Biology subjects

Johnson, M. B.

Publications and source records attributed to Johnson, M. B..

12 recordsLinked to original sources

bioRxiv2026

A Novel PD-L1 Splice Isoform Modulates β Cell Communication in Response to Interferon Signaling

{beta} cell expression of the immune checkpoint ligand PD-L1 (encoded by CD274) limits autoimmune {beta} cell destruction in type 1 diabetes (T1D). {beta} cell display PD-L1 not only at the cell surface but also secreted on extracellular vesicles (EVs), which bind PD-1 and restrain CD8+ T cell activation. {beta} cell IFN signaling is an early driver of T1D pathogenesis, yet the mechanisms linking IFN signaling to the fate of PD-L1 protein remain undefined. Here, we identify an IFN- and coxsackievirus-inducible alternatively spliced isoform of PD-L1, PD-L1{Delta}3, lacking cassette exon 3 and generated in human {beta} cells and islets in response to IFN signaling or viral infection. PD-L1{Delta}3 transcripts are elevated in islets from donors with single autoantibody positivity (AAB+) and T1D. Unlike full-length PD-L1, which localizes to the plasma membrane, PD-L1{Delta}3 is retained intracellularly and loses the capacity to bind PD-1. Functionally, PD-L1{Delta}3 fails to suppress CD8+ and CD4+ T cell proliferation, activation, and cytotoxic cytokine release, and is not efficiently sorted into EVs. Convergently, a germline CD274 splice-site variant (c.682+1G>A) found in siblings with neonatal T1D yields a protein with the same intracellular retention, reduced {beta} cell PD-1 binding, and reduced {beta} cell and circulating EV PD-L1. Together these findings define PD-L1{Delta}3 as an IFN-induced splice variant that diverts PD-L1 away from its immunoregulatory, EV-competent form, revealing a post-transcriptional axis that shapes {beta} cell immune communication.

cell biology↗

bioRxiv2026

Comparative analysis of microglia-targeted AAVs reveals capsid choice drives efficiency in vitro but has limited impact in vivo

Microglia play key roles in brain development, homeostasis, and neurodegeneration. Although multiple strategies for viral gene delivery to microglia have been reported, they have not been directly compared. Here, we developed microglia-targeting AAV capsids and benchmarked them against existing approaches. The novel capsids exhibit improved transduction efficiency in cultured mouse and human microglia, as well as neurons and astrocytes. However, when we compared microglial transduction efficiency of the novel capsids with published engineered and naturally occurring capsids after intracranial injection, all capsids achieved efficient and specific transduction when paired with a genome incorporating IBA1 promoter and miR-124 target sites. In contrast, CAG promoter did not support efficient microglial transduction. Moreover, blood-brain barrier- crossing capsids carrying IBA1 promoter and miR-124 target sites efficiently transduced microglia at high doses but exhibited off-target expression. Together, our work provides improved capsids for in vitro manipulation of microglia and establishes viral genome design, not capsid identity, as the principal determinant of efficient in vivo microglial targeting.

neuroscience↗

bioRxiv2026

iAstrocytes model cytokine influences on complement expression and neuronal network synchronization.

Astrocytes play essential roles in neuronal development, function, and disease, yet existing methods to derive astrocytes from human pluripotent stem cells (hPSCs) are complex and can involve months of in vitro maturation. We developed a genomic safe-harbor knock-in system for inducible expression of the astrogenic transcription factors NFIA, NFIB, and SOX9, enabling rapid and robust generation of functional induced astrocytes (iAstrocytes). Across five hPSC lines, NFIB-SOX9 and NFIA-NFIB-SOX9 combinations efficiently generated highly pure populations expressing astrocyte-specific and synaptogenic genes. iAstrocytes displayed cytokine-induced expression of complement factors C3 and C4 and were amenable to CRISPR interference (CRISPRi) gene expression knockdown. Optimization of culture conditions enabled survival of NFIB-SOX9 iAstrocytes in co-culture with human induced neurons (iNeurons). Through pharmacological and genetic perturbations, we uncovered a previously undescribed phenomenon in which co-culture with iAstrocytes promoted the development of synchronized iNeuron network calcium activity mediated by specific gap junction proteins. This rapid and genetically tractable iAstrocyte platform provides a robust model to dissect human genetic and environmental effects on astrocyte-neuron interactions.

neuroscience↗

bioRxiv2025

Spatially Organized DNA-templated Silver Nanoclusters as Potent Antimicrobial Agents for ESKAPE Infections

