Biology subjects

Wong, J.

Publications and source records attributed to Wong, J..

At least 73 records · Page 4Linked to original sources

bioRxiv2024

Human ancestry inference at scale, from genomic data

Ancestry information is essential to large cohort studies, yet it is often unavailable or inconsistently measured. For studies with a genome sequencing component, current ancestry prediction approaches are hindered by high computational demands and complex input requirements. We present ntRoot, a computationally-lightweight method for inferring human super-population-level ancestry from whole genome assemblies or raw short or long sequencing data. Utilizing an alignment-free variant detection framework, ntRoot employs a succinct Bloom filter data structure to efficiently query diverse genomic data inputs. Demonstrated on over 600 human genome sequencing datasets--including complete genomes, draft assemblies, and over 280 independently-generated datasets--ntRoot accurately predicts geographic labels, a descriptor of human populations, and shows high concordance with traditional methods such as ADMIXTURE (R2 = 0.9567) when predicting ancestry fractions. It achieves these predictions within 30 minutes for complete and draft genomes and within 1 hour and 15 minutes for 30X sequencing data, using a maximum of 13GB and 68GB of RAM, respectively. ntRoot offers both global and local ancestry inference, delivering high-resolution predictions across genomic loci. This paradigm fills a critical gap in cohort studies by enabling rapid, resource-efficient, and accurate ancestry inference at scale, advancing the characterization of continental-level ancestry in the genomic era. Author SummaryStudy concept: RLW. Software implementation: RLW, LC, JW, PK. Data analysis: RLW, LC. Manuscript development: RLW, LC. Manuscript editing: RLW, LC, JW, PK, IB. Funding acquisition: IB.

genetics↗

bioRxiv2024

Oncogenic GNAS drives a gastric pylorus program in intraductal papillary mucinous neoplasms of the pancreas

BACKGROUND & AIMSIntraductal Papillary Mucinous Neoplasms (IPMNs) are cystic lesions and bona fide precursors for pancreatic ductal adenocarcinoma (PDAC). Recent studies have shown that pancreatic precancer is characterized by a transcriptomic program similar to gastric metaplasia. The aims of this study were to assay IPMN for pyloric markers, to identify molecular drivers, and to determine a functional role for this program in the pancreas. METHODSPyloric marker expression was evaluated by RNA-seq and multiplex immunostaining in patient samples. Cell lines and organoids expressing KrasG12D +/- GNASR201C underwent RNA sequencing. A PyScenic-based regulon analysis was performed to identify molecular drivers, and candidates were evaluated by RNA-seq, immunostaining, and small interfering RNA knockdown. Glycosylation profiling was performed to identify GNASR201C-driven changes. Glycan abundance was evaluated in patient samples. RESULTSPyloric markers were identified in human IPMN. GNASR201Cdrove expression of this program as well as an indolent phenotype characterized by distinct glycosyltransferase changes. Glycan profiling identified an increase in LacdiNAcs and loss of pro-tumorigenic Lewis antigens. Knockdown of transcription factors Spdef or Creb3l1 or chitinase treatment reduced LacdiNAc deposition and reversed the indolent phenotype. LacdiNAc and 3-sulfoLeA/C abundance discriminated low from high grade patient IPMN. CONCLUSIONGNASR201C drives an indolent phenotype in IPMN by amplifying a differentiated, pyloric phenotype through SPDEF/CREB3L1 which is characterized by distinct glycans. Acting as a glycan rheostat, mutant GNAS elevates LacdiNAcs at the expense of pro-tumorigenic acidic Lewis epitopes, inhibiting cancer cell invasion and disease progression. LacdiNAc and 3-Sulfo-LeA/C are mutually exclusive and may serve as markers of disease progression.

cancer biology↗

bioRxiv2024

Gene Therapy Mediates Therapeutic Improvement in Cardiac Hypertrophy and Survival in a Murine Model of MYBPC3-Associated Cardiomyopathy

