened due to habitat destruction and pollution. We characterized the underwater soundscape, along with various ecological parameters, across five marshland habitats of B. splendens, from lentic waterbodies to small canals near a lake in Chiang Rai province (Thailand). All habitats exhibited common traits of low dissolved oxygen and dense herbaceous vegetation. Soundscapes were relatively quiet with Sound Pressure Level (SPL) around 102-105 dB re 1 {micro}Pa and most spectral energy below 1000 Hz. Sound recordings captured diverse biological sounds, including potential fish vocalizations, but primarily insect sounds. Based on Auditory Evoked Potential (AEP) recordings, we identified sex-specific differences and a best hearing range within 100-400 Hz. This low-frequency tuning highlights the potential susceptibility of B. splendens to anthropogenic noise activities. This study provides first characterization of the auditory sensitivity and natural soundscape of B. splendens, establishing an important ground for future hearing research in this species. The information provided on the auditory sensory adaptation of B. splendens emphasizes the importance of preserving quiet soundscapes from lentic freshwater ecosystems.

zoology↗

bioRxiv2024

CRISPR targeting of FOXL2 c.402C>G mutation reduces malignant phenotype in granulosa tumor cells and identifies anti-tumoral compounds

FOXL2 is a transcription factor essential for sex determination and ovary development and maintenance. Mutations in this gene are implicated in syndromes involving premature ovarian failure and granulosa cell tumors (GCTs). This rare cancer accounts for less than 5% of diagnosed ovarian cancers and is causally associated with the FOXL2 c.402C>G, p.C134W mutation in 97% of the adult cases (AGCTs). In this study, we employed CRISPR technology to specifically eliminate the FOXL2 c.402C>G mutation in granulosa tumor cells. Our results show that this Cas9-mediated strategy selectively targets the mutation without affecting the wild-type allele. Granulosa cells lacking the FOXL2 c.402C>G exhibit a reduced malignant phenotype, with significant changes in cell proliferation, invasion, and cell death. Furthermore, these modified cells are more susceptible to Dasatinib and Ketoconazole. Transcriptomic and proteomic analyses reveal that CRISPR-modified granulosa tumor cells shift their expression profiles towards a wild-type like phenotype. Additionally, this altered expression signature has led to the identification of new compounds with antiproliferative and pro-apoptotic effects on granulosa tumor cells. Our findings demonstrate the potential of CRISPR technology for the specific targeting and elimination of a mutation causing granulosa cell tumors, highlighting its therapeutic promise for treating this rare ovarian cancer. Simple SummaryAdult granulosa cell tumors (AGCTs), characterized by a specific point mutation (C134W) in the gene FOXL2, are less than 5% of all the diagnosed ovarian cancers. Surgery is the cornerstone treatment for AGCT even at relapse, with systemic therapy showing poor results. The aim of our study is to explore the potential therapeutic effect of the elimination of the C134W mutation. To achieve our goal, we have eliminated the mutant allele using CRISPR technology. Our results demonstrate that CRISPR-mediated elimination of FOXL2-C134W reduces the malignant phenotype of granulosa tumor cells, which change their transcriptional, proteomic, and cellular phenotype to a wild type like, granulosa type. Moreover, the induced changes allowed us to find new compounds with antitumoral activity. This work highlights the therapeutic potential of CRISPR mediated technology for the treatment of AGCT.

cancer biology↗

bioRxiv2024

Elaboration of the Homer1 Recognition Landscape Reveals Incomplete Divergence of Paralogous EVH1 Domains

