Proteomics as a quality control tool of pharmaceutical probiotic bacterial lysate products

Probiotic bacteria have a wide range of applications in veterinary and human therapeutics. Inactivated probiotics are complex samples and quality control (QC) should measure as many molecular features as possible. Capillary electrophoresis coupled to mass spectrometry (CE/MS) has been used as a multidimensional and high throughput method for the identification and validation of biomarkers of disease in complex biological samples such as biofluids. In this study we evaluate the suitability of CE/MS to measure the consistency of different lots of the probiotic formulation Pro-Symbioflor which is a bacterial lysate of heat-inactivated Escherichia coli and Enterococcus faecalis. Over 5000 peptides were detected by CE/MS in 5 different lots of the bacterial lysate and in a sample of culture medium. 71 to 75% of the total peptide content was identical in all lots. This percentage increased to 87–89% when allowing the absence of a peptide in one of the 5 samples. These results, based on over 2000 peptides, suggest high similarity of the 5 different lots. Sequence analysis identified peptides of both E. coli and E. faecalis and peptides originating from the culture medium, thus confirming the presence of the strains in the formulation. Ontology analysis suggested that the majority of the peptides identified for E. coli originated from the cell membrane or the fimbrium, while peptides identified for E. faecalis were enriched for peptides originating from the cytoplasm. The bacterial lysate peptides as a whole are recognised as highly conserved molecular patterns by the innate immune system as microbe associated molecular pattern (MAMP). Sequence analysis also identified the presence of soybean, yeast and casein protein fragments that are part of the formulation of the culture medium. In conclusion CE/MS seems an appropriate QC tool to analyze complex biological products such as inactivated probiotic formulations and allows determining the similarity between lots

Extending colonic mucosal microbiome analysis - Assessment of colonic lavage as a proxy for endoscopic colonic biopsies

This study was supported through GI Research funds and MRC Grant Ref: MR/M00533X/1 to GH.Peer reviewedPublisher PD

Treatment of COVID-19 with remdesivir in the absence of humoral immunity: a case report

The response to the coronavirus disease 2019 (COVID-19) pandemic has been hampered by lack of an effective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antiviral therapy. Here we report the use of remdesivir in a patient with COVID-19 and the prototypic genetic antibody deficiency X-linked agammaglobulinaemia (XLA). Despite evidence of complement activation and a robust T cell response, the patient developed persistent SARS-CoV-2 pneumonitis, without progressing to multi-organ involvement. This unusual clinical course is consistent with a contribution of antibodies to both viral clearance and progression to severe disease. In the absence of these confounders, we take an experimental medicine approach to examine the in vivo utility of remdesivir. Over two independent courses of treatment, we observe a temporally correlated clinical and virological response, leading to clinical resolution and viral clearance, with no evidence of acquired drug resistance. We therefore provide evidence for the antiviral efficacy of remdesivir in vivo, and its potential benefit in selected patients

A synthesis approach of mouse studies to identify genes and proteins in arterial thrombosis and bleeding.

Antithrombotic therapies reduce cardiovascular diseases by preventing arterial thrombosis and thromboembolism, but at expense of increased bleeding risks. Arterial thrombosis studies using genetically modified mice have been invaluable for identification of new molecular targets. Because of low sample sizes and heterogeneity in approaches or methodologies, a formal meta-analysis to compare studies of mice with single-gene defects encountered major limitations. To overcome these, we developed a novel synthesis approach to quantitatively scale 1514 published studies of arterial thrombus formation (in vivo and in vitro), thromboembolism, and tail-bleeding of genetically modified mice. Using a newly defined consistency parameter (CP), indicating the strength of published data, comparisons were made of 431 mouse genes, of which 17 consistently contributed to thrombus formation without affecting hemostasis. Ranking analysis indicated high correlations between collagen-dependent thrombosis models in vivo (FeCl3 injury or ligation/compression) and in vitro. Integration of scores and CP values resulted in a network of protein interactions in thrombosis and hemostasis (PITH), which was combined with databases of genetically linked human bleeding and thrombotic disorders. The network contained 2946 nodes linked to modifying genes of thrombus formation, mostly with expression in megakaryocytes. Reactome pathway analysis and network characteristics revealed multiple novel genes with potential contribution to thrombosis/hemostasis. Studies with additional knockout mice revealed that 4 of 8 (Apoe, Fpr2, Ifnar1, Vps13a) new genes were modifying in thrombus formation. The PITH network further: (i) revealed a high similarity of murine and human hemostatic and thrombotic processes and (ii) identified multiple new candidate proteins regulating these processes

Large Scale Association Analysis of Novel Genetic Loci for Coronary Artery Disease

