ResearchPad - forms-of-dna https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[ArdC, a ssDNA-binding protein with a metalloprotease domain, overpasses the recipient <i>hsdRMS</i> restriction system broadening conjugation host range]]> https://www.researchpad.co/article/elastic_article_7739 Horizontal gene transfer is the main mechanism by which bacteria acquire and disseminate new traits, such as antibiotic resistance genes, that allow adaptation and evolution. Here we identified a gene, ardC, that enables a plasmid to increase its conjugative host range, and thus positively contributes to plasmid fitness. The crystal structure of the antirestriction protein ArdC revealed a fold different from other antirestriction proteins. Our results have wide implications for understanding how a gene enlarges the environments a plasmid can colonize and point to new targets to harness the bacterial DNA uptake control.

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<![CDATA[Identification of early fruit development reference genes in plum]]> https://www.researchpad.co/article/N34728444-bb7f-4d99-8469-dd5c2a1110fc

An RNAseq study of early fruit development and stone development in plum, Prunus domestica, was mined to identify sets of genes that could be used to normalize expression studies in early fruit development. The expression values of genes previously identified from Prunus as reference genes were first extracted and found to vary considerably in endocarp tissue relative to whole fruit tissue. Nine other genes were chosen that varied less than 2-fold amongst the 20 RNAseq libraries of early fruit development and endocarp tissues. These gene were tested on a series of developmental plum fruit samples to determine if any could be used as a reference gene in the analyses of fruit-based tissues in plum. The three most stable genes as determined using RefFinder were IPGD (imidazole glycerol-phosphate dehydratase), HAM1 (histone acetyltransferase) and SNX1 (sorting nexin 1). These were further tested to analyze genes expressed differentially in endocarp tissue between normal and minimal endocarp cultivars. To determine the universality of those nine genes as fruit development reference genes, three other data sets of RNAseq from peach and apple were analyzed to determine the reference gene expression. Multiple genes exhibited tissue specific patterns of expression while one gene, the SNX1, emerged as possessing a universal pattern between the Rosaceae species, at all developmental stages, and tissue types tested. The results suggest that the use of existing RNAseq data to identify standard genes can provide stable reference genes for a specific tissues or experimental conditions under exploration.

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<![CDATA[Rectifying long-standing misconceptions about the ρ statistic for molecular dating]]> https://www.researchpad.co/article/5c75ac76d5eed0c484d08825

When divided by a given mutation rate, the ρ (rho) statistic provides a simple estimator of the age of a clade within a phylogenetic tree by averaging the number of mutations from each sample in the clade to its root. However, a long-standing critique of the use of ρ in genetic dating has been quite often cited. Here we show that the critique is unfounded. We demonstrate by a formal mathematical argument and illustrate with a simulation study that ρ estimates are unbiased and also that ρ and maximum likelihood estimates do not differ in any systematic fashion. We also demonstrate that the claim that the associated confidence intervals commonly estimate the uncertainty inappropriately is flawed since it relies on a means of calculating standard errors that is not used by any other researchers, whereas an established expression for the standard error is largely unproblematic. We conclude that ρ dating, alongside approaches such as maximum likelihood (ML) and Bayesian inference, remains a useful tool for genetic dating.

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<![CDATA[Secondary contact between diverged host lineages entails ecological speciation in a European hantavirus]]> https://www.researchpad.co/article/5c76fdefd5eed0c484e5b0f1

