Population-specific recombination sites within the human MHC region

Genetic rearrangement by recombination is one of the major driving forces for genome evolution, and recombination is known to occur in non-random, discreet recombination sites within the genome. Mapping of recombination sites has proved to be difficult, particularly, in the human MHC region that is complicated by both population variation and highly polymorphic HLA genes. To overcome these problems, HLA-typed individuals from three representative populations: Asian, European and African were used to generate phased HLA haplotypes. Extended haplotype homozygosity (EHH) plots constructed from the phased haplotype data revealed discreet EHH drops corresponding to recombination events and these signatures were observed to be different for each population. Surprisingly, the majority of recombination sites detected are unique to each population, rather than being common. Unique recombination sites account for 56.8% (21/37 of total sites) in the Asian cohort, 50.0% (15/30 sites) in Europeans and 63.2% (24/38 sites) in Africans. Validation carried out at a known sperm typing recombination site of 45 kb (HLA-F-telomeric) showed that EHH was an efficient method to narrow the recombination region to 826 bp, and this was further refined to 660 bp by resequencing. This approach significantly enhanced mapping of the genomic architecture within the human MHC, and will be useful in studies to identify disease risk genes.     

LXR and ABCA1 control cholesterol homeostasis in the proximal mouse epididymis in a cell-specific manner

Mammalian spermatozoa undergo important plasma membrane maturation steps during epididymal transit. Among these, changes in lipids and cholesterol are of particular interest as they are necessary for fertilization. However, molecular mechanisms regulating these transformations inside the epididymis are still poorly understood. Liver X receptors (LXRs), the nuclear receptors for oxysterols, are of major importance in intracellular cholesterol homeostasis, and LXR^−/−-deficient male mice have already been shown to have reduced fertility at an age of 5 months and complete sterility for 9-month-old animals. This sterility phenotype is associated with testes and caput epididymides epithelial defects. The research presented here was aimed at investigating how LXRs act in the male caput epididymidis by analyzing key actors in cholesterol homeostasis. We show that accumulation of cholesteryl esters in LXR^−/− male mice is associated with a specific loss of ABCA1 and an increase in apoptosis of apical cells of the proximal caput epididymidis. ATP-binding cassette G1 (ABCG1) and scavenger receptor B1 (SR-B1), two other cholesterol transporters, show little if any modifications. Our study also revealed that SR-B1 appears to have a peculiar expression pattern along the epididymal duct. These results should help in understanding the functional roles of LXR in cholesterol trafficking processes in caput epididymidis.     

Synthesis of Branched Oligoribonucleotides (“Lariat”)

Abstract

Four different strategies for the synthesis of branched oligoribonucleotides, from trimers to heptamer, are described.     

A Convergent Regiospecific Synthesis of the Lariat-Trinucleotides and A2′p 5′G and A2′p 5′G 3′p 5′C from A O4-2-Nitrophenyl) -Uridine Building Block

Abstract

Total synthesis of title compounds 1_ and 2_ from a common intermediate 7 is reported using the phosphotriester-phosphiteamidite approach. Appropriate NMR evidence has been presented in support of the regiospecific synthesis of target molecules in addition to enzymatic analysis. Present work clearly shows that the NMR evidence is mandatory to establish the isomeric purity of branched RNA molecules; enzymatic or/and electrophoretic analysis alone as tools for confirmation of branched RNA structures can be misleading.     

Phylogenomics of 8,839 Clostridioides difficile genomes reveals recombination-driven evolution and diversification of toxin A and B

Clostridioides difficile is the major worldwide cause of antibiotic-associated gastrointestinal infection. A pathogenicity locus (PaLoc) encoding one or two homologous toxins, toxin A (TcdA) and toxin B (TcdB), is essential for C. difficile pathogenicity. However, toxin sequence variation poses major challenges for the development of diagnostic assays, therapeutics, and vaccines. Here, we present a comprehensive phylogenomic analysis of 8,839 C. difficile strains and their toxins including 6,492 genomes that we assembled from the NCBI short read archive. A total of 5,175 tcdA and 8,022 tcdB genes clustered into 7 (A1-A7) and 12 (B1-B12) distinct subtypes, which form the basis of a new method for toxin-based subtyping of C. difficile. We developed a haplotype coloring algorithm to visualize amino acid variation across all toxin sequences, which revealed that TcdB has diversified through extensive homologous recombination throughout its entire sequence, and formed new subtypes through distinct recombination events. In contrast, TcdA varies mainly in the number of repeats in its C-terminal repetitive region, suggesting that recombination-mediated diversification of TcdB provides a selective advantage in C. difficile evolution. The application of toxin subtyping is then validated by classifying 351 C. difficile clinical isolates from Brigham and Women’s Hospital in Boston, demonstrating its clinical utility. Subtyping partitions TcdB into binary functional and antigenic groups generated by intragenic recombinations, including two distinct cell-rounding phenotypes, whether recognizing frizzled proteins as receptors, and whether it can be efficiently neutralized by monoclonal antibody bezlotoxumab, the only FDA-approved therapeutic antibody. Our analysis also identifies eight universally conserved surface patches across the TcdB structure, representing ideal targets for developing broad-spectrum therapeutics. Finally, we established an open online database (DiffBase) as a central hub for collection and classification of C. difficile toxins, which will help clinicians decide on therapeutic strategies targeting specific toxin variants, and allow researchers to monitor the ongoing evolution and diversification of C. difficile.     

