Rafael González-Alvarez

Molecular evolution and expression profile of the chemerine encoding gene RARRES2 in baboon and chimpanzee

BackgroundChemerin, encoded by the retinoic acid receptor responder 2 (RARRES2) gene is an adipocytesecreted protein with autocrine/paracrine functions in adipose tissue, metabolism and inflammation with a recently described function in vascular tone regulation, liver, steatosis, etc. This molecule is believed to represent a critical endocrine signal linking obesity to diabetes. There are no data available regarding evolution of RARRES2 in non-human primates and great apes. Expression profile and orthology in RARRES2 genes are unknown aspects in the biology of this multigene family in primates. Thus; we attempt to describe expression profile and phylogenetic relationship as complementary knowledge in the function of this gene in primates. To do that, we performed A RT-PCR from different tissues obtained during necropsies. Also we tested the hypotheses of positive evolution, purifying selection, and neutrality. And finally a phylogenetic analysis was made between primates RARRES2 protein.ResultsRARRES2 transcripts were present in liver, lung, adipose tissue, ovary, pancreas, heart, hypothalamus and pituitary tissues. Expression in kidney and leukocytes were not detectable in either species. It was determined that the studied genes are orthologous.ConclusionsRARRES2 evolution fits the hypothesis of purifying selection. Expression profiles of the RARRES2 gene are similar in baboons and chimpanzees and are also phylogenetically related.

Molecular Identification, Antifungal Susceptibility, and Geographic Origin of Clinical Strains of Sporothrix schenckii Complex in Mexico

Sporotrichosis is a subcutaneous mycosis caused by Sporothrix schenckii complex. The disease has been reported worldwide. However, the incidence of the etiological agent varies in its geographic distribution. We studied 39 clinical isolates of Sporothrix schenckii from diverse regions in Mexico, collected from 1998 to 2016. Molecular identification was performed by sequence analysis of the partial calmodulin gene. In vitro antifungal susceptibility to amphotericin B (AMB), itraconazole (ITC), voriconazole (VRC), posaconazole (PSC), fluconazole (FLC), terbinafine (TRB), caspofungin (CSF), anidulafungin (ANF), and micafungin (MCF) was evaluated. Thirty-eight isolates of S. schenckii complex were divided into five supported clades in a phylogenetic tree. The predominant clinical form was lymphocutaneous (92.3%), fixed cutaneous (5.1%), and disseminated (2.5%). Terbinafine exhibited the best in vitro antifungal activity, while fluconazole was ineffective against Sporothrix schenckii complex. Our results showed diverse geographic distribution of clinical isolates in eight states; definitive identification was done by CAL gen PCR-sequencing. In Mexico, S. schenckii is considered to be an etiological agent of human sporotrichosis cases, and lymphocutaneous is the most prevalent form of the disease. This study revealed four clades of S. schenckii sensu stricto by phylogenetic analysis. Furthermore, we report one case of S. globosa isolated from human origin from the North of Mexico.

Mining the Alkhumra Hemorrhagic Fever Virus Genomes in 2015

Abstract. Alkhurma hemorrhagic fever virus was first isolated at Jeddah, Saudi Arabia, in the early 1990s, and identified as a member of the Flaviviridae family. The virus has been confirmed only in the sand tampan tick, Ornithodoros savignyi (Audouin), and camel tick, Hyalomma dromedarii Koch. Symptoms of infection include headache, joint and muscle pain, vomiting, and thrombocytopenia, leading to hemorrhagic fever, which can cause death. The purpose of this article was to make a genomic-mined Alkhurma hemorrhagic fever virus from the genomic sequences previously reported in GenBank of NCBI. Genomes were divided into peptides, and different evolutionary parameters were derived using bioinformatics tools. The individual behavior of each of the peptides encoded in the genome of Alkhurma hemorrhagic fever virus is specific; the rate of change indicates different kinds of evolutionary pressure. Complete genome and NS3 protein are under positive selection (dN > dS, p < 0.05), while purifying selection is the main force that drives the evolution of envelope and NS5 proteins (dN < dS, p < 0.05). The spread of viruses such as Alkhurma hemorrhagic fever virus can be catastrophic if they affect the global population without antibodies. Genomic mining of Alkhurma hemorrhagic fever virus provides a powerful tool to design new strategies focused on variable regions of the genome in case of spread.