Short sequences that mediate interactions with modular binding domains are ubiquitous throughout eukaryotic proteomes. Networks of Short Linear Motifs (SLiMs) and their corresponding binding domains orchestrate many cellular processes, and the low mutational barrier to evolving novel interactions provides a way for biological systems to rapidly sample selectable phenotypes. Mapping SLiM binding specificity and the rules that govern SLiM evolution is fundamental to uncovering the pathways regulated by these networks and developing the tools to manipulate them. We used high-throughput screening of the human proteome to identify sequences that bind to the Enabled/VASP homology 1 (EVH1) domain of the postsynaptic density scaffolding protein Homer1. In doing so, we expanded current understanding of the determinants of Homer EVH1 binding preferences and defined a new motif that can facilitate the discovery of additional Homer-mediated interactions. Interestingly, the Homer1 EVH1 domain preferentially binds to sequences containing an N-terminally overlapping motif that is bound by the paralogous family of Ena/VASP actin polymerases, and many of these sequences can bind to EVH1 domains from both protein families. We provide evidence from orthologous EVH1 domains in pre-metazoan organisms that the overlap in human Ena/VASP and Homer binding preferences corresponds to an incomplete divergence from a common Ena/VASP ancestor. Given this overlap in binding profiles, promiscuous sequences that can be recognized by both families either achieve specificity through extrinsic regulatory strategies or may provide functional benefits via multi-specificity. This may explain why these paralogs incompletely diverged despite the accessibility of further diverged isoforms. Impact StatementShort linear motifs bind to structurally conserved domains to mediate many protein interactions. Current definitions of motifs, using regular expressions, are over simplified and do not capture what is required for binding. Unbiased proteome interaction screens can refine these definitions. For EVH1 domains considered in this work, screening elucidated Homer1 binding requirements and revealed a residual overlap of specificity of two anciently diverged families, highlighting both opportunities and challenges of multi-specificity in domain-motif interactions.

biochemistry↗

bioRxiv2023

Germline-targeting chimpanzee SIV Envelopes induce V2-apex broadly neutralizing-like B cell precursors in a rhesus macaque infection model

Eliciting broadly neutralizing antibodies-(bnAbs) remains a major goal of HIV-1 vaccine research. Previously, we showed that a soluble chimpanzee SIV Envelope-(Env) trimer, MT145K, bound several human V2-apex bnAb-precursors and stimulated an appropriate response in V2-apex bnAb precursor-expressing knock-in mice. Here, we tested the immunogenicity of three MT145 variants (MT145, MT145K, MT145K.dV5) expressed as chimeric simian-chimpanzee-immunodeficiency-viruses-(SCIVs) in rhesus macaques-(RMs). All three viruses established productive infections with high setpoint vRNA titers. RMs infected with the germline-targeting SCIV_MT145K and SCIV_MT145K.dV5 exhibited larger and more clonally expanded B cell lineages featuring long anionic heavy chain complementary-determining-regions-(HCDR3s) compared with wildtype SCIV_MT145. Moreover, antigen-specific B cell analysis revealed enrichment for long-CDHR3-bearing antibodies in SCIV_MT145K.dV5 infected animals with paratope features resembling prototypic V2-apex bnAbs and their precursors. Although none of the animals developed bnAbs, these results show that germline-targeting SCIVs can activate and preferentially expand B cells expressing V2-apex bnAb-like precursors, the first step in bnAb elicitation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=180 HEIGHT=200 SRC="FIGDIR/small/558743v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@17673c8org.highwire.dtl.DTLVardef@1ab61a8org.highwire.dtl.DTLVardef@98781forg.highwire.dtl.DTLVardef@1f63e9b_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

m6a methylation orchestrates IMP1 regulation of microtubules during human neuronal differentiation

Neuronal differentiation requires building a complex intracellular architecture, and therefore the coordinated regulation of defined sets of genes. RNA-binding proteins (RBPs) play a key role in this regulation. However, while their action on individual mRNAs has been explored in depth, the mechanisms used to coordinate expression of the gene programs shaping neuronal morphology are poorly understood. To address this, we analysed how the paradigmatic RBP IMP1 (IGF2BP1), an essential developmental factor, selects and regulates its RNA targets during the differentiation of human neurons. We performed a combination of system-wide and molecular analyses, revealing that IMP1 developmentally transitions to and directly regulates the expression of mRNAs encoding essential regulators of the microtubule network, a key component of neuronal morphology. Furthermore, we showed that m6A methylation drives the selection of specific IMP1 mRNA targets and their protein expression during the developmental transition from neural precursors to neurons, providing a molecular principle for the onset of target selectivity.

