Biology subjects

Lau, J.

Publications and source records attributed to Lau, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Cyanogenic clovers invest more in rhizobia but do not affect rhizobium evolution

Nitrogen governs the interactions between leguminous plants and their nitrogen-fixing resource mutualists, rhizobia. While most work has focused on how soil nitrogen availability shapes the ecology and evolution of the legume-rhizobium mutualism, the nitrogen demands of plants may also influence the mutualism. Plants differ in nitrogen demand, in part because of variation in specialized metabolite production, particularly in species that produce nitrogen-based defense compounds such as cyanogenic glycosides. Plants with high nitrogen demand might be expected to be more reliant on rhizobia, potentially investing more in rhizobia and exerting stronger preferences for high-quality mutualists. Here, we use white clover (Trifolium repens), which is polymorphic for cyanogenesis, from two biparental F3 mapping populations to examine whether nitrogen-based chemical defenses affect plant investment in the mutualism or rhizobium evolution. By inoculating cyanogenic and acyanogenic clover lines with a single strain of Rhizobium leguminosarum, we found that cyanogenic clovers produced larger root nodules than acyanogenic clover, suggesting that cyanogensis is associated with increased investment in rhizobia. Greater investment in rhizobia could select for more cooperative mutualists if accompanied by partner choice or sanctions allowing plants to preferentially reward more cooperative rhizobia. We conditioned soils with cyanogenic and acyanogenic clover lines for five plant growth cycles and compared the partner quality of rhizobia isolated from these soils. Rhizobia evolved in association with cyanogenic versus acyanogenic clover did not differ in quality. Overall, our results show that N-based anti-herbivore defense traits can influence plant investment in resource mutualists but suggest that differences in investment do not necessarily translate into long-term evolutionary changes in mutualist quality.

ecology↗

bioRxiv2026

Evaluation of a Novel Recombinant Human Protein Formula Compared to Donor Human Milk and Standard Formula in Neonatal Piglets

BackgroundDespite the advancements in infant nutrition, a gap still exists in the nutritional composition bioactive ingredients between infant formula and human milk. We developed a next-generation, proof-of-concept infant formula that contains recombinant human milk proteins. ObjectiveTo determine the impact of a novel infant formula (H1) on organ growth and development, and intestinal function compared to donor human milk (DHM) and standard infant formula (S) in a term piglet model. MethodsTerm piglets delivered via cesarean section were fed either a donor human milk (DHM) control, the investigational formula (H1), or infant formula (S) for 10 days. On d 10, a blood sample and tissues were collected. ResultsThere was no difference (P > 0.05) in piglet growth, although H1 piglets had a smaller relative stomach and liver than DHM and S piglets. H1 piglets had higher (P < 0.05) interleukins in the distal ileum, but no other systemic cytokines were elevated compared to the DHM and S piglets. H1 piglet small intestinal histology was similar (P > 0.05) to that of DHM and S piglets. Additionally, H1 piglets had either the same (P > 0.05) or higher (P < 0.05) amino acids in circulation compared to DHM and S piglets. Recombinant human proteins had either similar (P > 0.05) or lower (P < 0.05) activity compared to the native human proteins when assessing the individual ingredients in the H1 formula. ConclusionH1 formula was noninferior to DHM and S based on growth, small intestinal histology and plasma amino acid endpoints when fed to neonatal piglets. These findings warrant further studies to use the neonatal piglet as a model to evaluate more in-depth outcomes of health and safety for new infant formulas. Lay SummaryA novel piglet study shows a hypoallergenic, next-generation infant formula containing recombinant human milk proteins rivals donor human milk and standard formula for growth, gut health, and nutrient status.

physiology↗

bioRxiv2026

Rapid Determination of Drug-to-Antibody Ratios in Antibody Drug Conjugates Using Ultrafast Microdroplet Digestion Technology

