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Systemic sclerosis (SSc) can lead to pathological changes in the maxillofacial region, contributing to the violation of the microbiocenosis of the oral cavity with a predominance of pathogenic microflora.Objective: to study the composition of the oral microflora in patients with SSc. Patients and methods. The composition of the oral microflora was studied in 50 patients with SSc. The control group consisted of 50 subjects without rheumatic diseases. To assess the intensity of dental caries and the level of oral hygiene we used dental indices: the index of caries intensity (Decayed, Missing, and Filled Teeth (DMFT) and the hygienic index (OHI-S).Results and discussion. Microbiological examination in patients with SSc revealed pathogenic Staphylococcus aureus and Candida albicans > 10-6 CFU in equal percentage of cases (18.9%), which was significantly more frequent than in the control group (p=0.049). In the oral cavity in SSc, there were no representatives of normal microflora (lactobacilli). In patients with SSc, the DMFT index was 17.8±7.1 on average, and OHI-S – 2.3±0.7, which corresponds to a very high level of caries intensity and low indicators of oral hygiene, respectively. When analyzing the microflora of the oral cavity in 90% of cases, a dysbiotic shift of the 3rd degree was stated.Conclusion. It can hypothesized that the qualitative and quantitative composition of the microflora of the oral cavity affects the development and severity of inflammatory and destructive pathology of the periodontal and oral mucosa. It is necessary to develop and implement an adapted personal hygiene regimen, including cleansing of the tongue and administration of local probiotics, which, as part of complex therapy, can improve the results of SSc treatment.

Spiroacetals such as E-7-methyl-1,6-dioxaspiro[4,5]decane (trans-conophthorin; tC) and acetamides [predominantly N-(3-methylbutyl)acetamide; N3MBA], are two major groups of volatiles discovered in venoms of many Vespidae. In the course of testing the attractiveness of tC and N3MBA to Vespidae using Rescue® Wasp TrapStiks, a significant number of female milichiids, Desmometopa nearctica Sabrosky and D. sordida (Fallen) (Diptera: Milichiidae) were trapped as well. However, the attraction of vespid wasps was not significant at the dosages tested. We found a significant synergistic effect of tC and N3MBA in attracting Desmometopa flies. Both D. nearctica and D. sordida are kleptoparasitic species; and we conclude that females of these two milichiid flies use tC and N3MBA (and likely other volatiles) released from venom glands of the social vespids (yellowjackets, paper wasps and hornets) as kairomones to locate disturbed, injured, or freshly killed insects (vespids and/or their prey) as a protein-rich food source for egg development and production.

Mean sensitivity (MS) derived from a standard test grid using microperimetry is a sensitive outcome measure in clinical trials investigating new treatments for degenerative retinal diseases. This study hypothesizes that the functional decline is faster at the edge of the dense scotoma (eMS) than by using the overall MS.Multicenter, international, prospective cohort study: ProgStar Study.Stargardt disease type 1 patients (carrying at least 1 mutation in the ABCA4 gene) were followed over 12 months using microperimetry with a Humphrey 10-2 test grid. Customized software was developed to automatically define and selectively follow the test points directly adjacent to the dense scotoma points and to calculate their mean sensitivity (eMS).Among 361 eyes (185 patients), the mean age was 32.9 ± 15.1 years old. At baseline, MS was 10.4 ± 5.2 dB (n = 361), and the eMS was 9.3 ± 3.3 dB (n = 335). The yearly progression rate of MS (1.5 ± 2.1 dB/year) was significantly lower (β = -1.33; P.001) than that for eMS (2.9 ± 2.9 dB/year). There were no differences between progression rates using automated grading and those using manual grading (β = .09; P = .461).In Stargardt disease type 1, macular sensitivity declines significantly faster at the edge of the dense scotoma than in the overall test grid. An automated, time-efficient approach for extracting and grading eMS is possible and appears valid. Thus, eMS offers a valuable tool and sensitive outcome parameter with which to follow Stargardt patients in clinical trials, allowing clinical trial designs with shorter duration and/or smaller cohorts.

In recent years, scientists have increasingly focused on the microbial diversity of high-altitude hot springs to explore the biotechnological applications of extremophiles. In this regard, a total of 107 thermophilic bacilli were isolated from 9 high-altitude mineralized geothermal springs (of temperatures ranging from 27.5 to 70 °C) located within the territory of Armenia and Nagorno-Karabakh. The isolated bacilli were phylogenetically profiled and studied for their potential to produce extracellular hydrolytic enzymes (protease, amylase, and lipase). The identification of isolates based on 16S rRNA gene sequences revealed their relationship to members of more than 22 distinct species, of 8 different genera, namely Aeribacillus, Anoxybacillus, Bacillus, Brevibacillus, Geobacillus, Parageobacillus, Paenibacillus and Ureibacillus. Bacillus licheniformis, Parageobacillus toebii and Anoxybacillus flavithermus were found to be the most abundant species in the springs that were studied. Some of the isolated bacilli shared less than 91-97% sequence identity with their closest match in GenBank, indicating that Armenian geothermal springs harbor novel bacilli, at least at the species level. 71% of the isolates actively produced at least one or more extracellular proteases, amylases, or lipases. In total, 22 strains (28.6%) were efficient producers of all three types of thermostable enzymes.

Twelve thermophilic Anoxybacillus strains were isolated from sediment and water samples from a Karvachar hot spring located in the northern part of Nagorno-Karabakh. Based on phenotypic, chemotaxonomic and phylogenetic characteristics, one of the isolates, designated strain K1T, was studied in detail. The cells are straight, motile rods that are 0.2–0.4×2.3–7.2 µm in size. The strain is a Gram-stain-positive, moderately thermophilic facultative anaerobe with an optimum growth temperature of 60–65 °C and a growth temperature range of 45–70 °C. Growth of strain K1T was observed at pH 6–11 (optimum, pH 8–9) and was inhibited in the presence of NaCl concentrations above 2.5 % (optimum, 1–1.5 %). The isolate could utilize a wide variety of carbon sources, including d-arabinose, d-ribose, d-galactose, d-fructose, d-mannitol, maltose, aesculin, melibiose, sucrose, trehalose, raffinose, amidone, glycogen, turanose, d-lyxose, d-tagatose, potassium gluconate and 2-keto-gluconate. The strain was able to hydrolyse starch, casein and gelatin, was positive for oxidase and catalase, and reduced nitrate to nitrite, but was negative for H2S production. Production of urease and indole was not observed. The major cellular fatty acids were C15 : 0 iso, C16 : 0 and C17 : 0 iso (52.5, 13.6 and 19.6 % of total fatty acids, respectively). Strain K1T shares >99 % 16S rRNA sequence similarity and a genomic average nucleotide identity value of 94.5 % with its closest relative, Anoxybacillus flavithermus DSM 2641T, suggesting that it represents a separate and novel species, for which the name Anoxybacillus karvacharensis sp. nov. is proposed. The type strain of Anoxybacillus karvacharensis is K1T (=DSM 106524T=KCTC 15807T).

BackgroundRecently, there has been a push towards the extended barcode concept of utilising chloroplast genomes (cpGenome) and nuclear ribosomal DNA (nrDNA) sequences for molecular identification of plants instead of the standard barcode regions. These extended barcodes has a wide range of applications, including biodiversity monitoring and assessment, primer design, and evolutionary studies. However, these extended barcodes are not well represented in global reference databases. To fill this gap, we generated cpGenomes and nrDNA reference data from genome skims of 184 plant species collected in Denmark. We further explored the application of our generated reference data for molecular identifications of plants in an environmental DNA metagenomics study.ResultsWe assembled partial cpGenomes for 82.1% of sequenced species and full or partial nrDNA sequences for 83.7% of species. We added all assemblies to GenBank, of which chloroplast reference data from 101 species and nuclear reference data from 6 species were not previously represented. On average, we recovered 45 genes per species. The rate of recovery of standard barcodes was higher for nuclear barcodes (>89%) than chloroplast barcodes (< 60%). Extracted DNA yield did not affect assembly outcome, whereas high GC content did so negatively. For thein silicosimulation of metagenomic reads, taxonomic assignments using the reference data generated had better species resolution (94.9%) as compared to GenBank (18.1%) without any identification errors.ConclusionsGenome skimming generates reference data of both standard barcodes and other loci, contributing to the global DNA reference database for plants.

