Mutation rates and intraspecific divergence of the mitochondrial genome of Pristionchus pacificus

Evolutionary reconstruction of the natural history of an organism ultimately requires knowledge about the development, population genetics, ecology, and phylogeny of the species. Such investigations would benefit from studies of mutational processes because mutations are the source of natural variation. The nematode Pristionchus pacificus has been developed as a model organism in evolutionary biology by comparing its development with Caenorhabditis elegans. Pristionchus pacificus and related species are associated with scarab beetles, and their ecology and phylogeny are well known. More than 200 P. pacificus isolates from all over the world are available for this cosmopolitan species. We generated mutation accumulation (MA) lines in P. pacificus to study spontaneous mutation rates in the mitochondrial genome and compared mutation rate estimates with natural variation between nine representative isolates of the species. The P. pacificus mitochondrial genome is 15,955 bp in length and is typical for nematodes. Pristionchus pacificus has all known mitochondrial genes and contains an unusual suppressor transfer RNA (tRNA) for the codon UAA. This has most likely influenced the spectrum of observable mutations because 6 of 12 mutations found in the 82 MA lines analyzed are nonsense mutations that can be suppressed by the suppressor tRNA. The overall mutation rate in P. pacificus is 7.6 × 10?? per site per generation and is less than one order of magnitude different from estimates in C. elegans and Drosophila. Using this mutation rate estimate in a comparison of the mitochondrial genome of nine P. pacificus isolates, we calculate the minimum time to the most recent common ancestor at 10?-10? generations. The combination of mutation rate analysis with intraspecific divergence provides a powerful tool for the reconstruction of the natural history of P. pacificus, and we discuss the ecological implication of these findings.     

Both Baseline Clinical Factors and Genetic Polymorphisms Influence the Development of Severe Functional Status in Ankylosing Spondylitis

Functional severity in ankylosing spondylitis (AS) patients is variable and difficult to predict early. The aim of our study was to assess whether a combination of baseline clinical factors and genetic markers may predict the development of severe functional status in AS. We performed a cross-sectional association study on AS patients included in the Spanish National Registry of Spondyloarthropathies—REGISPONSER. Bath Ankylosing Spondylitis Functional Index (BASFI) was standardized by adjusting for disease duration since the first symptoms (BASFI/t). We considered as severe functional status the values of BASFI/t in the top of the 60th (p60), 65th (p65), 70th (p70), and 75th (p75) percentile. We selected 384 single nucleotide polymorphisms (SNPs) distributed in 190 genes to be analyzed. The study cohort included 456 patients with mean age 50.8(±10.5) years and with mean disease duration since first symptoms 24.7 (±10.1) years. Older age at disease onset and neck pain at baseline showed statistical significant association with severe BASFI/t. Polymorphisms associated in the allele frequencies test with severe BASFI/t in all classifications were: rs2542151 (p60 [P = .04], p65 [P = .04], p70 [P = .001] and p75 [P = .001]) and rs2254441 (p60 [P = .004], p65 [P = .02], p70 [P = .01] and p75 [P<.001]).. Genotype association, after adjustment for covariates, found an association in three of the four patients'' classifications for rs2542151 and in two of the classifications for rs2254441.Forward logistic regression did not identify any model with a good predictive power for severe functional development.In our study we identified clinical factors and 24 polymorphisms associated with development of severe functional status in AS patients. Validation of these results in other cohorts is required.     

The autoantigen Ro52 is an E3 ligase resident in the cytoplasm but enters the nucleus upon cellular exposure to nitric oxide

Patients with the systemic autoimmune diseases Sjögrens's syndrome and systemic lupus erythematosus often have autoantibodies against the intracellular protein Ro52. Ro52 is an E3 ligase dependent on the ubiquitin conjugation enzymes UBE2D1 and UBE2E1. While Ro52 and UBE2D1 are cytoplasmic proteins, UBE2E1 is localized to the nucleus. Here, we investigate how domains of human Ro52 regulate its intracellular localization. By expressing fluorescently labeled Ro52 and Ro52 mutants in HeLa cells, an intact coiled-coil domain was found to be necessary for the cytoplasmic localization of Ro52. The amino acids 381-470 of the B30.2 region were essential for translocation into the nucleus. Furthermore, after exposure of HeLa cells to the inflammatory mediator nitric oxide (NO), Ro52 translocated to the nucleus. A nuclear localization of Ro52 in inflamed tissue expressing inducible NO synthetase (iNOS) from cutaneous lupus patients was observed by immunohistochemistry and verified in NO-treated cultures of patient-derived primary keratinocytes. Our results show that the localization of Ro52 is regulated by endogenous sequences, and that nuclear translocation is induced by an inflammatory mediator. This suggests that Ro52 has both cytoplasmic and nuclear substrates, and that Ro52 mediates ubiquitination through UBE2D1 in the cytoplasm and through UBE2E1 in the nucleus.     

Effects of plasmid growth hormone-releasing hormone treatment during heat stress

A gene therapy treatment with plasmid-based growth hormone-releasing hormone (GHRH) delivered by electroporation (EP) was investigated during heat stress; 32 primiparous cows received 2.5 mg of a GHRH-expressing myogenic plasmid (pSP-HV-GHRH), while 20 were designated as controls. Offspring of treated animals showed a reduction in mortality (47%; p < 0.02), and survival from birth to 260 days was dramatically improved (0% mortality vs. 21% in controls) along with an increase in weight gain (p < 0.05). Milk production was increased compared to controls with an average yield gain of 421 kg/cow (p = 0.028). Prolactin (PRL) levels were also significantly increased compared to controls (p < 0.05). The second pregnancy rate was improved by GHRH treatment (53.3% vs. 30.8%). This study shows that the use of plasmid-mediated therapy delivered by EP can maintain health status during periods of heat stress, important for both animals and potentially humans in hot, challenging climates.     