Accurate determination of drug-to-antibody ratios (DARs) is essential for the development, quality control, and performance evaluation of antibody-drug conjugates (ADCs); yet conventional analytical approaches often require extensive sample preparation, long analysis time, and substantial sample consumption. The peak distribution of intact ADCs is highly complex due to inherent glycosylation heterogeneity and variable drug conjugation. By applying enzymatic digestion, ADC can be converted into smaller subunits or deglycosylated species, thereby significantly simplifying the mass spectral profile. This reduction in structural heterogeneity facilitates clearer peak assignment and enables more accurate and reliable DAR quantification. Herein, we report an ultrafast microdroplet digestion-mass spectrometry strategy for rapid DAR characterization of ADCs. Microdroplet enzymatic digestion of antibodies and ADCs occurs within microsecond-time scales during spray ionization, enabling direct online subunit analysis with minimal sample preparation. The method was validated using NIST monoclonal antibody (mAb) conjugated to ADC mimics spanning low to high DAR (0-14) ranges, Cetuximab-derived ADC mimics (DAR[~]5) with complex glycosylation, and the commercial ADC Kadcyla (DAR[~]3.5). Consistent DAR values were obtained across multiple enzymatic workflows (IdeS, EndoS2, and EndoF3) with good reproducibility (%CV typically <5%). This approach substantially reduces analysis time while maintaining analytical accuracy and structural specificity, providing a rapid, sensitive platform for high-throughput ADC characterization and process monitoring. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/729562v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@23145corg.highwire.dtl.DTLVardef@10ddd88org.highwire.dtl.DTLVardef@14b1370org.highwire.dtl.DTLVardef@1e94ad6_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Spatiotemporal profiling reveals the role of inflammatory niche in driving prostate cancer

Prostate cancer (PCa) is a lethal malignancy that displays profound resistance to immune checkpoint blockade (ICB), via mechanisms that are poorly understood. Here, we investigate the causes of CD8 T cell exhaustion and mechanisms of tumor progression in a PCa animal model, by single cell and spatial profiling, along a time course, following orthotopic transplantation of RB1/TP53/PTEN-deficient mouse organoids, competent to express neoantigens. The resulting tumors were castration resistant, consisting of largely basal and L2 malignant cells with upregulated inflammatory gene programs, and a specific spatial distribution of macrophages, cancer associated fibroblast (CAF) subtypes, and CD8 T-cells that was not previously reported. Using Zman-seq, we demonstrate that the effector function of tumor-infiltrating CD8 T cells was rapidly impaired as early as 24hrs after their infiltration, likely driven by signals from proinflammatory macrophages, Ccl2-Jak2+ inflammatory CAFs, and malignant basal cells, thus driving resistance to ICB. Interestingly, dual blockade of JAK1/2 and PD1 induced potent anti-tumor effects in tumor epithelial cells, decreased malignant epithelial cells and pro-inflammatory macrophages, and increased the proportion of normal (Pi16+) fibroblasts in the TME. Our results underscore the therapeutic potential of targeting JAK1/2 to enhance the efficacy of ICB, providing a rationale for clinical investigation of this combination in PCa.

cancer biology↗

bioRxiv2026

Blood Pressure Regulates Functional Coupling of L-Type Ca2+ Channels: Reimaging the Foundation of Cerebral Blood Flow Control.

The myogenic response is the key autoregulatory mechanism setting cerebral blood flow and its mechanistic foundation is intimately tied to depolarization and the voltage gating of L-type Ca2+ channels (CaV1.2). While critical, this study argues for an additional mechanism, that of pressure itself enhancing CaV1.2 activity via cooperative gating and perimembrane trafficking of channels subunits. These novel insights were pursued at the cell level using patch-clamp electrophysiology and advanced microscopy, and then functionally in pressurized arteries through measures of tone and intracellular [Ca2+]i. Key findings were confirmed in mutant mice with disrupted functional coupling and translated into arteries procured from human brain tissue. From cerebral blood flow simulations of semi-realistic microvascular networks, we predict that loss of this alternative mechanism leads to maldistribution of brain blood flow and potentially a diminishment of cognitive function. This study reveals previously unrecognized pressure-sensitive CaV1.2 regulatory mechanism that advances understanding of cerebral blood flow. SignificanceBlood pressure sets base arterial constriction - a response critical for blood flow control in brain. This response is tied to CaV1.2 channels and their presumptive and exclusive activation by voltage, reasoning now under great scrutiny. We establish herein with advance methods, a second mode of CaV1.2 pressure regulation, that of enhanced functional cooperativity among neighboring channels. This novel mechano-response is tied to PKC and its ability to set channel phosphorylation and CaV1.2 trafficking. CaV1.2 pressure regulation was observed in human tissues and its disruption (mutant mice) impaired myogenic tone in the presence of preserved voltage control. Cerebral microvascular modeling highlights that losing this mechanism destabilizes blood flow distribution in brain, the knock-on effect being comprised cognitive function.