Metastatic breast cancer causes most breast cancer-associated deaths, especially in triple negative breast cancers (TNBC). The metastatic drivers of TNBCs are still poorly understood, and effective treatment non-existent. Here we reveal that the presence of Aurora-A Kinase (AURKA) in the nucleus and metastatic dissemination are molecularly connected through HIF1 (Hypoxia-Inducible Factor-1) signaling. Nuclear AURKA activates transcription of "hypoxia-induced genes" under normoxic conditions (pseudohypoxia) and without upregulation of oxygen-sensitive HIF1A subunit. We uncover that AURKA preferentially binds to HIF1B and co-localizes with the HIF complex on DNA. The mass-spectrometry analysis of the AURKA complex further confirmed the presence of CBP and p300 along with other TFIIB/RNApol II components. Importantly, the expression of multiple HIF-dependent genes induced by nuclear AURKA (N-AURKA), including migration/invasion, survival/death, and stemness, promote early cancer dissemination. These results indicate that nuclear, but not cytoplasmic, AURKA is a novel driver of early metastasis. Analysis of clinical tumor specimens revealed a correlation between N-AURKA presence and decreased patient survival. Our results establish a mechanistic link between two critical pathways in cancer metastasis, identifying nuclear AURKA as a crucial upstream regulator of the HIF1 transcription complex and a target for anti-metastatic therapy.

The origins of viral pathogens and the age of their association with humans remains largely elusive. To date, there is no direct evidence about the diversity of viral infections in early modern humans pre-dating the Holocene. We recovered two near-complete genomes (5.2X and 0.7X) of human adenovirus C (HAdV-C), as well as low-coverage genomes from four distinct species of human herpesvirus obtained from two 31,630-year-old milk teeth excavated at Yana, in northeastern Siberia. Phylogenetic analysis of the two HAdV-C genomes suggests an evolutionary origin around 700,000 years ago consistent with a common evolutionary history with hominin hosts. Our findings push back the earliest direct molecular evidence for human viral infections by ∼25,000 years, and demonstrate that viral species causing common childhood viral infections today have been in circulation in humans at least since the Pleistocene.

Enigmatic phenomena have sparked the imagination of people around the globe into creating folkloric creatures. One prime example is Zana of Abkhazia (South Caucasus), a well-documented 19th century female who was captured living wild in the forest. Zana's appearance was sufficiently unusual, that she was referred to by locals as an Almasty-the analog of Bigfoot in the Caucasus. Although the exact location of Zana's burial site was unknown, the grave of her son, Khwit, was identified in 1971. The genomes of Khwit and the alleged Zana skeleton were sequenced to an average depth of ca. 3× using ancient DNA techniques. The identical mtDNA and parent-offspring relationship between the two indicated that the unknown woman was indeed Zana. Population genomic analyses demonstrated that Zana's immediate genetic ancestry can likely be traced to present-day East-African populations. We speculate that Zana might have had a genetic disorder such as congenital generalized hypertrichosis which could partially explain her strange behavior, lack of speech, and long body hair. Our findings elucidate Zana's unfortunate story and provide a clear example of how prejudices of the time led to notions of cryptic hominids that are still held and transmitted by some today.

In this study, we explore the Late Pleistocene (LP) vertebrate faunal diversity in south-eastern Lesser Caucasus based on morphological and genetic identification of fossil bones from Karin Tak cave. For the first time in this under-studied region, we used a bulk bone metabarcoding genetic approach to complement traditional morphology-based taxonomic identifications that are hampered by highly fragmented fossil bones. Excellent ancient DNA (aDNA) preservation allowed for a successful species identification of many bone remains and improved paleoenvironmental interpretations for the region. The aDNA identification of fossil bones revealed a high diversity of animal taxa inhabiting the region between ca. > 42,000 and 25,683–24,803 calibrated years before present (cal. BP).

The impact of the heavy metal contamination and acidity on the bacterial community was studied in samples collected from the Akhtala copper mine tailing using molecular approaches. The bacterial community structure analysis by PCR-DGGE fingerprinting revealed an abundance of Firmicutes, Acidobacteria, and Proteobacteria in different layers of the Akhtala tailing. 454 pyrotag sequence analyses revealed that a significant part of the sequences originated from Proteobacteria (49%) and Bacteroidetes (43%). Bacterial taxa are distributed also in phyla Saccharibacteria (2%), Verrucomicrobia (1.5%), Gammatimonadetes (1%), and some minor additional bacterial groups. The main primary producers in the Akhtala tailing appear to be obligate autotrophic Thiobacillus and Sulfuritalea species. Representatives of Lutibacter and Lysobacter genera are the most abundant acid-tolerant heterotrophs in the studied tailing. The presence of a large number of yet-uncultivated species emphasizes the importance of the future exploration of the tailing as an important source of novel bacteria.

Large vertebrates are extremely sensitive to anthropogenic pressure, and their populations are declining fast. The white rhinoceros (Ceratotherium simum) is a paradigmatic case: this African megaherbivore has suffered a remarkable decline in the last 150 years due to human activities. Its subspecies, the northern (NWR) and the southern white rhinoceros (SWR), however, underwent opposite fates: the NWR vanished quickly, while the SWR recovered after the severe decline. Such demographic events are predicted to have an erosive effect at the genomic level, linked to the extirpation of diversity, and increased genetic drift and inbreeding. However, there is little empirical data available to directly reconstruct the subtleties of such processes in light of distinct demographic histories. Therefore, we generated a whole-genome, temporal data set consisting of 52 resequenced white rhinoceros genomes, representing both subspecies at two time windows: before and during/after the bottleneck. Our data reveal previously unknown population structure within both subspecies, as well as quantifiable genomic erosion. Genome-wide heterozygosity decreased significantly by 10% in the NWR and 36% in the SWR, and inbreeding coefficients rose significantly by 11% and 39%, respectively. Despite the remarkable loss of genomic diversity and recent inbreeding it suffered, the only surviving subspecies, the SWR, does not show a significant accumulation of genetic load compared to its historical counterpart. Our data provide empirical support for predictions about the genomic consequences of shrinking populations, and our findings have the potential to inform the conservation efforts of the remaining white rhinoceroses., This work was supported by ERC Consolidator Grant 681396 “Extinction Genomics” to M.T.P.G. and by EMBO Short-Term Fellowship 7578 to F.S.B. The authors would like to acknowledge support from Science for Life Laboratory, the National Genomics Infrastructure (NGI), Sweden, the Knut and Alice Wallenberg Foundation and UPPMAX for providing assistance in massively parallel DNA sequencing and computational infrastructure.

Natural history museum collections worldwide represent a tremendous resource of information on past and present biodiversity. Fish, reptiles, amphibians and many invertebrate collections have often been preserved in ethanol for decades or centuries and our knowledge on the genomic and metagenomic research potential of such material is limited. Here, we use ancient DNA protocols, combined with shotgun sequencing to test the molecular preservation in liver, skin and bone tissue from five old (1842 to 1964) museum specimens of the common garter snake (Thamnophis sirtalis). When mapping reads to a T. sirtalis reference genome, we find that the DNA molecules are highly damaged with short average sequence lengths (38–64 bp) and high C-T deamination, ranging from 9% to 21% at the first position. Despite this, the samples displayed relatively high endogenous DNA content, ranging from 26% to 56%, revealing that genome-scale analyses are indeed possible from all specimens and tissues included here. Of the three tested types of tissue, bone shows marginally but significantly higher DNA quality in these metrics. Though at least one of the snakes had been exposed to formalin, neither the concentration nor the quality of the obtained DNA was affected. Lastly, we demonstrate that these specimens display a diverse and tissue-specific microbial genetic profile, thus offering authentic metagenomic data despite being submerged in ethanol for many years. Our results emphasize that historical museum collections continue to offer an invaluable source of information in the era of genomics.

