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Highly porous polypyrrole (PPy)-nanocellulose paper sheets have been evaluated as inexpensive and disposable electrochemically controlled three-dimensional solid phase extraction materials. The composites, which had a total anion exchange capacity of about 1.1 mol kg(-1), were used for extraction and subsequent release of negatively charged fluorophore tagged DNA oligomers via galvanostatic oxidation and reduction of a 30-50 nm conformal PPy layer on the cellulose substrate. The ion exchange capacity, which was, at least, two orders of magnitude higher than those previously reached in electrochemically controlled extraction, originated from the high surface area (i.e. 80 m(2) g(-1)) of the porous composites and the thin PPy layer which ensured excellent access to the ion exchange material. This enabled the extractions to be carried out faster and with better control of the PPy charge than with previously employed approaches. Experiments in equimolar mixtures of (dT)(6), (dT)(20), and (dT)(40) DNA oligomers showed that all oligomers could be extracted, and that the smallest oligomer was preferentially released with an efficiency of up to 40% during the reduction of the PPy layer. These results indicate that the present material is very promising for the development of inexpensive and efficient electrochemically controlled ion-exchange membranes for batch-wise extraction of biomolecules.

Background: Paper mulberry has been used for thousands of years in Asia and Oceania for making paper and bark-cloth, respectively. Museums around the world hold valuable collections of Polynesian bark-cloth. Genetic analysis of the plant fibers from which the textiles were made may answer a number of questions of interest related to provenance, authenticity or species used in the manufacture of these textiles. Recovery of nucleic acids from paper mulberry bark-cloth has not been reported before., Methodology: We describe a simple method for the extraction of PCR-amplifiable DNA from small samples of contemporary Polynesian bark-cloth (tapa) using two types of nuclear markers. We report the amplification of about 300 bp sequences of the ITS1 region and of a microsatellite marker., Conclusions: Sufficient DNA was retrieved from all bark-cloth samples to permit successful PCR amplification. This method shows a means of obtaining useful genetic information from modern bark-cloth samples and opens perspectives for the analyses of small fragments derived from ethnographic materials.

DNA extraction, a vital pre-requisite for most biological studies, continues to be studied extensively. According to some studies, DNA shows a certain degree of absorbability on filter paper made of plant fiber-based adsorbent material. However, the principle underlying such specific adsorption as well as plant species associated with plant fiber-based adsorbents and optimized extraction conditions have not yet been studied. This study demonstrates the tight correlation between crystallinity and hygroscopicity in plant fiber-based adsorbents used for DNA extraction and proposes the concept of DNA adsorption on plant fiber-based adsorbents, for the first time. We also explored optimal extracting and eluting conditions and developed a novel plant fiber-based DNA extraction method that was quadruple times more powerful than current approaches. Starting with the screening of various types of earthed plant fiber-based adsorbents, we went on to mine new plant fiber-based adsorbents, bamboo paper and degreased cotton, and succeeded in increasing their efficiency of DNA extraction to 4.2 times than that of current approaches. We found a very strong correlation between the crystallinity and hygroscopicity of plant fiber-based adsorbents which showed efficiency for DNA extraction, and thus propose a principle that potentially governs such specific adsorption processes, in the hope that this information may guide related multidisciplinary research studies in the future. Nanodrop, electrophoresis and PCR were selected to demonstrate the quantity, quality, integrity and utility of the extracted DNA. Furthermore, crystallinity, hygroscopicity, pore size distribution and composition of plant fiber-based adsorbents were studied to explore their correlation in an attempt to understand the principle underlying this particular type of adsorption. The findings of this study may be further extended to the extraction of other types of nucleic acids with similar biochemical properties. [ABSTRACT FROM AUTHOR]

Amphimeriasis, a fish-borne zoonotic disease caused by the liver fluke Amphimerus spp., has recently been reported as an emerging disease affecting an indigenous Ameridian group, the Chachi, living in Ecuador. The only method for diagnosing amphimeriasis was the microscopic detection of eggs from the parasite in patients' stool samples with very low sensitivity. Our group developed an ELISA technique for detection of anti-Amphimerus IgG in human sera and a molecular method based on LAMP technology (named LAMPhimerus) for specific and sensitive parasite DNA detection. The LAMPhimerus method showed to be much more sensitive than classical parasitological methods for amphimeriasis diagnosis using human stool samples for analysis. The objective of this work is to demonstrate the feasibility of using dried stool samples on filter paper as source of DNA in combination with the effectiveness of our previously designed LAMPhimerus assay for successfully Amphimerus sp. detection in clinical stool samples. A total of 102 untreated and undiluted stool samples collected from Chachi population were spread as thin layer onto common filter paper for easily transportation to our laboratory and stored at room temperature for one year until DNA extraction. When LAMPhimerus method was applied for Amphimerus sp. DNA detection, a higher number of positive results was detected (61/102; 59.80%) in comparison to parasitological methods (38/102; 37.25%), including 28/61 (45.90%) microscopy-confirmed Amphimerus sp. infections. The diagnostic parameters for the sensitivity and specificity werecalculated for our LAMPhimerus assay, which were 79.17% and 65.98%, respectively. We demonstrate, for the first time, that common filter paper is useful for easy collection and long-term storage of human stool samples for later DNA extraction and molecular analysis of human-parasitic trematode eggs. This simple, economic and easily handling method combined with the specific and sensible LAMPhimerus assay has the potential to beused as an effective molecular large-scale screening test for amphimeriasis-endemic areas. [ABSTRACT FROM AUTHOR]

Written communication plays a crucial role in the history of modern civilizations as manuscripts do not only exist contemporarily, but are passed on to subsequent generations. Besides a document’s content, information is stored in the materials used for its production. Analyses of the composition allow, for example, identifying the biological origins of materials, dating, and help to understand degradation patterns. A combination of microscopic and DNA approaches was applied in order to analyze various plant derived writing sheets. Given their diversity and abundance in museum collections, plant based writing supports are yet an underexplored target for DNA studies. DNA retrieval of paper is low compared to raw paper plant material, which is likely due to the loss of organic components during paper production. Optimizing DNA extraction for each respective material drastically increased DNA recovery. Finally, we present a non-invasive DNA sampling method that utilizes nylon membranes, commonly used for bacterial DNA sampling and that is applicable to delicate material. Although bacterial infestation was visible on one sample, as indicated by scanning electron microscopy, endogenous DNA was retrieved. The results presented here are promising as they extend the scope of sources for DNA analyses by demonstrating that DNA molecules can be retrieved from a variety of plant derived writing supports. In future, such analyses can help to explore the biological diversity not only of plants and of additives utilized for producing writing supports, but also of the plenty products made from paper. [ABSTRACT FROM AUTHOR]

Background: The alkalistable and thermostable xylanases are in high demand for pulp bleaching in paper industry and generating xylooligosaccharides by hydrolyzing xylan component of agro-residues. The compost-soil samples, one of the hot environments, are expected to be a rich source of microbes with thermostable enzymes. Methodology/Principal Findings: Metagenomic DNA from hot environmental samples could be a rich source of novel biocatalysts. While screening metagenomic library constructed from DNA extracted from the compost-soil in the p18GFP vector, a clone (TSDV-MX1) was detected that exhibited clear zone of xylan hydrolysis on RBB xylan plate. The sequencing of 6.321 kb DNA insert and its BLAST analysis detected the presence of xylanase gene that comprised 1077 bp. The deduced protein sequence (358 amino acids) displayed homology with glycosyl hydrolase (GH) family 11 xylanases. The gene was subcloned into pET28a vector and expressed in E. coli BL21 (DE3). The recombinant xylanase (rMxyl) exhibited activity over a broad range of pH and temperature with optima at pH 9.0 and 80uC. The recombinant xylanase is highly thermostable having T1/2 of 2 h at 80°C and 15 min at 90°C. Conclusion/Significance: This is the first report on the retrieval of xylanase gene through metagenomic approach that encodes an enzyme with alkalistability and thermostability. The recombinant xylanase has a potential application in paper and pulp industry in pulp bleaching and generating xylooligosaccharides from the abundantly available agro-residues. [ABSTRACT FROM AUTHOR]

