Alteration of hippocampal Egr3, GABA A receptors, Il-1ß, Il6 and Ccl3 expression in audiogenic tremor mice after seizure.

Neuroinflammation plays a protective role in the brain; however, in neurological diseases such as epilepsy, overactivated neuroinflammation, along with overexpression of inflammatory mediators, can cause neuronal tissue damage, which can trigger seizures due to loss of ionic or neurotransmitter homeostasis. Therefore, we aimed to evaluate mRNA expression levels of proinflammatory cytokines, early growth response factor 3 (Egr3), and GABA A receptors in the hippocampus of naive audiogenic mutant tremor mice, and stimulated tremor mice after a seizure. Gene expression of Il-1ß, Il-6, Tnf-α, Ccl2, Ccl3, Egr3, Gabra1, and Gabra4 from hippocampal samples of naive and stimulated tremor mice were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Relative to resistant mice, Ccl3 gene expression was increased and Il6 was decreased in the hippocampus of naïve tremor mice. Thirty minutes after a seizure, Ccl3 and Il-1ß mRNA expression were decreased (p < 0.0001; p = 0.0034, respectively) while Il6 was increased (p = 0.0052) in stimulated tremor mice, relative to naïve animals. In addition, Egr3, Gabra1, and Gabra4 mRNA expression was decreased in the hippocampus of naive tremor mice, relative to resistant mice, which increased 30 minutes after a seizure (p = 0.0496; p = 0.0447, and p = 0.0011, respectively), relative to naïve animals. In conclusion, overexpression of Ccl3 in the hippocampus of naive tremor mice, followed by downregulation soon after seizure in stimulated tremor mice, could be involved in changes in the blood-brain barrier (BBB) permeability in epilepsy. Il-1ß may be involved in hippocampal downregulation of GABA A receptors of naive tremor mice, characterizing an important mechanism in audiogenic seizures triggering. Hippocampal alterations of proinflammatory cytokines, Egr3, and GABA A receptors in tremor mice reinforce them as an alternative tool to modeling temporal lobe epilepsy.

Behavioral and neurochemical characterization of the spontaneous mutation tremor, a new mouse model of audiogenic seizures.

The tremor mutant phenotype results from an autosomal recessive spontaneous mutation arisen in a Swiss-Webster mouse colony. The mutant mice displayed normal development until three weeks of age when they began to present motor impairment comprised by whole body tremor, ataxia, and decreased exploratory behavior. These features increased in severity with aging suggesting a neurodegenerative profile. In parallel, they showed audiogenic generalized clonic seizures. Results from genetic mapping identified the mutation tremor on chromosome 14, in an interval of 5 cM between D14Mit37 (33.21 cM) and D14Mit115 (38.21 cM), making Early Growth Response 3 (Egr3) the main candidate gene. Comparing with wild type (WT) mice, the tremor mice showed higher hippocampal gene expression of Egr3 and Gabra1 and increased concentrations of noradrenalin (NOR; p = .0012), serotonin (5HT; p = .0083), 5-hydroxyindoleacetic acid (5-HIAA; p = .0032), γ-amino butyric acid (GABA; p = .0123), glutamate (p = .0217) and aspartate (p = .0124). In opposition, the content of glycine (p = .0168) and the vanillylmandelic acid (VMA)/NOR ratio (p = .032) were decreased. Regarding to dopaminergic system, neither dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) contents nor the turnover rate of DA showed statistically significant differences between WT and mutant mice. Data demonstrated that audiogenic seizures of tremor mice are associated with progressive motor impairment as well as to hippocampal alterations of the Egr3 and Gabra1 gene expression and amino acid and monoamine content. In addition, the tremor mice could be useful for study of neurotransmission pathways as modulators of epilepsy and the pathogenesis of epilepsies occurring with generalized clonic seizures.

Chemogenomic study of gemcitabine using Saccharomyces cerevisiae as model cell-molecular insights about chemoresistance.

