Biology subjects

Pennington, J.

Publications and source records attributed to Pennington, J..

5 recordsLinked to original sources

bioRxiv2026

A bistable type III secretion system is primed and loaded in Pseudomonas aeruginosa hypervirulent isolates with positively selected codon changes in exsA or exsD

Pseudomonas aeruginosa has multiple virulence factors that promote airway infections in people with immune deficiencies. Most clinical isolates from human infections have functional type III secretion systems (T3SSs) that are subject to bistable regulation and secrete the cytotoxic effector ExoS into neutrophils. During airway infection in people with cystic fibrosis (CF), P. aeruginosa maintains bistable regulation or genetically adapts by inactivating or hyperactivating T3SS expression. CF clinical isolates with hyperactive T3SSs have codon changes in the transcriptional regulators, exsA or exsD. It is not known how these mutations: 1) arise, 2) promote hypervirulence, or 3) impact bistable T3SS regulation. To understand how these mutants arise, ~24,000 genomes in the NCBI Pathogens database were searched for positively selected mutations in exs genes based on nonsynonomous to synonymous codon change ratios. Missense and nonsense mutations were detected in exsA (47) or exsD (45) the majority of which are predicted to be positively selected. To determine how one of these codon changes promotes hypervirulence, an exsAT48I isolate was used to establish lung infections in mice engineered with a CF genotype. The exsAT48I mutant and an exsA+ control had similar lung burdens, but the former caused exacerbated weight loss and lung inflammation, suggesting immunopathology from phagocyte death as the cause of hypervirulence. Two additional hyperactive mutants were identified by screening a small collection of isolates from children with CF. Immunoblotting and fluorescence microscopy showed that hyperactive exsA or exsD mutants increased production of T3SS injectisomes and effectors, indicating they have locked on (primed and loaded) phenotypes. ExsA dimer modeling revealed that codon changes like T48I in the {beta}-barrel domain cause a conformational change that hyperactivates the transcription factor. These results define that hyperactive mutants are positively selected, hypervirulent due to lung inflammation, and have primed and loaded T3SSs.

microbiology↗

bioRxiv2026

A System for Live Sorting of Neuronal Spiking Activity from Large-scale Recordings

Online monitoring of neuronal activity has tremendous value for closed-loop control in both experimental and clinical settings, yet established methods rely primarily on thresholded neural activity, rather than sorted single-neuron spikes. The recent introduction of large-scale electrophysiological tools has greatly expanded single-neuron recording capacity in animals and humans highlighting the need to sort spikes during data collection. Here, we describe a system for live spike sorting (LSS) populations of hundreds of neurons with millisecond-scale latency. Using neurophysiological recordings from macaque visual cortex with Neuropixels probes, we show that LSS closely replicates the temporal responses and tuning of single neurons obtained using offline sorting. We further show that decoding neural signals with LSS achieves the same performance as that obtained from offline sorting. Lastly, we demonstrate the capacity of LSS to enable closed-loop interventions based on the activity of specific neuronal subclasses.

neuroscience↗

bioRxiv2023

Activation of the aryl hydrocarbon receptor inhibits neuropilin-1 upregulation on IL-2 responding CD4+ T cells

Neuropilin-1 (Nrp1), a transmembrane protein expressed on CD4+ T cells, is mostly studied in the context of regulatory T cell (Treg) function. More recently, there is increasing evidence that Nrp1 is also highly expressed on activated effector T cells and that increases in these Nrp1-expressing CD4+ T cells correspond with immunopathology across several T cell-dependent disease models. Thus, Nrp1 may be implicated in the identification and function of immunopathologic T cells. Nrp1 downregulation in CD4+ T cells is one of the strongest transcriptional changes in response to immunoregulatory compounds that act though the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor. To better understand the link between AhR and Nrp1 expression on CD4+ T cells, Nrp1 expression was assessed in vivo and in vitro following AhR ligand treatment. In the current study, we identified that the percentage of Nrp1 expressing CD4+ T cells increases over the course of activation and proliferation in vivo. The actively dividing Nrp1+Foxp3- cells express the classic effector phenotype of CD44hiCD45RBlo, and the increase in Nrp1+Foxp3- cells is prevented by AhR activation. In contrast, Nrp1 expression is not modulated by AhR activation in non-proliferating CD4+ T cells. The downregulation of Nrp1 on CD4+ T cells was recapitulated in vitro in cells isolated from C57BL/6 and NOD (non-obese diabetic) mice. CD4+Foxp3- cells expressing CD25, stimulated with IL-2, or differentiated into Th1 cells, were particularly sensitive to AhR-mediated inhibition of Nrp1 upregulation. IL-2 was necessary for AhR-dependent downregulation of Nrp1 expression both in vitro and in vivo. Collectively, the data demonstrate that Nrp1 is a CD4+ T cell activation marker and that regulation of Nrp1 could be a previously undescribed mechanism by which AhR ligands modulate effector CD4+ T cell responses.