The diversity of viruses probably exceeds biodiversity of eukaryotes, but little is known about the origin and emergence of novel virus species. Experimentation and disease outbreak investigations have allowed the characterization of rapid molecular virus adaptation. However, the processes leading to the establishment of functionally distinct virus taxa in nature remain obscure. Here, we demonstrate that incipient speciation in a natural host species has generated distinct ecological niches leading to adaptive isolation in an RNA virus. We found a very strong association between the distributions of two major phylogenetic clades in Tula orthohantavirus (TULV) and the rodent host lineages in a natural hybrid zone of the European common vole (Microtus arvalis). The spatial transition between the virus clades in replicated geographic clines is at least eight times narrower than between the hybridizing host lineages. This suggests a strong barrier for effective virus transmission despite frequent dispersal and gene flow among local host populations, and translates to a complete turnover of the adaptive background of TULV within a few hundred meters in the open, unobstructed landscape. Genetic differences between TULV clades are homogenously distributed in the genomes and mostly synonymous (93.1%), except for a cluster of nonsynonymous changes in the 5′ region of the viral envelope glycoprotein gene, potentially involved in host-driven isolation. Evolutionary relationships between TULV clades indicate an emergence of these viruses through rapid differential adaptation to the previously diverged host lineages that resulted in levels of ecological isolation exceeding the progress of speciation in their vertebrate hosts.

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<![CDATA[Testing of library preparation methods for transcriptome sequencing of real life glioblastoma and brain tissue specimens: A comparative study with special focus on long non-coding RNAs]]> https://www.researchpad.co/article/5c6b26afd5eed0c484289e7d

Current progress in the field of next-generation transcriptome sequencing have contributed significantly to the study of various malignancies including glioblastoma multiforme (GBM). Differential sequencing of transcriptomes of patients and non-tumor controls has a potential to reveal novel transcripts with significant role in GBM. One such candidate group of molecules are long non-coding RNAs (lncRNAs) which have been proved to be involved in processes such as carcinogenesis, epigenetic modifications and resistance to various therapeutic approaches. To maximize the value of transcriptome sequencing, a proper protocol for library preparation from tissue-derived RNA needs to be found which would produce high quality transcriptome sequencing data and increase the number of detected lncRNAs. It is important to mention that success of library preparation is determined by the quality of input RNA, which is in case of real-life tissue specimens very often altered in comparison to high quality RNA commonly used by manufacturers for development of library preparation chemistry. In the present study, we used GBM and non-tumor brain tissue specimens and compared three different commercial library preparation kits, namely NEXTflex Rapid Directional qRNA-Seq Kit (Bioo Scientific), SENSE Total RNA-Seq Library Prep Kit (Lexogen) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB). Libraries generated using SENSE kit were characterized by the most normal distribution of normalized average GC content, the least amount of over-represented sequences and the percentage of ribosomal RNA reads (0.3–1.5%) and highest numbers of uniquely mapped reads and reads aligning to coding regions. However, NEBNext kit performed better having relatively low duplication rates, even transcript coverage and the highest number of hits in Ensembl database for every biotype of our interest including lncRNAs. Our results indicate that out of three approaches the NEBNext library preparation kit was most suitable for the study of lncRNAs via transcriptome sequencing. This was further confirmed by highly consistent data reached in an independent validation on an expanded cohort.

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<![CDATA[Low-cost cross-taxon enrichment of mitochondrial DNA using in-house synthesised RNA probes]]> https://www.researchpad.co/article/5c61e92ed5eed0c48496f93a

Hybridization capture with in-solution oligonucleotide probes has quickly become the preferred method for enriching specific DNA loci from degraded or ancient samples prior to high-throughput sequencing (HTS). Several companies synthesize sets of probes for in-solution hybridization capture, but these commercial reagents are usually expensive. Methods for economical in-house probe synthesis have been described, but they do not directly address one of the major advantages of commercially synthesised probes: that probe sequences matching many species can be synthesised in parallel and pooled. The ability to make “phylogenetically diverse” probes increases the cost-effectiveness of commercial probe sets, as they can be used across multiple projects (or for projects involving multiple species). However, it is labour-intensive to replicate this with in-house methods, as template molecules must first be generated for each species of interest. While it has been observed that probes can be used to enrich for phylogenetically distant targets, the ability of this effect to compensate for the lack of phylogenetically diverse probes in in-house synthesised probe sets has not been tested. In this study, we present a refined protocol for in-house RNA probe synthesis and evaluated the ability of probes generated using this method from a single species to successfully enrich for the target locus in phylogenetically distant species. We demonstrated that probes synthesized using long-range PCR products from a placental mammal mitochondrion (Bison spp.) could be used to enrich for mitochondrial DNA in birds and marsupials (but not plants). Importantly, our results were obtained for approximately a third of the cost of similar commercially available reagents.

