A live cell assay of GPCR coupling allows identification of optogenetic tools for controlling Go and Gi signaling

Abstract Background Animal opsins are light-sensitive G-protein-coupled receptors (GPCRs) that enable optogenetic control over the major heterotrimeric G-protein signaling pathways in animal cells. As such, opsins have potential applications in both biomedical research and therapy. Selecting the opsin with the best balance of activity and selectivity for a given application requires knowing their ability to couple to a full range of relevant Gι subunits. We present the GsX assay, a set of tools based on chimeric Gs subunits that transduce coupling of opsins to diverse G proteins into increases in cAMP levels,  measured with a real-time reporter in living cells. We use this assay to compare coupling to Gi/o/t across a panel of natural and chimeric opsins selected for potential application in gene therapy for retinal degeneration. Results Of the opsins tested, wild-type human rod opsin had the highest activity for chimeric Gs proxies for Gi and Gt (Gsi and Gst) and was matched in Go proxy (Gso) activity only by a human rod opsin/scallop opsin chimera. Rod opsin drove roughly equivalent responses via Gsi, Gso, and Gst, while cone opsins showed much lower activities with Gso than Gsi or Gst, and a human rod opsin/amphioxus opsin chimera demonstrated higher activity with Gso than with Gsi or Gst. We failed to detect activity for opsin chimeras bearing three intracellular fragments of mGluR6, and observed unexpectedly complex response profiles for scallop and amphioxus opsins thought to be specialized for Go. Conclusions These results identify rod opsin as the most potent non-selective Gi/o/t-coupled opsin, long-wave sensitive cone opsin as the best for selectively activating Gi/t over Go, and a rod opsin/amphioxus opsin chimera as the best choice for selectively activating Go over Gi/t

An RNAi-mediated screen identifies novel targets for next-generation antiepileptic drugs based on increased expression of the homeostatic regulator pumilio

Despite availability of a diverse range of anti-epileptic drugs (AEDs), only about two-thirds of epilepsy patients respond well to drug treatment. Thus, novel targets are required to catalyse the design of next-generation AEDs. Manipulation of neuron firing-rate homoeostasis, through enhancing Pumilio (Pum) activity, has been shown to be potently anticonvulsant in Drosophila. In this study, we performed a genome-wide RNAi screen in S2R + cells, using a luciferase-based dPum activity reporter and identified 1166 genes involved in dPum regulation. Of these genes, we focused on 699 genes that, on knock-down, potentiate dPum activity/expression. Of this subgroup, 101 genes are activity-dependent based on comparison with genes previously identified as activity-dependent by RNA-sequencing. Functional cluster analysis shows these genes are enriched in pathways involved in DNA damage, regulation of cell cycle and proteasomal protein catabolism. To test for anticonvulsant activity, we utilised an RNA-interference approach in vivo. RNAi-mediated knockdown showed that 57/101 genes (61%) are sufficient to significantly reduce seizure duration in the characterized seizure mutant, parabss. We further show that chemical inhibitors of protein products of some of the genes targeted are similarly anticonvulsant. Finally, to establish whether the anticonvulsant activity of identified compounds results from increased dpum transcription, we performed a luciferase-based assay to monitor dpum promoter activity. Third instar larvae exposed to sodium fluoride, gemcitabine, metformin, bestatin, WP1066 or valproic acid all showed increased dpum promoter activity. Thus, this study validates Pum as a favourable target for AED design and, moreover, identifies a number of lead compounds capable of increasing the expression of this homeostatic regulator.</p

All Original Data For "Insights into the proteolytic regulation of low-density lipoprotein receptor function by astacin proteases."

All original Western blots (both presented and unpresented repeats), corresponding lane layouts, data for presented graphs and corresponding analysis relating to the paper "Insights into the proteolytic regulation of low-density lipoprotein receptor function by astacin proteases" by Kellett et al., 2023

Optimisation of methods for bacterial skin microbiome investigation: primer selection and comparison of the 454 versus MiSeq platform

Abstract Background The composition of the skin microbiome is predicted to play a role in the development of conditions such as atopic eczema and psoriasis. 16S rRNA gene sequencing allows the investigation of bacterial microbiota. A significant challenge in this field is development of cost effective high throughput methodologies for the robust interrogation of the skin microbiota, where biomass is low. Here we describe validation of methodologies for 16S rRNA (ribosomal ribonucleic acid) gene sequencing from the skin microbiome, using the Illumina MiSeq platform, the selection of primer to amplify regions for sequencing and we compare results with the current standard protocols. Methods DNA was obtained from two low density mock communities of 11 diverse bacterial strains (with and without human DNA supplementation) and from swabs taken from the skin of healthy volunteers. This was amplified using primer pairs covering hypervariable regions of the 16S rRNA gene: primers 63F and 519R (V1-V3); and 347F and 803R (V3-V4). The resultant libraries were indexed for the MiSeq and Roche454 and sequenced. Both data sets were denoised, cleaned of chimeras and analysed using QIIME. Results There was no significant difference in the diversity indices at the phylum and the genus level observed between the platforms. The capture of diversity using the low density mock community samples demonstrated that the primer pair spanning the V3-V4 hypervariable region had better capture when compared to the primer pair for the V1-V3 region and was robust to spiking with human DNA. The pilot data generated using the V3-V4 region from the skin of healthy volunteers was consistent with these results, even at the genus level (Staphylococcus, Propionibacterium, Corynebacterium, Paracoccus, Micrococcus, Enhydrobacter and Deinococcus identified at similar abundances on both platforms). Conclusions The results suggest that the bacterial community diversity captured using the V3-V4 16S rRNA hypervariable region from sequencing using the MiSeq platform is comparable to the Roche454 GS Junior platform. These findings provide evidence that the optimised method can be used in human clinical samples of low bacterial biomass such as the investigation of the skin microbiota

