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AIMS/HYPOTHESIS: We aimed to investigate the genetic evidence that supports the repurposing of drugs already licensed or in clinical phases of development for prevention of type 1 diabetes. METHODS: We obtained genome-wide association study summary statistics for the risk of type 1 diabetes, whole-blood gene expression and serum protein levels and investigated genetic polymorphisms near seven potential drug target genes. We used co-localisation to examine whether the same genetic variants that are associated with type 1 diabetes risk were also associated with the relevant drug target genetic proxies and used Mendelian randomisation to evaluate the direction and magnitude of the associations. Furthermore, we performed Mendelian randomisation analysis restricted to functional variants within the drug target genes. RESULTS: Co-localisation revealed that the blood expression levels of IL2RA (encoding IL-2 receptor subunit α [IL2RA]), IL6R (encoding IL-6 receptor [IL6R]) and IL6ST (encoding IL-6 cytokine family signal transducer [IL6ST]) shared the same causal variant with type 1 diabetes liability near the corresponding genes (posterior probabilities 100%, 96.5% and 97.0%, respectively). The OR (95% CI) of type 1 diabetes per 1-SD increase in the genetically proxied gene expression of IL2RA, IL6R and IL6ST were 0.22 (0.17, 0.27), 1.98 (1.48, 2.65) and 1.90 (1.45, 2.48), respectively. Using missense variants, genetically proxied TYK2 (encoding tyrosine kinase 2) expression levels were associated with type 1 diabetes risk (OR 0.61 [95% CI 0.54, 0.69]). CONCLUSIONS/INTERPRETATION: Our findings support the targeting of IL-2, IL-6 and TYK2 signalling in prevention of type 1 diabetes. DATA AVAILABILITY: The analysis code is available at https://github.com/jkoskenniemi/T1DSCREEN , which also includes instructions on how to download the original GWAS summary statistics.

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Understanding protein-protein interactions is crucial for unravelling subcellular protein distribution, contributing to our understanding of cellular organisation. Moreover, interaction studies can reveal insights into the mechanisms that cover protein trafficking within cells. Although various techniques such as Förster resonance energy transfer (FRET), co-immunoprecipitation, and fluorescence microscopy are commonly employed to detect protein interactions, their limitations have led to more advanced techniques such as the in situ proximity ligation assay (PLA) for spatial co-localisation analysis. The PLA technique, specifically employed in fixed cells and tissues, utilises species-specific secondary PLA probes linked to DNA oligonucleotides. When proteins are within 40 nm of each other, the DNA oligonucleotides on the probes interact, facilitating circular DNA formation through ligation. Rolling-circle amplification then produces DNA circles linked to the PLA probe. Fluorescently labelled oligonucleotides hybridise to the circles, generating detectable signals for precise co-localisation analysis. We employed PLA to examine the co-localisation of dynein with the Kv7.4 channel protein in isolated vascular smooth muscle cells from rat mesenteric arteries. This method enabled us to investigate whether Kv7.4 channels interact with dynein, thereby providing evidence of their retrograde transport by the microtubule network. Our findings illustrate that PLA is a valuable tool for studying potential novel protein interactions with dynein, and the quantifiable approach offers insights into whether these interactions are changed in disease.

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Background: Whether there is an association between HF (HF) and cancer has not been conclusively established, and it is not clear whether patients with cancer can share similar hospitalization strategies and outcomes with patients with HF. Methods: Genome-wide association summary statistics were performed using a two-sample Mendelian randomization (MR) method for HF patients and cancer patients from the GWAS directory, with co-localization and Summary Data-Based Mendelian Randomization (SMR) analyses to identify HF-associated genes, and transcriptomic analyses to analyze the roles of these genes in the clinical diagnosis and targeted therapies of multiple cancer types. Results: Two-sample MR analysis showed that increased risk of HF was associated with decreased risk of cervical, brain, breast, colorectal, lung, and skin cancers, and co-localization combined with SMR analysis identified ABO and SURF1 as HF-associated genes, and transcriptomic analyses showed that ABO is a risk factor for HF and a protective factor against cancer, whereas SURF1 is a protective factor against HF and a protective factor against cancer. Conclusion: There was no causal relationship between heart failure and cancers (Cervical, brain, breast, colorectal, lung and skin cancers) risk factors, however there was a trend toward a negative causal relationship between heart failure and cancers (Cervical, brain, breast, colorectal, lung and skin cancers) occurrence.