physiology↗

bioRxiv2025

Repurposed endogenous virus-like vesicles mediate dendritic cell long-range antigen presentation and T cell activation for enhanced cancer vaccination

Dendritic cells (DCs) release extracellular vesicles (DEVs) that amplify antigen presentation while incorporating patient-derived, rapidly evolving antigens. By enriching peptide-MHC and co-stimulatory ligands orders-of-magnitude above donor-cell levels, DEVs emerge as potent vesicle-vaccines, although efficacy remains limited by unclear mechanisms. We show that DCs repurpose viral components to generate endogenous virus-like vesicles (VLVs) that preferentially carry peptide-MHC and high-density co-stimulatory ligands, intensifying and extending antigen presentation. Upon antigen exposure, Arc partners with endogenous envelope proteins to assemble VLVs that directly engage T cells and trigger intrinsic adjuvanticity via viral mimicry. Arc-/- DEVs failed to prime antigen-specific T cell responses, whereas Arc overexpression with its 5'-UTR stem-loop shifted DEVs toward VLVs that trafficked to lymphoid organs, drove rapid CD4-assisted priming and durable CD8-biased memory, suppressed melanoma, and prolonged survival. These reveal a viral-mimicry mechanism enabling long-range immune activation and support Arc+ VLVs as an antigen-agnostic vaccine for cancer immunotherapy. HighlightsO_LIArc+ VLVs intensify and extend DC antigen presentation in vivo C_LIO_LIArc+ VLVs directly engage T cells and trigger viral-mimic adjuvanticity C_LIO_LIArc-/- DEVs fail to prime antigen-specific T cell responses C_LIO_LIEngineered Arc+ VLVs drive durable CD8-biased memory and tumor control C_LIO_LIArc+ VLV vaccination provides long-range and cross-tumor protection in vivo C_LI

bioengineering↗

bioRxiv2025

LCI9 is required for normal pyrenoid starch sheath formation and efficiency of the CO2-concentrating mechanism in Chlamydomonas reinhardtii

O_LIPyrenoid -based CO2-concentrating mechanisms (pCCMs) boost photosynthesis by delivering elevated levels of CO2 to Rubisco. Pyrenoids are often surrounded by a starch sheath thought to enhance pCCM efficiency, but little is known about how the sheath is formed. Here we have assessed the role of the pyrenoid-associate protein LCI9 in starch sheath formation in Chlamydomonas reinhardtii. C_LIO_LIUsing an lci9 mutant we assessed the role of LCI9 in starch sheath morphology by fluorescence and electron microscopy, and its impact on pCCM function by CO2 uptake and growth assays. We determined potential interactors of LCI9 via TurboID proximity labeling. C_LIO_LIIn the absence of LCI9, cells fail to correctly assemble the starch sheath, have lower affinity for inorganic carbon and grow slower under CO2-limiting conditions. LCI9 localises to the starch sheath and is physically close to a range of starch metabolism enzymes and other potential scaffolding proteins. Finally, we show that LCI9 associates with starch in the land plant Arabidopsis thaliana. C_LIO_LILCI9 likely acts as a pyrenoid specific scaffold for starch metabolism enzymes. LCI9 could contribute to ongoing efforts to build a functional pyrenoid in a land plant. C_LI

plant biology↗

bioRxiv2025

Imaging CRISPR-Edited CAR-T Cell Therapies with Optical and Positron Emission Tomography Reporters