Antibiotic-resistant bacteria cause more than one million deaths annually worldwide. The rapid evolution and gene exchange among pathogens often render new antibiotic drugs ineffective soon after deployment, underscoring the urgent need for alternative therapeutic strategies. Nanoscale silver is well known for its innate bacteriostatic and bactericidal activity but typically requires high concentrations for efficacy that causes toxicities and limits broader clinical applications. To address this, we introduce programmable self-assembling DNA scaffolds that template, stabilize, and spatially organize multiple copies of monodisperse silver nanoclusters (DNA-AgNCs). These assemblies enhance antimicrobial potency of formulations while also exhibiting intrinsic fluorescence, providing dual functionality for therapeutic and bioimaging applications. Detailed characterization identified DNA-AgNC scaffolds with improved stability and enhanced activity against clinically relevant antibiotic-resistant planktonic ESKAPE pathogens. We also revealed the DNA-AgNC constructs that significantly lowered intracellular infections of human bone cells with Staphylococcus aureus. Collectively, the results highlight spatially organized DNA-AgNCs as a promising modular platform for next-generation antibacterial therapy and real-time bioimaging.

biochemistry↗

bioRxiv2025

Tribbles 3 blunts exercise-induced skeletal muscle adaptation

ABSTRACTTribbles 3 (TRB3) is a pseudokinase and its expression has been shown to disrupt glucose metabolism through the inhibition of Akt under obese and diabetic conditions. We recently found that overexpression of TRB3 in mouse skeletal muscle decreased skeletal muscle mass and function, leading to muscle atrophy. Here, we examined whether TRB3 affects exercise training-induced skeletal muscle adaptation. We trained muscle-specific TRB3 transgenic (TG) and wild-type (WT) littermates using a voluntary wheel running protocol for 6 weeks and found that TG mice ran significantly less weekly distances than WT littermates. To exclude the possibility that different skeletal muscle adaptations would be produced due to different training intensities, involuntary treadmill exercise (TM) was used as a training regimen. At the 5th week of training, we measured glucose tolerance and found that trained TG mice showed glucose intolerance compared to WT littermates. Furthermore, overexpression of TRB3 significantly suppressed the expression of genes needed for glucose uptake and mitochondrial biogenesis, independent of training status. To further determine the role of TRB3 in exercise-induced adaptation, TRB3 knockout (KO) mice were trained by voluntary and involuntary exercise protocols. KO mice presented improved glucose tolerance compared to WT littermates independent of training status. However, we did not observe significant change in the expression of markers for glucose uptake and mitochondrial biogenesis. Taken together, our results indicate that TRB3 in skeletal muscle blunts the benefits of exercise-induced skeletal muscle adaptation.

physiology↗

bioRxiv2025

Elephants inhabiting two forested sites in western Uganda exhibit contrasting patterns of species identity, density, and history of hybridization

Elephant populations across much of Africa face severe rates of decline due to poaching and habitat loss. The recent decision by the International Union for the Conservation of Nature (IUCN) to separately list African forest (Loxodonta cyclotis) and savanna (L. africana) elephants on the IUCN Red List both highlights the different threats of extinction faced by these two species and emphasizes the need for genetic data to classify taxonomically undefined populations across the continent. This includes western Uganda - a region that harbors the largest known modern hybrid zone between the two species. We combined a new high-throughput amplicon sequencing (HTAS) approach with fecal DNA-based Capture Mark Recapture (CMR) analysis to infer the population sizes and species compositions of elephants living in two forests. We demonstrate that Kibale National Park hosts a relatively large elephant population (573 individuals, 95% CI: 410 to 916; 0.72 elephants/km2) composed primarily of hybrids (81.5%) and savanna elephants (17.7%), while Bwindi Impenetrable National Park hosts a smaller population (96 individuals, 95% CI: 64 to 145; 0.29 elephants/km2) composed of forest elephants (86.8%) and hybrids (13.2%). We then sequenced maternally inherited (mtDNA) and paternally inherited (AMELY) genetic markers and found that the two parks populations exhibit different patterns of sex-linked genetic variation. The contrasting patterns of species identity and genetic variation between these parks demonstrate different histories of hybridization and highlight the importance of site-specific monitoring where elephants are taxonomically undefined.

ecology↗

bioRxiv2022

Phagocytosis via complement receptor 3 enables microbes to evade killing by neutrophils