Olfactomedin-like 2 A and B (OLFML2A and OLFML2B) expression profile in primates (human and baboon)

BackgroundThe olfactomedin-like domain (OLFML) is present in at least four families of proteins, including OLFML2A and OLFML2B, which are expressed in adult rat retina cells. However, no expression of their orthologous has ever been reported in human and baboon.ObjectiveThe aim of this study was to investigate the expression of OLFML2A and OLFML2B in ocular tissues of baboons (Papio hamadryas) and humans, as a key to elucidate OLFML function in eye physiology.MethodsOLFML2A and OLFML2B cDNA detection in ocular tissues of these species was performed by RT-PCR. The amplicons were cloned and sequenced, phylogenetically analyzed and their proteins products were confirmed by immunofluorescence assays.ResultsOLFML2A and OLFML2B transcripts were found in human cornea, lens and retina and in baboon cornea, lens, iris and retina. The baboon OLFML2A and OLFML2B ORF sequences have 96% similarity with their human’s orthologous. OLFML2A and OLFML2B evolution fits the hypothesis of purifying selection. Phylogenetic analysis shows clear orthology in OLFML2A genes, while OLFML2B orthology is not clear.ConclusionsExpression of OLFML2A and OLFML2B in human and baboon ocular tissues, including their high similarity, make the baboon a powerful model to deduce the physiological and/or metabolic function of these proteins in the eye.

Molecular Cloning, Sequence Analysis, and Gene Expression of the Circadian Clock Gene Period in Culex quinquefasciatus Say (Diptera: Culicidae)

Abstract. In prokaryotes and eukaryotes, the Period (PER) gene is important for the circadian clock that regulates some rhythmic biochemical, physiological, and behavioral processes. PER messenger amplification was done by RT-PCR from southern house mosquito, Culex quinquefasciatus Say, larvae collected at three locations in the State of Nuevo Leon, northeastern Mexico. The amplified products were cloned, sequenced, and compared/analyzed with those of other species of mosquitoes. A 3,093-bp amplified product was synthesized by PCR, which is the complete coding DNA sequence (3,027 bp) of PER encoding an open reading frame of 996 amino acid residues and much smaller than the orthologous in other mosquitoes analyzed. No other simultaneous amplification was visualized; thus, possible alternative splicings were discarded or at least not detected by the method. Because PER is a widely conserved gene in prokaryotes and eukaryotes and maintaining colonies of southern house mosquitoes is relativity easy, determining the sequence of the gene in the mosquito provides the possibility of using the gene as a model and generating data on the role in the circadian clock.

Molecular Cloning and Characterization of the Circadian Clock Timeless Gene in Culex quinquefasciatus Say (Diptera: Culicidae)

Abstract. In eukaryotes, the timeless (TIM) gene plays a fundamental role in the control of the circadian clock by regulating several biochemical, physiological, and behavioral processes. RT-PCR was used to amplify TIM mRNA from larval southern house mosquito, Culex quinquefasciatus Say, from three locations in the State of Nuevo Leon in northeastern Mexico. The 3062 bp TIM cDNA was assembled from three overlapping PCRs that were cloned and sequenced. DNA sequence analysis used amplified cDNA fragments to complete coding the DNA sequence (2991 bp). Tim ORF consisting of 996 codons was shorter than the orthologous genes from other mosquito species. A series of deletions in the core sequence of TIM were detected. No other amplification products were visualized, and thus, the possibility of alternative spliced mRNA species was discarded. Because TIM is a widely conserved gene in eukaryotes, and maintaining colonies of Cx. quinquefasciatus is easy, determining the sequence of the TIM gene in the mosquito provides the possibility of its use as a model for circadian clock experimentation.