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<![CDATA[Medieval mummies of Zeleny Yar burial ground in the Arctic Zone of Western Siberia]]> https://www.researchpad.co/article/5c79afdad5eed0c4841e3874

Notwithstanding the pioneering achievements of studies on arctic mummies in Siberia, there are insufficient data for any comprehensive understanding of the bio-cultural details of medieval people living in the region. In the Western Siberian arctic, permafrost mummies have been found in 12th to 13th century graves located in the Zeleny Yar (Z-Y) burial ground (66°19'4.54"С; 67°21'13.54"В). In 2013–2016, we were fortunate to be able to excavate that cemetery, locating a total of 47 burials, including cases of mummification. Some of these mummies had been wrapped in a multi-layered birch-bark cocoon. After removal of the cocoon, we conducted interdisciplinary studies using various scientific techniques. Gross anatomical examination and CT radiography showed that the internal organs were still well preserved inside the body cavities. Under light and electron microscopy, the histological findings were very similar to those for naturally mummified specimens discovered in other countries. Ancient DNA analysis showed that the Z-Y mummies’ mtDNA haplotypes belong to five different haplogroups, namely U5a (#34), H3ao (#53), D (#67–1), U4b1b1 (#67–2), and D4j8 (#68), which distinguish them for their unique combination of Western- and Eastern Siberia-specific mtDNA haplogroups. Our interdisciplinary study obtained fundamental information that will form the foundation of successful future investigations on medieval mummies found in the Western Siberian arctic.

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<![CDATA[Evaluation of a template for countering misinformation—Real-world Autism treatment myth debunking]]> https://www.researchpad.co/article/5c5b5287d5eed0c4842bcab4

Misinformation poses significant challenges to evidence-based practice. In the public health domain specifically, treatment misinformation can lead to opportunity costs or direct harm. Alas, attempts to debunk misinformation have proven sub-optimal, and have even been shown to “backfire”, including increasing misperceptions. Thus, optimized debunking strategies have been developed to more effectively combat misinformation. The aim of this study was to test these strategies in a real-world setting, targeting misinformation about autism interventions. In the context of professional development training, we randomly assigned participants to an “optimized-debunking” or a “treatment-as-usual” training condition and compared support for non-empirically-supported treatments before, after, and six weeks following completion of online training. Results demonstrated greater benefits of optimized debunking immediately after training; thus, the implemented strategies can serve as a general and flexible debunking template. However, the effect was not sustained at follow-up, highlighting the need for further research into strategies for sustained change.

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<![CDATA[Miniaturization and optimization of 384-well compatible RNA sequencing library preparation]]> https://www.researchpad.co/article/5c40f7c2d5eed0c48438688b

Preparation of high-quality sequencing libraries is a costly and time-consuming component of metagenomic next generation sequencing (mNGS). While the overall cost of sequencing has dropped significantly over recent years, the reagents needed to prepare sequencing samples are likely to become the dominant expense in the process. Furthermore, libraries prepared by hand are subject to human variability and needless waste due to limitations of manual pipetting volumes. Reduction of reaction volumes, combined with sub-microliter automated dispensing of reagents without consumable pipette tips, has the potential to provide significant advantages. Here, we describe the integration of several instruments, including the Labcyte Echo 525 acoustic liquid handler and the iSeq and NovaSeq Illumina sequencing platforms, to miniaturize and automate mNGS library preparation, significantly reducing the cost and the time required to prepare samples. Through the use of External RNA Controls Consortium (ERCC) spike-in RNAs, we demonstrated the fidelity of the miniaturized preparation to be equivalent to full volume reactions. Furthermore, detection of viral and microbial species from cell culture and patient samples was also maintained in the miniaturized libraries. For 384-well mNGS library preparations, we achieved cost savings of over 80% in materials and reagents alone, and reduced preparation time by 90% compared to manual approaches, without compromising quality or representation within the library.