Gemcitabine (GEM) is the drug used as first line to treat pancreatic cancer, one of the most devastating human tumors. This peculiar type of tumor develops resistance to several drugs, including GEM, due to its desmoplastic reaction and other features. The GEM chemoresistance has been investigated at molecular level aiming to find a pathway whose inhibition or activation should overcome it. Through next-generation sequencing was performed a chemogenomic assay of GEM using Saccharomyces cerevisiae as model cell and the results showed that more than 40% of genes related to GEM response in yeast possess unknown or dubious function. We choose two yeast mutants to individually validate the fitness defect results observed by chemogenomic assay, Δhmt1 and Δcsi1, and it was found that in addition to some already described pathways involved in GEM resistance, cells deficient in deneddylation enzyme Cop9 Signalosome Interactor 1 (Csi1p) presented a high sensitivity to GEM. This was confirmed by individual growth analyses of Δcsi1 cells exposed to GEM, and this phenotype was reverted with CSI1 complementation gene. Csi1p is a well-characterized homolog equivalent to human Csn6 subunit of COP9 signalosome (CSN) involved in deneddylation process. We highlighted too that epigenetic alterations, such as methylation mediated by protein arginine methyltransferase 1, play an important role in regulating gemcitabine treatment resistance. Our results point out new unexplored molecular pathways that can be used to overcome GEM resistance: the inhibition of CSN and the arginine methyltransferase activities.

Whole-exome sequencing reveals the impact of UVA light mutagenesis in xeroderma pigmentosum variant human cells.

UVA-induced mutagenesis was investigated in human pol eta-deficient (XP-V) cells through whole-exome sequencing. In UVA-irradiated cells, the increase in the mutation frequency in deficient cells included a remarkable contribution of C>T transitions, mainly at potential pyrimidine dimer sites. A strong contribution of C>A transversions, potentially due to oxidized bases, was also observed in non-irradiated XP-V cells, indicating that basal mutagenesis caused by oxidative stress may be related to internal tumours in XP-V patients. The low levels of mutations involving T induced by UVA indicate that pol eta is not responsible for correctly replicating T-containing pyrimidine dimers, a phenomenon known as the 'A-rule'. Moreover, the mutation signature profile of UVA-irradiated XP-V cells is highly similar to the human skin cancer profile, revealing how studies involving cells deficient in DNA damage processing may be useful to understand the mechanisms of environmentally induced carcinogenesis.

Behavioral and neurochemical characterization of the spontaneous mutation tremor, a new mouse model of audiogenic seizures

The tremor mutant phenotype results from an autosomal recessive spontaneous mutation arisen in a Swiss–Webster mouse colony. The mutant mice displayed normal development until three weeks of age when they began to present motor impairment comprised by whole body tremor, ataxia, and decreased exploratory behavior. These features increased in severity with aging suggesting a neurodegenerative profile. In parallel, they showed audiogenic generalized clonic seizures. Results from genetic mapping identified the mutation tremor on chromosome 14, in an interval of 5 cM between D14Mit37 (33.21cM) and D14Mit115 (38.21cM), making Early Growth Response 3 (Egr3) the main candidate gene. Comparing with wild type (WT) mice, the tremor mice showed higher hippocampal gene expression of Egr3 and Gabra1 and increased concentrations of noradrenalin (NOR; p=.0012), serotonin (5HT; p=.0083), 5-hydroxyindoleacetic acid (5-HIAA; p=.0032), gama-amino butyric acid (GABA; p=.0123), glutamate (p=.0217) and aspartate (p=.0124). In opposition, the content of glycine (p=.0168) and the vanillylmandelic acid (VMA)/NOR ratio (p=.032) were decreased. Regarding to dopaminergic system, neither dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) contents nor the turnover rate of DA showed statistically significant differences between WT and mutant mice. Data demonstrated that audiogenic seizures of tremor mice are associated with progressive motor impairment as well as to hippocampal alterations of the Egr3 and Gabra1 gene expression and amino acid and monoamine content. In addition, the tremor mice could be useful for study of neurotransmission pathways as modulators of epilepsy and the pathogenesis of epilepsies occurring with generalized clonic seizures.

