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The eradication of Helicobacter pylori is a valid strategy for preventing gastric cancer; however, the therapeutic failure of first-line treatments in Colombia is associated with high resistance to metronidazole and amoxicillin. This study explored alternative antibiotics and analyzed point mutations in resistance genes to furazolidone and rifampicin in order to include them in rescue therapy regimens. A total of 239 complete genomes of Helicobacter pylori Colombian strains were compared to that of the ATCC 26695 strain to identify mutations in the rpoB and porD genes for rifampicin and furazolidinone resistance, respectively. While rifampicin resistance mutations were not found, only 0.84% of the isolates showed the porD gene, suggesting that Helicobacter pylori is sensitive to these antibiotics. A phylogenomic analysis of Helicobacter pylori revealed an independent lineage in Colombia (hspColombia). The absence of point mutations in the rpoB gene, together with the scarce mutations identified in the porD gene of Helicobacter pylori , suggest that the hspColombia isolates are sensitive to rifampicin and furazolidone, which could be key to including these antibiotics in the rescue therapies against Helicobacter pylori .

The mussel Mytilus galloprovincialis is one of the most important aquaculture species in Europe. Its main production problem is the accumulation of toxins during coastal blooms, which prevents mussel commercialization. P-glycoprotein (ABCB1/MDR1/P-gp) is part of the multixenobiotic resistance system in aquatic organisms, and okadaic acid, the main DSP toxin, is probably a substrate of the P-gp-mediated efflux. In this study, the presence and possible role of P-gp in the okadaic acid detoxification process was studied in M. galloprovincialis . We identified, cloned, and characterized two complete cDNAs of mdr1 and mdr2 genes. MgMDR1 and MgMDR2 predicted proteins had the structure organization of ABCB full transporters, and were identified as P-gp/MDR/ABCB proteins. Furthermore, the expression of mdr genes was monitored in gills, digestive gland, and mantle during a cycle of accumulation-elimination of okadaic acid. Mdr1 significantly increased its expression in the digestive gland and gills, supporting the idea of an important role of the MDR1 protein in okadaic acid efflux out of cells in these tissues. The expression of M. galloprovincialis mrp2 , a multidrug associated protein (MRP/ABCC), was also monitored. As in the case of mdr1 , there was a significant induction in the expression of mrp2 in the digestive gland, as the content of okadaic acid increased. Thus, P-gp and MRP might constitute a functional defense network against xenobiotics, and might be involved in the resistance mechanisms to DSP toxins.

Some diatom species of the genus Pseudo-nitzschia produce the toxin domoic acid. The depuration rate of domoic acid in Pecten maximus is very low; for this reason, king scallops generally contain high levels of domoic acid in their tissues. A transcriptomic approach was used to identify the genes differentially expressed in the P. maximus digestive gland after the injection of domoic acid. The differential expression analysis found 535 differentially expressed genes (226 up-regulated and 309 down-regulated). Protein-protein interaction networks obtained with the up-regulated genes were enriched in gene ontology terms, such as vesicle-mediated transport, response to stress, signal transduction, immune system process, RNA metabolic process, and autophagy, while networks obtained with the down-regulated genes were enriched in gene ontology terms, such as response to stress, immune system process, ribosome biogenesis, signal transduction, and mRNA processing. Genes that code for cytochrome P450 enzymes, glutathione S-transferase theta-1, glutamine synthase, pyrroline-5-carboxylate reductase 2, and sodium- and chloride-dependent glycine transporter 1 were among the up-regulated genes. Therefore, a stress response at the level of gene expression, that could be caused by the domoic acid injection, was evidenced by the alteration of several biological, cellular, and molecular processes.