The paper wasp Polistes dominulus is unique among the social insects in that nearly one-third of co-foundresses are completely unrelated to the dominant individual whose offspring they help to rear and yet reproductive skew is high. These unrelated subordinates stand to gain direct fitness through nest inheritance, raising the question of whether their behaviour is adaptively tailored towards maximizing inheritance prospects. Unusually, in this species, a wealth of theory and empirical data allows us to predict how unrelated subordinates should behave. Based on these predictions, here we compare helping in subordinates that are unrelated or related to the dominant wasp across an extensive range of field-based behavioural contexts. We find no differences in foraging effort, defense behaviour, aggression or inheritance rank between unrelated helpers and their related counterparts. Our study provides no evidence, across a number of behavioural scenarios, that the behaviour of unrelated subordinates is adaptively modified to promote direct fitness interests. [ABSTRACT FROM AUTHOR]

DNA has been discussed as a potential medium for data storage. Potentially it could be denser, could consume less energy, and could be more durable than conventional storage media such as hard drives, solid-state storage, and optical media. However, performing computations on the data stored in DNA is a largely unexplored challenge. This paper proposes an integrated circuit (IC) based on microfluidics that can perform complex operations such as artificial neural network (ANN) computation on data stored in DNA. We envision such a system to be suitable for highly dense, throughput-demanding bio-compatible applications such as an intelligent Organ-on-Chip or other biomedical applications that may not be latency-critical. It computes entirely in the molecular domain without converting data to electrical form, making it a form of in-memory computing on DNA. The computation is achieved by topologically modifying DNA strands through the use of enzymes called nickases. A novel scheme is proposed for representing data stochastically through the concentration of the DNA molecules that are nicked at specific sites. The paper provides details of the biochemical design, as well as the design, layout, and operation of the microfluidics device. Benchmarks are reported on the performance of neural network computation. [ABSTRACT FROM AUTHOR]

Use of telomere length (TL) as a biomarker for various environmental exposures and diseases has increased in recent years. Various methods have been developed to measure telomere length. Polymerase chain reaction (PCR)-based methods remain wide-spread for population-based studies due to the high-throughput capability. While several studies have evaluated the repeatability and reproducibility of different TL measurement methods, the results have been variable. We conducted a literature review of TL measurement cross-method comparison studies that included a PCR-based method published between January 1, 2002 and May 25, 2020. A total of 25 articles were found that matched the inclusion criteria. Papers were reviewed for quality of methodologic reporting of sample and DNA quality, PCR assay characteristics, sample blinding, and analytic approaches to determine final TL. Overall, methodologic reporting was low as assessed by two different reporting guidelines for qPCR-based TL measurement. There was a wide range in the reported correlation between methods (as assessed by Pearson's r) and few studies utilized the recommended intra-class correlation coefficient (ICC) for assessment of assay repeatability and methodologic comparisons. The sample size for nearly all studies was less than 100, raising concerns about statistical power. Overall, this review found that the current literature on the relation between TL measurement methods is lacking in validity and scientific rigor. In light of these findings, we present reporting guidelines for PCR-based TL measurement methods and results of analyses of the effect of assay repeatability (ICC) on statistical power of cross-sectional and longitudinal studies. Additional cross-laboratory studies with rigorous methodologic and statistical reporting, adequate sample size, and blinding are essential to accurately determine assay repeatability and replicability as well as the relation between TL measurement methods. [ABSTRACT FROM AUTHOR]

This paper proposes multidimensional scaling (MDS) applied to high-throughput chromosome conformation capture (Hi-C) data on genomic interactions to visualize DNA loops. Currently, the mechanisms underlying the regulation of gene expression are poorly understood, and where and when DNA loops are formed remains undetermined. Previous studies have focused on reproducing the entire three-dimensional structure of chromatin; however, identifying DNA loops using these data is time-consuming and difficult. MDS is an unsupervised method for reconstructing the original coordinates from a distance matrix. Here, MDS was applied to high-throughput chromosome conformation capture (Hi-C) data on genomic interactions to visualize DNA loops. Hi-C data were converted to distances by taking the inverse to reproduce loops via MDS, and the missing values were set to zero. Using the converted data, MDS was applied to the log-transformed genomic coordinate distances and this process successfully reproduced the DNA loops in the given structure. Consequently, the reconstructed DNA loops revealed significantly more DNA-transcription factor interactions involved in DNA loop formation than those obtained from previously applied methods. Furthermore, the reconstructed DNA loops were significantly consistent with chromatin immunoprecipitation followed by sequencing (ChIP-seq) peak positions. In conclusion, the proposed method is an improvement over previous methods for identifying DNA loops., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Ryo Ishibashi. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The catalytic (CAT) domain is a key region of poly (ADP-ribose) polymerase 1 (PARP1), which has crucial interactions with inhibitors, DNA, and other domains of PARP1. To facilitate the development of potential inhibitors of PARP1, it is of great significance to clarify the differences in structural dynamics and key residues between CAT/inhibitors and DNA/PARP1/inhibitors through structure-based computational design. In this paper, conformational changes in PAPR1 and differences in key residue interactions induced by inhibitors were revealed at the molecular level by comparative molecular dynamics (MD) simulations and energy decomposition. On one hand, PARP1 inhibitors indirectly change some residues of the CAT domain which interact with DNA and other domains. Furthermore, the interaction between ligands and catalytic binding sites can be transferred to the DNA recognition domain of PARP1 by a strong negative correlation movement among multi-domains of PARP1. On the other hand, it is not reliable to use the binding energy of CAT/ligand as a measure of ligand activity, because it may in some cases differs greatly from the that of PARP1/DNA/ligand. For PARP1/DNA/ligand, the stronger the binding stability between the ligand and PARP1, the stronger the binding stability between PARP1 and DNA. The findings of this work can guide further novel inhibitor design and the structural modification of PARP1 through structure-based computational design., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Nucleic acid amplification is a powerful molecular biology tool, although its use outside the modern laboratory environment is limited due to the relatively cumbersome methods required to extract nucleic acids from biological samples. To address this issue, we investigated a variety of materials for their suitability for nucleic acid capture and purification. We report here that untreated cellulose-based paper can rapidly capture nucleic acids within seconds and retain them during a single washing step, while contaminants present in complex biological samples are quickly removed. Building on this knowledge, we have successfully created an equipment-free nucleic acid extraction dipstick methodology that can obtain amplification-ready DNA and RNA from plants, animals, and microbes from difficult biological samples such as blood and leaves from adult trees in less than 30 seconds. The simplicity and speed of this method as well as the low cost and availability of suitable materials (e.g., common paper towelling), means that nucleic acid extraction is now more accessible and affordable for researchers and the broader community. Furthermore, when combined with recent advancements in isothermal amplification and naked eye DNA visualization techniques, the dipstick extraction technology makes performing molecular diagnostic assays achievable in limited resource settings including university and high school classrooms, field-based environments, and developing countries. [ABSTRACT FROM AUTHOR]