immunology↗

bioRxiv2022

Nodavirus RNA Replication Crown Architecture Reveals Proto-Crown Precursor and Viral Protein A Conformational Switching

Positive-strand RNA viruses replicate their genomes in virus-induced membrane vesicles, and the resulting RNA replication complexes are a major target for virus control. Nodavirus studies first revealed viral RNA replication proteins forming a 12-fold symmetric "crown" at the vesicle opening to the cytosol, an arrangement recently confirmed to extend to distantly related alphaviruses. Using cryo-electron microscopy (cryo-EM), we show that mature nodavirus crowns comprise two stacked 12-mer rings of multi-domain viral RNA replication protein A. Each ring contains an ~19 nm circle of C-proximal polymerase domains, differentiated by strikingly diverged positions of N-proximal RNA capping/membrane binding domains. The lower ring is a "proto-crown" precursor that assembles prior to RNA template recruitment, RNA synthesis and replication vesicle formation. In this proto-crown, the N-proximal segments interact to form a toroidal central floor, whose 3.1 [A] resolution structure reveals many mechanistic details of the RNA capping/membrane binding domains. In the upper ring, cryo-EM fitting indicates that the N-proximal domains extend radially outside the polymerases, forming separated, membrane-binding "legs." The polymerase and N-proximal domains are connected by a long linker accommodating the conformational switch between the two rings and possibly also polymerase movements associated with RNA synthesis and non-symmetric electron density in the lower center of mature crowns. The results reveal remarkable viral protein multifunctionality, conformational flexibility and evolutionary plasticity and new insights into (+)RNA virus replication and control. SignificancePositive-strand RNA viruses - including coronaviruses, alphaviruses, flaviviruses and many other medically and economically important pathogens - replicate their RNA genomes by virus-encoded machinery that has been poorly characterized. Using an advanced nodavirus model, we identify a major precursor in RNA replication complex assembly and show it to be a 12-mer ring of viral RNA replication protein A, whose single particle cryo-EM structure reveals functional features of its membrane interaction, assembly, polymerase and RNA capping domains. We further show that fully functional RNA replication complexes acquire a second 12-mer ring of protein A in alternate conformation atop the first, and a central density likely to represent another polymerase conformation. These findings provide strong foundations for understanding, controlling and beneficially using such viruses.

microbiology↗

bioRxiv2019

Creatine transporter knockout mice (Slc6a8) show increases in serotonin-related proteins and are resilient to learned helplessness

Approximately 20% of adults in the U.S. will experience an affective disorder during their life. While it is well established that serotonin (5-HT) is a crucial factor in mood, impaired cellular bioenergetics are also implicated. Creatine (Cr), through the Cr/Phospho-Cr (PCr) shuttle, maintains high ATP concentrations in the neuron. This system may be implicated in the etiology of affective disorders, as reduced Cr, PCr, and ATP are often seen in the brains of affected patients. To address this issue, Cr transporter (Crt) deficient male mice (Slc6a8-/y) and female mice heterozygous for Crt expression (Slc6a8+/-) were used to evaluate how a Cr deficient system would alter affective-like behaviors. Slc6a8-/y and Slc6a8+/- mice had more escapes and faster escape latencies in learned helplessness, indicating a potential resilience to behavioral despair. Elevated zero maze and tail-suspension test performance matched that of wildtype mice, however. Slc6a8-/y mice have increased 5-hydroxyindoleacetic acid content in the hippocampus and striatum and increased monoamine oxidase protein and tryptophan hydroxylase-2 protein content in the hippocampus, while serotonin levels are unchanged. This indicates an increase in 5-HT turnover. Our results indicate that Cr plays a complex role in affective disorders and 5-HT neurotransmission, warranting further investigation.

animal behavior and cognition↗