Biology subjects

Berryman, S.

Publications and source records attributed to Berryman, S..

4 recordsLinked to original sources

bioRxiv2025

Targeting Tmem63b and Piezo2 in C-fiber low threshold mechanoreceptor: limitation of Vglut3-IRES-Cre

Peripheral somatosensory neurons in the dorsal root ganglia (DRG) transduce mechanical force in the skin and other organs into electrical signals using specialized mechanically activated (MA) ion channels that initiate neuronal activation in response to force. Increasing evidence highlights PIEZO2 as the primary transducer of low-threshold mechanical force in DRG neurons. However, in the absence of Piezo2, mice and humans still respond to noxious painful stimuli like pinch, suggesting that additional MA channel(s) likely exist in DRG neurons. Strategies to identify Cre lines and DRG subpopulations that select for non-PIEZO2 expressing neurons is therefore an ongoing effort in the field to discover unknown mechanosensors. Here, we investigated a Vglut3 labeled mouse line as a candidate to identify non-PIEZO2 MA channels in a subtype of DRG neurons called C-fiber low threshold mechanoreceptors (C-LTMRs). Our study carefully demonstrates that the Vglut3-IRES-Cre mouse line specifically and efficiently labels C-LTMR neurons of the DRG. Electrophysiological recordings using two different in vitro mechanical stimulation assays show that the genetically labelled Vglut3 neurons have robust indentation- and stretch-activated MA currents that are exclusively slowly or ultra-slowly adapting. To determine whether the Vglut3-IRES-Cre mouse line can be used to delete genes of interest and identify the underlying MA ion channels in C-LTMRs we attempted to generate a Tmem63b conditional knockout using this Cre-line but detected incomplete loss of Tmem63b transcript and lack of TMEM63B- dependent effect on C-LTMR MA currents. Together, our results emphasize that although the Vglut3-IRES-Cre line is robust in driving expression of a conditional reporter gene, it is inefficient in deleting genes like Tmem63b as well as Piezo2. Statement of SignificanceIn this study, we identify a previously uncharacterized Vglut3-IRES-Cre as a tool to uncover unknown mechanosensors in C-LTMRs of the DRG. We methodically demonstrate that the Vglut3-IRES-Cre line labels C-LTMRs and express two mechanosensors, Tmem63b and Piezo2. However, we were unable to discern which gene contributes to the MA currents due to poor knockout efficiency induced by the Vglut3-IRES-Cre. Our studies highlight the nuances and challenges associated with Cre lines and alert future investigations to exercise caution while using Cre lines to discover the contribution of specific genes to mechanosensation.

neuroscience↗

bioRxiv2025

ITAF45 is a Pervasive Trans Acting Factor for Picornavirus Type II IRES Elements

Viruses have evolved elaborate mechanisms to hijack the host mRNA translation machinery to direct viral protein synthesis. Picornaviruses, whose RNA genomes lack a cap structure, inhibit cap-dependent mRNA translation, and utilize an internal ribosome entry site (IRES) in the RNA 5'-UTR to recruit the 40S ribosomal subunit. IRES activity is stimulated by a set of host proteins termed IRES trans-acting factors (ITAFs). The cellular protein ITAF45 (also known as PA2G4 and EBP1) was identified as an essential ITAF for foot-and-mouth disease virus (FMDV), with no apparent role in cell-free systems for the closely related viruses harboring similar IRES elements such as encephalomyocarditis virus (EMCV) and Theilers murine encephalomyelitis virus (TMEV). Here, we demonstrate that ITAF45 is a pervasive host factor within cells for picornaviruses containing a Type II IRES. CRISPR/Cas9 knockout of ITAF45 in several human cell lines conferred resistance to infection with FMDV, EMCV, TMEV, and equine rhinitis A virus (ERAV). We show that ITAF45 enhances initiation of translation on type II IRESs in cell line models. This is mediated by the C-terminal lysine-rich region of ITAF45 known to enable binding to viral RNA. These findings challenge previous reports of a unique role for ITAF45 in FMDV infection, positioning ITAF45 as a promising antiviral target for various animal viruses and emerging human cardioviruses.

microbiology↗

bioRxiv2024

TMEM63A, associated with hypomyelinating leukodystrophies, is an evolutionarily conserved regulator of myelination

Infantile hypomyelinating leukodystrophy 19 (HLD19) is a rare genetic disorder where patients exhibit reduced myelin in central nervous system (CNS) white matter tracts and present with varied neurological symptoms. The causative gene TMEM63A encodes a mechanosensitive ion channel whose role in myelination has not been explored. Our study shows that TMEM63A is a major regulator of OL-driven myelination in the CNS. In mouse and zebrafish, Tmem63a inactivation led to early deficits in myelination, recapitulating the HLD19 phenotype. OL-specific conditional mouse knockouts of Tmem63a exhibited transient reductions in myelin, indicating that TMEM63A regulates myelination cell-autonomously. We show that TMEM63A is present at plasma membrane and on lysosomes and modulates myelin/myelin-associated protein production. Intriguingly, HLD19-associated TMEM63A variants from patients blocked trafficking to cell membrane. Together, our results reveal an ancient role for TMEM63A in fundamental aspects of myelination in vivo and highlight two exciting new models for the development of treatments for devastating hypomyelinating leukodystrophies.

neuroscience↗

bioRxiv2019

Detection of antibodies against a conserved capsid epitope as the basis of a novel universal serological test for foot-and-mouth disease

Diagnostic tests for foot-and-mouth disease (FMD) include the detection of antibodies against either the viral non-structural proteins or the capsid. The detection of antibodies against the structural proteins (SP) of the capsid can be used to monitor seroconversion in both infected and vaccinated animals. However, SP tests need to be tailored to the individual FMD virus serotype and their sensitivity performances may be affected by antigenic variability within each serotype and mismatching between tests reagents. As a consequence, FMD Reference Laboratories need to maintain contingency to employ multiple type-specific assays for large-scale serological surveillance and post-vaccination monitoring in the event of FMD outbreaks. In this study, a highly conserved region in the N terminus of FMDV capsid protein VP2 (VP2N) was characterised using a panel of intertypic-reactive monoclonal antibodies. This revealed a universal epitope in VP2N which could be used as a peptide antigen to detect FMDV-specific antibodies against all types of the virus. A VP2-peptide ELISA (VP2-ELISA) was optimised using experimental and reference antisera from immunized, convalescent and negative animals (n=172). The VP2-ELISA is universal, simple and provided sensitive (98.6 %) and specific (93%) detection of antibodies to all FMDV strains used in this study. We anticipate that this SP test could have utility for sero-surveillance during virus incursions in FMD-free countries and as an additional screening tool to assess FMD virus circulation in endemic countries.

immunology↗