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Immunohistochemical (IHC) localisation of protein expression is a widely used tool in pathology. This is semi-quantitative and exhibits substantial intra- and inter-observer variability. Digital approaches based on stain quantification applied to IHC are precise but still operator-dependent and time-consuming when regions of interest (ROIs) must be defined to quantify protein expression in a specific tissue area. This study aimed at developing an IHC quantification workflow that benefits from colour deconvolution for stain quantification and artificial intelligence for automatic ROI definition. The method was tested on 10 whole slide images (WSI) of alpha-smooth muscle actin (aSMA) stained mouse kidney sections. The task was to identify aSMA-positive areas within the glomeruli automatically. Total aSMA detection was performed using two channels (DAB, haematoxylin) colour deconvolution. Glomeruli segmentation within the same IHC WSI was performed by training a convolutional neural network with annotated examples of glomeruli. For both aSMA and glomeruli, binary masks were created. Co-localisation was performed by overlaying the masks and assigning red/green colours, with yellow indicative of a co-localised signal. The workflow described and exemplified using the case of aSMA expression in glomeruli can be applied to quantify the expression of IHC markers within different structures of immunohistochemically stained slides. The technique is objective, has a fully automated threshold approach (colour deconvolution phase) and uses AI to eliminate operator-dependent steps.

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Small extracellular vesicles (sEVs) have emerged as attractive drug delivery systems. However, the intracellular release of their cargoes is restricted. This study aimed to develop an efficient approach to re-engineer sEVs by hybridisation with pH-sensitive liposomes (PSLs) and investigate their endosome escape potential. MIA PaCa-2 cell-derived sEVs and PSLs were fused via three methods, and fusion efficiency (FE) was measured using a fluorescence resonance energy transfer assay and nanoparticle tracking analysis. Cellular uptake, intracellular trafficking, and cytotoxicity of doxorubicin-loaded vesicles (Dox@hybrids, Dox@sEVs, and Dox@PSLs) were investigated on MIA PaCa-2 cells. Among the three methods, Ca2+-mediated fusion was the simplest and led to a comparable FE with freeze-thaw method, which was significantly higher than PEG8000-mediated fusion. sEVs were more stable after hybridisation with PSLs. Confocal microscopy revealed that the hybrids internalised more efficiently than natural sEVs. While the internalised Dox@sEVs were primarily co-localised with endo/lysosomes even after 8 h, Dox from Dox@hybrids was found to escape from endosomes by 2 h and homogenously distributed in the cytosol before accumulated at nucleus, corresponding to the in vitro pH-responsive release profile. Consequently, Dox@hybrids enhanced cytotoxicity compared with Dox@sEVs, Dox@PSLs, or free drugs. Overall, the biomimetic nanosystem generated by simple Ca2+-mediated fusion was more stable and demonstrated higher efficiencies of cellular uptake and endosome escape compared to natural sEVs.

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Human mitochondria are highly dynamic organelles, fusing and budding to maintain reticular networks throughout many cell types. Although extending to the extremities of the cell, the majority of the network is concentrated around the nucleus in most of the commonly cultured cell lines. This organelle harbours its own genome, mtDNA, with a different gene content to the nucleus, but the expression of which is critical for maintaining oxidative phosphorylation. Recent advances in click chemistry have allowed us to visualise sites of mitochondrial protein synthesis in intact cultured cells. We show that the majority of translation occurs in the peri-nuclear region of the network. Further analysis reveals that whilst there is a slight peri-nuclear enrichment in the levels of mitoribosomal protein and mitochondrial rRNA, it is not sufficient to explain this substantial heterogeneity in the distribution of translation. Finally, we also show that in contrast, a mitochondrial mRNA does not show such a distinct gradient in distribution. These data suggest that the relative lack of translation in the peripheral mitochondrial network is not due to an absence of mitoribosomes or an insufficient supply of the mt-mRNA transcripts.