Some species of the genus Pseudo-nitzschia produce the toxin domoic acid, which causes amnesic shellfish poisoning (ASP). Given that bivalve mollusks are filter feeders, they can accumulate these toxins in their tissues. To elucidate the transcriptional response of the queen scallop Aequipecten opercularis after exposure to domoic acid-producing Pseudo-nitzschia , the digestive gland transcriptome was de novo assembled using an Illumina HiSeq 2000 platform. Then, a differential gene expression analysis was performed. After the assembly, 142,137 unigenes were obtained, and a total of 10,144 genes were differentially expressed in the groups exposed to the toxin. Functional enrichment analysis found that 374 Pfam (protein families database) domains were significantly enriched. The C1q domain, the C-type lectin, the major facilitator superfamily, the immunoglobulin domain, and the cytochrome P450 were among the most enriched Pfam domains. Protein network analysis showed a small number of highly connected nodes involved in specific functions: proteasome components, mitochondrial ribosomal proteins, protein translocases of mitochondrial membranes, cytochromes P450, and glutathione S-transferases. The results suggest that exposure to domoic acid-producing organisms causes oxidative stress and mitochondrial dysfunction. The transcriptional response counteracts these effects with the up-regulation of genes coding for some mitochondrial proteins, proteasome components, and antioxidant enzymes (glutathione S-transferases, thioredoxins, glutaredoxins, and copper/zinc superoxide dismutases).

Aim: To characterize punctual mutations in 23S rRNA gene of clarithromycin-resistant Helicobacter pylori ( H. pylori ) and determine their association with therapeutic failure., Methods: PCR products of 23S rRNA gene V domain of 74 H. pylori isolates; 34 resistant to clarithromycin (29 from a low-risk gastric cancer (GC) population: Tumaco-Colombia, and 5 from a high-risk population: Tuquerres-Colombia) and 40 from a susceptible population (28 from Tumaco and 12 from Túquerres) were sequenced using capillary electrophoresis. The concordance between mutations of V domain 23S rRNA gene of H. pylori and therapeutic failure was determined using the Kappa coefficient and McNemar's test was performed to determine the relationship between H. pylori mutations and clarithromycin resistance., Results: 23S rRNA gene from H. pylori was amplified in 56/74 isolates, of which 25 were resistant to clarithromycin (20 from Tumaco and 5 from Túquerres, respectively). In 17 resistant isolates (13 from Tumaco and 4 from Túquerres) the following mutations were found: A1593T1, A1653G2, C1770T, C1954T1, and G1827C in isolates from Tumaco, and A2144G from Túquerres. The mutations T2183C, A2144G and C2196T in H. pylori isolates resistant to clarithromycin from Colombia are reported for the first time. No association between the H. pylori mutations and in vitro clarithromycin resistance was found. However, therapeutic failure of eradication treatment was associated with mutations of 23S rRNA gene in clarithromycin-resistant H. pylori ( κ = 0.71)., Conclusion: The therapeutic failure of eradication treatment in the two populations from Colombia was associated with mutations of the 23S rRNA gene in clarithromycin-resistant H. pylori ., Competing Interests: Conflict-of-interest statement: The authors declare that there is no conflict of interest related to this study.