Only five species of the once-diverse Rhinocerotidae remain, making the reconstruction of their evolutionary history a challenge to biologists since Darwin. We sequenced genomes from five rhinoceros species (three extinct and two living), which we compared to existing data from the remaining three living species and a range of outgroups. We identify an early divergence between extant African and Eurasian lineages, resolving a key debate regarding the phylogeny of extant rhinoceroses. This early Miocene (∼16 million years ago [mya]) split post-dates the land bridge formation between the Afro-Arabian and Eurasian landmasses. Our analyses also show that while rhinoceros genomes in general exhibit low levels of genome-wide diversity, heterozygosity is lowest and inbreeding is highest in the modern species. These results suggest that while low genetic diversity is a long-term feature of the family, it has been particularly exacerbated recently, likely reflecting recent anthropogenic-driven population declines., The authors acknowledge support from the Science for Life Laboratory, the Garvan Institute of Medical Research, the Knut and Alice Wallenberg Foundation, and the National Genomics Infrastructure funded by the Swedish Research Council and Uppsala Multidisciplinary Center for Advanced Computational Science for assistance with massively parallel sequencing and access to the UPPMAX computational infrastructure. We thank the Natural History Museum at the University of Oslo for providing the Javan rhinoceros sample. We thank the Museum of the Institute of Plant and Animal Ecology (UB RAS, Ekaterinburg) for providing the sample of Siberian unicorn. M.T.P.G. was supported by European Research Council (ERC) Consolidator grant 681396 (Extinction Genomics). E.D.L. was supported by Independent Research Fund Denmark grant 8021-00218B. A.C. was supported by an Australian Research Council Laureate Fellowship (FL140100260). T.M.B. is supported by funding from the ERC under the European Union’s Horizon 2020 research and innovation program (grant agreement 864203), grant BFU2017-86471-P (MINECO/FEDER, UE), “Unidad de Excelencia María de Maeztu” funded by the AEI (CEX2018-000792-M), Howard Hughes International Early Career, and Secretaria d’Universitats i Recerca and CERCA Programme del Departament d’Economia i Coneixement de la Generalitat de Catalunya (GRC 2017 SGR 880). L.D. was supported by the Swedish Research Council (2017-04647) and Formas (2018-01640)., With funding from the Spanish government through the "Severo Ochoa Centre of Excellence" accreditation (CEX2018-000792-M).

The microbial diversity of high-altitude geothermal springs has been recently assessed to explore their biotechnological potential. However, little is known regarding the microbiota of similar ecosystems located on the Armenian Highland. This review summarizes the known information on the microbiota of nine high-altitude mineralized geothermal springs (temperature range 25.8–70 °C and pH range 6.0–7.5) in Armenia and Nagorno-Karabakh. All these geothermal springs are at altitudes ranging from 960–2090 m above sea level and are located on the Alpide (Alpine–Himalayan) orogenic belt, a seismically active region. A mixed-cation mixed-anion composition, with total mineralization of 0.5 mg/L, has been identified for these thermal springs. The taxonomic diversity of hot spring microbiomes has been examined using culture-independent approaches, including denaturing gradient gel electrophoresis (DGGE), 16S rRNA gene library construction, 454 pyrosequencing, and Illumina HiSeq. The bacterial phyla Proteobacteria, Bacteroidetes, Cyanobacteria, and Firmicutes are the predominant life forms in the studied springs. Archaea mainly include the phyla Euryarchaeota, Crenarchaeota, and Thaumarchaeota, and comprise less than 1% of the prokaryotic community. Comparison of microbial diversity in springs from Karvachar with that described for other terrestrial hot springs revealed that Proteobacteria, Bacteroidetes, Actinobacteria, and Deinococcus–Thermus are the common bacterial groups in terrestrial hot springs. Contemporaneously, specific bacterial and archaeal taxa were observed in different springs. Evaluation of the carbon, sulfur, and nitrogen metabolism in these hot spring communities has revealed diversity in terms of metabolic activity. Temperature seems to be an important factor in shaping the microbial communities of these springs. Overall, the diversity and richness of the microbiota are negatively affected by increasing temperature. Other abiotic factors, including pH, mineralization, and geological history, also impact the structure and function of the microbial community. More than 130 bacterial and archaeal strains (Bacillus, Geobacillus, Parageobacillus, Anoxybacillus, Paenibacillus, Brevibacillus Aeribacillus, Ureibacillus, Thermoactinomyces, Sporosarcina, Thermus, Rhodobacter, Thiospirillum, Thiocapsa, Rhodopseudomonas, Methylocaldum, Desulfomicrobium, Desulfovibrio, Treponema, Arcobacter, Nitropspira, and Methanoculleus) have been reported, some of which may be representative of novel species (sharing 91–97% sequence identity with their closest matches in GenBank) and producers of thermozymes and biomolecules with potential biotechnological applications. Whole-genome shotgun sequencing of T. scotoductus K1, as well as of the potentially new Treponema sp. J25 and Anoxybacillus sp. K1, were performed. Most of the phyla identified by 16S rRNA were also identified using metagenomic approaches. Detailed characterization of thermophilic isolates indicate the potential of the studied springs as a source of biotechnologically valuable microbes and biomolecules.

Biodiversity monitoring projects using environmental DNA techniques are becoming increasingly widespread. However, these techniques depend heavily on the quality and richness of the available DNA reference database against which the DNA sequences are queried. To create a comprehensive DNA sequence database for future DNA‐based biodiversity assessments in Denmark, a national DNA reference database, DNAmark, was established, which contains organellar and/or nuclear reference data from vouchered museum species of plants, animals, and fungi from Denmark. Here, we present full or partial mitochondrial genomes of 182 Danish vertebrate species representing ca. 22% of vertebrate species observed in Denmark. Further, we demonstrate that storage conditions of the specimens accounted for ca. 50% of the total variation in mitochondrial DNA (mtDNA) preservation while the age of museum specimens had little effect: ca. 4%. In addition, we roughly estimate the cost of sequencing to be 25 EUR per specimen for obtaining sufficient amounts of DNA reads (ca. 200‐fold coverage) for reliable mitogenome assemblies while also obtaining low coverage genomic data. The large number of mitogenomes of Danish vertebrate species represents the initial groundwork for DNA‐based biodiversity assessments of vertebrates in Denmark and paves the way for practitioners to freely choose mitochondrial DNA markers.

Metal-rich natural and artificial habitats are extreme environments for the development and evolution of unique microbial communities, which have adapted to the toxic levels of the metals. Diverse bacterial groups have developed abilities to deal with the toxic metals by bioaccumulation of the metal ions inside the cell actively or passively, extracellular precipitation, efflux of heavy metals outside to the microbial cell surface, biotransformation of toxic metals to less toxic forms, and metal adsorption on the cell wall. Metalophilic microbes are found in all bacterial and archaeal groups studied, but mostly appear among aerobic and facultative anaerobic chemoheterotrophic and chemolithoautotrophic microorganisms of the Bacillus, Pseudomonas, Staphylococcus, Actinobacteria, Cuprividus, Acidobacterium, Acidithiobacillus, Thiobacillus, Ferroplasma, and Sulfolobus genera. The phenomenon of microbial heavy metal resistance has fundamental importance and is particularly relevant in microbial ecology, especially in connection with the roles of microbes in biogeochemical cycling of heavy metals and in the bioremediation of metal-contaminated environments. The heavy metal resistance mechanisms and different applications of metal resistant/metalophilic bacteria and archaea have been expounded deeply in this chapter.