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<![CDATA[RNase H1 directs origin-specific initiation of DNA replication in human mitochondria]]> https://www.researchpad.co/article/5c37b7b0d5eed0c48449094a

Human mitochondrial DNA (mtDNA) replication is first initiated at the origin of H-strand replication. The initiation depends on RNA primers generated by transcription from an upstream promoter (LSP). Here we reconstitute this process in vitro using purified transcription and replication factors. The majority of all transcription events from LSP are prematurely terminated after ~120 nucleotides, forming stable R-loops. These nascent R-loops cannot directly prime mtDNA synthesis, but must first be processed by RNase H1 to generate 3′-ends that can be used by DNA polymerase γ to initiate DNA synthesis. Our findings are consistent with recent studies of a knockout mouse model, which demonstrated that RNase H1 is required for R-loop processing and mtDNA maintenance in vivo. Both R-loop formation and DNA replication initiation are stimulated by the mitochondrial single-stranded DNA binding protein. In an RNase H1 deficient patient cell line, the precise initiation of mtDNA replication is lost and DNA synthesis is initiated from multiple sites throughout the mitochondrial control region. In combination with previously published in vivo data, the findings presented here suggest a model, in which R-loop processing by RNase H1 directs origin-specific initiation of DNA replication in human mitochondria.

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<![CDATA[Geographic patterns of mtDNA and Z-linked sequence variation in the Common Chiffchaff and the ‘chiffchaff complex’]]> https://www.researchpad.co/article/5c390b90d5eed0c48491d417

The Common Chiffchaff Phylloscopus collybita is an abundant, polytypic Palearctic bird. Validity of some of its subspecies is controversial and birds from some parts of the species range remain unclassified taxonomically. The relationships among populations from different geographic areas have not been sufficiently explored with molecular data. In this study we analyzed the relationships among the four species in the ‘chiffchaff complex’ (Common Chiffchaff, Iberian Chiffchaff P. ibericus, Canary Islands Chiffchaff P. canariensis and Mountain Chiffchaff P. sindianus), and the patterns of intraspecific geographic variation in the mtDNA ND2 gene and intron 9 of the Z-linked aconitase gene (ACO1I9) across the Common Chiffchaff range, including a recently discovered population breeding on Mt. Hermon (Anti-Lebanon mountains). Our data supported the monophyly of the chiffchaff complex and its current systematics at the species level. Within the Common Chiffchaff, the Siberian race P. c. tristis was the most differentiated subspecies and may represent a separate or incipient species. Other Common Chiffchaff subspecies also were differentiated in their mtDNA, however, lineages of neighboring subspecies formed wide zones of introgression. The Mt. Hermon population was of mixed genetic origin but contained some birds with novel unique lineage that could not be assigned to known subspecies. All Common Chiffchaff lineages diverged at the end of the Ionian stage of Pleistocene. Lineage sorting of ACO1I9 alleles was not as complete as that of mtDNA. Chiffchaff species were mostly distinct at ACO1I9, except the Common and Canary Islands Chiffchaffs that shared multiple alleles. An AMOVA identified geographic structure in Common Chiffchaff ACO1I9 variation that was broadly consistent with that of mtDNA ND2 gene. The genetic and other data suggest the chiffchaff complex to be a group of evolutionarily young taxa that represent a paradigm of ‘species evolution in action’ from intergrading subspecies through to apparently complete biological speciation.