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AIMS/HYPOTHESIS: The aim of this study was to leverage human genetic data to investigate the cardiometabolic effects of glucose-dependent insulinotropic polypeptide (GIP) signalling. METHODS: Data were obtained from summary statistics of large-scale genome-wide association studies. We examined whether genetic associations for type 2 diabetes liability in the GIP and GIPR genes co-localised with genetic associations for 11 cardiometabolic outcomes. For those outcomes that showed evidence of co-localisation (posterior probability >0.8), we performed Mendelian randomisation analyses to estimate the association of genetically proxied GIP signalling with risk of cardiometabolic outcomes, and to test whether this exceeded the estimate observed when considering type 2 diabetes liability variants from other regions of the genome. RESULTS: Evidence of co-localisation with genetic associations of type 2 diabetes liability at both the GIP and GIPR genes was observed for five outcomes. Mendelian randomisation analyses provided evidence for associations of lower genetically proxied type 2 diabetes liability at the GIP and GIPR genes with lower BMI (estimate in SD units -0.16, 95% CI -0.30, -0.02), C-reactive protein (-0.13, 95% CI -0.19, -0.08) and triacylglycerol levels (-0.17, 95% CI -0.22, -0.12), and higher HDL-cholesterol levels (0.19, 95% CI 0.14, 0.25). For all of these outcomes, the estimates were greater in magnitude than those observed when considering type 2 diabetes liability variants from other regions of the genome. CONCLUSIONS/INTERPRETATION: This study provides genetic evidence to support a beneficial role of sustained GIP signalling on cardiometabolic health greater than that expected from improved glycaemic control alone. Further clinical investigation is warranted. DATA AVAILABILITY: All data used in this study are publicly available. The scripts for the analysis are available at: https://github.com/vkarhune/GeneticallyProxiedGIP .

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Using single-molecule fluorescence microscopes, individual biomolecules can be observed within live bacterial cells. Using differently coloured probes, physical associations between two different molecular species can be assessed through co-localisation measurements. However, bacterial cells are finite and small (~ 1 µm) relative to the resolution limit of optical microscopes (~ 0.25 µm). Furthermore, the images produced by optical microscopes are typically two-dimensional projections of three-dimensional objects. These limitations mean that a certain proportion of object pairs (molecules) will inevitably be assigned as being co-localised, even when they are distant at molecular distance scales (nm). What is this proportion? Here, we attack this problem, theoretically and computationally, by creating a model of the co-localisation expected purely due to chance. We thus consider a bacterial cell wherein objects are distributed at random and evaluate the co-localisation in a fashion that emulates an experimental analysis. We consider simplified geometries where we can most transparently investigate the effect of a finite size of the cell and the effect of probing a three-dimensional cell in only two dimensions. Coupling theory to simulations, we also study the co-localisation expected due to chance using parameters relevant to bacterial cells. Overall, we show that the co-localisation expected purely due to chance can be quite substantial and describe the parameters that it depends upon.