This paper presents a novel strategy for computing mathematical functions with molecular reactions, based on theory from the realm of digital design. It demonstrates how to design chemical reaction networks based on truth tables that specify analog functions, computed by stochastic logic. The theory of stochastic logic entails the use of random streams of zeros and ones to represent probabilistic values. A link is made between the representation of random variables with stochastic logic on the one hand, and the representation of variables in molecular systems as the concentration of molecular species, on the other. Research in stochastic logic has demonstrated that many mathematical functions of interest can be computed with simple circuits built with logic gates. This paper presents a general and efficient methodology for translating mathematical functions computed by stochastic logic circuits into chemical reaction networks. Simulations show that the computation performed by the reaction networks is accurate and robust to variations in the reaction rates, within a log-order constraint. Reaction networks are given that compute functions for applications such as image and signal processing, as well as machine learning: arctan, exponential, Bessel, and sinc. An implementation is proposed with a specific experimental chassis: DNA strand displacement with units called DNA "concatemers"., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Solanki et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Body size, nutrition and socioeconomic position (SEP) in early life have been associated with a wide range of long-term health effects. Epigenetics is one possible mechanism through which these early life exposures can impact later life health. We conducted a systematic review examining the observational evidence for the impact of body size, nutrition and SEP in early life on the epigenome in humans. Methods: This systematic review is registered with the PROSPERO database (registration number: CRD42016050193). Three datasets were simultaneously searched using Ovid and the resulting studies were evaluated by at least two independent reviewers. Studies measuring epigenetic markers either at the same time as, or after, the early life exposure and have a measure of body size, nutrition or SEP in early life (up to 12 years), written in English and from a community-dwelling participants were included. Results: We identified 90 eligible studies. Seventeen of these papers examined more than one early life exposure of interest. Fifty six papers examined body size, 37 nutrition and 17 SEP. All of the included papers examined DNA methylation (DNAm) as the epigenetic marker. Overall there was no strong evidence for a consistent association between these early life variables in DNAm which may be due to the heterogeneous study designs, data collection methods and statistical analyses. Conclusions: Despite these inconclusive results, the hypothesis that the early life environment can impact DNAm, potentially persisting into adult life, was supported by some studies and warrants further investigation. We provide recommendations for future studies. [ABSTRACT FROM AUTHOR]

This article deals with studying the dynamical behavior of the DNA model proposed by Peyrard and Bishop. The proposed model is investigated using the unified method (UM). Unified method successfully extracts solutions in the form of polynomial and rational functions. The solitary wave solutions and soliton solutions are constructed. An investigation of modulation instability is also presented in this paper. 3D and 2D plots are presented to exhibit the physical behavior of some of the obtained solutions., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Vivas-Cortez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The generation of massive data puts forward higher requirements for storage technology. DNA storage is a new storage technology which uses biological macromolecule DNA as information carrier. Compared with traditional silicon-based storage, DNA storage has the advantages of large capacity, high density, low energy consumption and high durability. DNA coding is to store data information with as few base sequences as possible without errors. Coding is a key technology in DNA storage, and its results directly affect the performance of storage and the integrity of data reading and writing. In this paper, a Levy Equilibrium Optimizer (LEO) algorithm is proposed to construct a DNA storage code set that satisfies combinatorial constraints. The performance of the proposed algorithm is tested on 13 benchmark functions, and 4 new global optima are obtained. Under the same constraints, the DNA storage code set is constructed. Compared with previous work, the lower bound of DNA storage code set is improved by 4-13%., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Jianxia Zhang. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Paper currency by its very nature is frequently transferred from one person to another and represents an important medium for human contact with—and potential exchange of—microbes. In this pilot study, we swabbed circulating $1 bills obtained from a New York City bank in February (Winter) and June (Summer) 2013 and used shotgun metagenomic sequencing to profile the communities found on their surface. Using basic culture conditions, we also tested whether viable microbes could be recovered from bills. Results: Shotgun metagenomics identified eukaryotes as the most abundant sequences on money, followed by bacteria, viruses and archaea. Eukaryotic assemblages were dominated by human, other metazoan and fungal taxa. The currency investigated harbored a diverse microbial population that was dominated by human skin and oral commensals, including Propionibacterium acnes, Staphylococcus epidermidis and Micrococcus luteus. Other taxa detected not associated with humans included Lactococcus lactis and Streptococcus thermophilus, microbes typically associated with dairy production and fermentation. Culturing results indicated that viable microbes can be isolated from paper currency. Conclusions: We conducted the first metagenomic characterization of the surface of paper money in the United States, establishing a baseline for microbes found on $1 bills circulating in New York City. Our results suggest that money amalgamates DNA from sources inhabiting the human microbiome, food, and other environmental inputs, some of which can be recovered as viable organisms. These monetary communities may be maintained through contact with human skin, and DNA obtained from money may provide a record of human behavior and health. Understanding these microbial profiles is especially relevant to public health as money could potentially mediate interpersonal transfer of microbes. [ABSTRACT FROM AUTHOR]

In this paper, the authors study the mathematical properties of a class of alternating links called polyhedral links which have been used to model DNA polyhedra. The motivation of such studies is to provide guidance and aid in the research of the properties of certain DNA molecules. For example, such studies can provide characterizations of the structural complexity of DNA molecules. In an earlier work, Cheng and Jin studied the mathematical properties of such polyhedral links and were able to determine the braid index of a double crossover polyhedral link with 4 turn. However, the braid index of a double crossover polyhedral link with 4.5 turn remained an unsolved problem to this date, even though the graphs that admit the double crossover polyhedral links with 4.5 turn have been synthesized. In this paper, we provide a complete formulation of the braid index of a double crossover polyhedral link with an arbitrary turn number. Our approach is more general and it allows us to completely determine the braid indices for a much larger class of links. In the case of the double crossover polyhedral links, our formulation of the braid index is a simple formula based on a simpler graph used as a template to build the double crossover polyhedral links. [ABSTRACT FROM AUTHOR]

To assess a species' impact on its environment–and the environment's impact upon a species–we need to pinpoint its links to surrounding taxa. The honeybee (Apis mellifera) provides a promising model system for such an exercise. While pollination is an important ecosystem service, recent studies suggest that honeybees can also provide disservices. Developing a comprehensive understanding of the full suite of services and disservices that honeybees provide is a key priority for such a ubiquitous species. In this perspective paper, we propose that the DNA contents of honey can be used to establish the honeybee's functional niche, as reflected by ecosystem services and disservices. Drawing upon previously published genomic data, we analysed the DNA found within 43 honey samples from Northern Europe. Based on metagenomic analysis, we find that the taxonomic composition of DNA is dominated by a low pathogenicity bee virus with 40.2% of the reads, followed by bacteria (16.7%), plants (9.4%) and only 1.1% from fungi. In terms of ecological roles of taxa associated with the bees or taxa in their environment, bee gut microbes dominate the honey DNA, with plants as the second most abundant group. A range of pathogens associated with plants, bees and other animals occur frequently, but with lower relative read abundance, across the samples. The associations found here reflect a versatile the honeybee's role in the North-European ecosystem. Feeding on nectar and pollen, the honeybee interacts with plants–in particular with cultivated crops. In doing so, the honeybee appears to disperse common pathogens of plants, pollinators and other animals, but also microbes potentially protective of these pathogens. Thus, honey-borne DNA helps us define the honeybee's functional niche, offering directions to expound the benefits and drawbacks of the associations to the honeybee itself and its interacting organisms. [ABSTRACT FROM AUTHOR]

The basidiomycete fungus Gloeophyllum trabeum causes a typical brown rot and is known to use reactive oxygen species in the degradation of cellulose. The extracellular Cel12A is one of the few endo-1,4-β-glucanase produced by G. trabeum. Here we cloned cel12A and heterologously expressed it in Aspergillus niger. The identity of the resulting recombinant protein was confirmed by mass spectrometry. We used the purified GtCel12A to determine its substrate specificity and basic biochemical properties. The G. trabeum Cel12A showed highest activity on β-glucan, followed by lichenan, carboxymethylcellulose, phosphoric acid swollen cellulose, microcrystalline cellulose, and filter paper. The optimal pH and temperature for enzymatic activity were, respectively, 4.5 and 50°C on β-glucan. Under these conditions specific activity was 239.2±9.1 U mg−1 and the half-life of the enzyme was 84.6±3.5 hours. Thermofluor studies revealed that the enzyme was most thermal stable at pH 3. Using β-glucan as a substrate, the Km was 3.2±0.5 mg mL−1 and the Vmax was 0.41±0.02 µmol min−1. Analysis of the effects of GtCel12A on oat spelt and filter paper by scanning electron microscopy revealed the morphological changes taking place during the process. [ABSTRACT FROM AUTHOR]