Complement receptor 3 (CR3; CD11b/CD18; m{beta}2 integrin) is a conserved phagocytic receptor. The active conformation of CR3 binds the iC3b fragment of complement C3 as well as many host and microbial ligands, leading to actin-dependent phagocytosis. There are conflicting reports about how CR3 engagement affects the fate of phagocytosed substrates. Using imaging flow cytometry, we confirmed that binding and internalization of iC3b-opsonized polystyrene beads by primary human neutrophils was CR3-dependent. iC3b-opsonized beads did not stimulate neutrophil reactive oxygen species (ROS), and most beads were found in primary granule-negative phagosomes. Similarly, Neisseria gonorrhoeae (Ngo) that does not express phase-variable Opa proteins suppresses neutrophil ROS and delays phagolysosome formation. Here, binding and internalization of Opa-deleted ({Delta}opa) Ngo by adherent human neutrophils was inhibited using blocking antibodies against CR3 and by adding neutrophil inhibitory factor, which targets the CD11b I-domain. Neutrophils did not produce detectable amounts of C3 to opsonize Ngo. Conversely, overexpressing CD11b in HL-60 promyelocytes enhanced {Delta}opa Ngo phagocytosis, which required CD11b I domain. Phagocytosis of Ngo was also inhibited in mouse neutrophils that were CD11b-deficient or treated with anti-CD11b. Phorbol ester treatment upregulated surface CR3 on neutrophils in suspension, enabling CR3-dependent phagocytosis of {Delta}opa Ngo. Neutrophils exposed to {Delta}opa Ngo had limited phosphorylation of Erk1/2, p38, and JNK. Neutrophil phagocytosis of unopsonized Mycobacterium smegmatis, which also resides in immature phagosomes, was CR3-dependent and did not elicit ROS. We suggest that CR3-mediated phagocytosis is a silent mode of entry into neutrophils, which is appropriated by diverse pathogens to subvert phagocytic killing.

microbiology↗

bioRxiv2020

CUB and Sushi Multiple Domains 1 (CSMD1) opposes the complement cascade in neural tissues

Schizophrenia risk is associated with increased gene copy number and brain expression of complement component 4 (C4). Because the complement system facilitates synaptic pruning, the C4 association has renewed interest in a hypothesis that excessive pruning contributes to schizophrenia pathogenesis. However, little is known about complement regulation in neural tissues or whether such regulation could be relevant to psychiatric illness. Intriguingly, common variation within CSMD1, which encodes a putative complement inhibitor, has consistently associated with schizophrenia at genome-wide significance. We found that Csmd1 is predominantly expressed in the brain by neurons, and is enriched at synapses; that human stem cell-derived neurons lacking CSMD1 are more vulnerable to complement deposition; and that mice lacking Csmd1 have increased brain complement activity, fewer synapses, aberrant complement-dependent development of a neural circuit, and synaptic elements that are preferentially engulfed by cultured microglia. These data suggest that CSMD1 opposes the complement cascade in neural tissues. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/291427v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@172c857org.highwire.dtl.DTLVardef@bee84corg.highwire.dtl.DTLVardef@184da10org.highwire.dtl.DTLVardef@10867c8_HPS_FORMAT_FIGEXP M_FIG Graphic Abstract.Our findings support a model in which CSMD1 opposes actions of the complement cascade in neural tissues (top left). We investigated two models in which Csmd1 was genetically ablated: human cortical neurons derived from embryonic stem cells, and a back-crossed C57bl6-Tac mouse line (top right). Csmd1 is normally expressed by neurons and present at synapses where it can protect them from complement (bottom left); in the absence of Csmd1 (bottom right), we find more deposition of complement (on cultured human cortical neurons and in the mouse visual system), reduced numbers of synapses (in the mouse visual system), and synaptic fractions that are more readily engulfed by microglia (ex vivo). Created with BioRender.com. C_FIG

neuroscience↗

bioRxiv2019

Abundance, density, and social structure of African forest elephants (Loxodonta cyclotis) in a human-modified landscape in southwestern Gabon