Bivalve molluscs are filter feeding species that can accumulate biotoxins in their body tissues during harmful algal blooms. Amnesic Shellfish Poisoning (ASP) is caused by species of the diatom genus Pseudo-nitzschia, which produces the toxin domoic acid. The Mytilus galloprovincialis digestive gland transcriptome was de novo assembled based on the sequencing of 12 cDNA libraries, six obtained from control mussels and six from mussels naturally exposed to domoic acid-producing diatom Pseudo-nitzschia australis. After de novo assembly 94,727 transcripts were obtained, with an average length of 1015 bp and a N50 length of 761 bp. The assembled transcripts were clustered (homology > 90%) into 69,294 unigenes. Differential gene expression analysis was performed (DESeq2 algorithm) in the digestive gland following exposure to the toxic algae. A total of 1158 differentially expressed unigenes (absolute fold change > 1.5 and p-value < 0.05) were detected: 686 up-regulated and 472 down-regulated. Several membrane transporters belonging to the family of the SLC (solute carriers) were over-expressed in exposed mussels. Functional enrichment was performed using Pfam annotations obtained from the genes differentially expressed, 37 Pfam families were found to be significantly (FDR adjusted p-value < 0.1) enriched. Some of these families (sulfotransferases, aldo/keto reductases, carboxylesterases, C1q domain and fibrinogen C-terminal globular domain) could be putatively involved in detoxification processes, in the response against of the oxidative stress and in immunological processes. Protein network analysis with STRING algorithm found alteration of the Notch signaling pathway under the action of domoic acid-producing Pseudo-nitzschia. In conclusion, this study provides a high quality reference transcriptome of M. galloprovincialis digestive gland and identifies potential genes involved in the response to domoic acid., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Aim: To evaluate effect of treatment failure on cagA and vacA genotypes in Helicobacter pylori ( H. pylori ) isolates from Colombia., Methods: One hundred and seventy-six participants infected with H. pylori from Colombia were treated during 14 d with the triple-standard therapy. Six weeks later, eradication was evaluated by 13 C-Urea breath test. Patients with treatment failure were subjected to endoscopy control; biopsies obtained were used for histopathology and culture. DNA from H. pylori isolates was amplified using primers specific for cagA and vacA genes. The phylogenetic relationships among isolates obtained before and after treatment were established by conglomerate analysis based on random amplified polymorphic DNA (RAPD) fingerprinting., Results: Treatment effectiveness was at 74.6%. Of the participants with treatment failure, 25 accepted subjected to a second endoscopy. Prevalence of post-treatment infection was 64% (16/25) and 40% (10/25) by histology and culture, respectively. Upon comparing the cagA and vacA genotypes found before and after therapy, multiple cagA genotypes ( cagA -positive and cagA -negative) were found before treatment; in contrast, cagA -negative genotypes decreased after treatment. vacA s1m1 genotype was highly prevalent in patients before and after therapy. The 3' cagA region was successfully amplified in 95.5% (21/22) of the isolates obtained before and in 81.8% (18/22) of the isolates obtained after treatment. In the isolates obtained from patients with treatment failure, it was found that 72.7% (16/22) presented alterations in the number of EPIYA motifs, compared to isolates found before treatment., Conclusion: Unsuccessful treatment limits colonization by low-virulence strains resulting in partial and selective eradication in mixed infections, and acts on the cagA -positive strains inducing genetic rearrangements in cagA variable region that produces a loss or gain of EPIYA repetitions., Competing Interests: Conflict-of-interest statement: The authors declare that there is no conflict of interest related to this study.

Aim: To compare the genomic variability and the multiple colonization of Helicobacter pylori ( H. pylori ) in patients with chronic gastritis from two Colombian populations with contrast in the risk of developing gastric cancer (GC): Túquerres-Nariño (High risk) and Tumaco-Nariño (Low risk)., Methods: Four hundred and nine patients from both genders with dyspeptic symptoms were studied. Seventy-two patients were included in whom H. pylori was isolated from three anatomic regions of the gastric mucosa, (31/206) of the high risk population of GC (Túquerres) and (41/203) of the low risk population of GC (Tumaco). The isolates were genotyped by PCR-RAPD. Genetic diversity between the isolates was evaluated by conglomerates analysis and multiple correspondence analyses., Results: The proportion of virulent genotypes of H. pylori was 99% in Túquerres and 94% in Tumaco. The coefficient of similarity of Nei-Li showed greater genetic diversity among isolates of Túquerres (0.13) than those of Tumaco (0.07). After adjusting by age, gender and type of gastritis, the multiple colonization was 1.7 times more frequent in Túquerres than in Tumaco ( P = 0.05)., Conclusion: In Túquerres, high risk of GC there was a greater probability of multiple colonization by H. pylori . From the analysis of the results of the PCR-RAPD, it was found higher genetic variability in the isolates of H. pylori in the population of high risk for the development of GC., Competing Interests: Conflict-of-interest statement: The authors declare that there is no conflict of interest related to this study.

The storage of data is a key process in the study of electrical power networks related to the search for harmonics and the finding of a lack of balance among phases. The presence of missing data of any of the main electrical variables (phase-to-neutral voltage, phase-to-phase voltage, current in each phase and power factor) affects any time series study in a negative way that has to be addressed. When this occurs, missing data imputation algorithms are required. These algorithms are able to substitute the data that are missing for estimated values. This research presents a new algorithm for the missing data imputation method based on Self-Organized Maps Neural Networks and Mahalanobis distances and compares it not only with a well-known technique called Multivariate Imputation by Chained Equations (MICE) but also with an algorithm previously proposed by the authors called Adaptive Assignation Algorithm (AAA). The results obtained demonstrate how the proposed method outperforms both algorithms., Competing Interests: The authors declare no conflict of interest.

