Phosphorylation of U24 from Human Herpes Virus type 6 (HHV-6) and its potential role in mimicking myelin basic protein (MBP) in multiple sclerosis

AbstractMyelin basic protein (MBP) from multiple sclerosis (MS) patients contains lower levels of phosphorylation at Thr97 than normal individuals. The significance of phosphorylation at this site is not fully understood, but it is proposed to play a role in the normal functioning of MBP. Human Herpesvirus Type 6 encodes the protein U24, which has tentatively been implicated in the pathology of MS. U24 shares a 7 amino acid stretch encompassing the Thr97 phosphorylation site of MBP: PRTPPPS. We demonstrate using a combination of mass spectrometry, thin layer chromatography and autoradiography, that U24 can be phosphorylated at the equivalent threonine. Phospho-U24 may confound signalling or other pathways in which phosphorylated MBP may participate, precipitating a pathological process.Structured summaryMINT-6613181:MAPK (uniprotkb:P28482) phosphorylates (MI:0217) MBP (uniprotkb:P02687) by protein kinase assay (MI:0424)MINT-6613171, MINT-6613190:MAPK (uniprotkb:P28482) phosphorylates (MI:0217) U24 (uniprotkb:Q69559) by protein kinase assay (MI:0424

Ubiquitination and proteasomal degradation of ATG12 regulates its proapoptotic activity

During macroautophagy, conjugation of ATG12 to ATG5 is essential for LC3 lipidation and autophagosome formation. Additionally, ATG12 has ATG5-independent functions in diverse processes including mitochondrial fusion and mitochondrial-dependent apoptosis. In this study, we investigated the regulation of free ATG12. In stark contrast to the stable ATG12–ATG5 conjugate, we find that free ATG12 is highly unstable and rapidly degraded in a proteasome-dependent manner. Surprisingly, ATG12, itself a ubiquitin-like protein, is directly ubiquitinated and this promotes its proteasomal degradation. As a functional consequence of its turnover, accumulation of free ATG12 contributes to proteasome inhibitor-mediated apoptosis, a finding that may be clinically important given the use of proteasome inhibitors as anticancer agents. Collectively, our results reveal a novel interconnection between autophagy, proteasome activity, and cell death mediated by the ubiquitin-like properties of ATG12

Diet and bone mineral density study in postmenopausal women from the TwinsUK registry shows a negative association with a traditional English dietary pattern and a positive association with wine

Background: The effect of diet on bone mineral density (BMD) remains controversial, mainly because of difficulties in isolating dietary factors from the confounding influences of age, lifestyle, and genetic factors. Objective: The aim of this study was to use a novel method to examine the relation between BMD and diet. Design: A co-twin control study design with linear regression modeling was used to test for associations between BMD and habitual intakes of calcium, vitamin D, protein, and alcohol plus 5 previously identified dietary patterns in postmenopausal women from the TwinsUK registry. This approach exploited the unique matching of twins to provide an estimate of an association that was not confounded by age, genetic background, or shared lifestyle. Results: In >2000 postmenopausal women (BMD data on 1019, 1218, and 1232 twin pairs at the hip neck, hip, and spine, respectively), we observed a positive association between alcohol intake (from wine but not from beer or spirits) and spine BMD (P = 0.01) and a negative association with a traditional 20th-century English diet at the hip neck (P = 0.01). Both associations remained borderline significant after adjustment for mean twin-pair intakes (P = 0.04 and P = 0.055, respectively). Other dietary patterns and intakes of calcium, vitamin D, and protein were unrelated to BMD. Conclusion: Our results showed that diet has an independent but subtle effect on BMD; wine intake was positively associated with spine BMD, whereas a traditional (20th-century) English diet had a negative association with hip BMD

<i>Trypanosoma evansi</i>: Genetic variability detected using amplified restriction fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) analysis of Kenyan isolates