RationaleChimeric antigen receptor (CAR) T cell therapies have shown remarkable success in treating hematological cancers and are increasingly demonstrating potential for solid tumors. CRISPR-based genome editing offers a promising approach to enhance the potency and safety of CAR-T cells. However, several challenges persist, including inefficient tumor homing and treatment-related toxicities in normal tissues, which continue to hinder widespread adoption. Advanced imaging technologies, including bioluminescence imaging (BLI) and positron emission tomography (PET), provide real-time insights into CAR-T cell distribution and activity in vivo, both in preclinical models and in patients. Here, we developed Trackable Reporter Adaptable CRISPR-Edited CAR (tRACE-CAR) T cells, a modular system for site-specific integration of CARs and imaging reporters. MethodsThe luciferase reporter AkaLuciferase (AkaLuc) or the human sodium iodide symporter (NIS) were cloned downstream of the CAR in adeno-associated virus (AAV) donors for BLI or PET tracking, respectively. CARs with imaging reporters were knocked into the TRAC locus of primary human T cells via CRISPR editing and AAV transduction. Editing efficiency was evaluated by flow cytometry and junction PCR. In vitro cytotoxicity was assessed by BLI using firefly luciferase (Fluc)-expressing cancer cells co-cultured with CAR-T cells at varying effector-to-target ratios. In vivo, BLI and PET imaging assessed CAR-AkaLuc and CAR-NIS T cell expansion and trafficking in Nod-SCID-gamma mice bearing xenograft tumors. ResultsT cell receptor (TCR) knockout efficiency exceeded 85%, with CAR expression observed in 70-80% of cells, depending on the reporter used. Reporter-engineered CAR-T cells retained functionality in vitro and exhibited significant cytotoxicity against target cancer cells, outperforming naive T cells. In vivo, AkaLuc BLI and 18F-tetrafluoroborate PET enabled non-invasive tracking of viable CAR-T cells. Notably, the route of administration (intravenous, peritumoral, or intraperitoneal) significantly influenced the distribution of CAR-T cells and their therapeutic effectiveness. ConclusiontRACE-CAR enabled precise optical and PET tracking of CAR-T cells in models of B cell leukemia and ovarian cancer, allowing dynamic, non-invasive monitoring of cell distribution in both tumors and off-target tissues. This imaging platform could lead to more personalized, effective CRISPR-edited CAR cell therapies.

immunology↗

bioRxiv2025

Promoter-centric gene regulation in drug-resistant cancer

In eukaryotic cells, gene-distal regulatory elements (REs) facilitate long-range gene regulation, ensuring cell type-specific transcriptional programs. This mechanism is frequently disrupted in cancer, often driven by transcription factors (TFs) that serve as targets for cancer therapy. However, targeting these TFs can lead to acquired resistance mechanisms that are not fully understood. We demonstrate that mesothelioma cancer cells, dependent on the oncogenic driver TF family TEAD, develop resistance to a pan-TEAD inhibitor and revert to an evolutionarily ancient, promoter-centric gene regulatory mechanism to recover gene expression following TEAD inhibition. Base-pair-resolution 3D chromatin conformation mapping reveals that RE-promoter interactions are disrupted in resistant cells, despite epigenetic and transcriptomic recovery. Mechanistically, in resistant cells, TF complexes, including resistance-specific FOSL1 and KLF4, preferentially bind and enhance promoter activity to recover gene expression, rendering distal REs dispensable. Our findings highlight promoter elements and promoter-specific TFs as potential therapeutic targets using a model of drug-resistant cancer.

cancer biology↗

bioRxiv2024

Comparative Analysis of Commercial Single-Cell RNA Sequencing Technologies

This study evaluates ten commercially available single-cell RNA sequencing (scRNA-seq) approaches across four technology groups: Emulsion-based kits from 10x Genomics and Fluent Biosciences; Microwell-based kits from Becton Dickinson, Honeycomb Technologies and Singerlon Technologies; Combinatorial-indexing kits from Parse Biosciences and Scale Biosciences; and a Matrigel-based kit from Scipio Biosciences. Peripheral blood mononuclear cells (PBMCs) from a single donor were used to assess analytical performance. Key features such as sample compatibility, cost, and experimental duration were also compared. Notably, superior analytical performance was demonstrated by the Chromium Fixed RNA Profiling kit from 10x Genomics, which uniquely features probe hybridization for transcript detection. Additionally, the Rhapsody WTA kit from Becton Dickinson provided a cost-effective balance of performance and expense per cell. With a rich dataset of 218,154 cells, this work provides a basis for differentiating commercial scRNA-seq technologies, which is intended to facilitate the effective application and further methodological development of single cell transcriptomics.