Whole-genome sequencing projects are increasingly populating the tree of life and characterizing biodiversity1–4. Sparse taxon sampling has previously been proposed to confound phylogenetic inference5, and captures only a fraction of the genomic diversity. Here we report a substantial step towards the dense representation of avian phylogenetic and molecular diversity, by analysing 363 genomes from 92.4% of bird families—including 267 newly sequenced genomes produced for phase II of the Bird 10,000 Genomes (B10K) Project. We use this comparative genome dataset in combination with a pipeline that leverages a reference-free whole-genome alignment to identify orthologous regions in greater numbers than has previously been possible and to recognize genomic novelties in particular bird lineages. The densely sampled alignment provides a single-base-pair map of selection, has more than doubled the fraction of bases that are confidently predicted to be under conservation and reveals extensive patterns of weak selection in predominantly non-coding DNA. Our results demonstrate that increasing the diversity of genomes used in comparative studies can reveal more shared and lineage-specific variation, and improve the investigation of genomic characteristics. We anticipate that this genomic resource will offer new perspectives on evolutionary processes in cross-species comparative analyses and assist in efforts to conserve species., A dataset of the genomes of 363 species from the Bird 10,000 Genomes Project shows increased power to detect shared and lineage-specific variation, demonstrating the importance of phylogenetically diverse taxon sampling in whole-genome sequencing.

The Javan rhinoceros (Rhinoceros sondaicus) is one of five extant rhinoceros species and among the rarest large mammals on Earth. Once widespread across Southeast Asia, it is now on the verge of extinction, with only one wild population remaining (estimated at ~60 individuals) on the island of Java, Indonesia. To assess the past genetic diversity of the female lineage of R. sondaicus, we generated mitochondrial genome data from eight museum specimens dating back to the 19th century, before the range of the Javan rhinoceros was dramatically reduced, for comparison against mitochondrial DNA (mtDNA) sequences of current R. sondaicus and other rhinoceros species. We succeeded in reconstructing five full and three partial ancient mitogenomes from the eight samples. We used BEAST to assess the phylogenetic relationship of the five extant rhinoceros species and the historical samples. The results show that the oldest and most diverse mtDNA lineages of R. sondaicus are found in historical samples, indicating a significant reduction of mtDNA diversity in modern Javan rhinos. We anticipate that the newly sequenced data will represent a useful resource for improving our understanding of evolutionary history of this species, should future studies be able to increase the available dataset. We hope this information may help in conservation efforts for this species.

Humans have a notable capacity to withstand the ravages of infectious diseases. Smallpox killed millions of people but drove Jenner's invention of vaccination, which eventually led to the annihilation of this virus, declared in 1980. To investigate the history of smallpox, Mühlemann et al. obtained high-throughput shotgun sequencing data from 1867 human remains ranging from >31,000 to 150 years ago (see the Perspective by Alcamí). Thirteen positive samples emerged, 11 of which were northern European Viking Age people (6th to 7th century CE). Although the sequences were patchy and incomplete, four could be used to infer a phylogenetic tree. This showed distinct Viking Age lineages with multiple gene inactivations. The analysis pushes back the date of the earliest variola infection in humans by ∼1000 years and reveals the existence of a previously unknown virus clade. This is the author’s version of the work. It is posted here by permission of the AAAS for personal use, not for redistribution.

Large vertebrates are extremely sensitive to anthropogenic pressures, and their populations are declining fast. The white rhinoceros (​Ceratotherium simum) is a paradigmatic case: this African megaherbivore suffered a remarkable population reduction in the last 150 years due to human activities. The two white rhino subspecies, the Northern (NWR) and the Southern white rhino (SWR), however, underwent opposite fates: the NWR vanished quickly after the onset of the decline, while the SWR recovered after a severe bottleneck. Such demographic events are predicted to have an erosive effect at the genomic level, linked to increased genetic drift and inbreeding. However, there is little empirical data available with which to reconstruct the subtleties of such processes. We therefore generated a whole-genome, temporal dataset consisting of 52 re-sequenced white rhino genomes, that represents both subspecies at two time windows throughout the past ~ 170 years. Our data not only reveals previously unknown population substructure within both subspecies, but allowed us to quantify the genomic erosion they underwent, with post-bottleneck white rhinos showing significantly lower levels of heterozygosity and higherinbreeding coefficients than pre-bottleneck individuals. Moreover, the effective population size (N​e​) has suffered a decrease of two and three orders of magnitude in the NWR and SWR, respectively. Our data provides empirical support for theoretical predictions about the genomic consequences ofshrinking populations, which is relevant for understanding the process of population extinction. Furthermore, our findings have the potential to inform tailored management approaches for the conservation of the remaining white rhinos.

The primary cilium is a ubiquitous organelle presented on most human cells. It is a crucial signaling hub for multiple pathways including growth factor and G-protein coupled receptors. Loss of primary cilia, observed in various cancers, has been shown to affect cell proliferation. Primary cilia formation is drastically decreased in glioblastoma (GBM), however, the role of cilia in normal astrocyte or glioblastoma proliferation has not been explored. Here, we report that loss of primary cilia in human astrocytes stimulates growth rate in a lysophosphatidic acid (LPA)-dependent manner. We show that lysophosphatidic acid receptor 1 (LPAR1) is accumulated in primary cilia. LPAR1 signaling through Gα12/Gαq was previously reported to be responsible for cancer cell proliferation. We found that in ciliated cells, Gα12 and Gαq are excluded from the cilium, creating a barrier against unlimited proliferation, one of the hallmarks of cancer. Upon loss of primary cilia, LPAR1 redistributes to the plasma membrane with a concomitant increase in LPAR1 association with Gα12 and Gαq. Inhibition of LPA signaling with the small molecule compound Ki16425 in deciliated highly proliferative astrocytes or glioblastoma patient-derived cells/xenografts drastically suppresses their growth both in vitro and in vivo. Moreover, Ki16425 brain delivery via PEG-PLGA nanoparticles inhibited tumor progression in an intracranial glioblastoma PDX model. Overall, our findings establish a novel mechanism by which primary cilium restricts proliferation and indicate that loss of primary cilia is sufficient to increase mitogenic signaling, and is important for the maintenance of a highly proliferative phenotype. Clinical application of LPA inhibitors may prove beneficial to restrict glioblastoma growth and ensure local control of disease.

Recent ancient DNA (aDNA) studies of human pathogens have provided invaluable insights into their evolutionary history and prevalence in space and time. Most of these studies were based on DNA extracted from teeth or postcranial bones. In contrast, no pathogen DNA has been reported from the petrous bone which has become the most desired skeletal element in ancient DNA research due to its high endogenous DNA content. To compare the potential for pathogenic aDNA retrieval from teeth and petrous bones, we sampled these elements from five ancient skeletons, previously shown to be carrying Yersinia pestis. Based on shotgun sequencing data, four of these five plague victims showed clearly detectable levels of Y. pestis DNA in the teeth, whereas all the petrous bones failed to produce Y. pestis DNA above baseline levels. A broader comparative metagenomic analysis of teeth and petrous bones from 10 historical skeletons corroborated these results, showing a much higher microbial diversity in teeth than petrous bones, including pathogenic and oral microbial taxa. Our results imply that although petrous bones are highly valuable for ancient genomic analyses as an excellent source of endogenous DNA, the metagenomic potential of these dense skeletal elements is highly limited. This trade‐off must be considered when designing the sampling strategy for an aDNA project.

Microorganisms are part of the natural environments and reflect the effects of different physical factors of surrounding environment, such as gamma (γ) radiation. This work was devoted to the study of the influence of low doses of γ radiation with the intensity of 2.56 μW (m2 s)−1 (absorbed doses were 3.8 mGy for the radiation of 15 min and 7.2 mGy—for 30 min) on Escherichia coli M-17 and Pseudomonas aeruginosa GRP3 wild type cells. The changes of bacterial, growth, survival, morphology, and membrane activity had been studied after γ irradiation. Verified microbiological (specific growth rate, lag phase duration, colony-forming units (CFU) number, and light microscopy digital image analysis), biochemical (ATPase activity of bacterial membrane vesicles), and biophysical (H+ fluxes throughout cytoplasmic membrane of bacteria) methods were used for assessment of radiation implications on bacteria. It was shown that growth specific rate, lag phase duration and CFU number of these bacteria were lowered after irradiation, and average cell surface area was decreased too. Moreover ion fluxes of bacteria were changed: for P. aeruginosa they were decreased and for E. coli—increased. The N,N′-dicyclohexylcarbodiimide (DCCD) sensitive fluxes were also changed which were indicative for the membrane-associated F0F1-ATPase enzyme. ATPase activity of irradiated membrane vesicles was decreased for P. aeruginosa and stimulated for E. coli. Furthermore, DCCD sensitive ATPase activity was also changed. The results obtained suggest that these bacteria especially, P. aeruginosa are sensitive to γ radiation and might be used for developing new monitoring methods for estimating environmental changes after γ irradiation.