We compared two methods to generate polymorphic markers to investigate the population genetics of Trypanosoma evansi; random amplified polymorphic DNA (RAPD) and amplified restriction fragment length polymorphism (AFLP) analyses. AFLP accessed many more polymorphisms than RAPD. Cluster analysis of the AFLP data showed that 12 T.evansi isolates were very similar (‘type A’) whereas 2 isolates differed substantially (‘type B’). Type A isolates have been generally regarded as genetically identical but AFLP analysis was able to identify multiple differences between them and split the type A T. evansi isolates into two distinct clades

A real-time PCR assay for accurate quantification of the individual members of the Altered Schaedler Flora microbiota in gnotobiotic mice

Changes in the gastrointestinal microbial community are frequently associated with chronic diseases such as Inflammatory Bowel Diseases. However, understanding the relationship of any individual taxon within the community to host physiology is made complex due to the diversity and individuality of the gut microbiota. Defined microbial communities such as the Altered Schaedler Flora (ASF) help alleviate the challenges of a diverse microbiota by allowing one to interrogate the relationship between individual bacterial species and host responses. An important aspect of studying these relationships with defined microbial communities is the ability to measure the population abundance and dynamics of each member. Herein, we describe the development of an improved ASF species-specific and sensitive real-time quantitative polymerase chain reaction (qPCR) for use with SYBR Green chemistry to accurately assess individual ASF member abundance. This approach targets hypervariable regions V1 through V3 of the 16S rRNA gene of each ASF taxon to enhance assay specificity. We demonstrate the reproducibility, sensitivity and application of this new method by quantifying each ASF bacterium in two inbred mouse lines. We also used it to assess changes in ASF member abundance before and after acute antibiotic perturbation of the community as well as in mice fed two different diets. Additionally, we describe a nested PCR assay for the detection of lowly abundant ASF members. Altogether, this improved qPCR method will facilitate gnotobiotic research involving the ASF community by allowing for reproducible quantification of its members under various physiological conditions

Experimental Evidence for Adaptation to Species-Specific Gut Microbiota in House Mice

The gut microbial communities of mammals have codiversified with host species, and changes in the gut microbiota can have profound effects on host fitness. Therefore, the gut microbiota may drive adaptation in mammalian species, but this possibility is underexplored. Here, we show that the gut microbiota has codiversified with mice in the genus Mus over the past 6 million years, and we present experimental evidence that the gut microbiota has driven adaptive evolution of the house mouse, Mus musculus domesticus. Phylogenetic analyses of metagenomeassembled bacterial genomic sequences revealed that gut bacterial lineages have been retained within and diversified alongside Mus species over evolutionary time. Transplantation of gut microbiotas from various Mus species into germfree M. m. domesticus showed that foreign gut microbiotas slowed growth rate and upregulated macrophage inflammatory protein in hosts. These results suggest adaptation by M. m. domesticus to its gut microbiota since it diverged from other Mus species

Macropinocytosis renders a subset of pancreatic tumor cells resistant to mTOR inhibition

Pancreatic ductal adenocarcinoma (PDAC) features a near-universal mutation in KRAS. Additionally, the tumor suppressor PTEN is lost in ∼10% of patients, and in mouse models, this dramatically accelerates tumor progression. While oncogenic KRAS and phosphatidylinositol 3-kinase (PI3K) cause divergent metabolic phenotypes individually, how they synergize to promote tumor metabolic alterations and dependencies remains unknown. We show that in KRAS-driven murine PDAC cells, loss of Pten strongly enhances both mTOR signaling and macropinocytosis. Protein scavenging alleviates sensitivity to mTOR inhibition by rescuing AKT phosphorylation at serine 473 and consequently cell proliferation. Combined inhibition of mTOR and lysosomal processing of internalized protein eliminates the macropinocytosis-mediated resistance. Our results indicate that mTORC2, rather than mTORC1, is an important regulator of protein scavenging and that protein-mediated resistance could explain the lack of effectiveness of mTOR inhibitors in certain genetic backgrounds. Concurrent inhibition of mTOR and protein scavenging might be a valuable therapeutic approach

The evolutionary dynamics of variant antigen genes in Babesia reveal a history of genomic innovation underlying host-parasite interaction