BackgroundHypertrophic cardiomyopathy (HCM) affects an estimated 600,000 people in the U.S. and is the leading cause of sudden cardiac arrest in those under 18. Loss-of-function mutations in Myosin Binding Protein C3, MYBPC3, are the most common genetic cause of HCM. The majority of MYBPC3 mutations causative for HCM result in truncations. The sarcomeric pathophysiology of the majority of HCM patients with MYBPC3 mutations appears to be due to haploinsufficiency, as the total amount of MYBPC3 protein incorporated into sarcomeres falls significantly below normal. MethodsA clear path for the treatment of haploinsufficiency is the restoration of the insufficient gene product; in this case wild-type MYBPC3. To achieve this, we engineered an AAV vector (TN-201) with superior properties for mediating cardiomyocyte-selective expression of MYBPC3 after systemic delivery. ResultsWe have demonstrated for the first time with AAV gene therapy the ability of both a mouse surrogate and TN-201, which encodes human MYBPC3 to reverse cardiac hypertrophy and systolic dysfunction and to improve diastolic dysfunction and survival in a symptomatic MYBPC3-deficient murine model of disease. Dose-ranging efficacy studies exhibited restoration of wild-type MYBPC3 protein levels and saturation of cardiac improvement at the clinically relevant dose of 3E13 vg/kg, outperforming a previously published construct. Further, we have established stable cardiac benefit for greater than one year post-injection, as well as reversal of cardiac dysfunction even in late-stage models of disease. ConclusionsOur data suggest that by restoring MYBPC3 to the sarcomere, TN-201 has the potential to slow and even reverse the course of the disease in patients with MYBPC3-associated HCM.

genetics↗

bioRxiv2024

Multi-genome synteny detection using minimizer graph mappings

In recent years, the landscape of reference-grade genome assemblies has seen substantial diversification. With such rich data, there is pressing demand for robust tools for scalable, multi-species comparative genomics analyses, including detecting genome synteny, which informs on the sequence conservation between genomes and contributes crucial insights into species evolution. Here, we introduce ntSynt, a scalable utility for computing large-scale multi-genome synteny blocks using a minimizer graph-based approach. Through extensive testing utilizing multiple [~]3 Gbp genomes, we demonstrate how ntSynt produces synteny blocks with coverages between 79-100% in at most 2h using 34 GB of memory, even for genomes with appreciable (>15%) sequence divergence. Compared to existing state-of-the-art methodologies, ntSynt offers enhanced flexibility to diverse input genome sequences and synteny block granularity. We expect the macrosyntenic genome analyses facilitated by ntSynt will have broad utility in generating critical evolutionary insights within and between species across the tree of life.

bioinformatics↗

bioRxiv2024

Characterization of the skeletal muscle arginine methylome in health and disease reveals remodeling in Amyotrophic Lateral Sclerosis

Arginine methylation is a protein post-translational modification important for the development of skeletal muscle mass and function. Despite this, our understanding of the regulation of arginine methylation under settings of health and disease remains largely undefined. Here, we investigated the regulation of arginine methylation in skeletal muscles in response to exercise and hypertrophic growth, and in diseases involving metabolic dysfunction and atrophy. We report a limited regulation of arginine methylation under physiological settings that promote muscle health, such as during growth and acute exercise, nor in disease models of insulin resistance. In contrast, we saw a significant remodeling of asymmetric dimethylation in models of atrophy characterized by the loss of innervation, including in muscle biopsies from patients with amyotrophic lateral sclerosis (ALS). Mass spectrometry-based quantification of the proteome and asymmetric arginine dimethylome of skeletal muscle from individuals with ALS revealed the largest compendium of protein changes with the identification of 793 regulated proteins, and novel site-specific changes in asymmetric dimethyl arginine (aDMA) of key sarcomeric and cytoskeletal proteins. Finally, we show that in vivo overexpression of PRMT1 and aDMA resulted in increased fatigue resistance and functional recovery in mice. Our study provides evidence for asymmetric dimethylation as a regulator of muscle pathophysiology and presents a valuable proteomics resource and rationale for numerous methylated and non-methylated proteins, including PRMT1, to be pursued for therapeutic development in ALS.

