Mitochondrial bioenergetics links inflammation and cardiac contractility in endotoxemia.

There is current awareness about the central role of mitochondrial dysfunction in the development of cardiac dysfunction in systemic inflammatory syndromes, especially in sepsis and endotoxemia. The aim of this work was to elucidate the mechanism that governs the link between the severity of the systemic inflammatory insult and mitochondrial function, analysing the consequences on heart function, particularly in cardiac contractile state. Female Sprague-Dawley rats were subjected to low-grade endotoxemia (i.p. injection LPS 0.5 mg kg-1 body weight) and severe endotoxemia (i.p. injection LPS 8 mg kg-1 body weight) for 6 h. Blood NO, as well as cardiac TNF-α and IL-1ß mRNA, were found increased as the severity of the endotoxemia increases. Cardiac relaxation was altered only in severe endotoxemia, although contractile and lusitropic reserves were found impaired in both treatments in response to work-overload. Cardiac ultrastructure showed disorientation of myofibrillar structure in both endotoxemia degrees, but mitochondrial swelling and cristae disruption were only observed in severe endotoxemia. Mitochondrial ATP production, O2 consumption and mitochondrial inner membrane potential decreases were related to blood NO levels and mitochondrial protein nitration, leading to diminished ATP availability and impairment of contractile state. Co-treatment with the NOS inhibitor L-NAME or the administration of the NO scavenger c-PTIO leads to the observation that mitochondrial bioenergetics status depends on the degree of the inflammatory insult mainly determined by blood NO levels. Unravelling the mechanisms involved in the onset of sepsis and endotoxemia improves the interpretation of the pathology, and provides new horizons for novel therapeutic targets.

Historical records under the genetic evidence: "Chiriguano" tribe genesis as a test case.

Historical records suggest that Chiriguano tribe is the result of a genetic admixture event. The process involved the arrival of Guaraní tribesmen descending from Amazonian region of Brazil along with groups of Arawak origin that inhabited the foothill plains of Bolivia. Later they arrived in Argentina at the beginning of the twentieth century. Aiming to test the historical records, we analysed a set of 46 samples collected at San Ramon de la Nueva Orán, Province of Salta, Argentina. A wide set of uni- and biparentally transmitted genetic markers were analysed, including 23 autosomal STRs; 46 AIM-DIPs and 24 AIM-SNPs all located at diverse autosomal chromosome locations; 23 Y-STRs and the entire mtDNA D-Loop sequence. Ancestry informative markers allowed for the detection of a strong Native American component in the genomes (> 94%), while all mtDNA haplotypes showed Native American characteristic motives, and 93% of Y-haplotypes belonged to the Q1a3a Y-haplogroup. The analysis of mitochondrial haplotypes and Y chromosome, although they did not match other populations, revealed a relationship between the Chiriguano and other groups of Guaraní and Arawak origin inhabiting Brazil and Bolivia, confirming, at least in part, the historical records describing the origins of Chiriguano tribal settlements in northwestern Argentina.

Development of a quantitation approach for total human and male DNA based on real time PCR followed by high resolution melting analysis.

We developed and validated a total human DNA quantitation technique that simultaneously allows male DNA detection. This assay, called Amel-Y, is a duplex Real Time PCR followed by HRM (high resolution melting) analysis using the intercalating dye SYTO9. Amel-Y duplex produces two amplicons, one for the amelogenin gene (106/112 bp, female/male) and another (84 bp) corresponding to human Y chromosome-specific fragment to detect male DNA. After HRM analysis, two melting peaks differing in 5.3°C-5.5°C are detected if both male and female DNA are present and only one if only female DNA is present. For specificity assessment, the inclusion of high concentrations of bacterial and fungal DNA in the quantitation reactions allowed discarding species cross-reactivity. A set of crime scene evidence from forensic casework has been quantified with commercial kits and compared with Amel-Y duplex. Our method detected male DNA from a concentration of 18 pg/µL and supports autosomal/Y DNA detection ratio up to 200:1. A limitation of the technique is its inability to quantify male and female donnors in a mixed sample. The Amel-Y duplex demonstrated to be an efficient system for quantifying total human DNA being a specific, rapid, sensitive, and cost-effective method suitable for mixed DNA samples and applicable to any field where human DNA quantification is required, such as molecular diagnosis, population genetics, and forensic identification.

Detection of a novel 16.3 variant allele at locus DYS533 in R1b males inhabiting southern South America: A 19-nucleotide insertion explains its origin based on Sanger sequencing results.

We typed 1541 Y-STR haplotypes from reference samples along forensic casework investigations. In three haplotypes, we detected a variant allele designed as 16.3 at locus DYS533. This was confirmed by amplification using two commercial kits. Sanger sequencing revealing a novel motif corresponding to [TATC]12 repeats with a 19-bp insertion in the flanking upstream region. We propose its origin as an insertion at - 9.1 upstream of the repeat motifs. We searched other local databases and found this allele in various geographical areas of Argentina and neighbouring countries. The haplotypes share a common core of 10 Y-STRs (DYS389-I/13; DYS389-II/30; DYS19/14; DYS481/22; DYS438/12; DYS437/16; DYS635/23; DYS392/13; DYS393/13; GATA H4/11) and belong to the R1b haplogroup. This 16.3 allele is restricted to southern South America, which allows us to propose a local and relatively recent origin. The sequence described herein constitutes a novelty that could be considered in future criteria for the nomenclature of STRs based on massively parallel sequencing.

Using long ssDNA polynucleotides to amplify STRs loci in degraded DNA samples.

Obtaining informative short tandem repeat (STR) profiles from degraded DNA samples is a challenging task usually undermined by locus or allele dropouts and peak-high imbalances observed in capillary electrophoresis (CE) electropherograms, especially for those markers with large amplicon sizes. We hereby show that the current STR assays may be greatly improved for the detection of genetic markers in degraded DNA samples by using long single stranded DNA polynucleotides (ssDNA polynucleotides) as surrogates for PCR primers. These long primers allow a closer annealing to the repeat sequences, thereby reducing the length of the template required for the amplification in fragmented DNA samples, while at the same time rendering amplicons of larger sizes suitable for multiplex assays. We also demonstrate that the annealing of long ssDNA polynucleotides does not need to be fully complementary in the 5' region of the primers, thus allowing for the design of practically any long primer sequence for developing new multiplex assays. Furthermore, genotyping of intact DNA samples could also benefit from utilizing long primers since their close annealing to the target STR sequences may overcome wrong profiling generated by insertions/deletions present between the STR region and the annealing site of the primers. Additionally, long ssDNA polynucleotides might be utilized in multiplex PCR assays for other types of degraded or fragmented DNA, e.g. circulating, cell-free DNA (ccfDNA).