Behavioral and neurochemical characterization of the spontaneous mutation tremor, a new mouse model of audiogenic seizures

The tremor mutant phenotype results from an autosomal recessive spontaneous mutation arisen in a Swiss–Webster mouse colony. The mutant mice displayed normal development until three weeks of age when they began to present motor impairment comprised by whole body tremor, ataxia, and decreased exploratory behavior. These features increased in severity with aging suggesting a neurodegenerative profile. In parallel, they showed audiogenic generalized clonic seizures. Results from genetic mapping identified the mutation tremor on chromosome 14, in an interval of 5 cM between D14Mit37 (33.21cM) and D14Mit115 (38.21cM), making Early Growth Response 3 (Egr3) the main candidate gene. Comparing with wild type (WT) mice, the tremor mice showed higher hippocampal gene expression of Egr3 and Gabra1 and increased concentrations of noradrenalin (NOR; p=.0012), serotonin (5HT; p=.0083), 5-hydroxyindoleacetic acid (5-HIAA; p=.0032), gama-amino butyric acid (GABA; p=.0123), glutamate (p=.0217) and aspartate (p=.0124). In opposition, the content of glycine (p=.0168) and the vanillylmandelic acid (VMA)/NOR ratio (p=.032) were decreased. Regarding to dopaminergic system, neither dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) contents nor the turnover rate of DA showed statistically significant differences between WT and mutant mice. Data demonstrated that audiogenic seizures of tremor mice are associated with progressive motor impairment as well as to hippocampal alterations of the Egr3 and Gabra1 gene expression and amino acid and monoamine content. In addition, the tremor mice could be useful for study of neurotransmission pathways as modulators of epilepsy and the pathogenesis of epilepsies occurring with generalized clonic seizures.

Draft genome sequences of KPC-2- and CTX-M-15-producing Klebsiella pneumoniae ST437 isolated from a clinical sample and urban rivers in Sao Paulo, Brazil.

OBJECTIVES: KPC-producing Klebsiella pneumoniae is considered one of the most worrisome multidrug-resistant micro-organisms in nosocomial infections. It has also been reported in wastewater and urban rivers in the city of Sao Paulo, Brazil. Here we report the draft genome sequences of three KPC-2- and CTX-M-15-producing K. pneumoniae sequence type 437 (ST437) isolates obtained from two urban rivers and from a clinical sample of a patient in Sao Paulo. METHODS: A genomic library was constructed using a Nextera XT Kit. An Illumina platform was used to perform whole-genome sequencing (WGS). RESULTS: WGS of environmental isolates Kp148/PINH-4900 and Kp196/TIET-4200 and clinical isolate Kp314/11 resulted in estimated genome sizes of 5464058, 5437723 and 5319218bp, respectively. Resistome analysis of the environmental and clinical strains revealed the presence of resistance genes to the following antimicrobials in all strains: aminoglycosides [aac(6')-Ib-cr]; ß-lactams (blaOXA-1, blaSHV-11, blaCTX-M-15 and blaKPC-2); fluoroquinolones [aac(6')-Ib-cr, oqxA and oqxB]; fosfomycin (fosAKP); macrolides [mph(A)]; phenicols (catB4); sulfonamides (sul1); and trimethoprim (dfrA30). The tetracycline resistance gene tetA was identified in Kp148/PINH-4900 and Kp314/11 only; the aminoglycoside resistance gene aph(3')-Ia was found only in environmental isolates, and aadA2 only in Kp314/11; and the phenicol resistance gene catA1 was identified only in Kp148/PINH-4900. CONCLUSIONS: The draft genome sequences of these strains help us to elucidate the dissemination of resistance genes in micro-organisms inside and outside the hospital and are useful for further comparisons between clinical and environmental strains.

Draft genome sequences of two fluoroquinolone-resistant CTX-M-15-producing Escherichia coli ST90 (ST23 complex) isolated from a calf and a dairy cow in South America.

