ALBERT

Description: Genes, Vol. 8, Pages 303: RNAi-Mediated Specific Gene Silencing as a Tool for the Discovery of New Drug Targets in Giardia lamblia; Evaluation Using the NADH Oxidase Gene Genes doi: 10.3390/genes8110303 Authors: Jaime Marcial-Quino Saúl Gómez-Manzo Francisco Fierro Yadira Rufino-González Daniel Ortega-Cuellar Edgar Sierra-Palacios America Vanoye-Carlo Abigail González-Valdez Angélica Torres-Arroyo Jesús Oria-Hernández Horacio Reyes-Vivas The microaerophilic protozoan Giardia lamblia is the agent causing giardiasis, an intestinal parasitosis of worldwide distribution. Different pharmacotherapies have been employed against giardiasis; however, side effects in the host and reports of drug resistant strains generate the need to develop new strategies that identify novel biological targets for drug design. To support this requirement, we have designed and evaluated a vector containing a cassette for the synthesis of double-stranded RNA (dsRNA), which can silence expression of a target gene through the RNA interference (RNAi) pathway. Small silencing RNAs were detected and quantified in transformants expressing dsRNA by a stem-loop RT-qPCR approach. The results showed that, in transformants expressing dsRNA of 100–200 base pairs, the level of NADHox mRNA was reduced by around 30%, concomitant with a decrease in enzyme activity and a reduction in the number of trophozoites with respect to the wild type strain, indicating that NADHox is indeed an important enzyme for Giardia viability. These results suggest that it is possible to induce the G. lamblia RNAi machinery for attenuating the expression of genes encoding proteins of interest. We propose that our silencing strategy can be used to identify new potential drug targets, knocking down genes encoding different structural proteins and enzymes from a wide variety of metabolic pathways.

Description: Fragile X syndrome (FXS) is the most common monogenetic cause of intellectual disability. The cognitive deficits in the mouse model for this disorder, the Fragile X Mental Retardation 1 (Fmr1) knockout (KO) mouse, have been restored by different pharmacological approaches, among those the blockade of cannabinoid type 1 (CB1) receptor. In this regard, our previous study showed that the CB1 receptor antagonist/inverse agonist rimonabant normalized a number of core features in the Fmr1 knockout mouse. Rimonabant was commercialized at high doses for its anti-obesity properties, and withdrawn from the market on the bases of mood-related adverse effects. In this study we show, by using electrophysiological approaches, that low dosages of rimonabant (0.1 mg/kg) manage to normalize metabotropic glutamate receptor dependent long-term depression (mGluR-LTD). In addition, low doses of rimonabant (from 0.01 mg/kg) equally normalized the cognitive deficit in the mouse model of FXS. These doses of rimonabant were from 30 to 300 times lower than those required to reduce body weight in rodents and to presumably produce adverse effects in humans. Furthermore, NESS0327, a CB1 receptor neutral antagonist, was also effective in preventing the novel object-recognition memory deficit in Fmr1 KO mice. These data further support targeting CB1 receptors as a relevant therapy for FXS.

Description: Bacteria of the genus Azospirillum colonize roots of important cereals and grasses, and promote plant growth by several mechanisms, notably phytohormone synthesis. The genomes of several Azospirillum strains belonging to different species, isolated from various host plants and locations, were recently sequenced and published. In this study, an additional genome of an A. brasilense strain, isolated from maize grown on an alkaline soil in the northeast of Mexico, strain CBG497, was obtained. Comparative genomic analyses were performed on this new genome and three other genomes (A. brasilense Sp245, A. lipoferum 4B and Azospirillum sp. B510). The Azospirillum core genome was established and consists of 2,328 proteins, representing between 30% to 38% of the total encoded proteins within a genome. It is mainly chromosomally-encoded and contains 74% of genes of ancestral origin shared with some aquatic relatives. The non-ancestral part of the core genome is enriched in genes involved in signal transduction, in transport and in metabolism of carbohydrates and amino-acids, and in surface properties features linked to adaptation in fluctuating environments, such as soil and rhizosphere. Many genes involved in colonization of plant roots, plant-growth promotion (such as those involved in phytohormone biosynthesis), and properties involved in rhizosphere adaptation (such as catabolism of phenolic compounds, uptake of iron) are restricted to a particular strain and/or species, strongly suggesting niche-specific adaptation.

