Biology subjects

Bott, R.

Publications and source records attributed to Bott, R..

3 recordsLinked to original sources

bioRxiv2024

Recombinant Pichinde reporter virus as a safe and suitable surrogate for high-throughput antiviral screening against highly pathogenic arenaviruses

Several arenaviruses, such as the Old World (OW) Lassa virus (LASV) and the New World (NW) Junin virus (JUNV), can cause severe and lethal viral hemorrhagic fevers in humans. Currently, no vaccines or specific antiviral therapies are FDA-approved for treating arenavirus infections. One major challenge for the development of new therapeutic candidates against these highly pathogenic viruses is that they are BSL-3/4 pathogens that need to be handled in high biocontainment laboratories. In this work, a recombinant non-pathogenic New World arenavirus, Pichinde virus (rPICV), was used for the development of a high-throughput screening (HTS) assay in the BSL-2 laboratory for the screening and identification of small molecule inhibitors against arenaviruses. The rPICV is a replication-competent virus expressing the firefly luciferase reporter gene in the infected cells proportionally to the infection rate. rPICV infection was optimized for an automated HTS in 384-well format with robust Z' scores, high signal-to-background ratios, and low intrinsic variance. Screening an established library allowed for the identification of five top hit compounds, which included ribavirin, a known inhibitor of arenaviral RNA synthesis, showing good potency and selectivity in inhibiting rPICV replication. The antiviral activity of the top hit compounds was further validated against another recombinant arenavirus, the OW lymphocytic choriomeningitis virus (rLCMV) and against laboratory strains of LASV (Josiah) and JUNV (Romero). The use of rPICV in the HTS-based antiviral assay under BSL-2 condition has proven to be safe and suitable for the identification of broad-spectrum small molecule inhibitors against highly pathogenic arenaviruses.

microbiology↗

bioRxiv2024

Molecular architecture of human dermal sleeping nociceptors

Human dermal sleeping nociceptors display ongoing activity in neuropathic pain, affecting 10% of the population. Despite advances in rodents, a molecular marker for these mechano-insensitive C-fibers (CMis) in human skin remains elusive, preventing targeted therapy. In this translational Patch-seq study, we combine single-cell transcriptomics following electrophysiological characterization with single-nucleus and spatial transcriptomics from pigs and humans. We functionally identified CMis in pig sensory neurons with patch-clamp using adapted protocols from human microneurography. We identified oncostatin-M-receptor (OSMR) and somatostatin (SST) as marker genes for CMis. Following dermal injection in healthy human volunteers, oncostatin-M, the ligand of OSMR, exclusively modulates CMis. We identified the entire molecular architecture of human dermal sleeping nociceptors, providing new therapeutic targets and the basis for a mechanistic understanding of neuropathic pain. One Sentence SummaryWe identify the molecular architecture and specifically OSMR and SST as molecular markers for human dermal sleeping nociceptors, key players in the generation of neuropathic pain. Short versionIn this Patch-seq study, we identify OSMR and SST as molecular markers for human dermal sleeping nociceptors, key players in the generation of neuropathic pain.

neuroscience↗

bioRxiv2022

Cold and warmth intensify pain-linked sodium channel gating effects and persistent currents

Voltage-gated sodium channels (Nav) are key players in excitable tissues with the capability to generate and propagate action potentials. Mutations in the genes encoding Navs can lead to severe inherited diseases, and some of these so-called channelopathies are showing temperature sensitive phenotypes, for example paramyotonia congenita, Brugada-syndrome, febrile seizure syndromes and inherited pain syndromes like erythromelalgia (IEM) and paroxysmal extreme pain disorder (PEPD). Nevertheless, most investigations of mutation-induced gating effects were conducted at room temperature and thus the role of cooling or warming in channelopathies remains poorly understood. Here, we investigated the temperature sensitivity of four Nav subtypes: Nav1.3, Nav1.5, Nav1.6, and Nav1.7 and two mutations in Nav1.7 causing IEM (Nav1.7/L823R) and PEPD (Nav1.7/I1461T), using an automated patch clamp system. Our experiments at 15 {degrees}C, 25 {degrees}C and 35 {degrees}C revealed a shift of the voltage dependence of activation to more hyperpolarized potentials with increasing temperature for all investigated subtypes. Nav1.3 exhibited strongly slowed inactivation kinetics compared to the other subtypes that resulted in enhanced persistent current especially at 15 {degrees}C, indicating a possible role in cold induced hyperexcitability. Impaired fast inactivation of Nav1.7/I1461T was significantly enhanced by cooling temperature to 15 {degrees}C. The subtype specific modulation as well as the intensified mutation induced gating changes stress the importance to consider temperature as regulator for channel gating and its impact on cellular excitability as well as disease phenotypes. SummaryActivation of the sodium channel subtypes Nav1.3, Nav1.5, Nav1.6, and Nav1.7 and two pain linked mutations is alleviated by warmth. Cooler temperatures, on the other hand, strongly enhance persistent currents of Nav1.3. The impaired fast inactivation of the pain-linked Nav1.7/I1461T mutation is further impaired by cooling, mimicking clinical findings.

physiology↗