Identification of Four Isoforms of Esterase-5A from Culex quinquefasciatus Say

Abstract. The southern house mosquito, Culex quinquefasciatus Say (Diptera: Culicidae), transmits pathogens and is one of the most important vectors of human disease. The carboxylesterase mosquito genes encoded esterase enzyme involved in the mechanism that provides resistance to insecticides. We amplified by RTPCR four alternative transcripts from the esterase gene not reported previously (A, B, C, and D) from Cx. quinquefasciatus larvae from Monterrey, N.L., northeastern Mexico. Through cDNA sequences, we predicted amino acid sequences used to model the three-dimensional structure of protein isoforms. We determined dN and dS ratios to identify evolutionary forces which lead the family. PCR products were obtained from 5A esterases (1839, 1898, 1894, and 1953 bp). The translation sequence of the PCR products showed polypeptides of 612, 473, 282, and 282 amino acids in length for isoforms A, B, C, and D, respectively. Three exons and two introns were obtained in the analyzed sequences. The study sequences were compared with those previously published showing 98.7, 98.5, 91.5, and 91.5% amino acid similarity for isoforms A, B, C, and D, respectively. The threedimensional models showed that only ORF-D had the three amino acids corresponding to the catalytic triad, while ORF-A and ORF-C lacked one or two of the three amino acids. Esterase genes are under purifying of selection; a clue the members are functional. The processes might be useful in monitoring evolutionary dynamics of known carboxylesterase genes and identifying new carboxylesterase splicing.

Genomic Mining, Evolution Analysis, and Structural Characterization of the Sepik Virus

Abstract. Sepik virus, a pathogen transmitted by arthropods to humans is in the flavivirus family. For this reason, the genomic study of the Sepik virus is relevant in vector-borne diseases field. Genomic mining of the virus and comparison of its coding regions were done to identify sequences that represent greater and lesser conservation than other phylogenetically correlated viruses and to identify regions of greater selection pressure within its genome. The Sepik genome was mapped and analyzed by alignment processes. Selection pressure was estimated using the Pamilio-Banachi-Li method. The genome of the Sepik virus is a 17-motif polyprotein. Within these motifs, the codifying regions of greatest conservation were immunoglobulin-like domain III (82.77%) and the DEAD domain (68.42%), while some domains indicated selection pressure. The selection pressure predicted for certain domains suggested evolutionary pressure on the virus that represented medical impact on virulence and biological risk of the virus.

Molecular cloning of the myo‑inositol oxygenase gene from the kidney of baboons

The enzyme myo-Inositol oxygenase (MIOX) is also termed ALDRL6. It is a kidney-specific member of the aldo-keto reductase family. MIOX catalyzes the first reaction involved in the myo-inositol metabolism signaling pathway and is fully expressed in mammalian tissues. MIOX catalyzes the oxidative cleavage of myo-Inositol and its epimer, D-chiro-Inositol to D-glucuronate. The dioxygen-dependent cleavage of the C6 and C1 bond in myo-Inositol is achieved by utilizing the Fe2+/Fe3+ binuclear iron center of MIOX. This enzyme has also been implicated in the complications of diabetes, including diabetic nephropathy. The MIOX gene was amplified with reverse transcription-polymerase chain reaction from baboon tissue samples, and the product was cloned and sequenced. MIOX expression in the baboon kidney is described in the present study. The percentages of nucleotide and amino acid similarities between baboons and humans were 95 and 96%, respectively. The MIOX protein of the baboon may be structurally identical to that of humans. Furthermore, the evolutionary changes, which have affected these sequences, have resulted from purifying forces.

PCR- RFLP for Identification of Bluetongue Virus Serotypes

Abstract. Bluetongue virus is the etiological agent of a disease characterized by bleeding in domestic and wild ruminants. Different viral strains can infect a ruminant species and severity of infection is variable. Transmission of the virus under natural conditions is through the bite of a mosquito of the genus Culicoides (Ceratopogonidae), but also can be transmitted by infected semen or embryos. The genus Culicoides is a vector of other diseases such as African horse sickness. Specimens are about 0.5 to 5 mm in size. There are about 1,400 species of Culicoides, only 32 of which can act as biological vectors. This is because of the receiver with the intestinal cells where amplification occurs in viral load. A segment 10 of the viral genome from 16 viral serotypes (of approximately 26 serotypes reported) was used. The sequences were aligned, and the consensus sequence was obtained. Subsequently, a pair of oligonucleotides was designed on a highly conserved region that theoretically allows co-amplification of the strains. Sites for restriction enzymes were identified to create a specific electrophoretic pattern for each serotype. It was possible to theoretically amplify a gene segment of 16 bluetongue virus serotypes with one set of oligonucleotides and the serotypes can be differentiated with two restriction enzymes. The proposed method could become a simple, inexpensive, and practical routine tool for type-specific identification of bluetongue virus.