Biology subjects

Holcomb, M.

Publications and source records attributed to Holcomb, M..

5 recordsLinked to original sources

bioRxiv2024

An mRNA Display Approach for Covalent Targeting of a Staphylococcus aureus Virulence Factor

Staphylococcus aureus (S. aureus) is an opportunistic human pathogen that causes over one million deaths around the world each year. We recently identified a family of serine hydrolases termed fluorophosphonate binding hydrolases (Fphs) that play important roles in lipid metabolism and colonization of a host. Because many of these enzymes are only expressed in Staphylococcus bacteria, they are valuable targets for diagnostics and therapeutics. Here we developed and screened highly diverse cyclic peptide libraries using mRNA display with a genetically encoded oxadiazolone (Ox) electrophile that was previously shown to potently and covalently inhibit multiple Fph enzymes. By performing multiple rounds of counter selections with WT and catalytic dead FphB, we were able to tune the selectivity of the resulting selected cyclic peptides containing the Ox residue towards the desired target. From our mRNA display hits, we developed potent and selective fluorescent probes that label the active site of FphB at single digit nanomolar concentrations in live S. aureus bacteria. Taken together, this work demonstrates the potential of using direct genetically encoded electrophiles for mRNA display of covalent binding ligands and identifies potent new probes for FphB that have the potential to be used for diagnostic and therapeutic applications.

microbiology↗

bioRxiv2024

Antibiotic treatment induces microbiome dysbiosis and reduction of neuroinflammation following traumatic brain injury in mice.

Traumatic brain injury (TBI) induces both neuroinflammation and gut microbiome dysbiosis, yet the influence of antibiotics (ABX) on TBI-related neuropathology remains unclear. We administered a broad-spectrum oral ABX regimen to deplete the gut microbiome in single and repeated TBI mouse models. In male mice, ABX treatment significantly reduced neuroinflammation and neurodegeneration post-TBI, with no effects observed in uninjured controls. ABX also altered microbiome composition and decreased serum and fecal short-chain fatty acid levels, while intestinal damage and dysbiosis were further exacerbated by TBI severity. Notably, germ-free male mice exhibited heightened neuroinflammation and larger lesion volumes following TBI, underscoring the microbiomes essential role in recovery. Metagenomic analyses revealed Parasutterella excrementihominis and Lactobacillus johnsonii as potential ABX-resistant taxa post-injury. These findings suggest that short-term ABX treatment may attenuate TBI-induced neuroinflammation by reshaping the gut microbiome, offering directions for microbiome-targeted therapies in TBI.

neuroscience↗

bioRxiv2024

Probiotic treatment causes sex-specific neuroprotection after traumatic brain injury in mice

BackgroundRecent studies have shed light on the potential role of gut dysbiosis in shaping traumatic brain injury (TBI) outcomes. Changes in the levels and types of Lactobacillus bacteria present might impact the immune system disturbances, neuroinflammatory responses, anxiety and depressive-like behaviors, and compromised neuroprotection mechanisms triggered by TBI. ObjectiveThis study aimed to investigate the effects of a daily pan-probiotic (PP) mixture in drinking water containing strains of Lactobacillus plantarum, L. reuteri, L. helveticus, L. fermentum, L. rhamnosus, L. gasseri, and L. casei, administered for either two or seven weeks before inducing TBI on both male and female mice. MethodsMice were subjected to controlled cortical impact (CCI) injury. Short-chain fatty acids (SCFAs) analysis was performed for metabolite measurements. The taxonomic profiles of murine fecal samples were evaluated using 16S rRNA V1-V3 sequencing analysis. Histological analyses were used to assess neuroinflammation and gut changes post-TBI, while behavioral tests were conducted to evaluate sensorimotor and cognitive functions. ResultsOur findings suggest that PP administration modulates the diversity and composition of the microbiome and increases the levels of SCFAs in a sex-dependent manner. We also observed a reduction of lesion volume, cell death, and microglial and macrophage activation after PP treatment following TBI in male mice. Furthermore, PP-treated mice show motor function improvements and decreases in anxiety and depressive-like behaviors. ConclusionOur findings suggest that PP administration can mitigate neuroinflammation and ameliorate motor and anxiety and depressive-like behavior deficits following TBI. These results underscore the potential of probiotic interventions as a viable therapeutic strategy to address TBI-induced impairments, emphasizing the need for gender-specific treatment approaches.