physiology↗

bioRxiv2023

PROTOSPACEJAM: AN OPEN-SOURCE, CUSTOMIZABLE AND WEB-ACCESSIBLE DESIGN PLATFORM FOR CRISPR/CAS9 INSERTIONAL KNOCK-IN

CRISPR/Cas9-mediated knock-in of DNA sequences enables precise genome engineering for research and therapeutic applications. However, designing effective guide RNAs (gRNAs) and homology-directed repair (HDR) donors remains a bottleneck. Here, we present protoSpaceJAM, an open-source algorithm to automate and optimize gRNA and HDR donor design for CRISPR/Cas9 insertional knock-in experiments. protoSpaceJAM utilizes biological rules to rank gRNAs based on specificity, distance to insertion site, and position relative to regulatory regions. protoSpaceJAM can introduce recoding mutations (silent mutations and mutations in non-coding sequences) in HDR donors to prevent re-cutting and increase knock-in efficiency. Users can customize parameters and design double-stranded or single-stranded donors. We validated protoSpaceJAMs design rules by demonstrating increased knock-in efficiency with recoding mutations and optimal strand selection for single-stranded donors. An additional module enables the design of genotyping primers for next-generation sequencing of edited alleles. Overall, protoSpaceJAM streamlines and optimizes CRISPR knock-in experimental design in a flexible and modular manner to benefit diverse research and therapeutic applications. protoSpaceJAM is available open-source as an interactive web tool at protospacejam.czbiohub.org or as a standalone Python package at github.com/czbiohub-sf/protoSpaceJAM.

bioinformatics↗

bioRxiv2023

Bacteroides thetaiotaomicron metabolic activity decreases with polysaccharide molecular weight

The human colon hosts hundreds of commensal bacterial species, many of which ferment complex dietary carbohydrates. To transform these fibers into metabolically-accessible compounds, microbes often express series of dedicated enzymes homologous to the starch utilization system (sus) encoded in polysaccharide utilization loci (PULs). The genome of Bacteroides thetaiotaomicron (Bt), a common member of the human gut microbiota, encodes nearly 100 PULs, conferring a strong metabolic versatility. While the structures and functions of individual enzymes within the PULs have been investigated, little is known about how polysaccharide complexity impacts the function of sus-like systems. We here show that the activity of sus-like systems depends on polysaccharide size, ultimately impacting bacterial growth. We demonstrate the effect of size-dependent metabolism in the context of dextran metabolism driven by the specific utilization system PUL48. We find that as molecular weight of dextran increases, Bt growth rate decreases and lag time increases. At the enzymatic level, the dextranase BT3087 is the main glycosylhydrolase for dextran utilization and that BT3087 and BT3088 contribute to Bt dextran metabolism in a size-dependent manner. Finally, we show that the polysaccharide size-dependent metabolism of Bt impacts its metabolic output in a way that modulates the composition of a producer-consumer community it forms with Bacteroides fragilis. Altogether, our results expose an overlooked aspect of Bt metabolism which can impact the composition and diversity of microbiota.

microbiology↗

bioRxiv2023

Genome assembly of the bearded iris Iris pallida Lam.

Irises are perennial plants, representing a large genus with hundreds of species. While cultivated extensively for their ornamental value, commercial interest in irises lies in the secondary metabolites present in their rhizomes. The Dalmatian Iris (Iris pallida Lam.) is an ornamental plant that also produces secondary metabolites with potential value to the fragrance and pharmaceutical industries. In addition to providing base notes for the fragrance industry, iris tissues and extracts possess anti-oxidant, anti- inflammatory, and immunomodulatory effects. However, study of these secondary metabolites has been hampered by a lack of genomic information, instead requiring difficult extraction and analysis techniques. Here, we report the genome sequence of Iris pallida Lam., generated with Pacific Bioscience long-read sequencing, resulting in a 10.04 Gbp assembly with a scaffold N50 of 14.34 Mbp and 91.8% complete BUSCOs. This reference genome will allow researchers to study the biosynthesis of these secondary metabolites in much greater detail, opening new avenues of investigation for drug discovery and fragrance formulations. Research area: Genetics and Genomics; Botany; Plant Genetics

plant biology↗

bioRxiv2023

Chemokine receptor 5 signaling in PFC mediates stress susceptibility in female mice