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AIMS/HYPOTHESIS: Given the potential shared aetiology between type 1 and type 2 diabetes, we aimed to identify any genetic regions associated with both diseases. For associations where there is a shared signal and the allele that increases risk to one disease also increases risk to the other, inference about shared aetiology could be made, with the potential to develop therapeutic strategies to treat or prevent both diseases simultaneously. Alternatively, if a genetic signal co-localises with divergent effect directions, it could provide valuable biological insight into how the association affects the two diseases differently. METHODS: Using publicly available type 2 diabetes summary statistics from a genome-wide association study (GWAS) meta-analysis of European ancestry individuals (74,124 cases and 824,006 controls) and type 1 diabetes GWAS summary statistics from a meta-analysis of studies on individuals from the UK and Sardinia (7467 cases and 10,218 controls), we identified all regions of 0.5 Mb that contained variants associated with both diseases (false discovery rate <0.01). In each region, we performed forward stepwise logistic regression to identify independent association signals, then examined co-localisation of each type 1 diabetes signal with each type 2 diabetes signal using coloc. Any association with a co-localisation posterior probability of ≥0.9 was considered a genuine shared association with both diseases. RESULTS: Of the 81 association signals from 42 genetic regions that showed association with both type 1 and type 2 diabetes, four association signals co-localised between both diseases (posterior probability ≥0.9): (1) chromosome 16q23.1, near CTRB1/BCAR1, which has been previously identified; (2) chromosome 11p15.5, near the INS gene; (3) chromosome 4p16.3, near TMEM129 and (4) chromosome 1p31.3, near PGM1. In each of these regions, the effect of genetic variants on type 1 diabetes was in the opposite direction to the effect on type 2 diabetes. Use of additional datasets also supported the previously identified co-localisation on chromosome 9p24.2, near the GLIS3 gene, in this case with a concordant direction of effect. CONCLUSIONS/INTERPRETATION: Four of five association signals that co-localise between type 1 diabetes and type 2 diabetes are in opposite directions, suggesting a complex genetic relationship between the two diseases.

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Autophagy flux is the rate at which cytoplasmic components are degraded through the entire autophagy pathway and is often measured by monitoring the clearance rate of autophagosomes. The specific means by which autophagy targets specific cargo has recently gained major attention due to the role of autophagy in human pathologies, where specific proteinaceous cargo is insufficiently recruited to the autophagosome compartment, albeit functional autophagy activity. In this context, the dynamic interplay between receptor proteins such as p62/Sequestosome-1 and neighbour of BRCA1 gene 1 (NBR1) has gained attention. However, the extent of receptor protein recruitment and subsequent clearance alongside autophagosomes under different autophagy activities remains unclear. Here, we dissect the concentration-dependent and temporal impact of rapamycin and spermidine exposure on receptor recruitment, clearance and autophagosome turnover over time, employing micropatterning. Our results reveal a distinct autophagy activity response profile, where the extent of autophagosome and receptor co-localisation does not involve the total pool of either entities and does not operate in similar fashion. These results suggest that autophagosome turnover and specific cargo clearance are distinct entities with inherent properties, distinctively contributing towards total functional autophagy activity. These findings are of significance for future studies where disease specific protein aggregates require clearance to preserve cellular proteostasis and viability and highlight the need of discerning and better tuning autophagy machinery activity and cargo clearance.

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Nanomaterial (NM) surface chemistry has an established and significant effect on interactions at the nano-bio interface, with important toxicological consequences for manufactured NMs, as well as potent effects on the pharmacokinetics and efficacy of nano-therapies. In this work, the effects of different surface modifications (PVP, Dispex AA4040, and Pluronic F127) on the uptake, cellular distribution, and degradation of titanium dioxide NMs (TiO2 NMs, ~10 nm core size) are assessed and correlated with the localization of fluorescently-labeled serum proteins forming their coronas. Imaging approaches with an increasing spatial resolution, including automated high throughput live cell imaging, correlative confocal fluorescence and reflectance microscopy, and dSTORM super-resolution microscopy, are used to explore the cellular fate of these NMs and their associated serum proteins. Uncoated TiO2 NMs demonstrate a rapid loss of corona proteins, while surface coating results in the retention of the corona signal after internalization for at least 24 h (varying with coating composition). Imaging with two-color super-resolution dSTORM revealed that the apparent TiO2 NM single agglomerates observed in diffraction-limited confocal microscopy are actually adjacent smaller agglomerates, and provides novel insights into the spatial arrangement of the initial and exchanged coronas adsorbed at the NM surfaces.

