Ancient DNA studies: new perspectives on old samples

In spite of past controversies, the field of ancient DNA is now a reliable research area due to recent methodological improvements. A series of recent large-scale studies have revealed the true potential of ancient DNA samples to study the processes of evolution and to test models and assumptions commonly used to reconstruct patterns of evolution and to analyze population genetics and palaeoecological changes. Recent advances in DNA technologies, such as next-generation sequencing make it possible to recover DNA information from archaeological and paleontological remains allowing us to go back in time and study the genetic relationships between extinct organisms and their contemporary relatives. With the next-generation sequencing methodologies, DNA sequences can be retrieved even from samples (for example human remains) for which the technical pitfalls of classical methodologies required stringent criteria to guaranty the reliability of the results. In this paper, we review the methodologies applied to ancient DNA analysis and the perspectives that next-generation sequencing applications provide in this field

Statistical analysis of post mortem DNA damage-derived miscoding lesions in Neandertal mitochondrial DNA

Background. We have analysed the distribution of post mortem DNA damage derived miscoding lesions from the datasets of seven published Neandertal specimens that have extensive cloned sequence coverage over the mitochondrial DNA (mtDNA) hypervariable region 1 (HVS1). The analysis was restricted to C → T and G → A miscoding lesions (the predominant manifestation of post mortem damage) that are seen at a frequency of more than one clone among sequences from a single PCR, but do not represent the true endogenous sequence. Findings. The data indicates an extreme bias towards C → T over G → A miscoding lesions (observed ratio of 67:2 compared to an expected ratio of 7:2), implying that the mtDNA Light strand molecule suffers proportionally more damage-derived miscoding lesions than the Heavy strand. Conclusion. The clustering of Cs in the Light strand as opposed to the singleton pattern of Cs in the Heavy strand could explain the observed bias, a phenomenon that could be further tested with non-PCR based approaches. The characterization of the HVS1 hotspots will be of use to future Neandertal mtDNA studies, with specific regards to assessing the authenticity of new positions previously unknown to be polymorphic

Genetic evidence for patrilocal mating behaviour among Neandertal groups

The remains of 12 Neandertal individuals have been found at the El Sidrón site (Asturias, Spain), consisting of six adults, three adolescents, two juveniles, and one infant. Archaeological, paleontological, and geological evidence indicates that these individuals represent all or part of a contemporaneous social group of Neandertals, who died at around the same time and later were buried together as a result of a collapse of an underground karst. We sequenced phylogenetically informative positions of mtDNA hypervariable regions 1 and 2 from each of the remains. Our results show that the 12 individuals stem from three different maternal lineages, accounting for seven, four, and one individual(s), respectively. Using a Y-chromosome assay to confirm the morphological determination of sex for each individual, we found that, although the three adult males carried the same mtDNA lineage, each of the three adult females carried different mtDNA lineages. These findings provide evidence to indicate that Neandertal groups not only were small and characterized by low genetic diversity but also were likely to have practiced patrilocal mating behavior.C.L.-F. also is supported by an Intramural Grant from the Consejo Superior de Investigaciones Cientificas. A.R., A.E., M.B., A.G.-T., and S.G.-V. are supported by Grant CGL2009-09013 from the Ministry of Science and Innovation of Spain.Peer reviewe

Rifaximin improves Clostridium difficile toxin A-induced toxicity in Caco-2 cells by the PXR-dependent TLR4/MyD88 /NF-?B pathway

Background: Clostridium difficile infections (CDIs) caused by Clostridium difficile toxin A (TcdA) lead to severe ulceration, inflammation and bleeding of the colon, and are difficult to treat. Aim: The study aimed to evaluate the effect of rifaximin on TcdA-induced apoptosis in intestinal epithelial cells and investigate the role of PXR in its mechanism of action. Methods: Caco‐2 cells were incubated with TcdA and treated with rifaximin (0.1−10 μM) with or without ketoconazole (10 μM). The transepithelial electrical resistance (TEER) and viability of the treated cells was determined. Also, the expression of zona occludens‐1 (ZO‐1), toll‐like receptor 4 (TLR4), Bcl‐2‐associated X protein (Bax), transforming growth factor‐β‐activated kinase‐1 (TAK1), myeloid differentiation factor 88 (MyD88) and nuclear factor‐kappaB (NF‐κB) was determined. Results Rifaximin treatment (0.1, 1.0 and 10 μM) caused a significant and concentration-dependent increase in the TEER of Caco-2 cells (360%, 480% and 680% vs TcdA treatment) 24 hours after the treatment and improved their viability (61%, 79% and 105%). Treatment also concentration-dependently decreased the expression of Bax protein (–29%, –65% and –77%) and increased the expression of ZO-1 (25%, 54% and 87%) and occludin (71%, 114% and 262%) versus TcdA treatment. The expression of TLR4 (–33%, –50% and –75%), MyD88 (–29%, –60% and –81%) and TAK1 (–37%, –63% and –79%) were also reduced with rifaximin versus TcdA treatment. Ketoconazole treatment inhibited these effects. Conclusions: Rifaximin improved TcdA‐induced toxicity in Caco‐2 cells by the PXR‐dependent TLR4/MyD88/NF‐κB pathway mechanism, and may be useful in the treatment of CDIs