Nowadays, data collection is a key process in the study of electrical power networks when searching for harmonics and a lack of balance among phases. In this context, the lack of data of any of the main electrical variables (phase-to-neutral voltage, phase-to-phase voltage, and current in each phase and power factor) adversely affects any time series study performed. When this occurs, a data imputation process must be accomplished in order to substitute the data that is missing for estimated values. This paper presents a novel missing data imputation method based on multivariate adaptive regression splines (MARS) and compares it with the well-known technique called multivariate imputation by chained equations (MICE). The results obtained demonstrate how the proposed method outperforms the MICE algorithm.

Homeobox-containing genes encode a set of transcription factors that have been shown to control spatial patterning mechanisms in bilaterian organism development. The homeobox gene Gbx, included in the EHGbox cluster, is implicated in the development of the nervous system. In this study, we surveyed five different families of Bivalvia for the presence of Gbx genes by means of PCR with degenerate primers. We were able to recover seven Gbx gene fragments from five bivalve species: Solen marginatus, Mimachlamys varia, Venerupis pullastra, Ostrea edulis and Mytilus galloprovincialis (the derived amino acid sequence were designated Sma-Gbx, Cva-Gbx, Vpu-Gbx, Oed-Gbx and Mga-Gbx, respectively). These genes are orthologous to various Gbx genes present in bilaterian genomes. The Gbx genes in four Bivalvia families, namely Solenidae, Veneridae, Ostreidae and Mytilidae, are newly reported here and we also showed additional information of the Gbx genes of Pectinidae. The phylogenetic analyses by neighbour-joining, UPGMA, maximum parsimony and Bayesian analysis clearly indicated that the Gbx sequences formed a well supported clade and assigned these Gbx genes to the Gbx family. These data permit to confirm that the homeodomain of the Gbx family is highly conserved among these five distinct families of bivalve molluscs., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The clustered Hox genes play a central role in the regulation of development in bilaterian animals. In this study, we analyzed the homeobox-containing genes in a bivalve mollusc, the mussel Mytilus galloprovincialis, an unsegmented spiralian lophotrochozoan. We isolated and characterized four Hox cluster genes using the polymerase chain reaction with specific primers. Molecular alignments and phylogenetic analysis indicate that these mussel genes are homologs of the anterior group (pb ortholog), paralog group 3, and central group (PG4/Dfd and PG5/Scr) genes. The putative homeodomain sequences were designated Mgox1, Mgox2, Mgox3, and Mgox4.

The aim of this study was to evaluate the ability of a nested PCR system to detect Salmonella senftenberg in raw oysters. The specific primers of the PCR were derived from the invA gene sequence, essential for Salmonella invasiveness into epithelial cells. First, for the extraction of DNA, four methods (guanidine isothiocyanate, E.Z.N.A. Mollusc Kit, Chelex-100, and lysis with detergents) were compared. A nested PCR method combined with 3.5 h pre-enrichment in buffered peptone water (BPW) and DNA extraction by the resin Chelex-100 is proposed for the detection of S. senftenberg in oyster samples. The detection limit of the method is less than 0.1 CFU/ml (<1 CFU/g of oyster). This procedure is shown to be an excellent tool for the sensitive detection of S. senftenberg from naturally contaminated oysters, with results being obtained within 8 h.

Homeobox genes encode a family of transcription factors that have essential roles in regulating the development of eukaryotes. Although they have been extensively studied in different phyla, relatively little is known about homeoboxcontaining genes and their function in molluscs. In this study, we used a polymerase chain reaction to investigate homeobox genes in the bivalve mollusc Pecten maximus. Four different homeobox sequences were identified; two were homologues of the non-Hox cluster gene caudal and the two remaining sequences had a significant homology to the ANT-C gene Proboscipedia. These sequences represent the first cad and pb homologues isolated from a member of the class Bivalvia, phylum Mollusca.

Sterol composition and content and their seasonal variations over 18 months were investigated in adductor muscle, digestive gland and gonads of Pecten maximus. Sterols were isolated by Silicagel 60 thin layer chromatography and identified by gas chromatography/mass spectrometry. Eleven sterols were identified, with cholesterol, brassicasterol, 24-methylenecholesterol and 22-trans-dehydrocholesterol being the principal components. The same sterols were found in all three tissues independent of season. The relative amounts of each sterol present in each tissue differed. Total sterol levels in gonad and muscle were higher than in digestive gland. Statistically significant differences (P<0.05) were found between the concentrations of each of the sterols isolated from the gonad or muscle and digestive gland. The seasonal variations in the sterol content of the gonad seem be related to the reproductive cycle, while the sterol content of the digestive gland appears to be linked to diet, mainly diatoms or dinoflagellates. The muscle sterol content showed minor changes throughout the year.