Determining the distribution of the Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis, YFP) in the Yangtze River has to date relied on traditional visual and counting methods, but such field surveys are time-consuming and expensive. Analyses using environmental DNA (eDNA) to investigate the presence and range of endangered aquatic species have proven to be more economical and effective detection methods, and are a non-invasive approach to sampling. A challenge of relying on eDNA for YFP monitoring is that the Yangtze River is characterized by high turbidity and a strong current. Here, we used an eDNA-based approach to estimate the presence of YFP at 18 sites in the Yangtze River in August 2017 and at an additional 11 sites in January 2018. At each sampling site, we filtered six 1 L water samples with 5 µm pore size filter paper and quantified the amount of YFP eDNA in each water sample using quantitative real-time polymerase chain reaction (qPCR). In addition, YFP eDNA was successfully detected in locations where we visually observed YFP, as well as in locations where YFP were not observed directly. We found that our eDNA-based method had higher detection rates than traditional field survey methods. Although YFP was visually observed in the Yangtze River in winter, water samples collected during the summer contained significantly higher YFP eDNA than winter water samples. Our results demonstrate the potential effectiveness of eDNA detection methods in determining the distribution of YFP in the Yangtze River. [ABSTRACT FROM AUTHOR]

SREBP1 and 2, are cholesterol sensors able to modulate cholesterol-related gene expression responses. SREBPs binding sites are characterized by the presence of multiple target sequences as SRE, NFY and SP1, that can be arranged differently in different genes, so that it is not easy to identify the binding site on the basis of direct DNA sequence analysis. This paper presents a complete workflow based on a one-dimensional Convolutional Neural Network (CNN) model able to detect putative SREBPs binding sites irrespective of target elements arrangements. The strategy is based on the recognition of SRE linked (less than 250 bp) to NFY sequences according to chromosomal localization derived from TF Immunoprecipitation (TF ChIP) experiments. The CNN is trained with several 100 bp sequences containing both SRE and NF-Y. Once trained, the model is used to predict the presence of SRE-NFY in the first 500 bp of all the known gene promoters. Finally, genes are grouped according to biological process and the processes enriched in genes containing SRE-NFY in their promoters are analyzed in details. This workflow allowed to identify biological processes enriched in SRE containing genes not directly linked to cholesterol metabolism and possible novel DNA patterns able to fill in for missing classical SRE sequences., Competing Interests: All the authors declare no conflict of interest regarding this submission.

Although many protocols have been previously developed for genomic DNA (gDNA) extraction from S. cerevisiae, to take advantage of recent advances in laboratory automation and DNA-barcode sequencing, there is a need for automated methods that can provide high-quality gDNA at high efficiency. Here, we describe and demonstrate a fully automated protocol that includes five basic steps: cell wall and RNA digestion, cell lysis, DNA binding to magnetic beads, washing with ethanol, and elution. Our protocol avoids the use of hazardous reagents (e.g., phenol, chloroform), glass beads for mechanical cell disruption, or incubation of samples at 100°C (i.e., boiling). We show that our protocol can extract gDNA with high efficiency both from cells grown in liquid culture and from colonies grown on agar plates. We also show results from gel electrophoresis that demonstrate that the resulting gDNA is of high quality. [ABSTRACT FROM AUTHOR]

N6-methyladenine (6mA) is an important DNA modification form associated with a wide range of biological processes. Identifying accurately 6mA sites on a genomic scale is crucial for under-standing of 6mA's biological functions. However, the existing experimental techniques for detecting 6mA sites are cost-ineffective, which implies the great need of developing new computational methods for this problem. In this paper, we developed, without requiring any prior knowledge of 6mA and manually crafted sequence features, a deep learning framework named Deep6mA to identify DNA 6mA sites, and its performance is superior to other DNA 6mA prediction tools. Specifically, the 5-fold cross-validation on a benchmark dataset of rice gives the sensitivity and specificity of Deep6mA as 92.96% and 95.06%, respectively, and the overall prediction accuracy is 94%. Importantly, we find that the sequences with 6mA sites share similar patterns across different species. The model trained with rice data predicts well the 6mA sites of other three species: Arabidopsis thaliana, Fragaria vesca and Rosa chinensis with a prediction accuracy over 90%. In addition, we find that (1) 6mA tends to occur at GAGG motifs, which means the sequence near the 6mA site may be conservative; (2) 6mA is enriched in the TATA box of the promoter, which may be the main source of its regulating downstream gene expression., Competing Interests: The authors have declared that no competing interests exist.

We present OpenAWSEM and Open3SPN2, new cross-compatible implementations of coarse-grained models for protein (AWSEM) and DNA (3SPN2) molecular dynamics simulations within the OpenMM framework. These new implementations retain the chemical accuracy and intrinsic efficiency of the original models while adding GPU acceleration and the ease of forcefield modification provided by OpenMM's Custom Forces software framework. By utilizing GPUs, we achieve around a 30-fold speedup in protein and protein-DNA simulations over the existing LAMMPS-based implementations running on a single CPU core. We showcase the benefits of OpenMM's Custom Forces framework by devising and implementing two new potentials that allow us to address important aspects of protein folding and structure prediction and by testing the ability of the combined OpenAWSEM and Open3SPN2 to model protein-DNA binding. The first potential is used to describe the changes in effective interactions that occur as a protein becomes partially buried in a membrane. We also introduced an interaction to describe proteins with multiple disulfide bonds. Using simple pairwise disulfide bonding terms results in unphysical clustering of cysteine residues, posing a problem when simulating the folding of proteins with many cysteines. We now can computationally reproduce Anfinsen's early Nobel prize winning experiments by using OpenMM's Custom Forces framework to introduce a multi-body disulfide bonding term that prevents unphysical clustering. Our protein-DNA simulations show that the binding landscape is funneled towards structures that are quite similar to those found using experiments. In summary, this paper provides a simulation tool for the molecular biophysics community that is both easy to use and sufficiently efficient to simulate large proteins and large protein-DNA systems that are central to many cellular processes. These codes should facilitate the interplay between molecular simulations and cellular studies, which have been hampered by the large mismatch between the time and length scales accessible to molecular simulations and those relevant to cell biology., Competing Interests: The authors have declared that no competing interests exist.

Determining the concentration of nucleic acids in biological samples precisely and reliably still is a challenge. In particular when only very small sample quantities are available for analysis, the established fluorescence-based methods give insufficient results. Photobleaching is seen as the main reason for this. In this paper we present a method to correct for the photobleaching effect. Using confocal microscopy with single molecule sensitivity, we derived calibration curves from DNA solutions with defined fragment length. We analyzed dilution series over a wide range of concentrations (1 pg/μl-1000 pg/μl) and measured their specific diffusion coefficients employing fluorescence correlation spectroscopy. Using this information, we corrected the measured fluorescence intensity of the calibration solutions for photobleaching effects. We evaluated our method by analyzing a series of DNA mixtures of varying composition. For fragments smaller than 1000 bp, our method allows to determine sample concentrations with high precision in very small sample quantities (< 2 μl with concentrations < 20 pg/μl). Once the technical parameters are determined and remain stable in an established process, our improved calibration method will make measuring molecular biological samples of unknown sequence composition more efficient, accurate and sample-saving than previous methods., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Stroke is a prominent cause of death, disability, and dementia in sub-Saharan Africa (SSA). The Stroke Investigative Research and Education Network works collaboratively with stroke survivors and individuals serving as community controls to comprehensively characterize the genomic, sociocultural, economic and behavioral risk factors for stroke in SSA. Purpose: In this paper, we aim to: i) explore the attitudes, beliefs, and practices related to stroke in Ghana and Nigeria using the process of qualitative description; and ii) propose actions for future research and community-based participation and education. Methods: Stroke survivors, their caregivers, health care professionals, and community representatives and faith-based leaders participated in one of twenty-six focus groups, which qualitatively explored community beliefs, attitudes and practices related to stroke in Ghana and Nigeria. Arthur Kleinman’s Explanatory Model of Illness and the Social Ecological Model guided the questions and/or thematic analysis of the qualitative data. We hereby describe our focus group methods and analyses of qualitative data, as well as the findings and suggestions for improving stroke outcomes. Results and discussion: The major findings illustrate the fears, causes, chief problems, treatment, and recommendations related to stroke through the views of the participants, as well as recommendations for working effectively with the SIREN communities. Findings are compared to SIREN quantitative data and other qualitative studies in Africa. As far as we are aware, this is the first paper to qualitatively explore and contrast community beliefs, attitudes, and practices among stroke survivors and their caregivers, community and faith-based leaders, and health professionals in multiple communities within Nigeria and Ghana. [ABSTRACT FROM AUTHOR]

