Spatial and temporal heterogeneity in human mobility patterns in Holocene Southwest Asia and the East Mediterranean.

We present a spatiotemporal picture of human genetic diversity in Anatolia, Iran, Levant, South Caucasus, and the Aegean, a broad region that experienced the earliest Neolithic transition and the emergence of complex hierarchical societies. Combining 35 new ancient shotgun genomes with 382 ancient and 23 present-day published genomes, we found that genetic diversity within each region steadily increased through the Holocene. We further observed that the inferred sources of gene flow shifted in time. In the first half of the Holocene, Southwest Asian and the East Mediterranean populations homogenized among themselves. Starting with the Bronze Age, however, regional populations diverged from each other, most likely driven by gene flow from external sources, which we term "the expanding mobility model." Interestingly, this increase in inter-regional divergence can be captured by outgroup-f3-based genetic distances, but not by the commonly used FST statistic, due to the sensitivity of FST, but not outgroup-f3, to within-population diversity. Finally, we report a temporal trend of increasing male bias in admixture events through the Holocene.

Testing a series of modifications on genomic library preparation methods for ancient or degraded DNA.

Technological advancements have revolutionized ancient and degraded DNA analysis, moving the field to the Next Generation Sequencing era. One of the advancements, the ancient DNA-oriented high-throughput library preparation methods, enabled the sequencing of more endogenous molecules. Although fairly optimized, both single- and double-stranded library preparation methods hold the potential for further improvement. Here, we test a series of modifications made at different steps of both single- and double-stranded library preparation methods. Given all the modifications tested, we found that two of them provide further benefits, including the use of Endonuclease VIII as a pre-treatment step before preparing single-stranded libraries and the use of a modified second adapter of the single stranded-libraries as an alternative option to enable sequencing of single stranded-libraries with the standard Illumina sequencing primer instead of the custom designed as described in the single stranded library preparation method. Furthermore, we propose uracil-DNA-glycosylase (UDG) could also be considered for both single- and double-stranded library preparation methods, although additional parameters should be taken into account depending on the sequencing strategy and the sample characteristics. Further modifications were also tested and although they were not advantageous, they could be considered as equivalent to the published options.

Identification and characterization of a rare variant in apolipoprotein A-IV, p.(V336M), and evaluation of HDL functionality in a Greek cohort with extreme HDL cholesterol levels.

High-Density Lipoprotein cholesterol (HDL-C) levels do not correlate well with Coronary Artery Disease (CAD) risk, while HDL functionality affects atherogenesis and is a better prognostic marker for CAD. Often, the extreme HDL-C levels have a multigenic origin. Here, we searched for single-nucleotide polymorphisms (SNPs) in ten genes of HDL metabolism in a Greek cohort with very low (<10th percentile, n = 13) or very high (>90th percentile, n = 21) HDL-C. We also evaluated the association between HDL-C levels, HDL functionality (anti-oxidant capacity) and CAD in the subjects of this cohort. Individuals with low HDL-C levels had higher triglyceride levels, lower apoA-I levels, decreased HDL anti-oxidant capacity and higher incidence of CAD compared with individuals with control or high HDL-C levels. With next generation sequencing we identified 18 exonic SNPs in 6 genes of HDL metabolism and for selected amino acid changes we performed computer-aided structural analysis and modeling. A previously uncharacterized rare apolipoprotein A-IV variant, apoA-IV [V336M], present in a subject with low HDL-C (14 mg/dL) and CAD, was expressed in recombinant form and structurally and functionally characterized. ApoA-IV [V336M] had similar α-helical content to WT apoA-IV but displayed a small thermodynamic stabilization by chemical unfolding analysis. ApoA-IV [V336M] was able to associate with phospholipids but presented reduced kinetics compared to WT apoA-IV. Overall, we identified a rare apoA-IV variant in a subject with low HDL levels and CAD with altered biophysical and phospholipid binding properties and showed that subjects with very low HDL-C presented with HDL dysfunction and higher incidence of CAD in a Greek cohort.

Molecular identification and geographic origin of a post-Medieval elephant finding from southwestern Portugal using high-throughput sequencing.

