Perilipin Isoforms and PGC-1α Are Regulated Differentially in Rat Heart during Pregnancy-Induced Physiological Cardiac Hypertrophy.

Background and Objectives: Perilipins 1-5 (PLIN) are lipid droplet-associated proteins that participate in regulating lipid storage and metabolism, and the PLIN5 isoform is known to form a nuclear complex with peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1α) to regulate lipid metabolism gene expression. However, the changes in PLIN isoforms' expression in response to pregnancy-induced cardiac hypertrophy are not thoroughly studied. The aim of this study was to quantify the mRNA expression of PLIN isoforms and PGC-1α along with total triacylglycerol (TAG) and cholesterol levels during late pregnancy and the postpartum period in the rat left ventricle. Materials and Methods: Female Sprague-Dawley rats were divided into three groups: non-pregnant, late pregnancy, and postpartum. The mRNA and protein levels were evaluated using quantitative RT-PCR and Western blotting, respectively. TAG and total cholesterol content were evaluated using commercial colorimetric methods. Results: The expression of mRNAs for PLIN1, 2, and 5 increased during pregnancy and the postpartum period. PGC-1α mRNA and protein expression increased during pregnancy and the postpartum period. Moreover, TAG and total cholesterol increased during pregnancy and returned to basal levels after pregnancy. Conclusions: Our results demonstrate that pregnancy upregulates differentially the expression of PLIN isoforms along with PGC-1α, suggesting that together they might be involved in the regulation of the lipid metabolic shift induced by pregnancy.

Characterization, Selection, and Trans-Species Polymorphism in the MHC Class II of Heermann's Gull (Charadriiformes).

The major histocompatibility complex (MHC) enables vertebrates to cope with pathogens and maintain healthy populations, thus making it a unique set of loci for addressing ecology and evolutionary biology questions. The aim of our study was to examine the variability of Heermann's Gull MHC class II (MHCIIB) and compare these loci with other Charadriiformes. Fifty-nine MHCIIB haplotypes were recovered from sixty-eight Heermann's Gulls by cloning, of them, twelve were identified as putative true alleles, forty-five as unique alleles, and two as pseudogenes. Intra and interspecific relationships indicated at least two loci in Heermann's Gull MHCIIB and trans-species polymorphism among Charadriiformes (coinciding with the documented evidence of two ancient avian MHCIIB lineages, except in the Charadriidae family). Additionally, sites under diversifying selection revealed a better match with peptide-binding sites inferred in birds than those described in humans. Despite the negative anthropogenic activity reported on Isla Rasa, Heermann's Gull showed MHCIIB variability consistent with population expansion, possibly due to a sudden growth following conservation efforts. Duplication must play an essential role in shaping Charadriiformes MHCIIB variability, buffering selective pressures through balancing selection. These findings suggest that MHC copy number and protected islands can contribute to seabird conservation.

Outer Membrane Vesicles of Gram-Negative Bacteria: An Outlook on Biogenesis.

Outer membrane vesicles (OMVs) from Gram-negative bacteria were first described more than 50 years ago. However, the molecular mechanisms involved in biogenesis began to be studied only in the last few decades. Presently, the biogenesis and molecular mechanisms for their release are not completely known. This review covers the most recent information on cellular components involved in OMV biogenesis, such as lipoproteins and outer membrane proteins, lipopolysaccharide, phospholipids, quorum-sensing molecules, and flagella.

Comparative proteomic analysis of outer membrane vesicles from Brucella suis, Brucella ovis, Brucella canis and Brucella neotomae.

Gram-negative bacteria release nanovesicles, called outer membrane vesicles (OMVs), from their outer membrane. Proteomics has been used to determine their composition. OMVs contain proteins able to elicit an immune response, so they have been proposed as a model to develop acellular vaccines. In this study, OMVs of Brucella suis, B. ovis, B. canis, and B. neotomae were purified and analyzed by SDS-PAGE, transmission electron microscopy and liquid chromatography coupled to mass spectrometry to determine the pan-proteome of these vesicles. In addition, antigenic proteins were detected by western blot with anti-Brucella sera. The in silico analysis of the pan-proteome revealed many homologous proteins, such as Omp16, Omp25, Omp31, SodC, Omp2a, and BhuA. Proteins contained in the vesicles from different Brucella species were detected by anti-Brucella sera. The occurrence of previously described immunogenic proteins derived from OMVs supports the use of these vesicles as candidates to be evaluated as an acellular brucellosis vaccine.

Outer Membrane Vesicles From Brucella melitensis Modulate Immune Response and Induce Cytoskeleton Rearrangement in Peripheral Blood Mononuclear Cells.