molecular biology↗

bioRxiv2022

The nuclear GAPDH-HMGB cascade in cortical microglia regulates cognitive flexibility

We report a mechanism that underlies stress-induced cognitive inflexibility at the molecular level. In a mouse model under subacute stress in which deficits in rule shifting tasks were elicited, the nuclear glyceraldehyde dehydrogenase (N-GAPDH) cascade was activated specifically in microglia in the prelimbic cortex. The cognitive deficits were normalized with a pharmacological intervention with a compound (the RR compound) that selectively blocked the initiation of N-GAPDH cascade without affecting glycolytic activity. The normalization was also observed with a microglia-specific genetic intervention targeting the N-GAPDH cascade. Furthermore, hyperactivation of the prelimbic layer 5 excitatory neurons, which are known to be a neuronal substrate of cognitive inflexibility, was also normalized by the pharmacological and microglia-specific genetic interventions. The RR compound may offer a mechanism-driven, translational opportunity against stress-induced cognitive inflexibility. Taken together, we show a pivotal role of cortical microglia and microglia-neuron interaction in stress-induced cognitive inflexibility. We underscore the N-GAPDH cascade in microglia, which causally mediates stress-induced cognitive alteration.

molecular biology↗

bioRxiv2022

Hormone levels increase in response to video playback in the Siamese fighting fish

The mechanisms of action of hormones regulating aggression in fish remain poorly understood. One possibly confounding variable is the lack of standardization in the type of stimuli used to elicit aggression. The presentation of controlled stimuli in videos, a.k.a. video playback, can provide better control of the fight components. However, this technique has produced conflicting results in animal behaviour studies and needs to be carefully validated. Here, the response of male Betta splendens to a matched for size conspecific fighting behind a one-way mirror presented live or through video playback was compared. Despite their non-interactive nature, both stimuli elicited a strong aggressive response by focal fish, which started with stereotypical threat displays and progressed to overt attacks. Overall, the frequency and duration of aggressive behaviours and swimming activity were similar towards live and video stimuli. Post-fight plasma levels of the androgen 11-ketotestosterone were elevated as compared to controls, regardless of the type of stimuli. Cortisol also increased in response to the video images, as previously described for interactive live fights in this species. The study shows for the first time in a fish a robust endocrine response to video stimuli and supports the use of this technique for researching aggressive behaviour in B. splendens.

animal behavior and cognition↗

bioRxiv2022

Leukemia-intrinsic determinants of CAR-T response revealed by in vivo genome-wide CRISPR screening.

CAR-T therapy is a promising new treatment modality for B-cell malignancies. However, the majority of patients inevitably go on to experience disease relapse through largely unknown means. To investigate leukemia-intrinsic CAR-T resistance mechanisms, we performed genome-wide CRISPR-Cas9 loss-of-function screens in an immunocompetent murine model of B-cell acute lymphoblastic leukemia (B-ALL) utilizing a novel, modular guide RNA library. We identified IFN{gamma}/JAK/STAT signaling and components of antigen processing and presentation pathway as key mediators of resistance to CAR-T therapy in vivo, but not in vitro. Transcriptional characterization of this model demonstrated an upregulation of these pathways in CAR-T treated relapsed tumors, and examination of data from CAR-T treated patients with B-ALL revealed an association between poor outcomes and increased expression of JAK/STAT/MHC-I in leukemia cells. Overall, our data identify an unexpected mechanism of resistance to CAR-T therapy in which tumor cell interaction with CAR-T cells in vivo induces expression of an adaptive T-cell resistance program in tumor cells.