neuroscience↗

bioRxiv2024

IP6 and PF74 affect HIV-1 Capsid Stability through Modulation of Hexamer-Hexamer Tilt Angle Preference

The HIV-1 capsid is an irregularly shaped complex of about 1200 protein chains containing the viral genome and several viral proteins. Together, these components are the key to unlocking passage into the nucleus, allowing for permanent integration of the viral genome into the host cell genome. Recent interest into the role of the capsid in viral replication has been driven by the approval of the first-in-class drug lenacapavir, which marks the first drug approved to target a non-enzymatic HIV-1 viral protein. In addition to lenacapavir, other small molecules such as the drug-like compound PF74, and the anionic sugar inositolhexakisphosphate (IP6), are known to impact capsid stability, and although this is widely accepted as a therapeutic effect, the mechanisms through which they do so remain unknown. In this study, we employed a systematic atomistic simulation approach to study the impact of molecules bound to hexamers at the central pore (IP6) and the FG-binding site (PF74) on capsid oligomer dynamics, compared to apo hexamers and pentamers. We found that neither small molecule had a sizeable impact on the free energy of binding of the interface between neighboring hexamers but that both had impacts on the free energy profiles of performing angular deformations to the pair of oligomers akin to the variations in curvature along the irregular surface of the capsid. The IP6 cofactor, on one hand, stabilizes a pair of neighboring hexamers in their flattest configurations, whereas without IP6, the hexamers prefer a high tilt angle between them. On the other hand, having PF74 bound introduces a strong preference for intermediate tilt angles. These results suggest that structural instability is a natural feature of the HIV-1 capsid which is modulated by molecules bound in either the central pore or the FG-binding site. Such modulators, despite sharing many of the same effects on non-bonded interactions at the various protein-protein interfaces, have decidedly different effects on the flexibility of the complex. This study provides a detailed model of the HIV-1 capsid and its interactions with small molecules, informing structure-based drug design, as well as experimental design and interpretation.

biophysics↗

bioRxiv2023

Sex-dependent improvement in traumatic brain injury outcomes after liposomal delivery of dexamethasone in mice.

Traumatic Brain Injury (TBI) can have long-lasting physical, emotional, and cognitive consequences due to the neurodegeneration caused by its robust inflammatory response. Despite advances in rehabilitation care, effective neuroprotective treatments for TBI patients are lacking. Furthermore, current drug delivery methods for TBI treatment are inefficient in targeting inflamed brain areas. To address this issue, we have developed a liposomal nanocarrier (Lipo) encapsulating dexamethasone (Dex), an agonist for the glucocorticoid receptor utilized to alleviate inflammation and swelling in various conditions. In vitro studies show that Lipo-Dex were well tolerated in human and murine neural cells. Lipo-Dex showed significant suppression of inflammatory cytokines, IL-6 and TNF-, release after induction of neural inflammation with lipopolysaccharide. Further, the Lipo-Dex were administered to young adult male and female C57BL/6 mice immediately after a controlled cortical impact injury. Our findings demonstrate that Lipo-Dex can selectively target the injured brain, thereby reducing lesion volume, cell death, astrogliosis, the release of proinflammatory cytokines, and microglial activation compared to Lipo-treated mice in a sex-dependent manner, showing a major impact only in male mice. This highlights the importance of considering sex as a crucial variable in developing and evaluating new nano-therapies for brain injury. These results suggest that Lipo-Dex administration may effectively treat acute TBI.

neuroscience↗