Similar to what has been described in other Gram-negative bacteria, Brucella melitensis releases outer membrane vesicles (OMVs). OMVs from B. melitensis 16M and the rough-mutant B. melitensis VTRM1 were able to induce a protective immune response against virulent B. melitensis in mice models. The presence of some proteins which had previously been reported to induce protection against Brucella were found in the proteome of OMVs from B. melitensis 16M. However, the proteome of OMVs from B. melitensis VTRM1 had not previously been determined. In order to be better understand the role of OMVs in host-cell interactions, the aim of this work was to compare the proteomes of OMVs from B. melitensis 16M and the derived rough-mutant B. melitensis VTRM1, as well as to characterize the immune response induced by vesicles on host cells. Additionally, the effect of SDS and proteinase K on the stability of OMVs was analyzed. OMVs from B. melitensis 16M (smooth strain) and the B. melitensis VTRM1 rough mutant (lacking the O-polysaccharide side chain) were analyzed through liquid chromatography-mass spectrometry (LC-MS/MS). OMVs were treated with proteinase K, sodium deoxycholate, and SDS, and then their protein profile was determined using SDS-PAGE. Furthermore, PBMCs were treated with OMVs in order to measure their effect on cytoskeleton, surface molecules, apoptosis, DNA damage, proliferation, and cytokine-induction. A total of 131 proteins were identified in OMVs from B. melitensis16M, and 43 in OMVs from B. melitensis VTRM1. Proteome comparison showed that 22 orthologous proteins were common in vesicles from both strains, and their core proteome contained Omp31, Omp25, GroL, and Omp16. After a subsequent detergent and enzyme treatment, OMVs from B. melitensis VTRM1 exhibited higher sensitive compared to OMVs from the B. melitensis 16M strain. Neither OMVs induced IL-17, proliferation, apoptosis or DNA damage. Nonetheless, OMVs from the smooth and rough strains induced overproduction of TNFα and IL-6, as well as actin and tubulin rearrangements in the cytoskeleton. Moreover, OMVs from both strains inhibited PD-L1 expression in T-cells. These data revealed significant differences in OMVs derived from the rough and smooth Brucella strains, among which, the presence or absence of complete LPS appeared to be crucial to protect proteins contained within vesicles and to drive the immune response.

Proteomic Analysis of Membrane Blebs of Brucella abortus 2308 and RB51 and Their Evaluation as an Acellular Vaccine.

Membrane blebs are released from Gram-negative bacteria, however, little is known about Brucella blebs. This work pursued two objectives, the first was to determine and identify the proteins in the membrane blebs by proteomics and in silico analysis. The second aim was to evaluate the use of membrane blebs of Brucella abortus 2308 and B. abortus RB51 as an acellular vaccine in vivo and in vitro. To achieve these aims, membrane blebs from B. abortus 2308 and RB51 were obtained and then analyzed by liquid chromatography coupled to mass spectrometry. Brucella membrane blebs were used as a "vaccine" to induce an immune response in BALB/c mice, using the strain B. abortus RB51 as a positive vaccine control. After subsequent challenge with B. abortus 2308, CFUs in spleens were determined; and immunoglobulins IgG1 and IgG2a were measured in murine serum by ELISA. Also, activation and costimulatory molecules induced by membrane blebs were analyzed in splenocytes by flow cytometry. Two hundred and twenty eight proteins were identified in 2308 membrane blebs and 171 in RB51 blebs, some of them are well-known Brucella immunogens such as SodC, Omp2b, Omp2a, Omp10, Omp16, and Omp19. Mice immunized with membrane blebs from rough or smooth B. abortus induced similar protective immune responses as well as the vaccine B. abortus RB51 after the challenge with virulent strain B. abortus 2308 (P < 0.05). The levels of IgG2a in mice vaccinated with 2308 membrane blebs were higher than those vaccinated with RB51 membrane blebs or B. abortus RB51 post-boosting. Moreover, mice immunized with 2308 blebs increased the percentage of activated B cells (CD19+CD69+) in vitro. Therefore, membrane blebs are potential candidates for the development of an acellular vaccine against brucellosis, especially those derived from the rough strains so that serological diagnostic is not affected.

Correct Identification of Ochrobactrum anthropi From Blood Culture Using 16rRNA Sequencing: A First Case Report in an Immunocompromised Patient in Mexico.

The present report describes the misidentification of Brucella spp. from a positive blood culture using traditional microbiology tests. A molecular test identified the bacterium as Ochrobactrum anthropi. According to the information available, this report is the first to include this type of case in Mexico.

Partial Characterization of Bacteriocin Produced by Halotolerant Pediococcus acidilactici Strain QC38 Isolated from Traditional Cotija Cheese.

During a screening of lactic acid bacteria producing bacteriocin from Cotija cheese, the strain QC38 was isolated. Based on the 16S rRNA gene nucleotide sequencing (516 pb accession no KJ210322) and phylogenetic analysis, the isolate was identified as Pediococcus acidilactici. Neutralized cell-free supernatant was tested for antimicrobial activity against 17 Gram-negative and Gram-positive pathogens. Growth inhibition was achieved against Listeria monocytogenes (supplier or indication or source), Staphylococcus aureus, Vibrio vulnificus, Vibrio cholerae O1 Ogawa, Vibrio cholerae NO 01 and Salmonella enterica subsp. Enterica serovar Typhimurium. Bacteriocin-like substance, after heating at 121°C for 15 min it remained stable and its antimicrobial activity was observed at pH ranging from 1.0 to 10.0 but inactivated by α-chymotrypsin and proteinase K. Strain QC38 was able to grow in 1-9% NaCl concentration. The plate overlay assay showed an approximate size of bacteriocin-like substance between 3.4 and 6.5 kDa. P. acidilactici QC38 harboured a plasmid that contains a gene for a pediocin (PA-1).