Background Among the huge diversity of thermophilic bacteria mainly bacilli have been reported as active thermostable lipase producers. Geothermal springs serve as the main source for isolation of thermostable lipase producing bacilli. Thermostable lipolytic enzymes, functioning in the harsh conditions, have promising applications in processing of organic chemicals, detergent formulation, synthesis of biosurfactants, pharmaceutical processing etc. Results In order to study the distribution of lipase-producing thermophilic bacilli and their specific lipase protein primary structures, three lipase producers from different genera were isolated from mesothermal (27.5–70 °C) springs distributed on the territory of Armenia and Nagorno Karabakh. Based on phenotypic characteristics and 16S rRNA gene sequencing the isolates were identified as Geobacillus sp., Bacillus licheniformis and Anoxibacillus flavithermus strains. The lipase genes of isolates were sequenced by using initially designed primer sets. Multiple alignments generated from primary structures of the lipase proteins and annotated lipase protein sequences, conserved regions analysis and amino acid composition have illustrated the similarity (98–99%) of the lipases with true lipases (family I) and GDSL esterase family (family II). A conserved sequence block that determines the thermostability has been identified in the multiple alignments of the lipase proteins. Conclusions The results are spreading light on the lipase producing bacilli distribution in geothermal springs in Armenia and Nagorno Karabakh. Newly isolated bacilli strains could be prospective source for thermostable lipases and their genes.

Background β2-Glycoprotein I (β2GPI) represents the major antigenic target for antiphospholipid antibodies (aPL), with domain 1 (D1) being identified as a risk factor for thrombosis and pregnancy complications in APS. We aimed to analyse the ability of aPL, and particularly anti-D1 β2GPI, to stimulate prothrombotic and proinflammatory activity of immune cells in vitro. Methods Peripheral blood mononuclear cells (PBMCs) from 11 healthy individuals were incubated with: (1) “anti-D1(+)”—pooled plasma derived from patients suspected of having APS contained anticardiolipin antibodies (aCL), lupus anticoagulant (LA), anti-β2GPI and anti-D1 β2GPI; (2) “anti-D1(−)”—pooled plasma from patients suspected of having APS contained aCL, LA, anti-β2GPI, and negative for anti-D1 β2GPI; (3) “seronegative”—negative for aPL. Results The presence of anti-D1(+) and anti-D1(−) plasma resulted in increased HLA-DR and CD11b on monocytes. While only anti-D1(+) plasma markedly increased the percentage and median fluorescence intensity (MFI) of CD142 (tissue factor, TF) on monocytes in comparison with those cultured with anti-D1(−) and seronegative plasma. Anti-D1(+) plasma resulted in increased percentage and MFI of activation marker CD69 on NK and T cytotoxic cells. Expression of IgG receptor FcγRIII(CD16) on monocytes and NK cells was down-regulated by the anti-D1(+) plasma. Conclusions Taking together, our study shows the ability of patient-derived aPL to induce immune cell activation and TF expression on monocytes. For the first time, we demonstrated the influence of anti-D1 β2GPI on the activation status of monocytes, NK and cytotoxic T cells. Our findings further support a crucial role of D1 epitope in the promotion of thrombosis and obstetrical complications in APS.

Northeastern Siberia has been inhabited by humans for more than 40,000 years but its deep population history remains poorly understood. Here we investigate the late Pleistocene population history of northeastern Siberia through analyses of 34 newly recovered ancient genomes that date to between 31,000 and 600 years ago. We document complex population dynamics during this period, including at least three major migration events: an initial peopling by a previously unknown Palaeolithic population of ‘Ancient North Siberians’ who are distantly related to early West Eurasian hunter-gatherers; the arrival of East Asian-related peoples, which gave rise to ‘Ancient Palaeo-Siberians’ who are closely related to contemporary communities from far-northeastern Siberia (such as the Koryaks), as well as Native Americans; and a Holocene migration of other East Asian-related peoples, who we name ‘Neo-Siberians’, and from whom many contemporary Siberians are descended. Each of these population expansions largely replaced the earlier inhabitants, and ultimately generated the mosaic genetic make-up of contemporary peoples who inhabit a vast area across northern Eurasia and the Americas. Analyses of 34 ancient genomes from northeastern Siberia, dating to between 31,000 and 600 years ago, reveal at least three major migration events in the late Pleistocene population history of the region.

As the only endemic neotropical parrot to have recently lived in the northern hemisphere, the Carolina parakeet (Conuropsis carolinensis) was an iconic North American bird. The last surviving specimen died in the Cincinnati Zoo in 1918 [1]. The cause of its extinction remains contentious: besides excessive mortality associated to habitat destruction and active hunting, their survival could have been negatively affected by its range having become increasingly patchy [2] or by the exposure to poultry pathogens [3, 4]. In addition, the Carolina parakeet showed a predilection for cockleburs, an herbaceous plant that contains a powerful toxin, carboxyatractyloside, or CAT [5], which did not seem to affect them but made the birds notoriously toxic to most predators [3]. To explore the demographic history of this bird, we generated the complete genomic sequence of a preserved specimen held in a private collection in Espinelves (Girona, Spain), as well as of a close extant relative, Aratinga solstitialis. We identified two non-synonymous genetic changes in two highly conserved proteins known to interact with CAT that could underlie a specific dietary adaptation to this toxin. Our genomic analyses did not reveal evidence of a dramatic past demographic decline in the Carolina parakeet; also, its genome did not exhibit the long runs of homozygosity that are signals of recent inbreeding and are typically found in endangered species. As such, our results suggest its extinction was an abrupt process and thus likely solely attributable to human causes. Gelabert et al. report the whole genome of the extinct Carolina parakeet and provide evidence of its phylogeny, adaptation to a toxic cocklebur diet, and demographic history. The lack of signs of recent inbreeding typically found in endangered species suggests its abrupt extinction was human mediated., This work was supported by ERC Consolidator Grant 681396 “Extinction Genomics” to M.T.P.G. and M.S.-V., Obra Social ‘‘La Caixa’’ and Secretaria d’Universitats i Recerca ( GRC2017-SGR880 ) to T.M.-B. and C.L.-F., BFU2017-86471-P and PGC2018-101927-B-I00 from MINECO/FEDER, UE to T.M.-B, and PGC2018-095931-B-100 from MINECO/FEDER, UE to C.L.-F. T.M.-B. is also supported by a U01 MH106874 grant and Howard Hughes International Early Career and CERCA Programme (Generalitat de Catalunya). A.A. is partially supported by the Strategic Funding UID/Multi/04423/2019 through national funds provided by the Portuguese Foundation for Science and Technology (FCT) and the European Regional Development Fund —program PT2020 , by the European Structural and Investment Funds —Competitiveness and Internationalization Operational Program-COMPETE 2020, and by National Funds through the FCT under the project PTDC/CTA-AMB/31774/2017(POCI-01-0145-FEDER/031774/2017 ).

Microorganisms have a large number of tools to withstand different, and sometimes strong, environmental stresses, including irradiation, but this ability should be further evaluated for certain applications. Growth inhibition and morphological alterations of Escherichia coli M-17 and Pseudomonas aeruginosa GRP3 wild-type cells caused by UV-A irradiation have been detected in the present study. Comparative analysis was carried out using well-established microbiological methods (determination of specific growth rate, growth lag phase duration, and colony-forming unit number-CFU) and computational approaches, employing light microscopy and digital image analysis to evaluate bacterial cell morphology. Decreases in the specific growth rate, prolonged lag-phases, and lowered CFUs were observed after 5 and 10 min of UV irradiation (approx. 40 Gy) compared to the control (nonirradiated) cells. Accordingly, two computational parameters-the average bacterial cell surface area and the bacterial cell perimeter (i.e., of the 2D projection of bacterial cells in microscopy image)-were reduced. The ratio of bacterial cell surface area (S) to the square of the perimeter (p (2) ) was reduced after 5 min of irradiation, but after 10 min of irradiation the studied bacterial cells became flat cylinders. The revealed findings are concluded to be highly useful in developing new, rapid analysis methods to monitor environmental and UV irradiation effects on bacteria and to detect bacterial cell morphology alterations.