Immunohistochemical detection of C9orf72 protein in Frontotemporal Lobar Degeneration and Motor Neurone Disease: patterns of immunostaining and an evaluation of commercial antibodies

We have employed as ‘gold standards’ two in-house, well-characterised and validated polyclonal antibodies, C9-L and C9-S, which detect the longer and shorter forms of C9orf72, and have compared seven other commercially available antibodies with these in order to evaluate the utility of the latter as credible tools for the demonstration of C9orf72. C9-L and C9-S antibodies immunostained cytoplasmic ‘speckles’, and the nuclear membrane, respectively, in cerebellar Purkinje cells of the cerebellum in patients with behavioural variant frontotemporal dementia (bvFTD) with amyotrophic lateral sclerosis (ALS), and in patients with ALS alone. Similar staining was seen in Purkinje cells in healthy control tissues and in other neurodegenerative disorders, and in pyramidal cells of CA4 and dentate gyrus of hippocampus. However, in the spinal cord there was little cytoplasmic staining with C9-L antibody. C9-S antibody immunostained the nuclear membrane of anterior horn cells in healthy neurons. In patients with bvFTD + ALS, or ALS alone, this C9-S nuclear staining was redistributed to the plasma membrane. In those patients with bvFTD + ALS or ALS bearing an expansion in C9orf72, none of the commercially available antibodies detected TDP-43 inclusions in anterior horn cells, nor were dipeptide repeat proteins demonstrated. Five of the commercial antibodies provided immunohistochemical staining patterns similar in morphological appearance to the in-house C9-L antibody, but distinct from C9-S antibody. However, only three showed sufficient specificity and intensity of staining for C9orf72 at acceptably low concentrations, to make them of practical value and sufficiently reliable for the detection of at least the longer form of C9orf72 protein.</p

SBF-SEM Mouse Achilles Tendon

Sample image volume to demonstrate capabilities of the Fibril Tracking Algorithm developed by Helena Raymond-Hayling

Supplementary Material for: Patterns of Microglial Cell Activation in Alzheimer Disease and Frontotemporal Lobar Degeneration

Aims: Microglia-driven neuroinflammation can play an important role in the pathophysiology of neurodegenerative disorders. In this study, we sought to characterize the distribution of microglial cell activation in 2 neurodegenerative dementias with distinct protein signatures, Alzheimer disease (AD) and frontotemporal lobar degeneration (FTLD) of the TDP subtype, and to determine if there was an anatomical correlation with the phenotypes most commonly associated with these conditions. Methods: The distribution and extent of microglial cell activation was assessed semiquantitatively in the hippocampal formation, cortical gray matter, and subcortical white matter of CD68-immunostained sections of the frontal, temporal, parietal, and occipital cortices from 15 pathologically confirmed cases of AD, 13 cases of FTLD, and 18 controls. Results: Significantly higher levels of microglial cell activation occurred in the subiculum in AD and FTLD than in controls. Additionally, AD had higher microglial activation in the CA1 and FTLD in the hippocampal white matter than the controls. Microglial activation was greater in the dentate gyrus molecular layer in AD than in FTLD. In the cortical regions, the 2 pathological groups differed only in frontal white matter, with the FTLD group showing higher microglial scores. FTLD showed higher microglial activation in the white matter compared to the respective gray matter in the entorhinal, temporal, and frontal regions. Conclusions: Our work expands the knowledge of the distribution and magnitude of microglial activation in these disorders. Additionally, we found some microglial circuit-specific patterns that could help to explain some of the clinical overlap between AD and FTLD-TDP, namely in memory deficits.</p

All data for "Inhibition of insulin-degrading enzyme in human neurons promotes amyloid-β deposition"

All original data files and images relating to the paper "Inhibition of insulin-degrading enzyme in human neurons promotes amyloid-β deposition" by Rowland et al

Eosinophils may play regionally disparate roles in influencing IgA+ plasma cell numbers during large and small intestinal inflammation

Abstract Background Eosinophils are innate immune cells present in the intestine during steady state conditions. An intestinal eosinophilia is a hallmark of many infections and an accumulation of eosinophils is also observed in the intestine during inflammatory disorders. Classically the function of eosinophils has been associated with tissue destruction, due to the release of cytotoxic granule contents. However, recent evidence has demonstrated that the eosinophil plays a more diverse role in the immune system than previously acknowledged, including shaping adaptive immune responses and providing plasma cell survival factors during the steady state. Importantly, it is known that there are regional differences in the underlying immunology of the small and large intestine, but whether there are differences in context of the intestinal eosinophil in the steady state or inflammation is not known. Results Our data demonstrates that there are fewer IgA+ plasma cells in the small intestine of eosinophil-deficient Î dblGATA-1 mice compared to eosinophil-sufficient wild-type mice, with the difference becoming significant post-infection with Toxoplasma gondii. Remarkably, and in complete contrast, the absence of eosinophils in the inflamed large intestine does not impact on IgA+ cell numbers during steady state, and is associated with a significant increase in IgA+ cells post-infection with Trichuris muris compared to wild-type mice. Thus, the intestinal eosinophil appears to be less important in sustaining the IgA+ cell pool in the large intestine compared to the small intestine, and in fact, our data suggests eosinophils play an inhibitory role. The dichotomy in the influence of the eosinophil over small and large intestinal IgA+ cells did not depend on differences in plasma cell growth factors, recruitment potential or proliferation within the different regions of the gastrointestinal tract (GIT). Conclusions We demonstrate for the first time that there are regional differences in the requirement of eosinophils for maintaining IgA+ cells between the large and small intestine, which are more pronounced during inflammation. This is an important step towards further delineation of the enigmatic functions of gut-resident eosinophils