This paper applies objective methods to explore the technological origins of the widely acclaimed CRISPR breakthrough in the technological domain of genome engineering. Previously developed patent search techniques are first used to recover a set of patents that well-represent the genome editing domain before CRISPR. Main paths are then determined from the citation network associated with this patent set allowing identification of the three major knowledge trajectories. The most significant of these trajectories for CRISPR involves the core of genome editing with less significant trajectories involving cloning and endonuclease specific developments. The major patents on the core trajectory are consistent with qualitative expert knowledge of the topical area. A second set of patents that we call the CRISPR roots are obtained by finding the patents directly cited by the recent CRISPR patents along with patents cited by that set of patents. We find that the CRISPR roots contain 8 key patents from the genome engineering main path associated with restriction endonucleases and the expected strong connection of CRISPR to prior genome editing technology such as Zn finger nucleases. Nonetheless, analysis of the full CRISPR roots shows that a very wide array of technological knowledge beyond genome engineering has contributed to achieving the CRISPR breakthrough. Such breadth in origins is not surprising since “spillover” is generally perceived as important and previous qualitative studies of CRISPR have shown not only technological breadth in origins but scientific breadth as well. In addition, we find that the estimated rate of functional performance improvement of the CRISPR roots set is about 9% per year compared to the genome engineering set (~4% per year). These estimates indicate below average rates of improvement and may indicate that CRISPR (and perhaps yet undiscovered) genome engineering developments could evolve in effectiveness over an upcoming long rather than short time period. [ABSTRACT FROM AUTHOR]

Fitting stochastic transmission models to electronic patient data can offer detailed insights into the transmission of healthcare-associated infections and improve infection control. Pathogen whole-genome sequencing may improve the precision of model inferences, but computational constraints have limited modelling applications predominantly to small datasets and specific outbreaks, whereas large-scale sequencing studies have mostly relied on simple rules for identifying/excluding plausible transmission. We present a novel approach for integrating detailed epidemiological data on patient contact networks in hospitals with large-scale pathogen sequencing data. We apply our approach to study Clostridioides difficile transmission using a dataset of 1223 infections in Oxfordshire, UK, 2007–2011. 262 (21% [95% credibility interval 20–22%]) infections were estimated to have been acquired from another known case. There was heterogeneity by sequence type (ST) in the proportion of cases acquired from another case with the highest rates in ST1 (ribotype-027), ST42 (ribotype-106) and ST3 (ribotype-001). These same STs also had higher rates of transmission mediated via environmental contamination/spores persisting after patient discharge/recovery; for ST1 these persisted longer than for most other STs except ST3 and ST42. We also identified variation in transmission between hospitals, medical specialties and over time; by 2011 nearly all transmission from known cases had ceased in our hospitals. Our findings support previous work suggesting only a minority of C. difficile infections are acquired from known cases but highlight a greater role for environmental contamination than previously thought. Our approach is applicable to other healthcare-associated infections. Our findings have important implications for effective control of C. difficile. Author summary: Preventing infections spreading in hospitals is a major priority for healthcare systems globally. Mathematical models can be used with electronic hospital records to reconstruct how infections spread, which in turn can help guide infection control interventions. Sequencing the genetic code (DNA) of the bacteria that cause infections can help to follow when transmission is occurring, as bacterial DNA from two patients infected from the same source will likely be very similar. Our paper describes a new statistical method for combining hospital records and sequencing data to track infections. We use our approach to study over 1200 infections of Clostridioides difficile (C. diff.), which is a common cause of diarrhoea in hospitals. We show that only a minority of infections are acquired from other unwell patients, but the amount of spread varies by the subtype of C. diff involved. We also show that different C. diff subtypes survive in the hospital environment for longer than others and may need enhanced control strategies. We also can detect differences in spread at different hospitals and show that by the end of the study we had largely eliminated transmission of C. diff from unwell patients in our hospitals. [ABSTRACT FROM AUTHOR]

The predominant clonal evolution (PCE) model seeks to formulate a common population genetics framework for all micropathogens (namely, parasitic protozoa, fungi and yeasts, bacteria, and viruses). It relies on a definition of clonality that is only based on population structure features (namely, strongly restrained genetic recombination). Its clear-cut properties make it of strong interest for applied and basic research, since it permits the definition of stable, clearly delimited units of analysis below the species level: clonal genotypes and discrete genetic subdivisions (“near-clades”). These units of analysis can be used for clinical and epidemiological studies, vaccine and drug design, species description, and evolutionary studies on natural and experimental populations. In this review, the evolutionary and population genetics background of the model will be only briefly mentioned, while considerable emphasis will be given to its practical significance for the study and control of neglected tropical diseases. The goal of the paper is to make this practical usefulness accessible to a broad audience of readers, including scientists who are not evolution specialists, such as epidemiologists, field scientists, and clinicians. For extensive developments about the evolutionary background of the model, see our previous papers [–]. Citations of these former articles lead to the many references quoted in them, which cannot be listed again here. [ABSTRACT FROM AUTHOR]

A working group established in the framework of the EUPHRESCO European collaborative project aimed to compare and validate diagnostic protocols for the detection of “Flavescence dorée” (FD) phytoplasma in grapevines. Seven molecular protocols were compared in an interlaboratory test performance study where each laboratory had to analyze the same panel of samples consisting of DNA extracts prepared by the organizing laboratory. The tested molecular methods consisted of universal and group-specific real-time and end-point nested PCR tests. Different statistical approaches were applied to this collaborative study. Firstly, there was the standard statistical approach consisting in analyzing samples which are known to be positive and samples which are known to be negative and reporting the proportion of false-positive and false-negative results to respectively calculate diagnostic specificity and sensitivity. This approach was supplemented by the calculation of repeatability and reproducibility for qualitative methods based on the notions of accordance and concordance. Other new approaches were also implemented, based, on the one hand, on the probability of detection model, and, on the other hand, on Bayes’ theorem. These various statistical approaches are complementary and give consistent results. Their combination, and in particular, the introduction of new statistical approaches give overall information on the performance and limitations of the different methods, and are particularly useful for selecting the most appropriate detection scheme with regards to the prevalence of the pathogen. Three real-time PCR protocols (methods M4, M5 and M6 respectively developed by Hren (2007), Pelletier (2009) and under patent oligonucleotides) achieved the highest levels of performance for FD phytoplasma detection. This paper also addresses the issue of indeterminate results and the identification of outlier results. The statistical tools presented in this paper and their combination can be applied to many other studies concerning plant pathogens and other disciplines that use qualitative detection methods. [ABSTRACT FROM AUTHOR]

Measurement of telomere length is crucial for the study of telomere maintenance and its role in molecular pathophysiology of diseases and in aging. Several methods are used to measure telomere length, the choice of which usually depends on the type and size of sample to be assayed, as well as cost and throughput considerations. The goal of this study was to investigate the factors that may influence the reliability of qPCR-based relative telomere length measurements in whole blood. Day to day intra-individual variability, types of blood anticoagulant, sample storage conditions, processing and site of blood draw were investigated. Two qPCR-based methods to measure telomere length (monoplex vs. multiplex) were also investigated and showed a strong correlation between them. Freezing and thawing of the blood and storage of the blood at 4°C for up to 4 days did not affect telomere length values. Telomere lengths in dried blood spots were significantly higher than both whole blood and peripheral mononuclear blood cells, and were highly correlated with both. We found that telomere length measurements were significantly higher in dried blood spots collected directly from fingertip prick compared to dried blood spots prepared with anticoagulated whole blood collected from the finger, and non-blotted whole blood taken from both finger and arm venipuncture. This suggests that DNA from cells blotted on paper is not equivalent to that collected from venipuncture whole blood, and caution should be taken when comparing between blood sample types. [ABSTRACT FROM AUTHOR]