Seasonal variations in lipid classes and fatty acid composition of triacylglycerols and phospholipids in the digestive gland of Pecten maximus were studied over a period of 16 months. Acylglycerols predominated (19-77% of total lipids), in accordance with the role of the digestive gland as an organ for lipid storage in scallops. Seasonal variations were mainly seen in the acylglycerol content, while phospholipids (2.5-10.0% of total lipids) and sterols (1.9-7.4% of total lipids) showed only minor changes. The most abundant fatty acids were 14:0, 16:0, 18:0, 16:1(n-7), 18:1(n-9), 18:1(n-7), 18:4(n-3), 20:5(n-3) and 22:6(n-3) and these showed similar seasonal profiles in both, triacylglycerol and phospholipid fractions. In contrast to the phospholipid fraction, the triacylglycerol fraction contained more 20:5(n-3) than 22:6(n-3). In three phospholipid samples we noted a high percentage of a 22-2-non-methylene-interrupted fatty acid, previously described to have a structural role in several bivalve species. The main polyunsaturated fatty acids displayed important seasonal variations parallel to those of the acylglycerols, suggesting good nutritional conditions. A positive correlation existed between the level of saturated fatty acids and temperature, whereas the levels of polyunsaturated fatty acids correlated negatively with temperature.

Previous cerebral ischemia studies have reported the limitations of restricted periods of postischemic hypothermia in producing long-term neuroprotection. The present experiment attempts to determine whether delayed treatment with the free radical scavenger N-tert-butyl-a-phenylnitrone (PBN) is protective at 2 months following transient global forebrain ischemia, and whether additive effects can be observed when PBN is administered in combination with moderate hypothermia. For this aim rats were subjected to 10 min of two-vessel forebrain ischemia followed by (a) 3 h of postischemic normothermia (37 degrees C); (b) 3 h of postischemic hypothermia (30 degrees C); (c) normothermic procedures combined with delayed injections of PBN (100 mg/kg) on days 3, 5 and 7 post-insult; (d) postischemic hypothermia combined with delayed PBN treatment; or (e) sham procedures. Outcome measures included cognitive behavioral testing and quantitative histopathological analysis at 2 months. Postischemic PBN injections induced a systemic hypothermia (1.5 degrees C-2.0 degrees C) that lasted for 2-2.5 h. Water maze testing revealed significant performance deficits relative to shams in the normothermic ischemic group, with the postischemic hypothermia and PBN groups showing intermediate values. A significant attenuation of cognitive deficits was observed in the animal group receiving the combination postischemic hypothermia and delayed PBN treatment. Quantitative CA1 hippocampal cell counts indicated that each of the ischemia groups exhibited significantly fewer viable CA1 neurons compared to sham controls. However, in rats receiving either delayed PBN treatment or 3 h of postischemic hypothermia, significant sparing of CA1 neurons relative to the normothermic ischemia group was observed. These data indicate that hypothermia combined with PBN treatment provides long-term cognitive improvement compared to nontreatment groups. PBN-induced mild hypothermia could contribute to the neuroprotective effects of this pharmacological strategy.