The TGF-β associated growth factor Nodal is highly expressed in aggressive metastatic melanoma. Determining the risk for melanomagenesis from Nodal expression in nevi prior to the development of melanoma may be useful for both the screening and prevention of melanoma. Tissue sections of human adult nevi with or without a history of melanoma were stained by immunohistochemistry (IHC) for Nodal, the Nodal co-receptor Cripto-1, and Notch4, which have previously been shown to be associated with Nodal expression in melanoma. The degree of Nodal, Cripto-1 and Notch4 staining was scored and correlated with available clinical data. Median IHC scores for Nodal, Cripto-1 and Notch4 expression were significantly higher in nevi removed from patients who eventually developed melanoma compared with nevi from patients with no history of melanoma. In addition, the degree of Nodal expression in nevi from patients who eventually developed melanoma correlated significantly with the Breslow depth of the melanoma. Expression of Nodal and components of its signaling pathway in nevi may represent a biomarker for selecting a unique subset of patients requiring increased surveillance for screening and prevention of melanoma.

Triple-negative breast cancer (TNBC) represents an aggressive cancer subtype characterized by the lack of expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). The independence of TNBC from these growth promoting factors eliminates the efficacy of therapies which specifically target them, and limits TNBC patients to traditional systemic neo/adjuvant chemotherapy. To better understand the growth advantage of TNBC - in the absence of ER, PR and HER2, we focused on the embryonic morphogen Nodal (associated with the cancer stem cell phenotype), which is re-expressed in aggressive breast cancers. Most notably, our previous data demonstrated that inhibition of Nodal signaling in breast cancer cells reduces their tumorigenic capacity. Furthermore, inhibiting Nodal in other cancers has resulted in improved effects of chemotherapy, although the mechanisms for this remain unknown. Thus, we hypothesized that targeting Nodal in TNBC cells in combination with conventional chemotherapy may improve efficacy and represent a potential new strategy. Our preliminary data demonstrate that Nodal is highly expressed in TNBC when compared to invasive hormone receptor positive samples. Treatment of Nodal expressing TNBC cell lines with a neutralizing anti-Nodal antibody reduces the viability of cells that had previously survived treatment with the anthracycline doxorubicin. We show that inhibiting Nodal may alter response mechanisms employed by cancer cells undergoing DNA damage. These data suggest that development of therapies which target Nodal in TNBC may lead to additional treatment options in conjunction with chemotherapy regimens - by altering signaling pathways critical to cellular survival.

The number of men and women with gastroesophageal reflux disease (GERB) is increasing each year; besides, the disease seeks rejuvenation. Because the oral cavity is the initial digestive system, gastrointestinal tract diseases (GT) and associated changes in the oral cavity are closely related.Was to assess the dental status of patients with gastroesophageal reflux disease depending on the use of proton pump inhibitors based on the comprehensive clinical and laboratory study. The study involved 100 people aged between 35-65 years, which were divided into 3 groups (30 people - I control group, 35 - II patients with GERD who did not take proton pump inhibitors and 35 - III patients with GERD who take proton pump inhibitors). To detect patients with a predisposition to GERB, all participants in the study answered the «GRDQ» questionnaire. The patient was referred to a gastroenterologist for consultation, if the final score was more than 8 points. To identify the dental status of the patients, the DMFT (Decayed/Missing/Filled Teeth) index and the periodontal PI index were determined, the pH of the saliva was measured, and also the PCR (polymerase chain reaction) method was used. According to the results of the study, the final score of the GRDQ questionnaire in group II was significantly higher than that of group I and group III. The study revealed a high level of caries intensity, deterioration of periodontal and hygienic status, high prevalence of periodontal pathogens:Число мужчин и женщин с гастроэзофагеальной рефлюксной болезнью (ГЭРБ) с каждым годом растет; кроме того, эта болезнь стремится к омоложению. В связи с тем что полость рта является начальным отделом пищеварительной системы, заболевания желудочно-кишечного тракта (ЖКТ) и соответствующие изменения в полости рта тесно взаимосвязаны.Оценка стоматологического статуса пациентов с ГЭРБ в зависимости от приема ингибиторов протонной помпы на основании комплексного клинико-лабораторного исследования.В исследовании приняли участие 100 человек в возрасте 35—65 лет, которые были распределены на три группы: 1-я (контрольная) — 30 человек; 2-я — 35 пациентов с ГЭРБ, не принимающих ингибиторы протонной помпы, 3-я — 35 пациентов с ГЭРБ, принимающих ингибиторы протонной помпы. Для выявления пациентов, имеющих предрасположенность к ГЭРБ, все участники исследования отвечали на опросник Gastroesophageal Reflux Disease Questionnaire (GRDQ). Если итоговая оценка была больше 8 баллов, пациента направляли на консультацию к гастроэнтерологу. Для оценки стоматологического статуса пациентов определяли индекс КПУ, пародонтальный индекс PI, измеряли pH слюны, а также использовали метод полимеразной цепной реакции для определения качественного и количественного состава пародонтопатогенов.Итоговая оценка по опроснику GRDQ у 2-й группы была значительно выше, чем у пациентов 1-й и 3-й групп. В ходе исследования выявлены высокая интенсивность кариеса, ухудшение пародонтального и гигиенического статусов, высокая распространенность пародонтопатогеновРезультаты исследования продемонстрировали снижение pH смешанной слюны во 2-й группе. Стоматологический статус у пациентов на фоне ГЭРБ ухудшается.

Human parvovirus B19 (B19V) is a ubiquitous human pathogen associated with a number of conditions, such as fifth disease in children and arthritis and arthralgias in adults. B19V is thought to evolve exceptionally rapidly among DNA viruses, with substitution rates previously estimated to be closer to those typical of RNA viruses. On the basis of genetic sequences up to similar to 70 years of age, the most recent common ancestor of all B19V has been dated to the early 1800s, and it has been suggested that genotype 1, the most common B19V genotype, only started circulating in the 1960s. Here we present 10 genomes (63.9-99.7% genome coverage) of B19V from dental and skeletal remains of individuals who lived in Eurasia and Greenland from similar to 0.5 to similar to 6.9 thousand years ago (kya). In a phylogenetic analysis, five of the ancient B19V sequences fall within or basal to the modern genotype 1, and five fall basal to genotype 2, showing a long-term association of B19V with humans. The most recent common ancestor of all B19V is placed similar to 12.6 kya, and we find a substitution rate that is an order of magnitude lower than inferred previously. Further, we are able to date the recombination event between genotypes 1 and 3 that formed genotype 2 to similar to 5.0-6.8 kya. This study emphasizes the importance of ancient viral sequences for our understanding of virus evolution and phylogenetics.

Type 2 diabetes mellitus (T2DM) is a severe health problem worldwide, reaching epidemic levels. High susceptibility to infections of T2DM patients indicates dysregulated immune responses to pathogens. However, innate immune responses, including monocyte functions, in T2DM are poorly investigated. Therefore, in this study we aimed to assess lipopolysaccharide- (LPS-) induced immune responses of circulating monocytes from T2DM patients. The results showed that monocytes from T2DM were hyporesponsive to LPS challenge as reflected by significantly suppressed secretion of TNFα (p<0.01) and expression of CD11b (p<0.001) and TLR4 (p<0.001) compared to those in monocytes from healthy subjects. Furthermore, LPS-induced IL-10 levels were similar in diabetic and healthy supernatants, while expression levels of CD163 were found to be downregulated on monocytes from T2DM (p<0.001) suggesting impaired ability of monocytes to switch their phenotype to anti-inflammatory. Taken together, our results suggest compromised function of monocytes in T2DM, which may explain, at least partly, high incidence of infection in these patients.