Description: Genes, Vol. 9, Pages 396: Genomic Diversity of Listeria monocytogenes Isolated from Clinical and Non-Clinical Samples in Chile Genes doi: 10.3390/genes9080396 Authors: Viviana Toledo Henk C. den Bakker Juan Carlos Hormazábal Gerardo González-Rocha Helia Bello-Toledo Magaly Toro Andrea I. Moreno-Switt Listeria monocytogenes is the causative agent of listeriosis, which is an uncommon but severe infection associated with high mortality rates in humans especially in high-risk groups. This bacterium survives a variety of stress conditions (e.g., high osmolality, low pH), which allows it to colonize different niches especially niches found in food processing environments. Additionally, a considerable heterogeneity in pathogenic potential has been observed in different strains. In this study, 38 isolates of L. monocytogenes collected in Chile from clinical samples (n = 22) and non-clinical samples (n = 16) were analyzed using whole genome sequencing (WGS) to determine their genomic diversity. A core genome Single Nucleotide Polymorphism (SNP) tree using 55 additional L. monocytogenes accessions classified the Chilean isolates in lineages I (n = 25) and II (n = 13). In silico, Multi-locus sequence typing (MLST) differentiated the isolates into 13 sequence types (ST) in which the most common were ST1 (15 isolates) and ST9 (6 isolates) and represented 55% of the isolates. Genomic elements associated with virulence (i.e., LIPI-1, LIPI-3, inlA, inlB, inlC, inlG, inlH, inlD, inlE, inlK, inlF, and inlJ) and stress survival (i.e., stress survival islet 1 and stress survival islet 2) were unevenly distributed among clinical and non-clinical isolates. In addition, one novel inlA premature stop codon (PMSC) was detected. Comparative analysis of L. monocytogenes circulating in Chile revealed the presence of globally distributed sequence types along with differences among the isolates analyzed at a genomic level specifically associated with virulence and stress survival.

Description: Genes, Vol. 9, Pages 427: Genetic Variants in pre-miR-146a, pre-miR-499, pre-miR-125a, pre-miR-605, and pri-miR-182 Are Associated with Breast Cancer Susceptibility in a South American Population Genes doi: 10.3390/genes9090427 Authors: Sebastián Morales Tomas De Mayo Felipe Andrés Gulppi Patricio Gonzalez-Hormazabal Valentina Carrasco José Miguel Reyes Fernando Gómez Enrique Waugh Lilian Jara Breast cancer (BC) is one of the most frequent tumors affecting women worldwide. microRNAs (miRNAs) single-nucleotide polymorphisms (SNPs) likely contribute to BC susceptibility. We evaluated the association of five SNPs with BC risk in non-carriers of the BRCA1/2-mutation from a South American population. The SNPs were genotyped in 440 Chilean BRCA1/2-negative BC cases and 1048 controls. Our data do not support an association between rs2910164:G>C or rs3746444:A>G and BC risk. The rs12975333:G>T is monomorphic in the Chilean population. The pre-miR-605 rs2043556-C allele was associated with a decreased risk of BC, both in patients with a strong family history of BC and in early-onset non-familial BC (Odds ratio (OR) = 0.5 [95% confidence interval (CI) 0.4–0.9] p = 0.006 and OR = 0.6 [95% CI 0.5–0.9] p = 0.02, respectively). The rs4541843-T allele is associated with increased risk of familial BC. This is the first association study on rs4541843 and BC risk. Previously, we showed that the TOX3-rs3803662:C>T was significantly associated with increased risk of familial BC. Given that TOX3 mRNA is a target of miR-182, and that both the TOX3 rs3803662-T and pri-miR-182 rs4541843-T alleles are associated with increased BC risk, we evaluated their combined effect. Risk of familial BC increased in a dose-dependent manner with the number of risk alleles (p-trend = 0.0005), indicating an additive effect.

Description: Genes, Vol. 9, Pages 222: Erratum: Jimena Soledad Cadona, et al.; Pathogenicity Islands Distribution in Non-O157 Shiga Toxin-Producing Escherichia coli (STEC). Genes 2018, 9, 81 Genes doi: 10.3390/genes9040222 Authors: Jimena Cadona Ana Bustamante Juliana González Andrea Sanso We wish to make the following correction to the paper by Soledad-Cadona et al.[...]

Description: Genes, Vol. 9, Pages 357: Genetic Targeting of GRP78 in the VMH Improves Obesity Independently of Food Intake Genes doi: 10.3390/genes9070357 Authors: Laura Liñares-Pose Eva Rial-Pensado Ánxela Estévez-Salguero Edward Milbank Ismael González-García Claudia Rodríguez Patricia Seoane-Collazo Noelia Martinez-Sánchez Rubén Nogueiras Dolores Prieto Carlos Diéguez Cristina Contreras Miguel López Recent data have demonstrated that the hypothalamic GRP78/BiP (glucose regulated protein 78 kDa/binding immunoglobulin protein) modulates brown adipose tissue (BAT) thermogenesis by acting downstream on AMP-activated protein kinase (AMPK). Herein, we aimed to investigate whether genetic over-expression of GRP78 in the ventromedial nucleus of the hypothalamus (VMH: a key site regulating thermogenesis) could ameliorate very high fat diet (vHFD)-induced obesity. Our data showed that stereotaxic treatment with adenoviruses harboring GRP78 in the VMH reduced hypothalamic endoplasmic reticulum ER stress and reversed vHFD-induced obesity. Herein, we also demonstrated that this body weight decrease was more likely associated with an increased BAT thermogenesis and browning of white adipose tissue (WAT) than to anorexia. Overall, these results indicate that the modulation of GRP78 in the VMH may be a target against obesity.