Chronic stress induces changes in the periphery and the central nervous system (CNS) that contribute to neuropathology and behavioral abnormalities associated with psychiatric disorders. In this study, we examined the impact of peripheral and central inflammation during chronic social defeat stress (CSDS) in female mice. Compared to male mice, we found that female mice exhibited heightened peripheral inflammatory response and identified C-C motif chemokine ligand 5 (CCL5), as a stress-susceptibility marker in females. Blocking CCL5 signaling in the periphery promoted resilience to CSDS. In the brain, stress-susceptible mice displayed increased expression of C-C chemokine receptor 5 (CCR5), a receptor for CCL5, in microglia in the prefrontal cortex (PFC). This upregulation was associated with microglia morphological changes, their increased migration to the blood vessels, and enhanced phagocytosis of synaptic components and vascular material. These changes coincided with neurophysiological alterations and impaired blood-brain barrier (BBB) integrity. By blocking CCR5 signaling specifically in the PFC were able to prevent stress-induced physiological changes and rescue social avoidance behavior. Our findings are the first to demonstrate that stress-mediated dysregulation of the CCL5-CCR5 axis triggers excessive phagocytosis of synaptic materials and neurovascular components by microglia, resulting in disruptions in neurotransmission, reduced BBB integrity, and increased stress susceptibility. Our study provides new insights into the role of cortical microglia in female stress susceptibility and suggests that the CCL5-CCR5 axis may serve as a novel sex-specific therapeutic target for treating psychiatric disorders in females.

animal behavior and cognition↗

bioRxiv2023

Evolutionary genomics identifies host-directed therapeutics to treat intracellular bacterial infections

Obligate intracellular bacteria shed essential biosynthetic pathways during their evolution towards host dependency, providing an opportunity for host-directed therapeutics. Using Rickettsiaceae as a model, we employed a novel computational pipeline called PoMeLo to systematically compare this cytosolic family of bacteria to the related Anaplasmataceae, which reside in a membrane-bound vacuole in the host cell. We identified 20 metabolic pathways that have been lost since the divergence of Anaplasmataceae and Rickettsiaceae, corresponding to the latters change to a cytosolic niche. We hypothesized that drug inhibition of these host metabolic pathways would reduce the levels of metabolites available to the bacteria, thereby inhibiting bacterial growth. We tested 22 commercially available inhibitors for 14 of the identified pathways and found that the majority (59%) reduced bacterial growth at concentrations that did not induce host cell cytotoxicity. Of these, 5 inhibitors with an IC50 under 5 M were tested to determine whether their mode of inhibition was bactericidal or bacteriostatic. Both mycophenolate mofetil, an inhibitor of inosine-5-monophosphate dehydrogenase in the purine biosynthesis pathway, and roseoflavin, an analog of riboflavin, displayed bactericidal activity. A complementary unbiased mass spectrometry-based metabolomics approach identified 14 pathways impacted by Rickettsia infection based on alterations in metabolite levels. Strikingly, 11 of these (79%) overlapped with those identified by our computational predictions. These in vitro validation studies support the feasibility of a novel evolutionary genomics-guided approach for host-directed antibiotic drug development against obligate pathogens. ImportanceMany pathogens have evolved to acquire essential metabolites from their host cell, while in turn shedding their own biosynthetic capacities. This leads to an interesting dilemma: on one hand, reduced genomes allow pathogens to save energy and replicate more quickly, while on the other hand, they become more dependent on the host cell for survival. This vulnerability can be exploited by identifying and therapeutically inhibiting the host pathways that are essential for pathogen survival. The significance of our research is in predicting the precise pathways lost during a pathogens evolutionary adaptation to parasitism and validating these predictions through targeted in vitro growth assays and an unbiased metabolomic survey of the host-pathogen interface.

genomics↗

bioRxiv2023

PoMeLo: a systematic computational approach to predicting metabolic loss in pathogen genomes