α-Linolenate reduces the dietary requirement for linoleate in the growing rat

Background: We hypothesized that due to the absence of a dietary source of omega-3 fatty acids, the essential fatty acid (EFA) deficiency model leads to an overestimate of linoleic acid (LA) requirements. Methods: over 7 wk, young rats consumed an EFA diet containing either 0 en% linoleate (0LA) and 0 en% α-linolenate (0LNA) or a diet containing 0.5 en% LNA plus one of seven levels of added LA (0.12–4.0 en%; n=6/group).Results: Rats consuming the 0LA–0LNA diet had the lowest final body weight, 34–68% lower LA and arachidonate in plasma and liver, 87% lower LA in epididymal fat, and an 8–20 fold higher eicosatrienoate in plasma, liver and muscle lipids. 0.5LNA completely prevented the lower growth and partly prevented the rise in eicosatrienoate seen in the 0LA–0LNA group.Conclusion: Providing dietary LNA at 0.5 en% reduces the rat's physiological requirement for LA by an estimated factor of at least four (0.5 en% instead of 2 en%). Since LA requirements in humans are also based on the same flawed model of EFA deficiency, it is plausible that they too have been overestimated and should therefore be reinvestigated.     

Disproportionate Cochlear Length in Genus Homo Shows a High Phylogenetic Signal during Apes’ Hearing Evolution

Changes in lifestyles and body weight affected mammal life-history evolution but little is known about how they shaped species’ sensory systems. Since auditory sensitivity impacts communication tasks and environmental acoustic awareness, it may have represented a deciding factor during mammal evolution, including apes. Here, we statistically measure the influence of phylogeny and allometry on the variation of five cochlear morphological features associated with hearing capacities across 22 living and 5 fossil catarrhine species. We find high phylogenetic signals for absolute and relative cochlear length only. Comparisons between fossil cochleae and reconstructed ape ancestral morphotypes show that Australopithecus absolute and relative cochlear lengths are explicable by phylogeny and concordant with the hypothetized ((Pan,Homo),Gorilla) and (Pan,Homo) most recent common ancestors. Conversely, deviations of the Paranthropus oval window area from these most recent common ancestors are not explicable by phylogeny and body weight alone, but suggest instead rapid evolutionary changes (directional selection) of its hearing organ. Premodern (Homo erectus) and modern human cochleae set apart from living non-human catarrhines and australopiths. They show cochlear relative lengths and oval window areas larger than expected for their body mass, two features corresponding to increased low-frequency sensitivity more recent than 2 million years ago. The uniqueness of the “hypertrophied” cochlea in the genus Homo (as opposed to the australopiths) and the significantly high phylogenetic signal of this organ among apes indicate its usefulness to identify homologies and monophyletic groups in the hominid fossil record.     

Effects of L-malate on mitochondrial oxidoreductases in liver of aged rats

Accumulation of oxidative damage has been implicated to be a major causative factor in the decline in physiological functions that occur during the aging process. The mitochondrial respiratory chain is a powerful source of reactive oxygen species (ROS), considered as the pathogenic agent of many diseases and aging. L-malate, a tricarboxylic acid cycle intermediate, plays an important role in transporting NADH from cytosol to mitochondria for energy production. Previous studies in our laboratory reported L-malate as a free radical scavenger in aged rats. In the present study we focused on the effect of L-malate on the activities of electron transport chain in young and aged rats. We found that mitochondrial membrane potential (MMP) and the activities of succinate dehydrogenase, NADH-cytochrome c oxidoreductase and cytochrome c oxidase in liver of aged rats were significantly decreased when compared to young control rats. Supplementation of L-malate to aged rats for 30 days slightly increased MMP and improved the activities of NADH-dehydrogenase, NADH-cytochrome c oxidoreductase and cytochrome c oxidase in liver of aged rats when compared with aged control rats. In young rats, L-malate administration increased only the activity of NADH-dehydrogenase. Our result suggested that L-malate could improve the activities of electron transport chain enzymes in aged rats.