cancer biology↗

bioRxiv2021

Prenatal immune stress induces a prolonged blunting of microglia activation that impacts striatal connectivity

Recent studies suggested that microglia, the primary brain immune cells, can affect circuit connectivity and neuronal function1-3. Microglia infiltrate the neuroepithelium early in embryonic development and are maintained in the brain throughout adulthood4,5. Several maternal environmental factors, such as aberrant microbiome, immune activation, and poor nutrition, can influence prenatal brain development6-8. Nevertheless, it is unknown how changes in the prenatal environment instruct the developmental trajectory of infiltrating microglia, which in turn affect brain development and function. Here we show that after maternal immune activation (MIA) microglia from the offspring have a long-lived decrease in immune reactivity (blunting) across the developmental trajectory. The blunted immune response was concomitant with changes in the chromatin accessibility and reduced transcription factor occupancy of the open chromatin. Single cell RNA sequencing revealed that MIA does not induce a distinct subpopulation but rather decreases the contribution to inflammatory microglia states. Prenatal replacement of MIA microglia with physiological infiltration of naive microglia ameliorated the immune blunting and restored a decrease in presynaptic vesicle release probability onto dopamine receptor type-two medium spiny neurons, indicating that aberrantly formed microglia due to an adverse prenatal environment impacts the long-term microglia reactivity and proper striatal circuit development.

neuroscience↗

bioRxiv2021

Functional connectomics spanning multiple areas of mouse visual cortex

To understand the brain we must relate neurons functional responses to the circuit architecture that shapes them. Here, we present a large functional connectomics dataset with dense calcium imaging of a millimeter scale volume. We recorded activity from approximately 75,000 neurons in primary visual cortex (VISp) and three higher visual areas (VISrl, VISal and VISlm) in an awake mouse viewing natural movies and synthetic stimuli. The functional data were co-registered with a volumetric electron microscopy (EM) reconstruction containing more than 200,000 cells and 0.5 billion synapses. Subsequent proofreading of a subset of neurons in this volume yielded reconstructions that include complete dendritic trees as well the local and inter-areal axonal projections that map up to thousands of cell-to-cell connections per neuron. Here, we release this dataset as an open-access resource to the scientific community including a set of tools that facilitate data retrieval and downstream analysis. In accompanying papers we describe our findings using the dataset to provide a comprehensive structural characterization of cortical cell types1-3 and the most detailed synaptic level connectivity diagram of a cortical column to date2, uncovering unique cell-type specific inhibitory motifs that can be linked to gene expression data4. Functionally, we identify new computational principles of how information is integrated across visual space5, characterize novel types of neuronal invariances6 and bring structure and function together to decipher a general principle that wires excitatory neurons within and across areas7, 8.

neuroscience↗

bioRxiv2021

CARPOOL: A library-based platform to rapidly identify next generation chimeric antigen receptors

CD19-targeted CAR therapies have successfully treated B cell leukemias and lymphomas, but many responders later relapse or experience toxicities. CAR intracellular domains (ICDs) are key to converting antigen recognition into anti-tumor effector functions. Despite the many possible immune signaling domain combinations that could be included in CARs, almost all CARs currently rely upon CD3{zeta}, CD28, and/or 4-1BB signaling. To explore the signaling potential of CAR ICDs, we generated a library of 700,000 CD19 CAR molecules with diverse signaling domains and developed a high throughput screening platform to enable optimization of CAR signaling for anti-tumor functions. Our strategy identifies CARs with novel signaling domain combinations that elicit distinct T cell behaviors from a clinically available CAR, including enhanced proliferation and persistence, lower exhaustion, potent cytotoxicity in an in vitro tumor rechallenge condition, and comparable tumor control in vivo. This approach is readily adaptable to numerous disease models, cell types, and selection conditions, making it a promising tool for rapidly improving adoptive cell therapies and expanding their utility to new disease indications.

immunology↗