A new method is introduced allowing seamless assembly of independent, functionally tested, blunt-end double strand nucleic acid parts (DNA fragments not supplied in vectors such as plasmids) into more complex biological devices (e.g. protein expression vectors) and higher order multi-device systems (e.g. biochemical pathways). Individual parts include bacterial selection markers and origins of replication, promoters useful in a variety of species, transcription terminators, shuttle sequences and a variety of "N" and "C" terminal solubility/affinity protein tags. Parts are not subjected to pre-assembly manipulation with nucleic acid modifying enzymes. Instead, they are simply mixed in appropriate pre-defined combinations and concentrations and then seamlessly linked into devices employing a specialized thermostable enzyme blend. Combinatorial assembly of parts is an inherent time-saving feature of the new method, in contrast to hierarchical binary assembly ("one part at a time") methods. This feature substantially simplifies and speeds optimization of device and system development. The versatility and functionality of the new method was shown by combinatorial assembly of parts into vector devices, one of which optimally expressed protein from a model gene. Also, a four-enzyme biosynthetic pathway system was re-created by combinatorial construction from parts and devices. Concepts discussed in this paper provide synthetic biologists, chemists and bio-engineers with improved and expanded capability to create novel biological molecules and systems., Competing Interests: The authors confirm that our commercial affiliation Agilent Technologies Inc does not alter our adherence to all PLOS ONE policies on sharing data and materials.

Bacteria contain several nucleoid-associated proteins that organize their genomic DNA into the nucleoid by bending, wrapping or bridging DNA. The Histone-like Nucleoid Structuring protein H-NS found in many Gram-negative bacteria is a DNA bridging protein and can structure DNA by binding to two separate DNA duplexes or to adjacent sites on the same duplex, depending on external conditions. Several nucleotide sequences have been identified to which H-NS binds with high affinity, indicating H-NS prefers AT-rich DNA. To date, highly detailed structural information of the H-NS DNA complex remains elusive. Molecular simulation can complement experiments by modelling structures and their time evolution in atomistic detail. In this paper we report an exploration of the different binding modes of H-NS to a high affinity nucleotide sequence and an estimate of the associated rate constant. By means of molecular dynamics simulations, we identified three types of binding for H-NS to AT-rich DNA. To further sample the transitions between these binding modes, we performed Replica Exchange Transition Interface Sampling, providing predictions of the mechanism and rate constant of H-NS binding to DNA. H-NS interacts with the DNA through a conserved QGR motif, aided by a conserved arginine at position 93. The QGR motif interacts first with phosphate groups, followed by the formation of hydrogen bonds between acceptors in the DNA minor groove and the sidechains of either Q112 or R114. After R114 inserts into the minor groove, the rest of the QGR motif follows. Full insertion of the QGR motif in the minor groove is stable over several tens of nanoseconds, and involves hydrogen bonds between the bases and both backbone and sidechains of the QGR motif. The rate constant for the process of H-NS binding to AT-rich DNA resulting in full insertion of the QGR motif is in the order of 10(6) M-1s-1, which is rate limiting compared to the non-specific association of H-NS to the DNA backbone at a rate of 10(8) M-1s-1., Competing Interests: The authors have declared that no competing interests exist.

PCR-based amplification of annotated genes has allowed construction of expression clones at genome-scale using classical and recombination-based cloning technologies. However, genome-scale expression and purification of proteins for down-stream applications is often limited by challenges such as poor expression, low solubility, large size of multi-domain proteins, etc. Alternatively, DNA fragment libraries in expression vectors can serve as the source of protein fragments with each fragment encompassing a function of its whole protein counterpart. However, the random DNA fragmentation and cloning result in only 1 out of 18 clones being in the correct open-reading frame (ORF), thus, reducing the overall efficiency of the system. This necessitates the selection of correct ORF before expressing the protein fragments. This paper describes a highly efficient ORF selection system for DNA fragment libraries, which is based on split beta-lactamase protein fragment complementation. The system has been designed to allow seamless transfer of selected DNA fragment libraries into any downstream vector systems using a restriction enzyme-free cloning strategy. The strategy has been applied for the selection of ORF using model constructs to show near 100% selection of the clone encoding correct ORF. The system has been further validated by construction of an ORF-selected DNA fragment library of 30 genes of M. tuberculosis. Further, we have successfully demonstrated the cytosolic expression of ORF-selected protein fragments in E. coli. [ABSTRACT FROM AUTHOR]

Liquid biopsy has become widely applied in clinical medicine along with the progress in innovative technologies, such as next generation sequencing, but the origin of circulating tumor DNA (ctDNA) has not yet been precisely established. We reported bimodal peaks of long fragment circulating free DNA (cfDNA) of 5 kb and short fragment cfDNA of 170 bp in patients with advanced lung cancer, and both contained ctDNA. In this paper, we demonstrate that the total amount of cfDNA is higher when patients with lung cancer have extrathoracic metastases, and the amount of long fragment cfDNA is significantly higher in those patients. To investigate the origin of long fragment cfDNA, conditioned media isolated from lung cancer cell lines was fractionated. Long fragment cfDNA was found concomitant with extracellular vesicles (EVs), but short fragment cfDNA was not observed in any fractions. However, in peripheral blood from a metastatic animal model both fragments were detected even with those same lung cancer cell lines. In human plasma samples, long fragment cfDNA was observed in the same fraction as that from conditioned media, and short fragment cfDNA existed in the supernatant after centrifugation at 100,000g. Concentration of ctDNA in the supernatant was two times higher than that in plasma isolated by the conventional procedure. Long fragment cfDNA associated with tumor progression might therefore be released into peripheral blood, and it is possible that the long fragment cfDNA escapes degradation by co-existing with EVs. Examination of the biological characteristics of long fragment cfDNA is a logical subject of further investigation. [ABSTRACT FROM AUTHOR]

The relevance of extracellular DNA (eDNA) in the soil ecosystem is becoming more and more evident to the scientific community by the progressive discovery of functions accompanying to natural gene transformation. However, despite the increased number of published articles dedicated to eDNA in soil, so far only few are focused on its single stranded form (eDNAss). The present paper is the first to investigate the quantitative relevance of eDNAss in the total soil eDNA pool, discriminating between its linear (eDNAssl) and circular (eDNAssc) forms and the respective weakly (wa) and tightly (ta) adsorbed fractions. The results showed the prevalence of eDNAss and its linear form in both the total soil eDNA pool and its wa and ta fractions. Both of the eDNAss fractions (linear and circular) were characterized by small fragments. [ABSTRACT FROM AUTHOR]

SCCmec elements are very important mobile genetic elements in Staphylococci that carry beta-lactam resistance genes mecA/mecC, recombinase genes and a variety of accessory genes. Twelve main types and a couple of variants have yet been described. In addition, there are also other SCC elements harbouring other markers. In order to subtype strains of methicillin-resistant S. aureus (MRSA) based on variations within their SCCmec elements, 86 markers were selected from published SCC sequences for an assay based on multiplexed primer extension reactions followed by hybridisation to the specific probes. These included mecA/mecC, fusC, regulatory genes, recombinase genes, genes from ACME and heavy metal resistance loci as well as several genes of unknown function. Hybridisation patterns for published genome or SCC sequences were theoretically predicted. For validation of the microarray based assay and for stringent hybridisation protocol optimization, real hybridization experiments with fully sequenced reference strains were performed modifying protocols until yielded the results were in concordance to the theoretical predictions. Subsequently, 226 clinical isolates from two hospitals in the city of Dresden, Germany, were characterised in detail. Beside previously described types and subtypes, a wide variety of additional SCC types or subtypes and pseudoSCC elements were observed as well as numerous composite elements. Within the study collection, 61 different such elements have been identified. Since hybridisation cannot recognise the localisation of target genes, gene duplications or inversions, this is a rather conservative estimate. Interestingly, some widespread epidemic strains engulf distinct variants with different SCCmec subtypes. Notable examples are ST239-MRSA-III, CC5-, CC22-, CC30-, and CC45-MRSA-IV or CC398-MRSA-V. Conversely, identical SCC elements were observed in different strains with SCCmec IVa being spread among the highest number of Clonal Complexes. The proposed microarray can help to distinguish isolates that appear similar or identical by other typing methods and it can be used as high-throughput screening tool for the detection of putative new SCC types or variants that warrant further investigation and sequencing. The high degree of diversity of SCC elements even within so-called strains could be helpful for epidemiological typing. It also raises the question on scale and speed of the evolution of SCC elements. [ABSTRACT FROM AUTHOR]