A large number of thermophilic representatives of the Geobacillus and Anoxybacillus genera have been isolated from geographically distant and physicochemically different environments, including high-, moderate-, and low-temperature habitats. However, terrestrial hot springs are the main habitats for Geobacillus and Anoxybacillus species. The members of these genera possess a variety of thermo-adaptive features that enable them to thrive at elevated temperatures. Due to their ability to withstand harsh environmental conditions, geobacilli and anoxybacilli are a valuable source for provision of thermostable enzymes, such as amylases, lipases, proteases, etc., and other components. Thermostable enzymes obtained from thermophilic bacilli have found a plethora of commercial applications due to their sturdiness and toughness in withstanding the heat generated in various biotechnological and industrial processes. This chapter contains a review of studies of geobacilli and anoxybacilli from terrestrial geothermal springs worldwide with special emphasis on their distribution and diversity, ecological significance, adaptive mechanisms, enzymes, and biotechnological potential.

CNS Primitive Neuroectodermal tumors (CNS-PNETs) are members of the embryonal family of malignant childhood brain tumors, which remain refractory to current therapeutic treatments. Current paradigm of brain tumorigenesis implicates brain tumor-initiating cells (BTIC) in the onset of tumorigenesis and tumor maintenance. However, despite their significance, there is currently no comprehensive characterization of CNS-PNETs BTICs. Recently, we described an animal model of CNS-PNET generated by orthotopic transplantation of human Radial Glial (RG) cells - the progenitor cells for adult neural stem cells (NSC) - into NOD-SCID mice brain and proposed that BTICs may play a role in the maintenance of these tumors. Here we report the characterization of BTIC lines derived from this CNS-PNET animal model. BTIC's orthotopic transplantation generated highly aggressive tumors also characterized as CNS-PNETs. The BTICs have the hallmarks of NSCs as they demonstrate self-renewing capacity and have the ability to differentiate into astrocytes and early migrating neurons. Moreover, the cells demonstrate aberrant accumulation of wild type tumor-suppressor protein p53, indicating its functional inactivation, highly up-regulated levels of onco-protein cMYC and the BTIC marker OCT3/4, along with metabolic switch to glycolysis - suggesting that these changes occurred in the early stages of tumorigenesis. Furthermore, based on RNA- and DNA-seq data, the BTICs did not acquire any transcriptome-changing genomic alterations indicating that the onset of tumorigenesis may be epigenetically driven. The study of these BTIC self-renewing cells in our model may enable uncovering the molecular alterations that are responsible for the onset and maintenance of the malignant PNET phenotype.

The heterogeneity and plasticity of aggressive melanoma presents for­midable challenges in the design of current therapies. Plasticity is defined as the phenotype of cancer cells expressing properties normally related to stem cells, including the expression of genes associated with multiple cellular phenotypes and appearing as undifferentiated, embryonic-like cells. The multipotent pheno­type of these tumor cells, expressing vascular, embryonic, and cancer stem cell (CSC) capabilities, offers new insights into their functional adaptation and resis­tance to current therapies. This chapter highlights major advances in research that (i) help clarify the underlying challenges associated with angiogenesis inhibitor therapy; (ii) discuss important implications of the discovery of reactivation of the normally dormant Nodal embryonic signaling pathway that underlies the CSC phenotype, unregulated tumor growth and metastasis, and resistance to current therapies; and (iii) demonstrate the advantage of using combinatorial strategies to effectively target heterogeneous melanoma subpopulations to eliminate relapse and disease progression.

Present-day hunter-gatherers (HGs) live in multilevel social groups essential to sustain a population structure characterized by limited levels of within-band relatedness and inbreeding. When these wider social networks evolved among HGs is unknown. Here, we investigate whether the contemporary HG strategy was already present in the Upper Paleolithic (UP), using complete genome sequences from Sunghir, a site dated to ~34 thousand years BP (kya) containing multiple anatomically modern human (AMH) individuals. We demonstrate that individuals at Sunghir derive from a population of small effective size, with limited kinship and levels of inbreeding similar to HG populations. Our findings suggest that UP social organization was similar to that of living HGs, with limited relatedness within residential groups embedded in a larger mating network.

Male infertility is a worldwide problem associated with genetic background, environmental factors, and diseases. One of the suspected contributing factors to male infertility is diabetes mellitus. We investigated the molecular and morphological changes in sperms and testicular tissue of diabetic males. The study was performed in streptozotocin-induced type 1 diabetes mouse model. Diabetes decreased sperm concentration and viability and increased sperm apoptosis. Changes in protamine 1/protamine 2 ratio indicated reduced sperm quality. The testicular tissue of diabetic males showed significant tissue damage, disruption of meiotic progression, and changes in the expression of genes encoding proteins important for spermiogenesis. Paternal diabetes altered sperm quality and expression pattern in the testes in offspring of two subsequent generations. Our study revealed that paternal diabetes increased susceptibility to infertility in offspring through gametic alternations. Our data also provide a mechanistic basis for transgenerational inheritance of diabetes-associated pathologies since protamines may be involved in epigenetic regulations.

Summary The South Caucasus, situated between the Black and Caspian Seas, geographically links Europe with the Near East and has served as a crossroad for human migrations for many millennia [1–7]. Despite a vast archaeological record showing distinct cultural turnovers, the demographic events that shaped the human populations of this region is not known [8, 9]. To shed light on the maternal genetic history of the region, we analyzed the complete mitochondrial genomes of 52 ancient skeletons from present-day Armenia and Artsakh spanning 7,800 years and combined this dataset with 206 mitochondrial genomes of modern Armenians. We also included previously published data of seven neighboring populations (n = 482). Coalescence-based analyses suggest that the population size in this region rapidly increased after the Last Glacial Maximum ca. 18 kya. We find that the lowest genetic distance in this dataset is between modern Armenians and the ancient individuals, as also reflected in both network analyses and discriminant analysis of principal components. We used approximate Bayesian computation to test five different demographic scenarios explaining the formation of the modern Armenian gene pool. Despite well documented cultural shifts in the South Caucasus across this time period, our results strongly favor a genetic continuity model in the maternal gene pool. This has implications for interpreting prehistoric migration dynamics and cultural shifts in this part of the world.

Recently, we described a new animal model of CNS primitive neuroectodermal tumors (CNS-PNET), which was generated by orthotopic transplantation of human Radial Glial (RG) cells into NOD-SCID mice’s brain sub-ventricular zone. In the current study we conducted comprehensive RNA-Seq analyses to gain insights on the mechanisms underlying tumorigenesis in this mouse model of CNS-PNET. Here we show that the RNA-Seq profiles derived from these tumors cluster with those reported for patients’ PNETs. Moreover, we found that (i) stabilization of HIF-1α and HIF-2α, which are involved in mediation of the hypoxic responses in the majority of cell types, (ii) up-regulation of MYCC, a key onco-protein whose dysregulation occurs in ~70% of human tumors, and (iii) accumulation of stabilized p53, which is commonly altered in human cancers, constitute hallmarks of our tumor model, and might represent the basis for CNS-PNET tumorigenesis in this model. We discuss the possibility that these three events might be interconnected. These results indicate that our model may prove invaluable to uncover the molecular events leading to MYCC and TP53 alterations, which would be of broader interest considering their relevance to many human malignancies. Lastly, this mouse model might prove useful for drug screening targeting MYCC and related members of its protein interaction network.

The origin of contemporary Europeans remains contentious. We obtained a genome sequence from Kostenki 14 in European Russia dating from 38,700 to 36,200 years ago, one of the oldest fossils of anatomically modern humans from Europe. We find that Kostenki 14 shares a close ancestry with the 24,000-year-old Mal’ta boy from central Siberia, European Mesolithic hunter-gatherers, some contemporary western Siberians, and many Europeans, but not eastern Asians. Additionally, the Kostenki 14 genome shows evidence of shared ancestry with a population basal to all Eurasians that also relates to later European Neolithic farmers. We find that Kostenki 14 contains more Neandertal DNA that is contained in longer tracts than present Europeans. Our findings reveal the timing of divergence of western Eurasians and East Asians to be more than 36,200 years ago and that European genomic structure today dates back to the Upper Paleolithic and derives from a metapopulation that at times stretched from Europe to central Asia.

