HLA alleles may serve as a tool to discriminate atypical type 2 diabetic patients

AIM: To investigate whether the presence of human leukocyte antigen(HLA) marker could add new information to discriminated atypical diabetic type 2 patients.METHODS: We analyzed 199 patients initially diagnosed as type 2 diabetes who are treated in special care diabetes clinics(3rd level). This population was classified in "atypical"(sample A) and "classic"(sample B) according to HLA typing. We consider "classic patient" when has absence of type 1 diabetes associated HLA alleles and no difficulties in their diagnosis and treatments. By the other hand, we considered "atypical patient" when show type 1 diabetes associated HLA alleles and difficulties in their diagnosis and treatments. The standard protocol Asociacion Latinoamericana de Diabetes 2006 was used for patients follow up. To analyze differences between both populations in paraclinical parameters we used unpaired t tests and contingence tables. Bivariate and multivariate analyses were carried out using the SPSS software program. In all studies we assume differences statistically significant, with a P-value < 0.05 corrected and 95%CI.RESULTS: The typing HLA in the "atypical" populations show that 92.47% patients presented at list one type 1 diabetes associated HLA alleles(DQB1*0201-0302 and DR 3-4) and 7.53% had two of its. The results showed for categorical variables(family history, presence or absence of hypertension and/or dyslipidemia, reason for initial consultation) the only difference found was at dyslipidemia(OR = 0.45, 0.243 < OD < 0.822(P < 0.001). In relation to continuous variables we found significant differences between atypical vs classic only in cholesterol(5.07 ± 1.1 vs 5.56 ± 1.5, P < 0.05), high density lipoproteins(1.23 ± 0.3 vs 1.33 ± 0.3, P < 0.05) and low density lipoproteins(2.86 ± 0.9 vs 3.38 ± 1.7, P < 0.01). None of the variables had discriminating power when logistic regression was done.CONCLUSION: We propose an algorithm including HLA genotyping as a tool to discriminate atypical patients, complementing international treatment guidelines for complex patients.

Association of TCF7L2 mutation and atypical diabetes in a Uruguayan population

AIM To investigate if mutations in TCF7 L2 are associated with "atypical diabetes" in the Uruguayan population.METHODS Healthy, nondiabetic controls(n = 133) and patients with type 2 diabetes(n = 177) were selected from among the presenting population at level-3 referral healthcare centers in Uruguay. Patients with type 2 diabetes were subgrouped according to "atypical diabetes"(n = 92) and "classical diabetes"(n = 85). Genotyping for the rs12255372 and rs7903146 single nucleotide polymorphisms(SNPs) in the TCFTL2 gene was carried out with Taq Man? probes. Random samples were sequenced by Macrogen Ltd.(South Korea). Statistical analysis of the SNP data was carried out with the SNPStats online tool(http://bioinfo.iconcologia.net/SNPstats). The best inheritance model was chosen according to the lowest values of Akaike's information criterion and Bayesian information criterion. Differences between groups were determined by unpaired t-tests after checking the normal distribution or were converted to normalize the data. The association of SNPs was tested for matched case-control samples by using χ2 analysis and calculation of odds ratios(ORs) with 95% confidence intervals(CIs). All statistical tests were performed using SPSS v10.0 and EpiI nfo7 statistical packages. Significant statistical differences were assumed in all cases showing adjusted P < 0.05.RESULTS We genotyped two TCF7 L2 SNPs(rs7903146 and rs12255372) in a population-based sample of 310 Uruguayan subjects, including 133 healthy control subjects and 177 clinical diagnosed with type 2 diabetes. For both SNPs analyzed, the best model was the dominant type: rs12255372 = G/G vs G/T+T/T, OR = 0.63, 95%CI: 0.40-0.98, P < 0.05 and rs7903146 = C/C vs C/T+T/T, OR = 0.79, 95%CI: 0.41-1.55, P = 0.3. The rs12255372 SNP showed high association with the type 2 diabetes cases(OR = 1.60, 95%CI: 1.20-2.51, P < 0.05). However, when the type 2 diabetics group was analyzed according to the atypical and classical subgroupings, the association with diabetes existed only for rs12255372 and the classical subgroup(vs controls: OR = 2.1, 95%CI: 1.21-3.75, P < 0.05); no significant differences were found for either SNP or atypical diabetes.CONCLUSION This is the first time SNPs_TCF7 L2 were genotyped in a diabetic population stratified by genotype instead of phenotype. Classical and atypical patients showed statistical differences.