OBJECTIVES: Farm animals have been recognised as important carriers and reservoirs of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli. The aim of this study was to report the draft genome sequences of two multidrug-resistant (MDR) CTX-M-15-producing E. coli strains (47VL and 13B) isolated from different bovine hosts (a calf and a dairy cow), housed separately in a commercial dairy farm in Brazil. METHODS: Total genomic DNA of the E. coli isolates was sequenced using an Illumina MiSeq paired-end 300-bp sequencing platform. Sequence reads were de novo assembled using the A5-miseq pipeline and polishing assembly in Geneious v.R9. The NCBI Prokaryotic Genome Annotation Pipeline v.3.2 was used for genome annotation, whereas whole-genome sequences were analysed using bioinformatic tools from the Center of Genomic Epidemiology and EnteroBase. RESULTS: E. coli 47VL generated a total of 3238770 and E. coli 13B a total of 1422808 paired-end reads of ca. 190× and ca. 80×, respectively. The resistome revealed that both isolates carried resistance genes to aminoglycosides, ß-lactams, macrolides, sulphonamides, trimethoprim and tetracycline. Comparative analyses revealed clonal relatedness. In fact, both isolates belonged to sequence type ST90 (clonal complex CC23) and phylogroup AxB1. CONCLUSION: To our knowledge, these are the first draft genome sequences of CTX-M-15-producing E. coli ST90 isolated from bovines in South America. These data can be used to elucidate genetic features that contribute to colonisation and adaptation of CTX-M-15-producing E. coli in dairy cattle.

Draft genome sequence of a multidrug-resistant KPC-2-producing Enterobacter aerogenes isolated from a hospitalised patient in Brazil.

OBJECTIVES: Multidrug-resistant (MDR) Enterobacter aerogenes strains are frequently associated with nosocomial infections and high mortality rates, representing a serious public health problem. The aim of this study was to present the draft genome sequence of a MDR KPC-2-producing E. aerogenes isolated from a perineal swab of a hospitalised patient in Brazil. METHODS: Genomic DNA was sequenced using an Illumina MiSeq platform. De novo genome assembly was carried out using the A5-Miseq pipeline, and whole-genome sequence analysis was performed using tools from the Center for Genomic Epidemiology. RESULTS: The strain harboured resistance genes to ß-lactams, aminoglycosides, sulphonamides and trimethoprim in addition to genes encoding multidrug efflux system proteins, a quaternary ammonium transporter and heavy metal efflux system proteins. In addition, the strain harboured genes encoding diverse virulence factors. CONCLUSION: These data might allow a better understanding of the genetic basis of antimicrobial resistance and virulence in E. aerogenes strains.

Draft genome sequence of a multidrug-resistant Aeromonas hydrophila ST508 strain carrying rmtD and blaCTX-M-131 isolated from a bloodstream infection.

Here we report the draft genome sequence of a multidrug-resistant (MDR) Aeromonas hydrophila strain belonging to sequence type 508 (ST508) isolated from a human bloodstream infection. Assembly and annotation of this draft genome resulted in 5028498bp and revealed the presence of 16S rRNA methylase rmtD and blaCTX-M-131 genes encoding high-level resistance to aminoglycosides and cephalosporins, respectively, as well as multiple virulence genes. This draft genome can provide significant information for understanding mechanisms on the establishment and treatment of infections caused by this pathogen.

Draft genome sequence of an environmental multidrug-resistant Klebsiella pneumoniae ST340/CC258 harbouring blaCTX-M-15 and blaKPC-2 genes.

Anthropogenic activities, including the release of wastewater and sewage from hospitals, have contributed to the contamination of aquatic environments, raising a concern to public health. In this study, we present the first draft genome sequence of a Klebsiella pneumoniae strain (Kp171, TIET-4200) belonging to the high-risk hospital-associated clonal lineage ST340/CC258, which was recovered from a water sample collected in an urban river in Brazil.

Draft genome sequence of an aminoglycoside-resistant RmtG-producing Pseudomonas aeruginosa ST235 isolated from a cystic fibrosis patient.

Cystic fibrosis (CF) patients are often chronically colonised or infected by non-fermenting Gram-negative bacilli, with Pseudomonas aeruginosa being the most prevalent. In this study, we report the draft genome sequence of a multidrug-resistant P. aeruginosa strain belonging to sequence type ST235, isolated from the respiratory tract of a CF patient with chronic colonisation. Whole-genome sequencing analysis revealed a 6.7Mb genome size and the presence of 12 antibiotic resistance genes, including the rmtG gene conferring high-level aminoglycoside resistance, located on the chromosome.

Draft Genome Sequence of a Hospital-Associated Clone of Klebsiella pneumoniae ST340/CC258 Coproducing RmtG and KPC-2 Isolated from a Pediatric Patient.