Tracing the origin of the East-West population admixture in the Altai region (Central Asia)

A recent discovery of Iron Age burials (Pazyryk culture) in the Altai Mountains of Mongolia may shed light on the mode and tempo of the generation of the current genetic east-west population admixture in Central Asia. Studies on ancient mitochondrial DNA of this region suggest that the Altai Mountains played the role of a geographical barrier between West and East Eurasian lineages until the beginning of the Iron Age. After the 7th century BC, coinciding with Scythian expansion across the Eurasian steppes, a gradual influx of East Eurasian sequences in Western steppes is detected. However, the underlying events behind the genetic admixture in Altai during the Iron Age are still unresolved: 1) whether it was a result of migratory events (eastward firstly, westward secondly), or 2) whether it was a result of a local demographic expansion in a 'contact zone' between European and East Asian people. In the present work, we analyzed the mitochondrial DNA lineages in human remains from Bronze and Iron Age burials of Mongolian Altai. Here we present support to the hypothesis that the gene pool of Iron Age inhabitants of Mongolian Altai was similar to that of western Iron Age Altaians (Russia and Kazakhstan). Thus, this people not only shared the same culture (Pazyryk), but also shared the same genetic east-west population admixture. In turn, Pazyryks appear to have a similar gene pool that current Altaians. Our results further show that Iron Age Altaians displayed mitochondrial lineages already present around Altai region before the Iron Age. This would provide support for a demographic expansion of local people of Altai instead of westward or eastward migratory events, as the demographic event behind the high population genetic admixture and diversity in Central Asia

Rifaximin, a non-absorbable antibiotic, inhibits the release of pro-angiogenic mediators in colon cancer cells through a pregnane X receptor-dependent pathway

Activation of intestinal human pregnane X receptor (PXR) has recently been proposed as a promising strategy for the chemoprevention of inflammation-induced colon cancer. The present study was aimed at evaluating the effect of rifaximin, a non-absorbable antibiotic, in inhibiting angiogenesis in a model of human colorectal epithelium and investigating the role of PXR in its mechanism of action. Caco-2 cells were treated with rifaximin (0.1, 1.0 and 10.0 μM) in the presence or absence of ketoconazole (10 μM) and assessed for cell proliferation, migration and expression of proliferating cell nuclear antigen (PCNA). The release of vascular endothelial growth factor (VEGF) and nitric oxide (NO), expression of Akt, mechanistic target of rapamycin (mTOR), p38 mitogen activated protein kinases (MAPK), nuclear factor κB (NF-κB) and metalloproteinase-2 and -9 (MMP-2 and -9) were also evaluated. Treatment with rifaximin 0.1, 1.0 and 10.0 μM caused significant and concentration-dependent reduction of cell proliferation (-25, -40 and -68%), cell migration (-18, -30 and -46%) and PCNA expression (-29, -53 and -76%) in the Caco-2 cells vs. untreated cells. Treatment downregulated VEGF secretion (-32, -45 and -72%), NO release (-40, -69 and -87%), VEGFR-2 expression (-33, -58 and -65%) and MMP-2 (-25, -62 and -87%) and MMP-9 expression (-38, -56 and -78%) vs. untreated cells. Rifaximin treatment also resulted in a concentration-dependent decrease in the phosphorylation of Akt (-50, -75 and -86%), mTOR (-38, -56 and -78%), p38MAPK (-24, -62 and -71%) and inhibition of HIF-1α (-65, -82 and -92%), p70S6K (-27, -55 and -85%) and NF-κB (-29, -55 and -61%). Ketoconazole (PXR antagonist) treatment inhibited these effects. These findings demonstrated that rifaximin causes PXR-mediated inhibition of angiogenic factors in Caco-2 cell line and may be a promising anticancer tool

HIV-1 Tat-induced diarrhea evokes an enteric glia-dependent neuroinflammatory response in the central nervous system

Despite the effectiveness of combined anti-retroviral therapy, human immunodeficiency virus (HIV) infected-patients frequently report diarrhea and neuropsychological deficits. It is claimed that the viral HIV-1 Trans activating factor (HIV-1 Tat) protein is responsible for both diarrhea and neurotoxic effects, but the underlying mechanisms are not known. We hypothesize that colonic application of HIV-1 Tat activates glial cells of the enteric nervous system (EGCs), leading to a neuroinflammatory response able to propagate to the central nervous system. We demonstrated that HIV-1 Tat-induced diarrhea was associated with a significant activation of glial cells within the colonic wall, the spinal cord and the frontal cortex, and caused a consistent impairment of the cognitive performances. The inhibition of glial cells activity by lidocaine, completely abolished the above-described effects. These observations point out the role of glial cells as putative effectors in HIV-1 Tat-associated gastrointestinal and neurological manifestations and key regulators of gut-brain signaling