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Inhibition of viral replication by icIgA antibodies has only been observed with in vitro studies using epithelial cell lines in transwell cultures. This effect appears to involve an interaction between polymeric immunoglobulin A (pIgA) and viral particles within an intracellular compartment, since IgA is transported across polarized cells. Polyclonal guinea pig antisera against purified influenza A virus and mouse antisera prepared against Influenza A/H3N2 hemagglutinin (HA0) cleavage loop peptides, were used in confocal fluorescence microscopy to show specific staining of wild-type influenza H1N1 and H3N2 viruses in clinical specimens. The HA0 cleavage loop peptides used for intranasal immunization of mice were designed and synthesized from specific conserved regions of influenza A/H1N1 & A/H3N2 viruses. Anti-human secretory IgA antibodies were used to show co-localisation of influenza A virus and icIgA. The results showed specific immunofluorescent staining of influenza A/H3N2 (X31) (HA0 uncleaved)-infected MDCK cells and the presence of icIgA in respiratory exudate cells of infected patients. Both results confirm specific co-localisation and suggest interaction between influenza A virus and icIgA in patients' respiratory exudate cells. Importantly, antisera to the mouse anti-HA0 cleavage site were specific for wild-type virus in clinical specimens, indicating that the conserved region of HA0 was present in the uncleaved form. Similar staining and colocalization patterns between icIgA and virus were observed with polyclonal guinea pig antisera against influenza A virus. These are the first observations of co-localization of influenza A virus and intracellular IgA in clinical specimens. Role of icIgA: This report shows the co-localization of influenza A virus HA0 and icIgA antibodies in respiratory exudate cells of patients who were culture and viral RNA positive, suggesting that icIgA directed against the conserved HA0 site may have a privileged and unique opportunity to act on immature virus and thus prevent HA0 cleavage, maturation and subsequent cycles of viral replication. The precise mechanism by which icIgA mediates intracellular viral neutralization remains to be fully elucidated. SIGNIFICANCE: The above findings in clinical specimens would contribute strongly to our understanding of the mechanisms and kinetics of icIgA neutralization in relation to viral entry and early replication steps of mucosal viral infections. A rapid, objective and sensitive assay - by ex vivo enumeration of respiratory epithelial cells that have co-localized influenza virus and icIgA - would contribute to further mucosal immunity studies and inform the design of more effective vaccines against influenza and other viral infections transmitted via the mucosal route e.g. respiratory syncytial virus, rotavirus.

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To provide a better understanding of the genetic architecture of fruiting body formation of Lentinula edodes, quantitative trait loci (QTLs) mapping was employed to uncover the loci underlying seven fruiting body-related traits (FBRTs). An improved L. edodes genetic linkage map, comprising 572 markers on 12 linkage groups with a total map length of 983.7 cM, was constructed by integrating 82 genomic sequence-based insertion-deletion (InDel) markers into a previously published map. We then detected a total of 62 QTLs for seven target traits across two segregating testcross populations, with individual QTLs contributing 5.5 %-30.2 % of the phenotypic variation. Fifty-three out of the 62 QTLs were clustered in six QTL hotspots, suggesting the existence of main genomic regions regulating the morphological characteristics of fruiting bodies in L. edodes. A stable QTL hotspot on MLG2, containing QTLs for all investigated traits, was identified in both testcross populations. QTLs for related traits were frequently co-located on the linkage groups, demonstrating the genetic basis for phenotypic correlation of traits. Meta-QTL (mQTL) analysis was performed and identified 16 mQTLs with refined positions and narrow confidence intervals (CIs). Nine genes, including those encoding MAP kinase, blue-light photoreceptor, riboflavin-aldehyde-forming enzyme and cyclopropane-fatty-acyl-phospholipid synthase, and cytochrome P450s, were likely to be candidate genes controlling the shape of fruiting bodies. The study has improved our understanding of the genetic architecture of fruiting body formation in L. edodes. To our knowledge, this is the first genome-wide QTL detection of FBRTs in L. edodes. The improved genetic map, InDel markers and QTL hotspot regions revealed here will assist considerably in the conduct of future genetic and breeding studies of L. edodes.

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