The mitochondrion is the power plant of the eukaryotic cell, and tRNAs are the fundamental components of its translational machinery. In the present paper, the evolution of mitochondrial tRNAs was investigated in the Cetacea, a clade of Cetartiodactyla that retuned to water and thus had to adapt its metabolism to a different medium than that of its mainland ancestors. Our analysis focussed on identifying the factors that influenced the evolution of Cetacea tRNA double-helix elements, which play a pivotal role in the formation of the secondary and tertiary structures of each tRNA and consequently manipulate the whole translation machinery of the mitochondrion. Our analyses showed that the substitution pathways in the stems of different tRNAs were influenced by various factors, determining a molecular evolution that was unique to each of the 22 tRNAs. Our data suggested that the composition, AT-skew, and GC-skew of the tRNA stems were the main factors influencing the substitution process. In particular, the range of variation and the fluctuation of these parameters affected the fate of single tRNAs. Strong heterogeneity was observed among the different species of Cetacea. Finally, it appears that the evolution of mitochondrial tRNAs was also shaped by the environments in which the Cetacean taxa differentiated. This latter effect was particularly evident in toothed whales that either live in freshwater or are deep divers. [ABSTRACT FROM AUTHOR]

Stock theft is a persistent and widespread problem affecting farmers in the Eastern Cape Province of South Africa. This study aimed to explore farmers' perceptions of stock theft in the region. A mixed methods approach was used to collect data. 192 pre-tested questionnaires were collected from a sample of farmers in three districts in the province. The descriptive and chi-square test was used to test the associations between demographic profile statistically, knowledge of stock theft, reported stock theft cases, the economic impact of stock theft, and stock theft control. According to the findings, stock theft is significantly more likely to occur during the winter season (P < 0.05). About 94.8% of farmers are in the communal farming sector in the three districts visited. Furthermore, 81.2% of the respondents believe that the government needs to do more to combat the spread of stock theft. This study also revealed that most respondents (88.6%) agree that branding and tattooing should be made available to all registered farmers, while 53.1% believe that forensic deoxyribonucleic acid should not be used to control stock theft at crime scenes. This study highlights farmers' perceptions and knowledge of stock theft to enable policymakers to develop targeted interventions and strategies. Policing strategy must be adaptive and technology-driven to fast-track detection, prevention, and reduction of stuck theft crime. [ABSTRACT FROM AUTHOR]

In this paper we have theoretically predicted a novel pathway for the mutagenic tautomerization of the classical A∙T DNA base pairs in the free state, the Watson-Crick A·Т(WC), reverse Watson-Crick A·Т(rWC), Hoogsteen A·Т(H) and reverse Hoogsteen A·Т(rH) pairs, via sequential proton transfer accompanied by a significant change in the mutual orientation of the bases. Quantum-mechanical (QM) calculations were performed at the MP2/aug-cc-pVDZ//B3LYP/6-311++G(d,p) level in vacuum phase, along with Bader's quantum theory of Atoms in Molecules (QTAIM). These processes involve transition states (TSs) with quasi-orthogonal structures (symmetry C1), which are highly polar, tight ion pairs (A-, N6H2-deprotonated)∙(T+, O4/O2-protonated). Gibbs free energies of activation for the A∙T(WC) / A∙T(rWC) ↔ A*∙Т(rwWC) / A*∙Т(wWC) tautomeric transitions (~43.5 kcal∙mol-1) are lower than for the A∙T(H) / A∙T(rH) ↔ A*N7∙Т(rwH) / A*N7∙Т(wH) tautomerisations (~53.0 kcal∙mol-1) (rare tautomers are marked by an asterisk; w-wobble configured tautomerisation products). The (T)N3+H⋯N1-(A), (T)O4+H⋯N1-(A) / (T)N3+H⋯N1-(A) and (T)O2+H⋯N1-(A) H-bonds are found in the transition states TSA-·T+A·T(WC)↔A*·T(rwWC) / TSA-·T+A·T(rWC)↔A*·T(wWC). However, in the transition state TSA-·T+A·Т(H)↔A*N7·T(rwH) / TSA-·T+A·Т(rH)↔A*N7·T(wH), the (T)N3+H⋯N7-(A), (T)O4+H⋯N7-(A) / (T)N3+H⋯N7-(A) and (T)O2+H⋯N7-(A) H-bonds are supplemented by the attractive (T)O4+/O2+⋯N6-(A) van der Waals contacts. It was demonstrated that the products of the tautomerization of the classical A∙T DNA base pairs-A*∙Т(rwWC), A*N7∙Т(rwH) and A*N7∙Т(wH) (symmetry Cs)-further transform via double proton transfer into the energetically favorable wobble A∙T*(rwWC), A∙T*(rwH) and A∙T*O2(wH) base mispairs (symmetry Cs)., Competing Interests: The authors have declared that no competing interests exist.

There is a growing necessity to integrate morphological and genetic studies. This paper proposes a new technique that allows DNA extraction of arthropods while still keeping intact the entire morphology of the specimens. The technique uses Proteinase K to dissolve protein tissues and preserve the chitinous exoskeleton of specimens. The method is fast, cheap, non-toxic, and allows for good morphological preparations of specimens retaining much of their tridimensional structure. The methodology works fine with specimens preserved in different kinds of media, such as for dry (pinned) specimens, and specimens preserved in Ethanol. In addition, it allows the extraction of DNA from fresh specimens, as well as from specimens preserved for a long time. The technique works well for morphological studies alone, but allows the generation of an associated genomic library at an individual-scale. Among the advantages of the new technique is the possibility of extracting DNA from the entire specimen (necessary for the study of diseases transmitted by arthropod vectors), while still keeping the morphology intact for correct taxonomic identification. In addition, in comparison with methods that extract DNA from small tissue samples (e.g., from legs or wings), the method allows for the extraction of a larger amount of DNA and is better suited for small specimens.

DNA variants that alter gene expression contribute to variation in many phenotypic traits. In particular, trans-acting variants, which are often located on different chromosomes from the genes they affect, are an important source of heritable gene expression variation. However, our knowledge about the identity and mechanism of causal trans-acting variants remains limited. Here, we developed a fine-mapping strategy called CRISPR-Swap and dissected three expression quantitative trait locus (eQTL) hotspots known to alter the expression of numerous genes in trans in the yeast Saccharomyces cerevisiae. Causal variants were identified by engineering recombinant alleles and quantifying the effects of these alleles on the expression of a green fluorescent protein-tagged gene affected by the given locus in trans. We validated the effect of each variant on the expression of multiple genes by RNA-sequencing. The three variants differed in their molecular mechanism, the type of genes they reside in, and their distribution in natural populations. While a missense leucine-to-serine variant at position 63 in the transcription factor Oaf1 (L63S) was almost exclusively present in the reference laboratory strain, the two other variants were frequent among S. cerevisiae isolates. A causal missense variant in the glucose receptor Rgt2 (V539I) occurred at a poorly conserved amino acid residue and its effect was strongly dependent on the concentration of glucose in the culture medium. A noncoding variant in the conserved fatty acid regulated (FAR) element of the OLE1 promoter influenced the expression of the fatty acid desaturase Ole1 in cis and, by modulating the level of this essential enzyme, other genes in trans. The OAF1 and OLE1 variants showed a non-additive genetic interaction, and affected cellular lipid metabolism. These results demonstrate that the molecular basis of trans-regulatory variation is diverse, highlighting the challenges in predicting which natural genetic variants affect gene expression. Author summary: Differences in the DNA sequence of individual genomes contribute to differences in many traits, such as appearance, physiology, and the risk for common diseases. An important group of these DNA variants influences how individual genes across the genome are turned on or off. In this paper, we describe a strategy for identifying such "trans-acting" variants in different strains of baker's yeast. We used this strategy to reveal three single DNA base changes that each influences the expression of dozens of genes. These three DNA variants were very different from each other. Two of them changed the protein sequence, one in a transcription factor and the other in a sugar sensor. The third changed the expression of an enzyme, a change that in turn caused other genes to alter their expression. One variant existed in only a few yeast isolates, while the other two existed in many isolates collected from around the world. This diversity of DNA variants that influence the expression of many other genes illustrates how difficult it is to predict which DNA variants in an individual's genome will have effects on the organism. [ABSTRACT FROM AUTHOR]