Molecular species identification plays a crucial role in archaeology and palaeontology, especially when diagnostic morphological characters are unavailable. Molecular markers have been used in forensic science to trace the geographic origin of wildlife products, such as ivory. So far, only a few studies have applied genetic methods to both identify the species and circumscribe the provenance of historic wildlife trade material. Here, by combining ancient DNA methods and genome skimming on a historical elephantid tooth found in southwestern Portugal, we aimed to identify its species, infer its placement in the elephantid phylogenetic tree, and triangulate its geographic origin. According to our results the specimen dates back to the eighteenth century CE and belongs to a female African forest elephant (non-hybrid Loxodonta cyclotis individual) geographically originated from west-west-central Africa, from areas where one of the four major mitochondrial clades of L. cyclotis is distributed. Historical evidence supports our inference, pointing out that the tooth should be considered as post-Medieval raw ivory trade material between West Africa and Portugal. Our study provides a comprehensive approach to study historical products and artefacts using archaeogenetics and contributes towards enlightening cultural and biological historical aspects of ivory trade in western Europe.

Transcriptome analysis of the adipose tissue in a mouse model of metabolic syndrome identifies gene signatures related to disease pathogenesis.

The white adipose tissue (WAT) contributes to the metabolic imbalance observed in obesity and the metabolic syndrome (MetS) by mechanisms that are poorly understood. The aim of this study was to monitor changes in the transcriptome of epididymal WAT during the development of MetS. ApoE3L.CETP mice were fed a high fat (HFD) or a low-fat (LFD) diet for different time periods. Adipose RNA was analyzed by microarrays. We found an increasing number of differentially expressed transcripts during MetS development. In mice with MetS, 1396 transcripts were differentially expressed including transcripts related to immune/inflammatory responses and extracellular matrix enzymes, suggesting significant inflammation and tissue remodeling. The top list of pathways included focal adhesion, chemokine, B and T cell receptor and MAPK signaling. The data identify for the first time adipose gene signatures in apoE3L.CETP mice with diet-induced MetS and might open new avenues for investigation of potential biomarkers or therapeutic targets.

Significant changes in hepatic transcriptome and circulating miRNAs are associated with diet-induced metabolic syndrome in apoE3L.CETP mice.

Long-term exposure to excess dietary fat leads to obesity and the metabolic syndrome (MetS). The purpose of the present study was to identify global changes in liver gene expression and circulating miRNAs in a humanized mouse model of diet-induced MetS. Male apoE3L.CETP mice received a high-fat diet (HFD) or a low-fat diet (LFD) for different time periods and the progression of MetS pathology was monitored. A separate group of mice was divided into responders (R) or nonresponders (NR) and received HFD for 16 weeks. We found that mice receiving the HFD developed manifestations of MetS and displayed an increasing number of differentially expressed transcripts at 4, 8, and 12 weeks compared with mice receiving the LFD. Significantly changed genes were functionally annotated to metabolic diseases and pathway analysis revealed the downregulation of genes in cholesterol and fatty acid biosynthesis and upregulation of genes related to lipid droplet formation, which was in line with the development of hepatic steatosis. In the serum of the apoE3L.CETP mice we identified three miRNAs that were upregulated specifically in the HFD group. We found that responder mice have a distinct gene signature that differentiates them from nonresponders. Comparison of the two diet intervention studies revealed a limited number of common differentially expressed genes but the expression of these common genes was affected in a similar way in both studies. In conclusion, the characteristic hepatic gene signatures and serum miRNAs identified in the present study provide novel insights to MetS pathology and could be exploited for diagnostic or therapeutic purposes.

A Novel Hantavirus of the European Mole, Bruges Virus, Is Involved in Frequent Nova Virus Coinfections.

Hantaviruses are zoonotic viruses with a complex evolutionary history of virus-host coevolution and cross-species transmission. Although hantaviruses have a broad reservoir host range, virus-host relationships were previously thought to be strict, with a single virus species infecting a single host species. Here, we describe Bruges virus, a novel hantavirus harbored by the European mole (Talpa europaea), which is the well-known host of Nova virus. Phylogenetic analyses of all three genomic segments showed tree topology inconsistencies, suggesting that Bruges virus has emerged from cross-species transmission and ancient reassortment events. A high number of coinfections with Bruges and Nova viruses was detected, but no evidence was found for reassortment between these two hantaviruses. These findings highlight the complexity of hantavirus evolution and the importance of further investigation of hantavirus-reservoir relationships.