Poly(ADP-ribose),adherens junctions,vinculin and the actin cytoskeleton:Current evidence,future perspectives and implications

Poly(ADP-ribose)(PAR)is a highly negatively charged polymer.PAR is synthesized by poly(ADP-ribose)polymerases(PARPs)and is involved in the assembly and stabilization of macromolecular complexes.Here,the presence and putative roles of poly(ADP-ribosyl)ation(PARylation)associated to adherens junctions(AJ)and the actin cytoskeleton in epithelial and Schwann cells,is reviewed.The hypothesis generated by analogy,stating that PAR is associated to AJ in other cell types,is postulated.According to this hypothesis,PAR associated to puncta adherentia in chemical synapses would participate in plasticity,learning and memory.In turn,PAR associated to fascia adherens in cardiomyocytes,would affect heart beating.PARP inhibitors are currently under development and clinical testing.Basic research in different tissues will probably influence their clinical uses.

Characterizing proteases in an Antarctic Janthinobacterium sp. isolate: Evidence of a protease horizontal gene transfer event

We report the isolation of a cold-adapted bacterium belonging to the genus Janthinobacterium （named AU 11）, from a water sample collected in Lake Uruguay （King George Island, South Shetlands）. AUI 1 （growth between 4℃ and 30℃） produces a single cold-active extracellular protease （ExPAU11）, differentially expressed at low temperature. ExPAU11 was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-ToF MS） as an alkaline metallo-protease （70% coverage with an extracellular protease of Janthinobacterium sp. PI12）, and by protease-inhibitor screening identified as a serine-protease. To the best of our knowledge this is the first experimental evidence of a cold-active extracellular protease produced by Janthinobacterium. Furthermore, we identified a serine-protease gene （named JSP8A） showing 60% identity （98% query coverage） to subtilisin peptidases belonging to the $8 family （S8A subfamily） of many cyanobacteria. A phylogenetic analysis of the JSP8A protease, along with related bacterial protein sequences, confirms that JSP8A clusters with S8A subtilisin sequences from different cyanobacteria, and is clearly separated from SSA bacterial sequences of other phyla （including its own）. An analysis of the genomic organization around JSP8A suggests that this protease gene was acquired in an event that duplicated a racemase gene involved in transforming L- to D-amino acids. Our results suggest that AU11 probably acquired this subtilisin-like protease gene by horizontal gene transfer （HGT） from a cyanobacterittrn. We discuss the relevance of a bacterial protease-HGT in the Antarctic environment in light of this hypothesis.

Habitat Preference and Effects of Coastal Fragmentation in the Sand-Dwelling Spider <i>Allocosa brasiliensis</i>(Lycosidae, Allocosinae)

Allocosa brasiliensis is a sand-dwelling wolf spider considered a good bioindicator to evaluate the quality of coastal dune ecosystems from Uruguay. Habitat fragmentation and human activities have impacted and reduced the Southern Uruguayan coast during the last decades. The aim of the present study was to evaluate the density, surface activity and habitat preference of A. brasiliensis in beaches with different human impact. For that purpose, we sampled during the night with head lamps and applied the capture-mark-recapture method. Females were more abundant than males and were the most recaptured sex. The most fragmented beaches showed lower number of burrows and individuals, especially in immatures stages. We found that the most suitable sandy habitat for A. brasiliensis should present a vegetation cover optimum of 25% - 50% of the surface. This study could provide tools for the implementation of future management conservation plans of the Southern Uruguayan coastline.