Hydroxymethylcytosine (5hmC) methylation is a well-known epigenetic mark that is involved in gene regulation and may impact genome stability. To investigate a possible role of 5hmC in cancer development and progression, one must be able to detect and quantify its level first. In this paper, we address the issue of 5hmC detection at a single base resolution, starting with consideration of the well-established 5hmC measure Δβ and, in particular, with an analysis of its properties, both analytically and empirically. Then we propose several alternative hydroxymethylation measures and compare their properties with those of Δβ. In the absence of a gold standard, the (pairwise) resemblance of those 5hmC measures to Δβ is characterized by means of a similarity analysis and relative accuracy analysis. All results are illustrated on matched healthy and cancer tissue data sets as derived by means of bisulfite (BS) and oxidative bisulfite converting (oxBS) procedures. [ABSTRACT FROM AUTHOR]

The BARD1 protein, which heterodimerizes with BRCA1, is encoded by a known breast cancer susceptibility gene. While several BARD1 variants have been identified as pathogenic, many more missense variants exist that do not occur frequently enough to assign a clinical risk. In this paper, whole exome sequencing of over 10,000 cancer samples from 33 cancer types identified from somatic mutations and loss of heterozygosity in tumors 76 potentially cancer-associated BARD1 missense and truncation variants. These variants were tested in a functional assay for homology-directed repair (HDR), as HDR deficiencies have been shown to correlate with clinical pathogenicity for BRCA1 variants. From these 76 variants, 4 in the ankyrin repeat domain and 5 in the BRCT domain were found to be non-functional in HDR. Two known benign variants were found to be functional in HDR, and three known pathogenic variants were non-functional, supporting the notion that the HDR assay can be used to predict the clinical risk of BARD1 variants. The identification of HDR-deficient variants in the ankyrin repeat domain indicates there are DNA repair functions associated with this domain that have not been closely examined. In order to examine whether BARD1-associated loss of HDR function results in DNA damage sensitivity, cells expressing non-functional BARD1 variants were treated with ionizing radiation or cisplatin. These cells were found to be more sensitive to DNA damage, and variations in the residual HDR function of non-functional variants did not correlate with variations in sensitivity. These findings improve the understanding of BARD1 functional domains in DNA repair and support that this functional assay is useful for predicting the cancer association of BARD1 variants. [ABSTRACT FROM AUTHOR]

Context: Over the last decade, design patterns have been used extensively to generate reusable solutions to frequently encountered problems in software engineering and object oriented programming. A design pattern is a repeatable software design solution that provides a template for solving various instances of a general problem. Objective: This paper describes a new method for pattern mining, isolating design patterns and relationship between them; and a related tool, DLA-DNA for all implemented pattern and all projects used for evaluation. DLA-DNA achieves acceptable precision and recall instead of other evaluated tools based on distributed learning automata (DLA) and deoxyribonucleic acid (DNA) sequences alignment. Method: The proposed method mines structural design patterns in the object oriented source code and extracts the strong and weak relationships between them, enabling analyzers and programmers to determine the dependency rate of each object, component, and other section of the code for parameter passing and modular programming. The proposed model can detect design patterns better that available other tools those are Pinot, PTIDEJ and DPJF; and the strengths of their relationships. Results: The result demonstrate that whenever the source code is build standard and non-standard, based on the design patterns, then the result of the proposed method is near to DPJF and better that Pinot and PTIDEJ. The proposed model is tested on the several source codes and is compared with other related models and available tools those the results show the precision and recall of the proposed method, averagely 20% and 9.6% are more than Pinot, 27% and 31% are more than PTIDEJ and 3.3% and 2% are more than DPJF respectively. Conclusion: The primary idea of the proposed method is organized in two following steps: the first step, elemental design patterns are identified, while at the second step, is composed to recognize actual design patterns. [ABSTRACT FROM AUTHOR]

Approximate string matching with k-differences has a number of practical applications, ranging from pattern recognition to computational biology. This paper proposes an efficient memory-access algorithm for parallel approximate string matching with k-differences on Graphics Processing Units (GPUs). In the proposed algorithm, all threads in the same GPUs warp share data using warp-shuffle operation instead of accessing the shared memory. Moreover, we implement the proposed algorithm by exploiting the memory structure of GPUs to optimize its performance. Experiment results for real DNA packages revealed that the performance of the proposed algorithm and its implementation archived up to 122.64 and 1.53 times compared to that of sequential algorithm on CPU and previous parallel approximate string matching algorithm on GPUs, respectively.

Insertion sequence elements (IS elements) are proposed to play major roles in shaping the genetic and phenotypic landscapes of prokaryotic cells. Recent evidence has raised the possibility that environmental stress conditions increase IS hopping into new sites, and often such hopping has the phenotypic effect of relieving the stress. Although stress-induced targeted mutations have been reported for a number of E. coli genes, the glpFK (glycerol utilization) and the cryptic bglGFB (β-glucoside utilization) systems are among the best characterized where the effects of IS insertion-mediated gene activation are well-characterized at the molecular level. In the glpFK system, starvation of cells incapable of utilizing glycerol leads to an IS5 insertion event that activates the glpFK operon, and enables glycerol utilization. In the case of the cryptic bglGFB operon, insertion of IS5 (and other IS elements) into a specific region in the bglG upstream sequence has the effect of activating the operon in both growing cells, and in starving cells. However, a major unanswered question in the glpFK system, the bgl system, as well as other examples, has been why the insertion events are promoted at specific locations, and how the specific stress condition (glycerol starvation for example) can be mechanistically linked to enhanced insertion at a specific locus. In this paper, we show that a specific DNA structural feature (superhelical stress-induced duplex destabilization, SIDD) is associated with "stress-induced" IS5 insertion in the glpFK, bglGFB, flhDC, fucAO and nfsB systems. We propose a speculative mechanistic model that links specific environmental conditions to the unmasking of an insertional hotspot in the glpFK system. We demonstrate that experimentally altering the predicted stability of a SIDD element in the nfsB gene significantly impacts IS5 insertion at its hotspot.

The environmental DNA (eDNA) method is a detection technique that is rapidly gaining credibility as a sensitive tool useful in the surveillance and monitoring of invasive and threatened species. Because eDNA analysis often deals with small quantities of short and degraded DNA fragments, methods that maximize eDNA recovery are required to increase detectability. In this study, we performed experiments at different stages of the eDNA analysis to show which combinations of methods give the best recovery rate for eDNA. Using Oriental weatherloach (Misgurnus anguillicaudatus) as a study species, we show that various combinations of DNA capture, preservation and extraction methods can significantly affect DNA yield. Filtration using cellulose nitrate filter paper preserved in ethanol or stored in a -20°C freezer and extracted with the Qiagen DNeasy kit outperformed other combinations in terms of cost and efficiency of DNA recovery. Our results support the recommendation to filter water samples within 24hours but if this is not possible, our results suggest that refrigeration may be a better option than freezing for short-term storage (i.e., 3-5 days). This information is useful in designing eDNA detection of low-density invasive or threatened species, where small variations in DNA recovery can signify the difference between detection success or failure.