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<![CDATA[Insights into evolving global populations of Phytophthora infestans via new complementary mtDNA haplotype markers and nuclear SSRs]]> https://www.researchpad.co/article/5c3667f9d5eed0c4841a6bc6

In many parts of the world the damaging potato late blight pathogen, Phytophthora infestans, is spread as a succession of clonal lineages. The discrimination of genetic diversity within such evolving populations provides insights into the processes generating novel lineages and the pathways and drivers of pathogen evolution and dissemination at local and global scales. This knowledge, in turn, helps optimise management practices. Here we combine two key methods for dissecting mitochondrial and nuclear diversity and resolve intra and inter-lineage diversity of over 100 P. infestans isolates representative of key clonal lineages found globally. A novel set of PCR primers that amplify five target regions are provided for mitochondrial DNA sequence analysis. These five loci increased the number of mtDNA haplotypes resolved from four with the PCR RFLP method to 37 (17, 6, 8 and 4 for Ia, Ib, IIa, and IIb haplotypes, respectively, plus 2 Herb-1 haplotypes). As with the PCR RFLP method, two main lineages, I and II were defined. Group I contained 25 mtDNA haplotypes that grouped broadly according to the Ia and Ib types and resolved several sub-clades amongst the global sample. Group II comprised two distinct clusters with four haplotypes corresponding to the RFLP type IIb and eight haplotypes resolved within type IIa. The 12-plex SSR assay revealed 90 multilocus genotypes providing accurate discrimination of dominant clonal lineages and other genetically diverse isolates. Some association of genetic diversity and geographic region of contemporary isolates was observed; US and Mexican isolates formed a loose grouping, distinct from isolates from Europe, South America and other regions. Diversity within clonal lineages was observed that varied according to the age of the clone. In combination, these fine-scale nuclear and maternally inherited mitochondrial markers enabled a greater level of discrimination among isolates than previously available and provided complementary perspectives on evolutionary questions relating to the diversity, phylogeography and the origins and spread of clonal lineages of P. infestans.

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<![CDATA[Leprosy at the edge of Europe—Biomolecular, isotopic and osteoarchaeological findings from medieval Ireland]]> https://www.researchpad.co/article/5c2d2e50d5eed0c484d9931e

Relatively little is known of leprosy in Medieval Ireland; as an island located at the far west of Europe it has the potential to provide interesting insights in relation to the historical epidemiology of the disease. To this end the study focuses on five cases of probable leprosy identified in human skeletal remains excavated from inhumation burials. Three of the individuals derived from the cemetery of St Michael Le Pole, Golden Lane, Dublin, while single examples were also identified from Ardreigh, Co. Kildare, and St Patrick’s Church, Armoy, Co. Antrim. The individuals were radiocarbon dated and examined biomolecularly for evidence of either of the causative pathogens, M. leprae or M. lepromatosis. Oxygen and strontium isotopes were measured in tooth enamel and rib samples to determine where the individuals had spent their formative years and to ascertain if they had undertaken any recent migrations. We detected M. leprae DNA in the three Golden Lane cases but not in the probable cases from either Ardreigh Co. Kildare or Armoy, Co. Antrim. M. lepromatosis was not detected in any of the burals. DNA preservation was sufficiently robust to allow genotyping of M. leprae strains in two of the Golden Lane burials, SkCXCV (12-13th century) and SkCCXXX (11-13th century). These strains were found to belong on different lineages of the M. leprae phylogenetic tree, namely branches 3 and 2 respectively. Whole genome sequencing was also attempted on these two isolates with a view to gaining further information but poor genome coverage precluded phylogenetic analysis. Data from the biomolecular study was combined with osteological, isotopic and radiocarbon dating to provide a comprehensive and multidisciplinary study of the Irish cases. Strontium and oxygen isotopic analysis indicate that two of the individuals from Golden Lane (SkCXLVIII (10-11th century) and SkCXCV) were of Scandinavian origin, while SkCCXXX may have spent his childhood in the north of Ireland or central Britain. We propose that the Vikings were responsible for introducing leprosy to Ireland. This work adds to our knowledge of the likely origins of leprosy in Medieval Ireland and will hopefully stimulate further research into the history and spread of this ancient disease across the world.