Background-Combined analysis of 2 genome-wide association studies in cases enriched for family history recently identified 7 loci (on 1p13.3, 1q41, 2q36.3, 6q25.1, 9p21, 10q11.21, and 15q22.33) that may affect risk of coronary artery disease (CAD). Apart from the 9p21 locus, the other loci await substantive replication. Furthermore, the effect of these loci on CAD risk in a broader range of individuals remains to be determined.Methods and Results-We undertook association analysis of single nucleotide polymorphisms at each locus with CAD risk in 11 550 cases and 11 205 controls from 9 European studies. The 9p21.3 locus showed unequivocal association (rs1333049, combined odds ratio [OR]=1.20, 95% CI [1.16 to 1.25], probability value=2.81x10(-21)). We also confirmed association signals at 1p13.3 (rs599839, OR=1.13 [1.08 to 1.19], P=1.44x10(-7)), 1q41 (rs3008621, OR=1.10 [1.04 to 1.17], P=1.02x10(-3)), and 10q11.21 (rs501120, OR=1.11 [1.05 to 1.18], P=4.34x10(-4)). The associations with 6q25.1 (rs6922269, P=0.020) and 2q36.3 (rs2943634, P=0.032) were borderline and not statistically significant after correction for multiple testing. The 15q22.33 locus did not replicate. The 10q11.21 locus showed a possible sex interaction (P = 0.015), with a significant effect in women (OR=1.29 [1.15 to 1.45], P=1.86x10(-5)) but not men (OR=1.03 [0.96 to 1.11], P=0.387). There were no other strong interactions of any of the loci with other traditional risk factors. The loci at 9p21, 1p13.3, 2q36.3, and 10q11.21 acted independently and cumulatively increased CAD risk by 15% (12% to 18%), per additional risk allele. ConclusionsThe findings provide strong evidence for association between at least 4 genetic loci and CAD risk. Cumulatively, these novel loci have a significant impact on risk of CAD at least in European populations. (Arterioscler Thromb Vasc Biol. 2009; 29: 774-780.

Hundreds of variants clustered in genomic loci and biological pathways affect human height

Most common human traits and diseases have a polygenic pattern of inheritance: DNA sequence variants at many genetic loci influence the phenotype. Genome-wide association (GWA) studies have identified more than 600 variants associated with human traits, but these typically explain small fractions of phenotypic variation, raising questions about the use of further studies. Here, using 183,727 individuals, we show that hundreds of genetic variants, in at least 180 loci, influence adult height, a highly heritable and classic polygenic trait. The large number of loci reveals patterns with important implications for genetic studies of common human diseases and traits. First, the 180 loci are not random, but instead are enriched for genes that are connected in biological pathways (P = 0.016) and that underlie skeletal growth defects (P < 0.001). Second, the likely causal gene is often located near the most strongly associated variant: in 13 of 21 loci containing a known skeletal growth gene, that gene was closest to the associated variant. Third, at least 19 loci have multiple independently associated variants, suggesting that allelic heterogeneity is a frequent feature of polygenic traits, that comprehensive explorations of already-discovered loci should discover additional variants and that an appreciable fraction of associated loci may have been identified. Fourth, associated variants are enriched for likely functional effects on genes, being over-represented among variants that alter amino-acid structure of proteins and expression levels of nearby genes. Our data explain approximately 10% of the phenotypic variation in height, and we estimate that unidentified common variants of similar effect sizes would increase this figure to approximately 16% of phenotypic variation (approximately 20% of heritable variation). Although additional approaches are needed to dissect the genetic architecture of polygenic human traits fully, our findings indicate that GWA studies can identify large numbers of loci that implicate biologically relevant genes and pathways.

Inherited Variation in Vitamin D Genes Is Associated With Predisposition to Autoimmune Disease Type 1 Diabetes

Objective: Vitamin D deficiency (25-hydroxyvitamin D [25(OH)D] <50 nmol/L) is commonly reported in both children and adults worldwide, and growing evidence indicates that vitamin D deficiency is associated with many extraskeletal chronic disorders, including the autoimmune diseases type 1 diabetes and multiple sclerosis. Research Design and Methods: We measured 25(OH)D concentrations in 720 case and 2,610 control plasma samples and genotyped single nucleotide polymorphisms from seven vitamin D metabolism genes in 8,517 case, 10,438 control, and 1,933 family samples. We tested genetic variants influencing 25(OH)D metabolism for an association with both circulating 25(OH)D concentrations and disease status. Results: Type 1 diabetic patients have lower circulating levels of 25(OH)D than similarly aged subjects from the British population. Only 4.3 and 18.6% of type 1 diabetic patients reached optimal levels ($\geq$75 nmol/L) of 25(OH)D for bone health in the winter and summer, respectively. We replicated the associations of four vitamin D metabolism genes (GC, DHCR7, CYP2R1, and CYP24A1) with 25(OH)D in control subjects. In addition to the previously reported association between type 1 diabetes and CYP27B1 (P = 1.4 × 10$^{−4}$), we obtained consistent evidence of type 1 diabetes being associated with DHCR7 (P = 1.2 × 10$^{−3}$) and CYP2R1 (P = 3.0 × 10$^{−3}$). Conclusions: Circulating levels of 25(OH)D in children and adolescents with type 1 diabetes vary seasonally and are under the same genetic control as in the general population but are much lower. Three key 25(OH)D metabolism genes show consistent evidence of association with type 1 diabetes risk, indicating a genetic etiological role for vitamin D deficiency in type 1 diabetes