Biology subjects

Hopkins, D.

Publications and source records attributed to Hopkins, D..

3 recordsLinked to original sources

bioRxiv2025

Structural and Evolutionary Constraints of Organophosphate Resistance in Dipteran Carboxylesterases

Enzymatic detoxification of organophosphate (OP) insecticides can confer resistance in some insects, yet the precise molecular basis of this trait, and how it has evolved, remains poorly understood. In certain dipteran species, a G[->]D mutation in the oxyanion hole of -carboxylesterases (CBEs) enhances OP hydrolysis, yet this adaptation is not widespread despite the presence of orthologous CBEs in other insect species that are also exposed to OPs. The extent, and molecular basis, of evolutionary contingency and epistasis in this catalytic OP resistance has not been explored, and how further mutations might optimize OP detoxification in the future is not clear. Here, we systematically compare OP hydrolysis and analyse structures of CBE orthologs across several dipteran species, revealing that the success of the G137D mutation is sequence context-dependent. We employed laboratory-directed evolution to enhance OP turnover over 1000-fold vs. the wild-type enzyme and tested these variants in transgenic Drosophila melanogaster, demonstrating that improved catalytic rates do not directly translate to increased resistance. By highlighting the trade-off between organophosphate affinity and turnover, this work further clarifies the complex evolutionary trajectories determining why a particular resistance mechanism may evolve in some species but not others. SignificanceThis study reveals the intricate evolutionary path to insecticide resistance in insects, highlighting why a potent resistance mutation is effective in some species but not others. We show that the mutations success is contingent on the enzymes pre-existing structural features, highlighting the strong intramolecular epistasis. Using laboratory evolution, we enhanced the enzymes detoxification activity over 1000-fold, yet discovered this did not translate to increased resistance in transgenic flies. This surprising result demonstrates that effective real-world resistance requires a delicate balance between an enzymes ability to bind an insecticide (affinity) and its speed at breaking it down (turnover), providing crucial insights into the constraints governing molecular adaptation.

biochemistry↗

bioRxiv2025

NAMPT activity plays a key role in driving autoimmune processes that characterize type 1 diabetes development in mice

Type 1 diabetes (T1D) is characterised by destruction of pancreatic beta cells by islet-infiltrating cytotoxic lymphocytes, and elevated intra-islet secretion of pro-inflammatory cytokines. However, the underlying pathophysiological mechanisms remain incompletely understood. We hypothesised that abnormal elevation of islet NAD, via activation of NAMPT, plays a key role in driving islet autoimmune processes in T1D. Here, we report that NAMPT inhibition protects against pro-inflammatory cytokine (IL-1{beta}, TNF and IFN{gamma}) mediated beta-cell dysfunction and apoptosis in isolated mouse and human islets. RNAseq revealed that NAMPT inhibition blocked cytokine-mediated gene expression linked to pro-inflammatory responses and leukocyte migration. In vivo, diabetes was induced in CD1 mice via multiple low dose streptozotocin (MLDS) injection. MLDS mice were administered the NAMPT inhibitor FK866 (10 mg/kg; IP) or saline equivalent for 16 days. These experiments demonstrated that NAMPT inhibition improved glycaemic control and beta-cell function and insulin content in MLDS mice. FK866 also reduced proportions of islet-residing TNF-producing CD4+T-cells and F4/80+macrophages, proliferation of spleen-derived CD4+ and CD8+T-cells, and proliferation of islet-derived CD4+T-cells and F4/80+macrophages. Finally, we report that NAMPT inhibition was able to block pro-inflammatory cytokine-mediated migration of cytotoxic CD8+T-cells into isolated islets, using an in vitro transwell platform. This data supports a key immunomodulatory role for NAMPT in islet autoimmunity. NAMPT inhibition may represent a novel therapeutic approach for T1D. The effects of increased NAD levels on islet inflammation require in-depth characterisation, and caution should be exercised with regard to use of NAD boosting supplements, particularly in individuals at risk of developing T1D.

cell biology↗

bioRxiv2024

Rat anterior cingulate neurons responsive to rule or strategy changes are modulated by the hippocampal theta rhythm and sharp-wave ripples

To better understand neural processing during adaptive learning of stimulus-response-reward contingencies, we recorded synchrony of neuronal activity in anterior cingulate cortex (ACC) with hippocampal rhythms in male rats acquiring and switching between spatial and visual discrimination tasks in a Y-maze. ACC population and single unit activity responded shortly after task rule changes, or just before the rats adopted different task strategies. Hippocampal theta oscillations (associated with memory encoding) modulated an elevated proportion of rule-change responsive neurons (70%), but other neurons that were correlated with strategy-change, strategy value, and reward-rate were not. However, hippocampal sharp wave-ripples modulated significantly higher proportions of rule-change, strategy-change and reward-rate responsive cells during post-session sleep but not pre-session sleep. This suggests an underestimated mechanism for hippocampal mismatch and contextual signals to facilitate ACC detection of contingency changes for cognitive flexibility, a function that is attenuated after it is damaged.

neuroscience↗