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<![CDATA[Antimicrobial resistance, plasmid, virulence, multilocus sequence typing and pulsed-field gel electrophoresis profiles of Salmonella enterica serovar Typhimurium clinical and environmental isolates from India]]> https://www.researchpad.co/article/5c1ab858d5eed0c484027ad1

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a common serovar associated with non-typhoidal salmonellosis globally. However, there is insufficient data on molecular characterization of S. Typhimurium isolates from India. This study was undertaken to determine the antimicrobial resistance (AMR), plasmid, virulence profiles and molecular subtypes of S. Typhimurium Indian isolates (n = 70) of clinical and environmental origin isolated during 2010–2017. Antimicrobial susceptibility and minimum inhibitory concentrations were determined by disc diffusion and E-test methods respectively. Plasmid extraction was done following standard protocol. AMR genes, virulence genes and plasmid incompatibility types were detected by PCR; Pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) were used for molecular subtyping. Majority (57%) of the study isolates was pan susceptible; five AMR profiles were observed among the resistant (43%) isolates. AMR was significantly (p = 0.004) associated with extra-intestinal isolates than intestinal isolates.The class 1 integron and plasmid-mediated quinolone resistance genes (qnrB1, qnrS1) in the resistant isolates were transferable by conjugation. Plasmids (≥1) ranging from 1.9 to 254kb size and of IncFIIS and/or FIB type were found in most isolates. A total of 39 pulsotypes by PFGE and four sequence types by MLST like ST36 (55.7%), ST19 (32.9%), ST313 (10%) and ST213 (1.4%) were observed. ST36 and ST19 were found circulating in both clinical and environmental host, while ST313 isolates had an exclusive clinical origin. All ST19 isolates (100%) were drug-resistant, while isolates belonging to ST313 (100%), ST213 (100%) and ST36 (82%) were pan susceptible. The virulence plasmid (VP) genes (spvB- spvC) were present in all genotypes except ST36. The VP was significantly (p<0.001) associated with extra-intestinal than intestinal isolates. Some environmental and clinical isolates were clonal indicating their zoonotic transmission. Knowledge on the molecular subtypes and AMR profiles of locally prevalent Salmonella serotypes is important for effective control of spread of resistant organisms. The MLST of S. Typhimurium isolates and its association with AMR, virulence profiles was not reported earlier from India.

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<![CDATA[Deleterious mitochondrial DNA point mutations are overrepresented in Drosophila expressing a proofreading-defective DNA polymerase γ]]> https://www.researchpad.co/article/5bfc6269d5eed0c484ec8fa8

Mitochondrial DNA (mtDNA) mutations cause severe maternally inherited syndromes and the accumulation of somatic mtDNA mutations is implicated in aging and common diseases. However, the mechanisms that influence the frequency and pathogenicity of mtDNA mutations are poorly understood. To address this matter, we created a Drosophila mtDNA mutator strain expressing a proofreading-deficient form of the mitochondrial DNA polymerase. Mutator flies have a dramatically increased somatic mtDNA mutation frequency that correlates with the dosage of the proofreading-deficient polymerase. Mutator flies also exhibit mitochondrial dysfunction, shortened lifespan, a progressive locomotor deficit, and loss of dopaminergic neurons. Surprisingly, the frequency of nonsynonymous, pathogenic, and conserved-site mutations in mutator flies exceeded predictions of a neutral mutational model, indicating the existence of a positive selection mechanism that favors deleterious mtDNA variants. We propose from these findings that deleterious mtDNA mutations are overrepresented because they selectively evade quality control surveillance or because they are amplified through compensatory mitochondrial biogenesis.

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<![CDATA[Synthetic STARR-seq reveals how DNA shape and sequence modulate transcriptional output and noise]]> https://www.researchpad.co/article/5c256c83d5eed0c484474f5a