molecular biology↗

bioRxiv2024

Overloading And unpacKing (OAK) - droplet-based combinatorial indexing for ultra-high throughput single-cell multiomic profiling

Multiomic profiling of single cells by sequencing is a powerful technique for investigating cellular diversity in complex biological systems. Although the existing droplet-based microfluidic methods have advanced single-cell sequencing, they produce a plethora of cell-free droplets and underutilize barcoding capacities due to their low cell concentration prerequisites. Meanwhile, combinatorial indexing on microplates can index cells in a more effective way; however, it requires time-consuming and laborious protocols involving multiple splitting and pooling steps. Addressing these constraints, we have developed "Overloading And unpacKing" (OAK). With reduced labor intensity, OAK can provide cost-effective multiomic profiling for hundreds of thousands of cells, offering detection sensitivity on par with commercial droplet-based methods. To demonstrate OAKs versatility, we conducted single-cell RNA sequencing (scRNA-Seq) as well as joint single-nucleus RNA sequencing (snRNA-Seq) and single-nucleus Assay for Transposase Accessible Chromatin with sequencing (snATAC-Seq) using cell lines. We further showcased OAKs performance on more complex samples, including in vitro differentiated bronchial epithelial cells and primary retinal tissues. Finally, we examined transcriptomic responses of 408,000 melanoma cells across around 1,000 starting lineages over a 90-day treatment with a RAF inhibitor, belvarafenib. We discovered a rare cell population (0.12%) that underwent a sequence of transcriptomic changes, resulting in belvarafenib resistance. Ultra-high throughput, broad compatibility with diverse molecular modalities, high detection sensitivity, and simplified experimental procedures distinguish OAK from previous methods, and render OAK a powerful tool for large-scale analysis of molecular signatures, even for rare cells.

molecular biology↗

bioRxiv2023

KRASG12D-mediated PanIN progression in mice is affected by gene driving cre recombinase expression

The fundamental biology of pancreatic ductal adenocarcinoma has been greatly impacted by the characterization of genetically modified mouse models that allow temporal and spatial activation of oncogenic KRAS (KRASG12D). The most commonly used model involves targeted insertion of a cre recombinase into the Ptf1a gene. However, this approach disrupts the Ptf1a gene, resulting in haploinsufficiency that likely affects sensitivity to oncogenic KRAS (KRASG12D). The goal of this study was to determine if Ptf1a haploinsufficiency affected the acinar cell response to KRASG12D before and after induction of pancreatic injury. We performed morphological and molecular analysis of three mouse lines that express a tamoxifen-inducible cre recombinase to activate KRASG12D in acinar cells of the pancreas. The cre-recombinase was targeted to the acinar-specific transcription factor genes, Ptf1a and Mist1/Bhlha15, or expressed within a BAC-derived Elastase transgene. Up to two months after tamoxifen induction of KRASG12D, morphological changes were negligible. However, induction of pancreatic injury by cerulein resulted in stark differences in tissue morphology between lines within seven days, which were maintained for at least five weeks after injury. Ptf1acreERT pancreata showed widespread PanIN lesions and fibrosis, while the Mist1creERT and Ela-creERT models showed reduced amounts of pre-neoplastic lesions. RNA-seq analysis prior to inducing injury suggested Ptf1acreERT and Mist1creERT lines have unique profiles of gene expression that predict a differential response to injury. Multiplex analysis of pancreatic tissue confirmed different inflammatory responses between the lines. These findings suggest understanding the mechanisms underlying the differential response to KRASG12D will help in further defining the intrinsic KRAS-driven mechanisms of neoplasia initiation.

cancer biology↗

bioRxiv2023

Distinct human stem cell subpopulations drive adipogenesis and fibrosis in musculoskeletal injury