BackgroundGenome streamlining, the process by which genomes become smaller and encode fewer genes over time, is a common phenomenon among pathogenic bacteria. This reduction is driven by selection for faster replication and minimized energy expenditure in a nutrientrich environment. As pathogens evolve to become more reliant on the host, metabolic genes and resulting capabilities are lost in favor of siphoning metabolites from the host. Characterizing genome streamlining, gene loss, and pathway degradation can be useful in assessing pathogen metabolic dependency on host metabolism and identifying potential targets for host-directed therapeutics. ResultsPoMeLo (Predictor of Metabolic Loss) is a novel evolutionary genomics-guided computational approach for identifying metabolic gaps in the genomes of pathogenic bacteria. PoMeLo leverages a centralized public database of high-quality genomes and annotations and allows the user to compare an unlimited number of genomes across individual genes and pathways. PoMeLo runs locally using user-friendly prompts in a matter of minutes and generates tabular and visual outputs for users to compare predicted metabolic capacity between groups of bacteria and individual species. Each pathway is assigned a Predicted Metabolic Loss (PML) score to assess the magnitude of genome streamlining. Optionally, PoMeLo places results in an evolutionary context by including phylogenetic relationships in visual outputs. It can also initially compute phylogenetically-weighted mean genome sizes to identify genome streamlining events. Here we describe PoMeLo and demonstrate its use in identifying metabolic gaps in genomes of pathogenic Treponema species. ConclusionsPoMeLo represents an advance over existing methods for identifying metabolic gaps in genomic data, allowing comparison across large numbers of genomes and placing the resulting data in a phylogenetic context. PoMeLo is freely available for academic and nonacademic use at https://github.com/czbiohub-sf/pomelo.

bioinformatics↗

bioRxiv2023

Interdependence of a microtubule polymerase and a motor protein in establishment of kinetochore end-on attachments

Faithful segregation of chromosomes into daughter cells during mitosis requires formation of attachments between kinetochores and mitotic spindle microtubules. Chromosome alignment on the mitotic spindle, also referred to as congression, is facilitated by translocation of side-bound chromosomes along the microtubule surface, which allows the establishment of end-on attachment of kinetochores to microtubule plus ends. Spatial and temporal constraints hinder observation of these events in live cells. Therefore, we used our previously developed reconstitution assay to observe dynamics of kinetochores, the yeast kinesin-8, Kip3, and the microtubule polymerase, Stu2, in lysates prepared from metaphase-arrested budding yeast, Saccharomyces cerevisiae. Using total internal reflection fluorescence (TIRF) microscopy to observe kinetochore translocation on the lateral microtubule surface toward the microtubule plus end, motility was shown to be dependent on both Kip3, as we reported previously, and Stu2. These proteins were shown to have distinct dynamics on the microtubule. Kip3 is highly processive and moves faster than the kinetochore. Stu2 tracks both growing and shrinking microtubule ends but also colocalizes with moving lattice-bound kinetochores. In cells, we observed that both Kip3 and Stu2 are important for establishing chromosome biorientation, Moreover, when both proteins are absent, biorientation is completely defective. All cells lacking both Kip3 and Stu2 had declustered kinetochores and about half also had at least one unattached kinetochore. Our evidence argues that despite differences in their dynamics, Kip3 and Stu2 share roles in chromosome congression to facilitate proper kinetochore-microtubule attachment.

cell biology↗

bioRxiv2023

Accurate identification of structural variations from cancer samples

Structural variations (SVs) are commonly found in cancer genomes. They can cause gene amplification, deletion, and fusion, among other functional consequences. With an average read length of hundreds of kilobases, nano-channel-based optical DNA mapping is powerful in detecting large SVs. However, existing SV calling methods are not tailored for cancer samples, which have special properties such as mixed cell types and sub-clones. Here we propose the COMSV method that is specifically designed for cancer samples. It shows high sensitivity and specificity in benchmark comparisons. Applying to cancer cell lines and patient samples, COMSV identifies hundreds of novel SVs per sample.

genomics↗

bioRxiv2023

Vascular Endothelial-derived SPARCL1 Exacerbates Viral Pneumonia Through Pro-Inflammatory Macrophage Activation