MinePath: Mining for Phenotype Differential Sub-paths in Molecular Pathways.

Pathway analysis methodologies couple traditional gene expression analysis with knowledge encoded in established molecular pathway networks, offering a promising approach towards the biological interpretation of phenotype differentiating genes. Early pathway analysis methodologies, named as gene set analysis (GSA), view pathways just as plain lists of genes without taking into account either the underlying pathway network topology or the involved gene regulatory relations. These approaches, even if they achieve computational efficiency and simplicity, consider pathways that involve the same genes as equivalent in terms of their gene enrichment characteristics. Most recent pathway analysis approaches take into account the underlying gene regulatory relations by examining their consistency with gene expression profiles and computing a score for each profile. Even with this approach, assessing and scoring single-relations limits the ability to reveal key gene regulation mechanisms hidden in longer pathway sub-paths. We introduce MinePath, a pathway analysis methodology that addresses and overcomes the aforementioned problems. MinePath facilitates the decomposition of pathways into their constituent sub-paths. Decomposition leads to the transformation of single-relations to complex regulation sub-paths. Regulation sub-paths are then matched with gene expression sample profiles in order to evaluate their functional status and to assess phenotype differential power. Assessment of differential power supports the identification of the most discriminant profiles. In addition, MinePath assess the significance of the pathways as a whole, ranking them by their p-values. Comparison results with state-of-the-art pathway analysis systems are indicative for the soundness and reliability of the MinePath approach. In contrast with many pathway analysis tools, MinePath is a web-based system (www.minepath.org) offering dynamic and rich pathway visualization functionality, with the unique characteristic to color regulatory relations between genes and reveal their phenotype inclination. This unique characteristic makes MinePath a valuable tool for in silico molecular biology experimentation as it serves the biomedical researchers' exploratory needs to reveal and interpret the regulatory mechanisms that underlie and putatively govern the expression of target phenotypes.

Dysregulated production of interleukin-1ß upon activation of the NLRP3 inflammasome in patients with familial Mediterranean fever.

Familial Mediterranean fever (FMF) is caused by mutations in pyrin, a protein expressed in innate immune cells that interacts with caspase-1 and other inflammasome components to regulate interleukin (IL)-1ß maturation. Since NLRP3 inflammasome represents major source of IL-1ß, we studied its protein expression and function in FMF. We isolated peripheral white blood cells (WBCs) from 20 symptoms-free FMF patients and 21 healthy individuals. Intracellular protein expression of NLRP3, caspase-1, IL-1ß at baseline and after LPS/ATP sequential treatment for NLRP3 activation was assessed by immunoblotting. Secreted IL-1ß was quantified by ELISA. THP-1 cells were transfected with wild-type or mutant pyrin and IL-1ß secretion was measured. FMF WBCs exhibited lower NLRP3 and active caspase-1 protein expression compared to healthy individuals, and LPS/ATP treatment resulted in significantly lower intracellular IL-1ß levels in FMF patients. Likewise, LPS/ATP induced caspase-1-dependent IL-1ß release at significantly lower amounts in the FMF group (1182±192 versus 2134±245pg/mL in controls, p=0.004). Consistently, THP-1 cells transfected with FMF-associated M694V mutant pyrin displayed lower LPS/ATP-induced IL-1ß compared with wild-type pyrin-transfected cells. FMF WBCs demonstrate reduced NLRP3-mediated IL-1ß production. Additional studies are needed to define whether this finding represents a compensatory mechanism to control inflammation or is directly linked to disease pathogenesis.

On the identification of circulating tumor cells in breast cancer.

Breast cancer is a highly heterogeneous disease and very common among western women. The main cause of death is not the primary tumor but its metastases at distant sites, such as lymph nodes and other organs (preferentially lung, liver, and bones). The study of circulating tumor cells (CTCs) in peripheral blood resulting from tumor cell invasion and intravascular filtration highlights their crucial role concerning tumor aggressiveness and metastasis. Genomic research regarding CTCs monitoring for breast cancer is limited due to the lack of indicative genes for their detection and isolation. Instead of direct CTC detection, in our study, we focus on the identification of factors in peripheral blood that can indirectly reveal the presence of such cells. Using selected publicly available breast cancer and peripheral blood microarray datasets, we follow a two-step elimination procedure for the identification of several discriminant factors. Our procedure facilitates the identification of major genes involved in breast cancer pathology, which are also indicative of CTCs presence.