Spontaneous and Bleomycin-Induced <i>&gamma;</i>H2AX Signals in CHO9 Metaphase Chromosomes

In eukaryotes, a cascade of events named DNA damage response (DDR) has evolved to handle DNA lesions. DDR engages the recruitment of signaling, checkpoint control, repair and chromatin remodeling protein complexes, allowing cell cycle delay, DNA repair or induction of apoptosis. An early DDR event involves the phosphorylation of the histone variant γH2AX on serine 139 (H2AX139 phosphorylation) originating the so-called γH2AX. DDR-related H2AX139 phosphorylation have been extensively studied in interphase nuclei. More recently, γH2AX signals on mitotic chromosomes of asynchronously growing cell cultures were observed. We performed a quantitative analysis of γH2AX signals on γH2AX immunolabeled cytocentrifuged metaphase spreads, analyzing the γH2AX signal distributions of CHO9 chromosomes harboring homologous regions both in control and bleomycin (BLM)-treated cultures. We detected γH2AX signals in CHO9 chromosomes of controls which significantly increase after BLM-exposure. γH2AX signals were uniformly distributed in chromosomes of controls. However, the γH2AX signal distribution in BLM exposed cells was significantly different between chromosomes and among chromosome regions, with few signals near the centromeres and a tendency to increase towards the telomeres. Interestingly, both basal and BLM-induced γH2AX signal distribution were statistically equal between CHO9 homologous chromosome regions. Our results suggest that BLM exerts an effect on H2AX139 phosphorylation, prevailing towards acetylated and gene-rich distal chromosome segments. The comparable H2AX139 phosphorylation of homologous regions puts forward its dependence on chromatin structure or function and its independence of the position in the karyotype.

Five-year bio-monitoring of aquatic ecosystems near Artigas Antarctic Scientific Base, King George Island

Fildes Peninsula, in King George Island, Antarctica, has a great concentration of international facilities, and it has clearly been affected by human activities. The objective of this 5-year study was to assess the impact of anthropogenic activities on the bacterial abundance in water bodies close to Artigas Antarctic Scientific Base （BCAA, in Spanish Base Cientifica Antdrtica Artigas）. Water samples from areas under different human influence （Uruguay Lake, nearby ponds, and meltwater from Collins Glacier） were aseptically collected and refrigerated until processed. The number of heterotrophic bacteria and Pseudomonas spp. was analyzed using a culture-dependent approach. Physico-chemical properties of the water samples （temperature, pH, and conductivity） were also determined. Results showed that water from the highly affected area, Uruguay Lake, where the pump that provides water to the BCAA is located, did not suffer significant fluctuations in heterotrophie bacterial abundance （10^4- 10^5 CFU.mL^-1）; however, Pseudomonas abundance increased until becoming the predominant population. In other water samples, the number of heterotrophie bacteria and Pseudomonas gradually increased during this 5-year study, by 2014 reaching similar values to those observed for Uruguay Lake. The implications of human activities on Antarctic bacterial abundance are discussed.

Detection of integron integrase genes on King George Island, Antarctica

The presence and diversity of class 1 integrase gene （intl） sequences were evaluated by PCR using previously designed primers. Two clone libraries were constructed from DNA in sediment and microbial mat samples collected on Fildes Peninsula, King George Island, Antarctica.The libraries constructed from samples collected at Halfthree Point （HP） and Norma Cove （NC） contained 62 and 36 partial intl sequences, respectively. These sequences clustered into 10 different groups with 〈 95% amino acid identity. Alignment of the deduced amino acid sequences with those from recognized integron-encoded integrases demonstrated the presence of highly conserved motifs characteristic of intl integrases. The HP library contained 42 nucleotide sequences identical to the class 1 intl gene found in a collection of trimethoprim-resistant （Tmpr） Antarctic Enterobacter sp. isolates, previously collected in the same area. These integrons, located on plasmids, had a genetic organization similar to that of pKOX105 from Klebsiella oxytoca. The 20 remaining HP and NC library sequences were similar to integrase sequences previously determined in a metagenomic analysis of environmental samples. We have demonstrated the presence of integron integrase genes in Antarctic sediment samples. About half these genes were very similar to the class 1 integrons found in human- associated microbiota, suggesting that they originated from human-dominated ecosystems. The remaining integrase genes were probably associated with endemic bacteria.