Inflammation upon infectious lung injury is a double-edged sword: while tissue-infiltrating immune cells and cytokines are necessary to control infection, these same factors often aggravate injury. Full appreciation of both the sources and targets of inflammatory mediators is required to facilitate strategies to maintain antimicrobial effects while minimizing off-target epithelial and endothelial damage. Recognizing that the vasculature is centrally involved in tissue responses to injury and infection, we observed that pulmonary capillary endothelial cells (ECs) exhibit dramatic transcriptomic changes upon influenza injury punctuated by profound upregulation of Sparcl1. Endothelial deletion and overexpression of SPARCL1 implicated this secreted matricellular protein in driving key pathophysiologic symptoms of pneumonia, which we demonstrate result from its effects on macrophage polarization. SPARCL1 induces a shift to a pro-inflammatory "M1-like" phenotype (CD86+CD206-), thereby increasing associated cytokine levels. Mechanistically, SPARCL1 acts directly on macrophages in vitro to induce the pro-inflammatory phenotype via activation of TLR4, and TLR4 inhibition in vivo ameliorates inflammatory exacerbations caused by endothelial Sparcl1 overexpression. Finally, we confirmed significant elevation of SPARCL1 in COVID-19 lung ECs in comparison with those from healthy donors. Survival analysis demonstrated that patients with fatal COVID-19 had higher levels of circulating SPARCL1 protein compared to those who recovered, indicating the potential of SPARCL1 as a biomarker for prognosis of pneumonia and suggesting that personalized medicine approaches might be harnessed to block SPARCL1 and improve outcomes in high-expressing patients.

immunology↗

bioRxiv2023

A Conserved Core Region of the Scaffold NEMO is Essential for Signal-induced Conformational Change and Liquid-liquid Phase Separation

Scaffold proteins help mediate interactions between protein partners, often to optimize intracellular signaling. Herein, we use comparative, biochemical, biophysical, molecular, and cellular approaches to investigate how the scaffold protein NEMO contributes to signaling in the NF-{kappa}B pathway. Comparison of NEMO and the related protein optineurin from a variety of evolutionarily distant organisms revealed that a central region of NEMO, called the Intervening Domain (IVD), is conserved between NEMO and optineurin. Previous studies have shown that this central core region of the IVD is required for cytokine-induced activation of I{kappa}B kinase (IKK). We show that the analogous region of optineurin can functionally replace the core region of the NEMO IVD. We also show that an intact IVD is required for the formation of disulfide-bonded dimers of NEMO. Moreover, inactivating mutations in this core region abrogate the ability of NEMO to form ubiquitin-induced liquid-liquid phase separation droplets in vitro and signal-induced puncta in vivo. Thermal and chemical denaturation studies of truncated NEMO variants indicate that the IVD, while not intrinsically destabilizing, can reduce the stability of surrounding regions of NEMO, due to the conflicting structural demands imparted on this region by flanking upstream and downstream domains. This conformational strain in the IVD mediates allosteric communication between N- and C-terminal regions of NEMO. Overall, these results support a model in which the IVD of NEMO participates in signal-induced activation of the IKK/NF-{kappa}B pathway by acting as a mediator of conformational changes in NEMO.

molecular biology↗

bioRxiv2023

aaHash: recursive amino acid sequence hashing

MotivationK-mer hashing is a common operation in many foundational bioinformatics problems. However, generic string hashing algorithms are not optimized for this application. Strings in bioinformatics use specific alphabets, a trait leveraged for nucleic acid sequences in earlier work. We note that amino acid sequences, with complexities and context that cannot be captured by generic hashing algorithms, can also benefit from a domain-specific hashing algorithm. Such a hashing algorithm can accelerate and improve the sensitivity of bioinformatics applications developed for protein sequences. ResultsHere, we present aaHash, a recursive hashing algorithm tailored for amino acid sequences. This algorithm utilizes multiple hash levels to represent biochemical similarities between amino acids. aaHash performs [~]10X faster than generic string hashing algorithms in hashing adjacent k-mers. Availability and implementationaaHash is available online at https://github.com/bcgsc/btllib and is free for academic use.

bioinformatics↗

bioRxiv2023

Inhibition of dopamine neurons prevents incentive value encoding of a reward cue:With revelations from deep phenotyping