Description: The nascent RNA can reinvade the DNA double helix to form a structure termed the R-loop, where a single-stranded DNA (ssDNA) is accompanied by a DNA-RNA hybrid. Unresolved R-loops can impede transcription and replication processes and lead to genomic instability by a mechanism still not fully understood. In this sense, a connection between R-loops and certain chromatin markers has been reported that might play a key role in R-loop homeostasis and genome instability. To counteract the potential harmful effect of R-loops, different conserved messenger ribonucleoprotein (mRNP) biogenesis and nuclear export factors prevent R-loop formation, while ubiquitously-expressed specific ribonucleases and DNA-RNA helicases resolve DNA-RNA hybrids. However, the molecular events associated with R-loop sensing and processing are not yet known. Given that R-loops hinder replication progression, it is plausible that some DNA replication-associated factors contribute to dissolve R-loops or prevent R-loop mediated genome instability. In support of this, R-loops accumulate in cells depleted of the BRCA1, BRCA2 or the Fanconi anemia (FA) DNA repair factors, indicating that they play an active role in R-loop dissolution. In light of these results, we review our current view of the role of replication-associated DNA repair pathways in preventing the harmful consequences of R-loops.

Description: Genes, Vol. 9, Pages 277: Size and Content of the Sex-Determining Region of the Y Chromosome in Dioecious Mercurialis annua, a Plant with Homomorphic Sex Chromosomes Genes doi: 10.3390/genes9060277 Authors: Paris Veltsos Guillaume Cossard Emmanuel Beaudoing Genséric Beydon Dessislava Savova Bianchi Camille Roux Santiago C. González-Martínez John R. Pannell Dioecious plants vary in whether their sex chromosomes are heteromorphic or homomorphic, but even homomorphic sex chromosomes may show divergence between homologues in the non-recombining, sex-determining region (SDR). Very little is known about the SDR of these species, which might represent particularly early stages of sex-chromosome evolution. Here, we assess the size and content of the SDR of the diploid dioecious herb Mercurialis annua, a species with homomorphic sex chromosomes and mild Y-chromosome degeneration. We used RNA sequencing (RNAseq) to identify new Y-linked markers for M. annua. Twelve of 24 transcripts showing male-specific expression in a previous experiment could be amplified by polymerase chain reaction (PCR) only from males, and are thus likely to be Y-linked. Analysis of genome-capture data from multiple populations of M. annua pointed to an additional six male-limited (and thus Y-linked) sequences. We used these markers to identify and sequence 17 sex-linked bacterial artificial chromosomes (BACs), which form 11 groups of non-overlapping sequences, covering a total sequence length of about 1.5 Mb. Content analysis of this region suggests that it is enriched for repeats, has low gene density, and contains few candidate sex-determining genes. The BACs map to a subset of the sex-linked region of the genetic map, which we estimate to be at least 14.5 Mb. This is substantially larger than estimates for other dioecious plants with homomorphic sex chromosomes, both in absolute terms and relative to their genome sizes. Our data provide a rare, high-resolution view of the homomorphic Y chromosome of a dioecious plant.

Description: Stem-loop quantitative reverse transcription PCR (RT-qPCR) is a molecular technique used for identification and quantification of individual small RNAs in cells. In this work, we used a Universal ProbeLibrary (UPL)-based design to detect—in a rapid, sensitive, specific, and reproducible way—the small nucleolar RNA (snoRNA) GlsR17 and its derived miRNA (miR2) of Giardia lamblia using a stem-loop RT-qPCR approach. Both small RNAs could be isolated from both total RNA and small RNA samples. Identification of the two small RNAs was carried out by sequencing the PCR-amplified small RNA products upon ligation into the pJET1.2/blunt vector. GlsR17 is constitutively expressed during the 72 h cultures of trophozoites, while the mature miR2 is present in 2-fold higher abundance during the first 48 h than at 72 h. Because it has been suggested that miRNAs in G. lamblia have an important role in the regulation of gene expression, the use of the stem-loop RT-qPCR method could be valuable for the study of miRNAs of G. lamblia. This methodology will be a powerful tool for studying gene regulation in G. lamblia, and will help to better understand the features and functions of these regulatory molecules and how they work within the RNA interference (RNAi) pathway in G. lamblia.