The binding of transcription factors to short recognition sequences plays a pivotal role in controlling the expression of genes. The sequence and shape characteristics of binding sites influence DNA binding specificity and have also been implicated in modulating the activity of transcription factors downstream of binding. To quantitatively assess the transcriptional activity of tens of thousands of designed synthetic sites in parallel, we developed a synthetic version of STARR-seq (synSTARR-seq). We used the approach to systematically analyze how variations in the recognition sequence of the glucocorticoid receptor (GR) affect transcriptional regulation. Our approach resulted in the identification of a novel highly active functional GR binding sequence and revealed that sequence variation both within and flanking GR’s core binding site can modulate GR activity without apparent changes in DNA binding affinity. Notably, we found that the sequence composition of variants with similar activity profiles was highly diverse. In contrast, groups of variants with similar activity profiles showed specific DNA shape characteristics indicating that DNA shape may be a better predictor of activity than DNA sequence. Finally, using single cell experiments with individual enhancer variants, we obtained clues indicating that the architecture of the response element can independently tune expression mean and cell-to cell variability in gene expression (noise). Together, our studies establish synSTARR as a powerful method to systematically study how DNA sequence and shape modulate transcriptional output and noise.

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<![CDATA[Complete mitochondrial genome of Benthodytes marianensis (Holothuroidea: Elasipodida: Psychropotidae): Insight into deep sea adaptation in the sea cucumber]]> https://www.researchpad.co/article/5c0ae44ad5eed0c48458950b

Complete mitochondrial genomes play important roles in studying genome evolution, phylogenetic relationships, and species identification. Sea cucumbers (Holothuroidea) are ecologically important and diverse members, living from the shallow waters to the hadal trench. In this study, we present the mitochondrial genome sequence of the sea cucumber Benthodytes marianensis collected from the Mariana Trench. To our knowledge, this is the first reported mitochondrial genome from the genus Benthodytes. This complete mitochondrial genome is 17567 bp in length and consists of 13 protein-coding genes, two ribosomal RNA genes and 22 transfer RNA genes (duplication of two tRNAs: trnL and trnS). Most of these genes are coded on the positive strand except for one protein-coding gene (nad6) and five tRNA genes which are coded on the negative strand. Two putative control regions (CRs) have been found in the B. marianensis mitogenome. We compared the order of genes from the 10 available holothurian mitogenomes and found a novel gene arrangement in B. marianensis. Phylogenetic analysis revealed that B. marianensis clustered with Peniagone sp. YYH-2013, forming the deep-sea Elasipodida clade. Positive selection analysis showed that eleven residues (24 S, 45 S, 185 S, 201 G, 211 F and 313 N in nad2; 108 S, 114 S, 322 C, 400 T and 427 S in nad4) were positively selected sites with high posterior probabilities. We predict that nad2 and nad4 may be the important candidate genes for the further investigation of the adaptation of B. marianensis to the deep-sea environment.

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<![CDATA[Acquisition and transfer of antibiotic resistance genes in association with conjugative plasmid or class 1 integrons of Acinetobacter baumannii]]> https://www.researchpad.co/article/5c12cfa2d5eed0c484914ace

Conjugation is a type of horizontal gene transfer (HGT) that serves as the primary mechanism responsible for accelerating the spread of antibiotic resistance genes in Gram-negative bacteria. The present study aimed to elucidate the mechanisms underlying the conjugation-mediated gene transfer from the extensively drug-resistant Acinetobacter baumannii (XDR-AB) and New Delhi Metallo-beta-lactamase-1-producing Acinetobacter baumannii (NDM-AB) to environmental isolates of Acinetobacter spp. Conjugation experiments demonstrated that resistance to ticarcillin and kanamycin could be transferred from four donors to two sodium azide-resistant A. baumannii strains, namely, NU013R and NU015R. No transconjugants were detected on Mueller-Hinton Agar (MHA) plates containing tetracycline. Plasmids obtained from donors as well as successful transconjugants were characterized by PCR-based replicon typing and S1-nuclease pulsed-field gel electrophoresis (S1-PFGE). Detection of antibiotic resistance genes and integrase genes (int) was performed using PCR. Results revealed that the donor AB364 strain can transfer the blaOXA-23 and blaPER-1 genes to both recipients in association with int1. A 240-kb plasmid was successfully transferred from the donor AB364 to recipients. In addition, the aphA6 and blaPER-1 genes were co-transferred with the int1 gene from the donor strains AB352 and AB405. The transfer of a 220-kb plasmid from the donors to recipient was detected. The GR6 plasmid containing the kanamycin resistance gene (aphA6) was successfully transferred from the donor strain AB140 to both recipient strains. However, the blaNDM-1 and tet(B) genes were not detected in all transconjugants. Our study is the first to demonstrate successful in vitro conjugation, which indicated that XDR-AB contained combination mechanisms of the co-transfer of antimicrobial resistance elements with integron cassettes or with the plasmid group GR6. Thus, conjugation could be responsible for the emergence of new types of antibiotic-resistant strains.