The survival of an organism is dependent on their ability to respond to cues in the environment. Such cues can attain control over behavior as a function of the value ascribed to them. Some individuals have an inherent tendency to attribute reward-paired cues with incentive motivational value, or incentive salience. For these individuals, termed sign-trackers, a discrete cue that precedes reward delivery becomes attractive and desirable in its own right. Prior work suggests that the behavior of sign-trackers is dopamine-dependent, and cue-elicited dopamine in the nucleus accumbens is believed to encode the incentive value of reward cues. Here we exploited the temporal resolution of optogenetics to determine whether selective inhibition of ventral tegmental area (VTA) dopamine neurons during cue presentation attenuates the propensity to sign-track. Using male tyrosine hydroxylase (TH)-Cre Long Evans rats it was found that, under baseline conditions, [~]84% of TH-Cre rats tend to sign-track. Laser-induced inhibition of VTA dopamine neurons during cue presentation prevented the development of sign-tracking behavior, without affecting goal-tracking behavior. When laser inhibition was terminated, these same rats developed a sign-tracking response. Video analysis using DeepLabCut revealed that, relative to rats that received laser inhibition, rats in the control group spent more time near the location of the reward cue even when it was not present and were more likely to orient towards and approach the cue during its presentation. These findings demonstrate that cue-elicited dopamine release is critical for the attribution of incentive salience to reward cues. Significance StatementActivity of dopamine neurons in the ventral tegmental area (VTA) during cue presentation is necessary for the development of a sign-tracking, but not a goal-tracking, conditioned response in a Pavlovian task. We capitalized on the temporal precision of optogenetics to pair cue presentation with inhibition of VTA dopamine neurons. A detailed behavioral analysis with DeepLabCut revealed that cue-directed behaviors do not emerge without VTA dopamine. Importantly, however, when optogenetic inhibition is lifted, cue-directed behaviors increase, and a sign-tracking response develops. These findings confirm the necessity of VTA dopamine during cue presentation to encode the incentive value of reward cues.

neuroscience↗

bioRxiv2023

Long-term survival and induction of operational tolerance to murine islet allografts through the co-transplantation of cyclosporine A eluting microparticles

One strategy to prevent islet rejection, is to create a favorable immune-protective local environment at the transplant site. Herein, we utilize localized cyclosporine A (CsA) delivery to islet grafts via poly(lactic-co-glycolic acid) (PLGA) microparticles to attenuate allograft rejection. CsA microparticles alone significantly delayed islet allograft rejection compared to islets alone (p<0.05). Over 50% (6/11) of recipients receiving CsA microparticles and short-term cytotoxic T lymphocyte-associated antigen 4-Ig (CTLA4-Ig) therapy displayed prolonged allograft survival for 214 days, compared to 25% (2/8) receiving CTLA4-Ig alone (p>0.05). CsA microparticles + CTLA4-Ig islet allografts exhibited reduced T-cell (CD4+ and CD8+ cells) and macrophage (CD68+ cells) infiltration compared to islets alone. We observed reduced mRNA expression of proinflammatory cytokines (IL-6, IL-10, INF-{gamma} & TNF-; p<0.05) and chemokines (CCL2, CCL5, CCL22, and CXCL10; p<0.05) in CsA microparticles + CTLA4-Ig allografts compared to islets alone. Long-term islet allografts contained insulin+ and intra-graft FoxP3+ T regulatory cells. Rapid rejection of third-party skin grafts (C3H) in islet allograft recipients suggested that CsA microparticles + CTLA4-Ig therapy induced donor specific operational tolerance. This study demonstrates that localized CsA drug delivery plus short-course systemic immunosuppression promotes an immune protective transplant niche for allogeneic islets. Article HighlightsO_LISystemic immunosuppression limits patient inclusion for beta cell replacement therapies C_LIO_LILocalized islet graft immunosuppression may reduce drug toxicity and improve graft survival C_LIO_LICyclosporine eluting microparticles + CTLA4-Ig therapy induced donor specific operational tolerance C_LIO_LIGraft localized drug delivery can create an immune protective transplant niche C_LI

immunology↗