Fibroadipogenic progenitors (FAPs) maintain healthy skeletal muscle in homeostasis but drive muscle degeneration in chronic injuries by promoting adipogenesis and fibrosis. To uncover how these stem cells switch from a pro-regenerative to pro-degenerative role we perform single-cell mRNA sequencing of human FAPs from healthy and injured human muscles across a spectrum of injury, focusing on rotator cuff tears. We identify multiple subpopulations with progenitor, adipogenic, or fibrogenic gene signatures. We utilize full spectrum flow cytometry to identify distinct FAP subpopulations based on highly multiplexed protein expression. Injury severity increases adipogenic commitment of FAP subpopulations and is driven by the downregulation of DLK1. Treatment of FAPs both in vitro and in vivo with DLK1 reduces adipogenesis and fatty infiltration, suggesting that during injury, reduced DLK1 within a subpopulation of FAPs may drive degeneration. This work highlights how stem cells perform varied functions depending on tissue context, by dynamically regulating subpopulation fate commitment, which can be targeted improve patient outcomes after injury.

cell biology↗

bioRxiv2023

A shift in the mechanisms controlling hippocampal engram formation during brain maturation

The ability to form precise, episodic memories develops with age, with young children only able to form gist-like memories that lack precision. The cellular and molecular events in the developing hippocampus that underlie the emergence of precise, episodic-like memory formation are unclear. In mice, the absence of a competitive neuronal engram allocation process in the immature hippocampus precluded the formation of sparse engrams and precise memories until the fourth postnatal week, when inhibitory circuits in the hippocampus mature. This age-dependent shift in precision of episodic-like memories involved the functional maturation of parvalbumin-expressing interneurons in subfield CA1 by extracellular perineuronal nets which is necessary and sufficient for the onset of competitive neuronal allocation, sparse engram formation, and memory precision. One-Sentence SummaryEpisodic-like memory precision requires maturation of hippocampal inhibitory interneurons by the extracellular matrix.

neuroscience↗

bioRxiv2022

Developing best practices for genotyping-by-sequencing analysis using linkage maps as benchmarks

BackgroundGenotyping-by-Sequencing (GBS) provides affordable methods for genotyping hundreds of individuals using millions of markers. However, this challenges bioinformatic procedures that must overcome possible artifacts such as the bias generated by PCR duplicates and sequencing errors. Genotyping errors lead to data that deviate from what is expected from regular meiosis. This, in turn, leads to difficulties in grouping and ordering markers resulting in inflated and incorrect linkage maps. Therefore, genotyping errors can be easily detected by linkage map quality evaluations. ResultsWe developed and used the Reads2Map workflow to build linkage maps with simulated and empirical GBS data of diploid outcrossing populations. The workflows run GATK, Stacks, TASSEL, and Freebayes for SNP calling and updog, polyRAD, and SuperMASSA for genotype calling, and OneMap and GUSMap to build linkage maps. Using simulated data, we observed which genotype call software fails in identifying common errors in GBS sequencing data and proposed specific filters to better handle them. We tested whether it is possible to overcome errors in a linkage map using genotype probabilities from each software or global error rates to estimate genetic distances with an updated version of OneMap. We also evaluated the impact of segregation distortion, contaminant samples, and haplotype-based multiallelic markers in the final linkage maps. Through our evaluations, we observed that some of the approaches produce different results depending on the dataset (dataset-dependent) and others produce consistent advantageous results among them (dataset-independent). ConclusionsWe set as default in the Reads2Map workflows the approaches that showed to be dataset-independent for GBS datasets according to our results. This reduces the number required of tests to identify optimal pipelines and parameters for other empirical datasets. Using Reads2Map, users can select the pipeline and parameters that best fit their data context. The Reads2MapApp shiny app provides a graphical representation of the results to facilitate their interpretation.

bioinformatics↗

bioRxiv2022

Individual differences in oxycodone addiction-like behaviors in a large cohort of heterogeneous stock (HS) rats