Revealing Toxin Signatures in Cyanobacteria: Report of Genes Involved in Cylindrospermopsin Synthesis from Saxitoxin-Producing <i>Cylindrospermopsis raciborskii</i>

Cyanotoxins are distinctive molecules in Cyanobacteria whose evolutionary origin, radiation and ecological role are still controversial. The cyanobacterium Cylindrospermopsis raciborskii is alternately capable of producing two types of potent toxins, cylindrospermopsin (CYN) or saxitoxin and analogues (SAX). It has been proposed that this species spread to all continents early in its evolutionary history and biogeographical differences in toxin production are found between populations. Most reports indicate that American strains are able to produce SAX but not CYN, while Australian strains are described to produce CYN but not SAX. Here we describe the presence of three genes belonging to the cylindrospermopsin cluster (cyr), cyrA, cyrB and cyrC, in two SAX producing South American C. raciborskii strains, MVCC14 and MVCC19, which due to their differences in morphology, growth preferences, SAX production and genetic context are defined as different ecotypes. No CYN production was detected in either strain (by ELISA) after growth under nitrogen replete or nitrogen-free nutrient conditions. Phylogenetic analyses of cyrA, cyrB and cyrC partial sequences from both strains showed high similarity (>99%) with CYN genes belonging to C. raciborskii strains from Australia and Germany and to Aphanizomenon strains. This is the first report of the presence of cyr genes in strains known to produce only SAX.

Chemosensitizer Effect of Violacein on Cisplatin-treated Bladder Cancer Cells

Background:Bladder cancer is the tenth most common cancer worldwide.Considering its high prevalence(vulnerability to multiple recurrences and progression despite local therapy),which leads to a substantial health service burden,it becomes necessary to develop new strategies to increase the effectiveness of bladder tumor therapy.Natural compounds with antiproliferative effect on cancer cells could be a good choice for co-adjuvant chemotherapy.Microorganisms are one of the main sources for natural compounds.Pigments extracted from the cold-adapted microorganisms can contribute to the development of a broader range of applications in biotechnology.Violacein is a purple pigment commonly produced by many bacterial strains.We have previously shown that very low concentrations of violacein extracted from Janthinobacterium sp.produced an antiproliferative effect on HeLa cells.Objective:With the aim to determine if violacein has an antiproliferative activity on bladder cancer cells,as well as to test if it has synergistic effects on cisplatin treated cells in vitro,T24 and 253J cell lines(derived bladder cancer cells from carcinoma in situ and retroperitoneal metastasis,respectively)were exposed to different concentrations of violacein in the presence or absence of cisplatin.Methods:i)Resazurin assay and flow cytometry were performed in two bladder cancer-derived cell lines,namely T24 and 253J,to see if violacein affects cell viability and induce cell death.ii)To find out whether violacein sensitizes bladder cancer cells to cisplatin,the drug interaction among different doses of cisplatin and violacein was analyzed,as well their combination index was determined.iii)The effect of violacein to induce primary genetic damage was determined through the analysis of induced micronuclei frequency and𝛾H2AX foci,as well as performing the comet assay.Results:The half-maximal inhibitory concentration of violacein at 24 h for both cell lines were around 500 nM,and decreased below 400 nM in combination with 10μM of cisplatin,indicating antiproliferative and sensitizing effects of violacein to cisplatin in both cell lines tested.A clear cell cycle delay,as well as an increase in the percentage of cell death was observed by flow cytometry at 300 nM of violacein,either alone or in combination with cisplatin.On the other hand,the analysis of the micronucleus frequency did not evidence an increase in genetic damage.Moreover,in combined treatments with cisplatin there was a slight decrease on micronucleus induction.Besides,the induction of genetic damage was not observed through comet assay when cells were treated with violacein alone,however,when cells were treated with violacein in the presence of cisplatin(10μM).The production of genetic damage was diminished in T24 or 253J cells.By the same token,increase in the frequency of𝛾H2AX foci by violacein was not observed at any tested dose in both cell lines.Conclusion:It was shown that violacein has an in vitro antiproliferative effect in bladder cancer cell lines,sensitizing them to cisplatin.Interestingly,at doses tested,violacein did not induce genotoxicity and reduce the genotoxic effect produced by cisplatin.