Enantiomerically pure quaternary ammonium salts with a chiral alkyl chain N(CH3)(n-C3H7)2(sec-C4H9)I: synthesis and physical studies

A pair of enantiomerically pure quaternary ammonium salts with a chiral side chain, methyl-(R)-(1-methylpropyl)di(n-propyl)ammonium iodide 1 and methyl-(S)-(1-methylpropyl)di(n-propyl)ammonium iodide 2, and the related racemate, methyl-(rac)-(1-methylpropyl)di(n-propyl)ammonium iodide 3, were synthesized through a reductive alkylation procedure, starting from enantiomerically pure and, also, racemic forms of (rac)-(1-methylpropyl)amine. A spectroscopic chiroptical signature in solution was provided by the Raman optical activity spectra of compounds 1 and 2. The crystallographic structures of 1, 2, and 3 were examined by single crystal X-ray diffraction. 1 crystallizes in the tetragonal space group P4(3)2(1)2 (no. 96), a = b = 12.826 (2) A, c = 17.730 (2) A, V = 2916.9 (5) A(3), Z = 8, Flack coefficient 0.04 (2). 2 crystallizes in the tetragonal space group P4(1)2(1)2 (no. 92), a = b = 12.842 (1) A, c = 17.749 (2) A, V = 2927.0 (5) A(3), Z = 8, Flack coefficient 0.05 (2). The crystal structures and space groups for 1 and 2 are enantiomorphs and the crystallographic investigation confirmed the absolute configuration of the stereocenter in both compounds. 3 crystallizes in the monoclinic space group P2(1)/n(no. 14), a = 8.178 (1) A, b = 14.309 (2) A, c = 12.328 (2) A, beta = 96.811 (6) degrees, V = 1432.4 (2) A(3), Z = 4.     

Higher-order repeats in the satellite DNA of the cave beetle Pholeuon proserpinae glaciale (Coleoptera: Cholevidae)

The present study characterizes the satellite DNA of the cave beetle Pholeuon proserpinae glaciale which represent about 3-5 % of its genome, and which is composed of monomers of 266 bp and 70.5 % A-T. Concerted evolution seems to act on a higher-order repeat, a dimer, composed of two types of 266-bp monomers that differ in three diagnostic sites. These dimers show a striking nucleotide identity (98.7 % similarity) suggesting strong homogenization processes. The presence of particular mutations shared by several dimers represents an early expansion of these types of repeats as proposed by the molecular drive model. Moreover, evidence of gene conversion tracts in P. proserpinae glaciale, which also could be the result of unequal sister chromatid exchange, would suggest that recombination is involved in the homogenization of stDNA sequences. The presence of a 17-bp-motif repeated six times, along another one of 31-bp repeated twice which also have embedded one 17-bp-motif, suggest that monomers have originated from those basic motifs.     

Characterization of guanylate kinase from gram positive and gram negative microorganisms; preliminary results

Guanylate kinase is a member of the nucleoside monophosphate (NMP) kinase family, a family of enzymes that despite having a low primary structure identity share a similar fold, which consists of three structurally distinct regions termed the CORE, LID, and NMP-binding regions. Guanylate kinase (GMPK) is an essential enzyme for the biosynthesis of GTP and dGTP by catalyzing the phosphoryl transfer from ATP to (d)GMP resulting in ADP and (d)GDP. Despite the similar fold of the monomer there is an important difference between GMPKs from prokaryotes and eukaryotes: eukaryotes GMPK are monomers while prokaryotes GMPK are dimmers, tetramers or hexamers. For this reason bacterial GMPKs are possible targets for new antibacterial drugs. Finding new targets for antibacterial therapies is a prior subject in today's medical research. The purpose of this work was to characterize guanylate kinases from both gram positive and gram negative pathogenic bacteria. We started with GMPK from Enterococcus faecalis as gram positive microorganism and Pseudomonas aeruginosa as gram negative representative.     

The Pathological Links between Adiposity and the Carpal Tunnel Syndrome

An association between obesity and carpal tunnel syndrome is found in many epidemiological studies. Therefore, there is a need to evaluate the physiopathological links that could explain the association between these two entities. Ectopic adipose tissue is responsible for metabolic syndrome and inflammation, and is a major risk factor for diabetes and cardiovascular diseases. Taking these elements into consideration, we conducted an extensive literature revision of the subject, considering as ectopic fat-related mechanisms the following: (a) the direct compression and the association with the metabolic syndrome of the fat deposition around the wrist, (b) the insulin resistance, dyslipidemia, inflammatory, and oxidative mechanisms related to the central deposition of the fat, (c) the impaired muscle contraction and metabolism related to myosteatosis. Each section presents the cellular pathways which are modified by the ectopic deposition of the adipose tissue and the impact in the pathogeny of the carpal tunnel syndrome. In conclusion, the experimental and clinical data support the epidemiological findings. Efforts to reduce the obesity epidemics will improve not only cardio-metabolic health but will reduce the burden of the disability-free life expectancy due to the carpal tunnel syndrome.