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<![CDATA[How many individuals share a mitochondrial genome?]]> https://www.researchpad.co/article/5c16a4a2d5eed0c4844e0b84

Mitochondrial DNA (mtDNA) is useful to assist with identification of the source of a biological sample, or to confirm matrilineal relatedness. Although the autosomal genome is much larger, mtDNA has an advantage for forensic applications of multiple copy number per cell, allowing better recovery of sequence information from degraded samples. In addition, biological samples such as fingernails, old bones, teeth and hair have mtDNA but little or no autosomal DNA. The relatively low mutation rate of the mitochondrial genome (mitogenome) means that there can be large sets of matrilineal-related individuals sharing a common mitogenome. Here we present the mitolina simulation software that we use to describe the distribution of the number of mitogenomes in a population that match a given mitogenome, and investigate its dependence on population size and growth rate, and on a database count of the mitogenome. Further, we report on the distribution of the number of meioses separating pairs of individuals with matching mitogenome. Our results have important implications for assessing the weight of mtDNA profile evidence in forensic science, but mtDNA analysis has many non-human applications, for example in tracking the source of ivory. Our methods and software can also be used for simulations to help validate models of population history in human or non-human populations.

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<![CDATA[Absence of IL-10 production by human PBMCs co-cultivated with human cells expressing or secreting retroviral immunosuppressive domains]]> https://www.researchpad.co/article/5b5ff78d463d7e28ade495c1

Immunosuppression by retroviruses including the human immunodeficiency virus—1 (HIV-1) is well known, however the mechanisms how retroviruses induce this immunosuppression is not fully investigated. It was shown that non-infectious retroviral particles as well as retroviral or recombinant retroviral transmembrane envelope (TM) proteins demonstrated immunosuppressive properties. The same was shown for peptides corresponding to a highly conserved domain in the TM protein. This domain is called immunosuppressive (ISU) domain and it induces modulation of the cytokine release of peripheral blood mononuclear cells (PBMCs) from healthy donors. In addition, it changes the gene expression of these cells. Common indications for the immunosuppressive activity were tumour growth in vivo and interleukin—10 (IL-10) release from human PBMCs in vitro. Single mutations in the ISU domain abrogated the immunosuppressive activity. In order to develop a new model system for the expression of the ISU domain and presentation to PBMCs which is not prone to possible endotoxin contaminations, two expression systems were developed. In the first system, designated pOUT, retroviral proteins containing the ISU domain were expressed and released into the cell culture medium, and in the second system, tANCHOR, the ISU domain was presented by a tetraspanin-anchored sequence on the cell surface of human cells. Both systems were exploited to express the wild-type (wt) ISU domains of HIV-1, of the porcine endogenous retrovirus (PERV) and of the murine leukaemia virus (MuLV) as well as to express mutants (mut) of these ISU domains. PERV is of special interest in the context of virus safety of xenotransplantation using pig organs. Expression of the TM proteins was demonstrated by confocal laser scanning microscopy, ELISA and Western blot analyses using specific antibodies. However, when cells expressing and releasing the ISU were co-incubated with human PBMCs, no increased production of IL-10 was observed when compared with the mutants. Similar results were obtained when the released TM proteins were concentrated by immunoprecipitation and added to PBMCs. We suggest that the absence of IL-10 induction can be explained by a low amount of protein, by the lack of a biologically active conformation or the absence of additional factors.

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