Diversity and expression of P450 genes from Dendroctonus valens LeConte (Curculionidae: Scolytinae) in response to different kairomones.

Bark beetles (Curculionidae: Scolytinae) are major cause of woody plants death in the world. They colonize the stem and other parts of trees recognizing host-produced specific compounds (kairomones) and insect pheromones. Bark beetle's antennae and alimentary canal participate in the host selection identifying chemical compounds produced by trees and insects, and also in the metabolism and detoxification of these compounds. The red turpentine beetle (RTB), Dendroctonus valens LeConte, is an unaggressive species that colonize > 40 pine species (Pinaceae) in North and Central America. Several studies suggest that bark beetle cytochrome P450 enzymes are involved in monoterpene oxidation. In this study we identified by means of PCR, cloning, sequencing, and bioinformatic analysis, eleven full-length genes: five CYP4, four CYP6, and two CYP9 in the antennae and gut region of RTB, after stimulation with vapors of monoterpenes: (±)-α-pinene, (R)-(+)-α-pinene, (S)-(-)-ß-pinene, (S)-(-)-α-pinene and (+)-3-carene; pine trees volatiles used by RTB as kairomones. The recovered cDNA of these genes vary from 1.5 kb to 1.8 kb and the open frame encodes from 496 to 562 amino acid proteins. The bioinformatic analysis suggests that the majority of P450 proteins encoded by these genes are membrane anchored in the endoplasmic reticulum. RT-qPCR assays showed differential expression of all CYP genes between male and female. The gene expression was dependent of monoterpenes and exposure time, with some of them sex, antennae and gut region specific. Significant differences among monoterpenes, gut region, antennae and exposure time were found. Our results suggest that some of these genes may be involved in the detoxification process of these compounds during tree colonization.

Historical demography and phylogeography of a specialist bark beetle, Dendroctonus pseudotsugae Hopkins (Curculionidae: Scolytinae).

Contemporary distribution of North American species has been shaped by past glaciation events during the Quaternary period. However, their effects were not as severe in the southern Rocky Mountains and Northern Mexico as elsewhere in North America. In this context, we test hypotheses about the historical demography of Dendroctonus pseudotsugae, based on 136 haplotypes of mitochondrial cytochrome oxidase I. The phylogenetic analysis yielded four haplogroups corresponding to northwestern United States and southwestern Canada (NUS), southwestern United States (Arizona, SUS), northwestern Mexico (Sierra Madre Occidental, SMOC), and northeastern Mexico (Sierra Madre Oriental, SMOR). Predictions of demographic expansion were examined through neutrality tests against population growth and mismatch distribution. Results showed that the NUS and SMOC haplogroups have experienced demographic expansion events, whereas the SUS and SMOR haplogroups have not. Divergence times between pairs of haplogroups were estimated from early to middle Pleistocene. The longer divergence time between NUS and all other haplogroups could be the result of refugia within the Pacific Northwest and northern Rocky Mountains and long-term isolation from southernmost populations in Mexico. The results obtained in this study are in agreement with the evolutionary history of the host Douglas-fir, as the warmer climates of interglacial periods pushed conifers northward of Colorado, New Mexico, and Arizona, whereas environmental changes reduced the population size of Douglas-fir and forced fragmentation of distribution range southward into northern Mexico.

Effect of geographic isolation on genetic differentiation in Dendroctonus pseudotsugae (Coleoptera: Curculionidae).

Genetic structure of phytophagous insects has been widely studied, however, relative influence of the effect of geographic isolation, the host plant or both has been subject of considerable debate. Several studies carried out on bark beetles in the genus Dendroctonus evaluated these factors; nonetheless, recent evidence has shown that genetic structuring is a more complex process. Our goal was to examine the effect of geographic isolation on genetic structure of the Douglas-fir beetle Dendroctonus pseudotsugae. We used mtDNA cytochrome oxidase I (COI) sequences and RAPD markers. One hundred-seventy-two individuals were obtained from 17 populations, for which we analyzed 60 haplotypes (among 172 sequences of COI gene, 550 bp long) and 232 RAPD markers (7 primers). Analyses of molecular variance (AMOVA and SAMOVA), F-statistics and linear regressions suggest that the genetic structure of D. pseudotsugae is strongly influenced by geographic distance. We found that D. pseudotsugae has high intra- and inter-population genetic variation compared with several other bark beetles. Genetic differences among populations based on COI and RAPD markers were correlated with geographic distance. The observed genetic differences between northern (Canada-USA) and southern (Mexico) populations on Pseudotsuga menziesii var. glauca confirm that these two sets of populations correspond to previously assigned subspecies.