Gonadotropin-releasing hormones (GnRH) constitute a family of neuropeptides which are important regulators of reproduction in vertebrates. The effect of mammalian GnRH (mGnRH), salmon GnRH, chicken GnRH-I, chicken GnRH-II, and lamprey GnRH-I on [3H]thymidine incorporation into DNA of dissociated gonadal cells of marine bivalves has been studied. The incorporation of [3H]thymidine is linear between 1.5 and 8 h of incubation. All five GnRHs significantly increased DNA synthesis in gonial cells of Crassostrea gigas. The maximal activation was about of 135-140% above control. The activation is dose dependent, over the range 10(-11) to 10(-6) M, but is modulated by the physiological condition of the cells and the stage of sexual maturity of the gonad. mGnRH has also a mitogenic effect in dissociated mantle cells of Mytilus edulis. The effect of mGnRH is blocked by a GnRH antagonist ([D-pGlu1,D-Phe2, D-Trp3,6]GnRH, 5 x 10(-6)M) in C. gigas as well as in M. edulis, suggesting that the GnRH action in the gonad is mediated by specific receptors for GnRH or GnRH-like peptides. The existence of GnRH-immunoreactive neurons and fibers in the cerebral and pedal ganglia of M. edulis was demonstrated by immunocytochemistry. They are located principally in the anterior internal area of the cerebral ganglia, close to the cerebral commissure and in the posterior part of the pedal ganglia. The presence of GnRH-responsive cells and GnRH-like immunoreactive material suggests that peptides of the GnRH-like family are present and functional in bivalve molluscs., (Copyright 1999 Academic Press.)

The present study was designed to determine whether postischemic hypothermia, delayed MK-801 (dizocilpine) administration, or a combination of these treatments can provide lasting neurobehavioral protection following transient global ischemia in rats. Rats were subjected to 10 min of normothermic (37 degrees C) ischemia induced by 2-vessel occlusion and hypotension (50 mmHg) or sham procedures. Ischemia was followed by either: (a) 3 h at normothermic brain temperatures, (b) 3 h of postischemic brain hypothermia at 30 degrees C, (c) hypothermia coupled with MK-801 (4 mg/kg, i.p.) on postischemic days 3, 5 and 7, or (d) postischemic MK-801 treatment alone. Neurobehavioral evaluation 6-8 weeks following surgery showed that normothermic ischemia (NI) was associated with water maze navigational deficits, including performance on a simple place task involving finding a hidden platform maintained in one position for 6 days, and a learning set task in which the platform was moved to a different location each day (both P's < 0.02 vs. sham). NI was also associated with increased locomotion in an open field (P < 0.01 vs. sham). A combination of postischemic hypothermia and delayed MK-801 injections provided partial protection from ischemic-associated hyperactivity in the open field (P < 0.02 vs. NI), and robust protection from simple place task deficits (P < 0.02 vs. NI). Evidence for significant protective effects of MK-801 or hypothermia alone was observed in the learning set, during the final trial blocks each day. These results provide further evidence for neuroprotective effects of these treatments at chronic survival intervals, and indicate that the therapeutic window for attenuating ischemic damage is considerably longer than has heretofore been appreciated.

Previous work has demonstrated that damage to the primary somatosensory cortex produces substantial deficits in a vibrissal cue-dependent discrimination task which recover gradually over the course of post-injury testing. The present study was designed to evaluate the possible site(s) and mechanisms underlying behavioral recovery in this task. Wistar rats were trained under red light in a T-maze to produce ipsilateral turns depending upon the presence of a vibrissal cue. Animals were then subjected to photothrombotic infarctions of either the ipsilateral medial parietal cortex, the ipsilateral primary and secondary somatosensory cortex (SI/SII), the primary and secondary somatosensory cortices of both hemispheres (bilateral SI/SII) or sham surgical procedures. Behavioral testing resumed 24 hours following surgery, and continued for a total of 60 days. The performance of animals with infarcts restricted to the medial parietal cortex did not differ from that of sham-operated controls. Animals with either unilateral or bilateral SI/SII infarcts exhibited a significant decrease in percent correct responding as compared to shams and rats in the medial parietal group. These deficits recovered to pre-infarct levels over approximately 35-40 days. This rate of recovery was slower than the recovery exhibited by animals given medial parietal infarcts which spared the primary barrelfield cortex. The results of this study suggest that neither the contralateral somatosensory cortex nor the vibrissal representation within ipsilateral SII cortex play a critical role in the recovery process. The possibility that subcortical structures underlie the deficits observed following barrelfield cortical damage is discussed.

Rats with one olfactory bulb removed were trained to detect 0.5%-0.005% concentrations of amyl acetate. These rats had only a slight decrement in performance when the naris ipsilateral to the intact olfactory bulb was closed. Thus, vapors inhaled through one naris can stimulate olfactory receptor neurons in the contralateral nasal vault. This internasal stimulation is probably mediated by the nasopharyngeal canal or nasopharynx.