Opioids increase bladder cancer cell migration via bradykinin B2 receptors.

Data relating opioid treatment and modification of cancer cell migration (a prerequisite of metastasis) both in vitro and in vivo are diverging. In the present report we show that opioids increase the migratory activity of bladder cancer cells (T24 and EJ) and we provide a new mechanistic insight, explaining (at least partially) their action: we report that the enhanced opioid-related cell migration is controlled (in the absence of opioid receptors) through their interaction with bradykinin B2 receptors. Indeed, in these cell lines, opioids increase migration, adhesion, spreading and invasion by re-arranging actin cytoskeleton, increasing MMP-2 and -9 secretion and triggering specific intracellular signaling cascades in a non-opioid receptor mediated manner. An interaction, albeit with low affinity, of opioids with the bradykinin B2 receptor is reported, resulting in the increase of migration, while B2 antagonists revert this action. A systematic assay of different human epithelial cancer cell lines confirmed that only the B2-positive/opioid receptor-negative bladder cancer cells present this opioid-related increased migration/invasive phenotype. We suggest that opioid administration in cancer patients should be re-evaluated, keeping in mind that they may have other beneficial (protection) or adverse effects (spreading of cancer cells), in spite of their unique role in pain relief.

The role of the pro-apoptotic protein Siva in the pathogenesis of Familial Mediterranean fever: A structural and functional analysis.

Familial Mediterranean fever (FMF) is an autosomal, recessive disease, attributed to mutations in MEFV gene encoding pyrin, which is characterized by recurrent, acute and self-limiting attacks of fever as well as an increased neutrophil and monocyte apoptosis. Most disease-associated mutations in MEFV gene reside on the C-terminal PRYSPRY (B30.2) domain of pyrin, an area found to interact with the pro-apoptotic protein Siva. Because apoptotic events may be contributing to endogenous inflammation we hypothesized that mutations in pyrin may affect Siva-mediated apoptosis. The confirmation of this hypothesis would be of a great biological significance since it would be demonstrated a connection between apoptosis and inflammation. We used homology modeling to construct a 3-D model of Siva protein and the constructed model of Siva defined structural elements with potential of binding other proteins to induce apoptosis. Given that Siva protein binds pyrin as shown by transfection and immunoprecipitation experiments, apoptosis was assessed by FACS and Western blotting. No differences in rates of apoptosis in myeloid cells (THP-1) upon transfection with either wt pyrin or mutant forms of pyrin were found. Patients with FMF did not display any mutations in the Siva-1 (full length) gene. Siva-1 was not linked to pyrin in the major predicted FMF gene network constructed using a literature-curated gene signature for FMF. These results suggest that Siva-mediated unprovoked apoptosis is not likely to be involved in the pathogenesis of FMF.

Opioids modulate constitutive B-lymphocyte secretion.

The opioid system plays a major role in immunomodulation, while its action on cells of the immune system may be opioid receptor-mediated or not. Opioid effects on B-lymphocytes are considered as indirect, attributed to an interplay between distinct cell populations. The aim of the present study was to investigate whether opioid agonists (morphine, alpha(S1)-casomorphin and ethylketocyclazocine) may have a direct action on the secretion of antibodies and cytokines by multiple myeloma-derived cell lines and normal CD19+ B-lymphocytes. Our results show that opioids modulate antibody and cytokine secretion by multiple myeloma cells in a cell line-dependent and opioid receptor-independent manner, while they decrease antibody secretion by normal B-lymphocytes. Furthermore, they decrease the proliferation rate of multiple myeloma cells through opioid receptor activation. Our data suggest two different mechanisms of action of opioids, mediated by different signaling pathways: an early non-opioid receptor-related effect, modulating the constitutive immunoglobulin and cytokine secretion, and a long-term receptor-mediated action on cell growth. These data suggest a further opioid implication in the control of humoral immunity.