Babesia spp. are tick-borne, intraerythrocytic hemoparasites that use antigenic variation to resist host immunity, through sequential modification of the parasite-derived variant erythrocyte surface antigen (VESA) expressed on the infected red blood cell surface. We identified the genomic processes driving antigenic diversity in genes encoding VESA (ves1) through comparative analysis within and between three Babesia species, (B. bigemina, B. divergens and B. bovis). Ves1 structure diverges rapidly after speciation, notably through the evolution of shortened forms (ves2) from 5′ ends of canonical ves1 genes. Phylogenetic analyses show that ves1 genes are transposed between loci routinely, whereas ves2 genes are not. Similarly, analysis of sequence mosaicism shows that recombination drives variation in ves1 sequences, but less so for ves2, indicating the adoption of different mechanisms for variation of the two families. Proteomic analysis of the B. bigemina PR isolate shows that two dominant VESA1 proteins are expressed in the population, whereas numerous VESA2 proteins are co-expressed, consistent with differential transcriptional regulation of each family. Hence, VESA2 proteins are abundant and previously unrecognized elements of Babesia biology, with evolutionary dynamics consistently different to those of VESA1, suggesting that their functions are distinct

A gut pathobiont synergizes with the microbiota to instigate inflammatory disease marked by immunoreactivity against other symbionts but not itself

Inflammatory bowel diseases (IBD) are likely driven by aberrant immune responses directed against the resident microbiota. Although IBD is commonly associated with a dysbiotic microbiota enriched in putative pathobionts, the etiological agents of IBD remain unknown. Using a pathobiont-induced intestinal inflammation model and a defined bacterial community, we provide new insights into the immune-microbiota interactions during disease. In this model system, the pathobiont Helicobacter bilis instigates disease following sub-pathological dextran sulfate sodium treatment. We show that H. bilis causes mild inflammation in mono-associated mice, but severe disease in the presence of a microbiota, demonstrating synergy between the pathobiont and microbiota in exacerbating pathology. Remarkably, inflammation depends on the presence of H. bilis, but is marked by a predominant Th17 response against specific members of the microbiota and not the pathobiont, even upon the removal of the most immune-dominant taxa. Neither increases in pathobiont burden nor unique changes in immune-targeted microbiota member abundances are observed during disease. Collectively, our findings demonstrate that a pathobiont instigates inflammation without being the primary target of a Th17 response or by altering the microbiota community structure. Moreover, our findings point toward monitoring pathobiont-induced changes in microbiota immune targeting as a new concept in IBD diagnotics

Commensal \u3ci\u3eEscherichia coli\u3c/i\u3e Strains Can Promote Intestinal Inflammation via Differential Interleukin-6 Production

Escherichia coli is a facultative anaerobic symbiont found widely among mammalian gastrointestinal tracts. Several human studies have reported increased commensal E. coli abundance in the intestine during inflammation; however, host immunological responses toward commensal E. coli during inflammation are not well-defined. Here, we show that colonization of gnotobiotic mice with different genotypes of commensal E. coli isolated from healthy conventional microbiota mice and representing distinct populations of E. coli elicited strain-specific disease phenotypes and immunopathological changes following treatment with the inflammatory stimulus, dextran sulfate sodium (DSS). Production of the inflammatory cytokines GM-CSF, IL-6, and IFN-y was a hallmark of the severe inflammation induced by E. coli strains of Sequence Type 129 (ST129) and ST375 following DSS administration. In contrast, colonization with E. coli strains ST150 and ST468 caused mild intestinal inflammation and triggered only low levels of pro-inflammatory cytokines, a response indistinguishable from that of E. coli-free control mice treated with DSS. The disease development observed with ST129 and ST375 colonization was not directly associated with their abundance in the GI tract as their levels did not change throughout DSS treatment, and no major differences in bacterial burden in the gut were observed among the strains tested. Data mining and in vivo neutralization identified IL-6 as a key cytokine responsible for the observed differential disease severity. Collectively, our results show that the capacity to exacerbate acute intestinal inflammation is a strain-specific trait that can potentially be overcome by blocking the pro-inflammatory immune responses that mediate intestinal tissue damage