The noninvasive monitoring of population size and demography is critical to effective conservation, but forest living taxa can be difficult to directly observe due to elusiveness and/or inaccessible habitat. This has been true of African forest elephants (Loxodonta cyclotis), for which we have limited information regarding population size and social behavior despite their threatened conservation status. In this study, we estimated demographic parameters focusing specifically on population size and density using genetic capture-recapture of forest elephants in the southern Industrial Corridor of the Gamba Complex of Protected Areas, which is considered a global stronghold for forest elephants in southwestern Gabon. Additionally, we examined forest elephant sociality through analysis of social networks, predicting that we would find matrilineal structure as exhibited by savanna elephants and other forest elephants. Given 95% confidence intervals, we estimate the size of the population in the sampled area to be between 754 and 1,502 individuals and our best density estimate ranges from 0.47 to 0.80 elephants per km2. When extrapolated across the entire Industrial Corridor, this estimate suggests an elephant population size of 3,033 to 6,043 in this area based on abundance or 1,684 to 2,832 based on density, which is 40 - 83% smaller than previously suggested. Furthermore, our social network analysis revealed approximately half of network components included females with different mitochondrial haplotypes; this suggests a wider range of variation in forest elephant sociality than has previously been reported. This study emphasizes the threatened status of forest elephants and demonstrates the need to further refine baseline estimates of population size and knowledge on social behavior in this taxon, both of which will aid in determining how population dynamics in this keystone species may be changing through time in relation to increasing conservation threats.

genetics↗

bioRxiv2019

SavvyCNV: genome-wide CNV calling from off-target reads

Identifying copy number variants (CNVS) can provide diagnoses to patients and provide important biological insights into human health and disease. Current exome and targeted sequencing approaches cannot detect clinically and biologically-relevant CNVs outside their target area. We present SavvyCNV, a tool which uses off-target read data to call CNVs genome-wide. Up to 70% of sequencing reads from exome and targeted sequencing fall outside the targeted regions - SavvyCNV exploits this free data.\n\nWe benchmarked SavvyCNV using truth sets generated from genome sequencing data and Multiplex Ligation-dependent Probe Amplification assays. SavvyCNV called CNVs with high precision and recall, outperforming five state-of-the-art CNV callers at calling CNVs genome-wide using off-target or on-target reads from targeted panel and exome sequencing. Furthermore SavvyCNV was able to call previously undetected clinically-relevant CNVs from targeted panel data highlighting the utility of this tool within the diagnostic setting. SavvyCNV is freely available.

bioinformatics↗

bioRxiv2018

Correlates of cannabinoid concentrations, real-world driving, and driving-related skills

Research on the relationship between cannabis use and safe driving has produced mixed results. Most studies have focused exclusively on the presence or concentration of delta-9-tetrahydrocannabinol (THC), the primary psychoactive ingredient in the drug. However, cannabis is a complex substance, and both toxicological research and user experience suggests that some cannabis strains--often those with at least moderate levels of cannabidiol (CBD)-- produce a different, more sedating \"high\" than cannabis strains with no or low levels of CBD. We hypothesize that the sedating properties of some high-CBD cannabis strains has potential to impair driving and driving-related skills above and beyond the effects of THC intoxication. Three studies--one instrumented vehicle driving study and two laboratory-style epidemiological studies--examined real driving and computerized task performance as a function THC and CBD concentrations (and their interactions). In all three studies, higher CBD levels predicted greater impairment. There was relatively little evidence of impairment when CBD was zero, even at high THC levels. The results suggest that THC concentrations alone are not sufficient to predict impairment due to cannabis use. Results are interpreted in the context of drug tolerance.

epidemiology↗

bioRxiv2017

Heterozygous RFX6 protein truncating variants cause Maturity-Onset Diabetes of the Young (MODY) with reduced penetrance

Finding new genetic causes of monogenic diabetes can help to understand development and function of the human pancreas. We aimed to find novel protein-truncating variants causing Maturity-Onset Diabetes of the Young (MODY), a subtype of monogenic diabetes. We used a combination of next-generation sequencing of MODY cases with unknown aetiology along with comparisons to the ExAC database to identify new MODY genes. In the discovery cohort of 36 European patients, we identified two probands with novel RFX6 heterozygous nonsense variants. RFX6 protein truncating variants were enriched in the MODY discovery cohort compared to the European control population within ExAC (odds ratio, OR=131, P=lxl0-4). We found similar results in non-Finnish European (n=348, OR=43, P=5xl0-5) and Finnish (n=80, OR=22, P=1xl0-6) replication cohorts. The overall meta-analysis OR was 34 (P=lxl0-16). RFX6 heterozygotes had reduced penetrance of diabetes compared to common HNF1A and HNF4A-MODY mutations (27%, 70% and 55% at 25 years of age, respectively). The hyperglycaemia resulted from beta-cell dysfunction and was associated with lower fasting and stimulated gastric inhibitory polypeptide (GIP) levels. Our study demonstrates that heterozygous RFX6 protein truncating variants are associated with MODY with reduced penetrance.

genetics↗