The liver, as the central metabolic organ, plays an important role in metabolism of carbohydrates, lipids and proteins. In the environment, there are many hepatotoxic xenobiotics. CCl4 is a free radical toxic for organelles of hepatocytes. The presented work was studying the character of ultrastructural changes in CCl4-intoxicated hepatocytes as well as the tissue after treatment by grape seed extract (GSE), α-tocopherol (α-T). As a result of cell bioremediation effect and reactions against corruptive factors, the liver has a high regeneration ability.

Aims and objectives Despite the successes in managing drug-susceptible TB, drug-resistant (DR) tuberculosis is a major challenge to the effectiveness of National Tuberculosis Program in Armenia, placing the country in the list of 18 high-burden countries for MDR-TB in the WHO European Region. Estimated burden of MDR-TB in 2012 was 9.4 (7–12) and 43 (38–49) among retreatment TB cases. A total of 92 laboratory confirmed cases had been reported to the WHO (57 new and 35 previously treated) out of 511 cases tested for MDR-TB. GenoType MTBDRsl is a new molecular kit designed for rapid identification of the resistance to the second-line antituberculosis drugs with a single strip. The aim of this study was to identify the mutation that confers resistance to ethambutol (EMB) in Mycobacterium tuberculosis in comparison with the phenotypic drug susceptibility test (DST). Ethambutol is used in M/XDR-TB regimens only if it is susceptible by DST results. Methods A set of 173 drug resistances TB isolates during 2011 and 2012 period, being either acid fast bacterium positive or negative but culture positive resistant to isoniazid, rifampin, or both according to the GenoType MTBDR plus assay were consecutively tested, using the GenoType MTBDRsl (MTBDR plus version 1.0 and MTBDRsl version 2.0 Hain Lifescience, Nehren). embB gene analysis and the results were compared to phenotypic EMB testing (DST). The DNA preparation method used as described recommended by the manufacturer. Results Genotypic analysis identified mutations at codon 306 of the embB gene in 20.8% (36/173) and miss band of wild type in 12.71% (22/173) of the resistant isolates in comparison to only 14.45% (25/173) of those that were phenotypically resistant to EMB by DST MGIT liquid media. Mutation locus were identified in embB MUT1B and embB MUT1A 8.67% (15/173) and 12.13% (21/173), simultaneously. Phenotypic retesting in MGIT demonstrated that 45 (306 of the embB gene and miss band of wild type together) genotypically resistant isolates were phenotypically resistant to EMB. This implies that 58.62% (33/58) of EMB resistance had been phenotypically missed by routine laboratory procedures. EMB resistance was closely linked to multidrug resistance (MDR); 70.69% (41/58) of the EMB-resistant isolates were resistant to both isoniazid and rifampicin. Conclusion Implementation of more accurate diagnosis of EMB resistance may enhance patient management in Armenia, as standardized treatment of MDR-TB with second-line drugs is currently dependent on the outcome of the EMB resistance test.

Crop wild relatives provide a useful source of genetic variation and represent a large pool of genetic diversity for new allelic variation required in breeding programs. Armenia is an important center of origin both for cultivated Vitis vinifera ssp. sativa and wild Vitis vinifera ssp. sylvestris. Owing to recent prospection in Armenian woods and river floodplains many forms of wild grapevine were discovered and inventoried, which is an important prerequisite to unlock their breeding potential in the future. The fact that some genotypes of V. sylvestris can withstand the diseases is likely to be due to a more efficient basal immunity. The overall goal of the proposed research was to characterize the diversity of V. sylvestris from Armenia with respect to its capacity for stilbene biosynthesis, which might be exploited as genetic resource for resistance breeding. The realized research stimulates the recovery, characterization and preservation of wild grape germplasm, presently at risk of extinction. The recovery and characterization of wild genotypes will be the base of selection of genetic traits important in breeding programs for the generation of biotic and changing climate tolerant grapevine varieties and rootstocks, both necessary for the future of viticulture in Armenia and in Europe.

The brown marmorated stink bug (BMSB), Halyomorpha halys (Stal), native to Northeastern Asia, is a serious invasive pest in the United States, Canada, Switzerland, Germany and France. Several common essential oils and their compositions were tested against BMSBs as potential repellents. All the tested individual essential oils and a ternary oil blend showed sig- nificant repellency to both BMSB nymphs and adults. Clove oil, lemon- grass oil, spearmint oil, ylang-ylang oil, and the ternary oil mixture (clove, lemongrass and spearmint) almost completely blocked attraction of BMSBs to the stink bug attractant-baited traps; whereas wintergreen oil, geranium oil, pennyroyal oil and rosemary oil resulted in 60-85% trap catch reductions. Over 20 BMSB antennally active compounds were iden- tified from SPME headspace samples of the eight repellent essential oils using GC-EAD and GC-MS techniques. Among the synthetic EAD-active compounds tested in the field, eugenol, l-carvone, p/l-menthone, pule- gone, methyl salicylate, trans/cis-citral, methyl benzoate and b-caryophyl- lene significantly reduced trap catches of BMSBs by 72-99%; these compounds are likely responsible for the repellency of their corresponding essential oils. Surprisingly, a synthetic mixture of the predacious spined soldier bug (SSB) (Podisus maculiventris (Say)) aggregation pheromone (trans-2-hexenal, a-terpineol and benzyl alcohol) also showed a signifi- cant inhibition of BMSB response to its attractants. These repellent essen- tial oils and their active compounds, as well as the synthetic SSB pheromone, are potentially useful as part of an efficient, environmentally sound semiochemical-based IPM programme to combat this serious inva- sive stink bug.

Embryonic neural tumors are responsible for a disproportionate number of cancer deaths in children. Although dramatic improvements in survival for pediatric malignancy has been achieved in previous years advancements seem to be slowing down. For the development of new enhanced therapy and an increased understanding of the disease, pre-clinical models better capturing the neoplastic niche are essential. Tumors of early childhood present in this respect a particular challenge. Here, we explore how components of the embryonic process in stem‑cell induced mature teratoma can function as an experimental in vivo microenvironment instigating the growth of injected childhood neuroblastoma (NB) cell lines. Three human NB cell lines, IMR-32, Kelly and SK-N-BE(2), were injected into mature pluripotent stem cell‑induced teratoma (PSCT) and compared to xenografts of the same cell lines. Proliferative NB cells from all lines were readily detected in both models with a typical histology of a poorly differentiated NB tumor with a variable amount of fibrovascular stroma. Uniquely in the PSCT microenvironment, NB cells were found integrated in a non‑random fashion. Neuroblastoma cells were never observed in areas with well-differentiated somatic tissue i.e. bone, muscle, gut or areas of other easily identifiable tissue types. Instead, the three cell lines all showed initial growth exclusively occurring in the embryonic loose mesenchymal stroma, resulting in a histology recapitulating NB native presentation in vivo. Whether this reflects the 'open' nature of loose mesenchyme more easily giving space to new cells compared to other more dense tissues, the rigidity of matrix providing physical cues modulating NB characteristics, or if embryonic loose mesenchyme may supply developmental cues that attracted or promoted the integration of NB, remains to be tested. We tentatively hypothesize that mature PSCT provide an embryonic niche well suited for in vivo studies on NB.

Tetrabromobisphenol A (TBBPA) is a substance widely used in industry as a flame retardant. TBBPA was found in the environment and was detected even in the human body. The effect of this chemical was observed in different cell lines in vitro and it is supposed that TBBPA may affect various hormonal systems in vivo. In this study we examined the effect of TBBPA on the reproductive parameters of two generations of outbred mice in vivo. Experimental and control animals of F1 generation were bred in various conditions to enable evaluation of the possible trans-generational effect. An increased incidence of apoptosis in the testes and changes in the morphometry of seminiferous tubules was detected in the experimental animals. In addition, changes in the expression pattern of selected genes encoding proteins that play an important role during spermatogenesis were observed. In contrast, sperm quality and reproduction were not affected by TBBPA.