We report here the draft genome sequence of a Klebsiella pneumoniae strain 1194/11, belonging to the hospital-associated sequence type 340 (ST340; clonal complex CC258), isolated from a catheter tip culture from a pediatric patient. The multidrug-resistant strain coproduced the 16S rRNA methyltransferase rRNA RmtG and ß-lactamases KPC-2 and CTX-M-15.

Draft genome sequence of a CTX-M-15-producing Klebsiella pneumoniae sequence type 340 (clonal complex 258) isolate from a food-producing animal.

Klebsiella pneumoniae carrying blaCTX-M-15 have been widely disseminated in hospital settings. In this regard, most clinically important strains belong to clonal complex 28 (CC258), which includes sequence type 340 (ST340). In this study, we present the draft genome sequence of a CTX-M-15-producing ST340 K. pneumoniae strain isolated from a food-producing animal in Brazil.

A novel ABCG-like transporter of Trypanosoma cruzi is involved in natural resistance to benznidazole.

Benznidazole (BZ) is one of the two drugs used for Chagas disease treatment. Nevertheless therapeutic failures of BZ have been reported, which were mostly attributed to variable drug susceptibility among Trypanosoma cruzi strains. ATP-binding cassette (ABC) transporters are involved in a variety of translocation processes and some members have been implicated in drug resistance. Here we report the characterisation of the first T. cruzi ABCG transporter gene, named TcABCG1, which is over-expressed in parasite strains naturally resistant to BZ. Comparison of TcABCG1 gene sequence of two TcI BZ-resistant strains with CL Brener BZ-susceptible strain showed several single nucleotide polymorphisms, which determined 11 amino acid changes. CL Brener transfected with TcI transporter genes showed 40-47% increased resistance to BZ, whereas no statistical significant increment in drug resistance was observed when CL Brener was transfected with the homologous gene. Only in the parasites transfected with TcI genes there was 2-2.6-fold increased abundance of TcABCG1 transporter protein. The analysis in wild type strains also suggests that the level of TcABCG1 transporter is related to BZ natural resistance. The characteristics of untranslated regions of TcABCG1 genes of BZ-susceptible and resistant strains were investigated by computational tools.

A novel ABCG-like transporter of Trypanosoma cruzi is involved in natural resistance to benznidazole

Benznidazole (BZ) is one of the two drugs used for Chagas disease treatment. Nevertheless therapeutic failures of BZ have been reported, which were mostly attributed to variable drug susceptibility among Trypanosoma cruzi strains. ATP-binding cassette (ABC) transporters are involved in a variety of translocation processes and some members have been implicated in drug resistance. Here we report the characterisation of the first T. cruzi ABCG transporter gene, named TcABCG1, which is over-expressed in parasite strains naturally resistant to BZ. Comparison of TcABCG1 gene sequence of two TcI BZ-resistant strains with CL Brener BZ-susceptible strain showed several single nucleotide polymorphisms, which determined 11 amino acid changes. CL Brener transfected with TcI transporter genes showed 40-47% increased resistance to BZ, whereas no statistical significant increment in drug resistance was observed when CL Brener was transfected with the homologous gene. Only in the parasites transfected with TcI genes there was 2-2.6-fold increased abundance of TcABCG1 transporter protein. The analysis in wild type strains also suggests that the level of TcABCG1 transporter is related to BZ natural resistance. The characteristics of untranslated regions of TcABCG1 genes of BZ-susceptible and resistant strains were investigated by computational tools.

ABCG-like transporter of Trypanosoma cruzi involved in benznidazole resistance: gene polymorphisms disclose inter-strain intragenic recombination in hybrid isolates.