Family and twin studies demonstrate that genetic factors determine 20-60% of the vulnerability to opioid use disorder. However, the genes/alleles that mediate the risk of developing addiction-related behaviors, including the sensitivity to the analgesic efficacy of opioids, the development of tolerance, dependence, and escalation of oxycodone taking and seeking, have been ill-defined, thus hindering efforts to design pharmacological interventions to enable precision medicine strategies. Here we characterized oxycodone addiction-like behaviors in heterogeneous stock (HS) rats, that show high genetic diversity that mimics the high genetic variability in humans. HS rats were allowed to self-administer oxycodone for two h/daily for four days (ShA) and then moved to 12h/daily (LgA) for 14 days. Animals were screened for motivation to self-administer oxycodone using a progressive-ratio (PR) schedule of reinforcement and for the development of withdrawal-induced hyperalgesia and tolerance to the analgesic effects of oxycodone using the von-Frey and tail immersion tests, respectively. To reduce cohort-specific effects, we used cohorts of 46-60 rats and normalized the response level within cohorts using a Z-score. To take advantage of the four opioid-related behaviors and further identify subjects that are consistently vulnerable vs. resilient to compulsive oxycodone use, we computed an Addiction Index by averaging normalized responding (Z-scores) for the four behavioral tests. Results showed high individual variability between vulnerable and resilient rats, likely to facilitate the detection of gene variants associated with vulnerable vs. resilient individuals. Such data will have considerable translational value for designing follow-up studies in humans.

neuroscience↗

bioRxiv2021

A connectomic study of a petascale fragment of human cerebral cortex

We acquired a rapidly preserved human surgical sample from the temporal lobe of the cerebral cortex. We stained a 1 mm3 volume with heavy metals, embedded it in resin, cut more than 5000 slices at [~]30 nm and imaged these sections using a high-speed multibeam scanning electron microscope. We used computational methods to render the three-dimensional structure containing 57,216 cells, hundreds of millions of neurites and 133.7 million synaptic connections. The 1.4 petabyte electron microscopy volume, the segmented cells, cell parts, blood vessels, myelin, inhibitory and excitatory synapses, and 104 manually proofread cells are available to peruse online. Many interesting and unusual features were evident in this dataset. Glia outnumbered neurons 2:1 and oligodendrocytes were the most common cell type in the volume. Excitatory spiny neurons comprised 69% of the neuronal population, and excitatory synapses also were in the majority (76%). The synaptic drive onto spiny neurons was biased more strongly toward excitation (70%) than was the case for inhibitory interneurons (48%). Despite incompleteness of the automated segmentation caused by split and merge errors, we could automatically generate (and then validate) connections between most of the excitatory and inhibitory neuron types both within and between layers. In studying these neurons we found that deep layer excitatory cell types can be classified into new subsets, based on structural and connectivity differences, and that chandelier interneurons not only innervate excitatory neuron initial segments as previously described, but also each others initial segments. Furthermore, among the thousands of weak connections established on each neuron, there exist rarer highly powerful axonal inputs that establish multi-synaptic contacts (up to [~]20 synapses) with target neurons. Our analysis indicates that these strong inputs are specific, and allow small numbers of axons to have an outsized role in the activity of some of their postsynaptic partners.

neuroscience↗

bioRxiv2021

Functional, metabolic and transcriptional maturation of stem cell derived beta cells

Transplantation of pancreatic islet cells derived from human pluripotent stem cells is a promising treatment for diabetes. Despite progress in stem cell-derived islet (SC-islet) generation, detailed characterization of their functional properties has not been conducted. Here, we generated functionally mature SC-islets using an optimized protocol and comprehensively benchmarked them against primary adult islets. Biphasic glucose stimulated insulin secretion developed during in vitro maturation, associated with cytoarchitectural reorganization and increased alpha cells. Electrophysiology and exocytosis of SC-islets were comparable to adult islets. Glucose-responsive insulin secretion was achieved despite differences in glycolytic and mitochondrial glucose metabolism. Single-cell transcriptomics of SC-islets in vitro and throughout 6 months of murine engraftment revealed a continuous maturation trajectory culminating in a transcriptional landscape closely resembling that of primary islets. Our thorough evaluation of SC-islet maturation highlights their advanced degree of functionality and supports their use in further efforts to understand and combat diabetes.

developmental biology↗