Benznidazole (BZ) is one of the two drugs for Chagas disease treatment. In a previous study we showed that the Trypanosoma cruzi ABCG-like transporter gene, named TcABCG1, is over-expressed in parasite strains naturally resistant to BZ and that the gene of TcI BZ-resistant strains exhibited several single nucleotide polymorphisms (SNPs) as compared to the gene of CL Brener BZ-susceptible strain. Here we report the sequence of TcABCG1 gene of fourteen T. cruzi strains, with diverse degrees of BZ sensitivity and belonging to different discrete typing units (DTUs) and Tcbat group. Although DTU-specific SNPs and amino acid changes were identified, no direct correlation with BZ-resistance phenotype was found. Thus, it is plausible that the transporter abundance is a determinant factor for drug resistance, as pointed out above. Sequence data were used for Bayesian phylogenies and network genealogy analysis. The network showed a high degree of reticulation suggesting genetic exchange between the parasites. TcI and TcII clades were clearly separated. Tcbat sequences were close to TcI. A fourth clade clustered TcABCG1 haplotypes of TcV, TcVI and TcIII strains, with closer proximity to TcI. Analysis of the recombination patterns indicated that hybrid strains contain haplotypes that are mosaics most likely derived by intragenic recombination of parental sequences. The data confirm that TcII and TcIII as the parentals of TcV and TcVI DTUs. Since genetic fingerprint of TcI was found in TcIII, we sustain the previously proposed "Two Hybridization model" for the origin of hybrid strains. Among the twenty best BLASTP hits in databases, orthologues of TcABCG1 transporter were found in Leishmania spp. and African trypanosomes, though their function remains undescribed.

Auxin production by the plant trypanosomatid Phytomonas serpens and auxin homoeostasis in infected tomato fruits.

Previously we have characterized the complete gene encoding a pyruvate decarboxylase (PDC)/indolepyruvate decarboxylase (IPDC) of Phytomonas serpens, a trypanosomatid highly abundant in tomato fruits. Phylogenetic analyses indicated that the clade that contains the trypanosomatid protein behaves as a sister group of IPDCs of γ-proteobacteria. Since IPDCs are key enzymes in the biosynthesis of the plant hormone indole-3-acetic acid (IAA), the ability for IAA production by P. serpens was investigated. Similar to many microorganisms, the production of IAA and related indolic compounds, quantified by high performance liquid chromatography, increased in P. serpens media in response to amounts of tryptophan. The auxin functionality was confirmed in the hypocotyl elongation assay. In tomato fruits inoculated with P. serpens the concentration of free IAA had no significant variation, whereas increased levels of IAA-amide and IAA-ester conjugates were observed. The data suggest that the auxin produced by the flagellate is converted to IAA conjugates, keeping unaltered the concentration of free IAA. Ethanol also accumulated in P. serpens-conditioned media, as the result of a PDC activity. In the article we discuss the hypothesis of the bifunctionality of P. serpens PDC/IPDC and provide a three-dimensional model of the enzyme.

Horizontal gene transfer confers fermentative metabolism in the respiratory-deficient plant trypanosomatid Phytomonas serpens.

Among trypanosomatids, the genus Phytomonas is the only one specifically adapted to infect plants. These hosts provide a particular habitat with a plentiful supply of carbohydrates. Phytomonas sp. lacks a cytochrome-mediated respiratory chain and Krebs cycle, and ATP production relies predominantly on glycolysis. We have characterised the complete gene encoding a putative pyruvate/indolepyruvate decarboxylase (PDC/IPDC) (548 amino acids) of P. serpens, that displays high amino acid sequence similarity with phytobacteria and Leishmania enzymes. No orthologous PDC/IPDC genes were found in Trypanosoma cruzi or T. brucei. Conservation of the PDC/IPDC gene sequence was verified in 14 Phytomonas isolates. A phylogenetic analysis shows that Phytomonas protein is robustly monophyletic with Leishmania spp. and C. fasciculata enzymes. In the trees this clade appears as a sister group of indolepyruvate decarboxylases of γ-proteobacteria. This supports the proposition that a horizontal gene transfer event from a donor phytobacteria to a recipient ancestral trypanosome has occurred prior to the separation between Phytomonas, Leishmania and Crithidia. We have measured the PDC activity in P. serpens cell extracts. The enzyme has a Km value for pyruvate of 1.4mM. The acquisition of a PDC, a key enzyme in alcoholic fermentation, explains earlier observations that ethanol is one of the major end-products of glucose catabolism under aerobic and anaerobic conditions. This represents an alternative and necessary route to reoxidise part of the NADH produced in the highly demanding glycolytic pathway and highlights the importance of this type of event in metabolic adaptation.