DNA Sequence Research Articles

A new species of Pythium hydnosporum as a pathogen associated with vine decline of melon (Cucumis melo L.) in Iraq

Several pathogenic fungi have become an important vine decline of cantaloupe melone (Cucumis melo L.). In 2021, root rot on cantaloupe was observed during the harvest phase in the fields in Penjwen and Shahrazoor in Sulaimani governorate of Northern Kurdistan region Iraq. This present study was conducted to isolate the causal agent of fungi in lesions of root rot of cantaloupe. A total of 16 fungal isolates were obtained of which isolates Monosporascus cannonballus, M. eutypoides, Acremonium vitellinium, Fusarium oxysporium, F. equiseti, Macrophomina phaseolina, Saccharomyces kudriavzevii, F. robinianum, Rhizopus arrhizus, Botrytis cinerea, Cytospora eucalypticola, F. falciforme, Pythium hydnosporum, Alternaria tenuissima, Rhizoctonia solani and Phytophthora colocasiae were obtained from root melon. Identification of all fungal isolates was based on using both morphological characteristics and molecular analysis, on internal transcribed spacer (ITS1, ITS4, LSU) are primers that are used for identification. DNA sequences of the fungal pathogen were identified as Pythium hydnosporum a new pathogenic fungi causal agent of cantaloupe vine decline. A pathogenicity test was conducted to verify Koch's postulates and P. hydnosporum was observed to cause root rot of melon, symptoms of the disease were similar to those seen in the field.

Bacterial and Symbiodiniaceae communities’ variation in corals with distinct traits and geographical distribution

Coral microbiomes play crucial roles in holobiont homeostasis and adaptation. The host’s ability to populate broad ecological niches and to cope with environmental changes seems to be related to the flexibility of the coral microbiome. By means of high-throughput DNA sequencing we characterized simultaneously both bacterial (16S rRNA) and Symbiodiniaceae (ITS2) communities of four reef-building coral species (Mussismilia braziliensis, Mussismilia harttii, Montastraea cavernosa, and Favia gravida) that differ in geographic distribution and niche specificity. Samples were collected in a marginal reef system (Abrolhos, Brazil) in four sites of contrasting irradiance and turbidity. Biological filters governed by the host are important in shaping corals’ microbiome structure. More structured associated microbial communities by reef site tend to occur in coral species with broader geographic and depth ranges, especially for Symbiodiniaceae, whereas the endemic and habitat-specialist host, M. braziliensis, has relatively more homogenous bacterial communities with more exclusive members. Our findings lend credence to the hypothesis that higher microbiome flexibility renders corals more adaptable to diverse environments, a trend that should be investigated in more hosts and reef areas.

RNAi epimutations conferring antifungal drug resistance are inheritable.

Epimutations modify gene expression and lead to phenotypic variation while the encoding DNA sequence remains unchanged. Epimutations mediated by RNA interference (RNAi) and/or chromatin modifications can confer antifungal drug resistance and may impact virulence traits in fungi (1-5). However, whether these epigenetic modifications can be transmitted across generations following sexual reproduction was unclear. This study demonstrates that RNAi epimutations conferring antifungal drug resistance are transgenerationally inherited in the human fungal pathogen Mucor circinelloides . Our research revealed that RNAi-based antifungal resistance follows a DNA sequence-independent, non-Mendelian inheritance pattern. Small RNAs (sRNAs) are the exclusive determinants of inheritance, transmitting drug resistance independently of other known repressive epigenetic modifications. Unique sRNA signature patterns can be traced through inheritance from parent to progeny, further supporting RNA as an alternative molecule for transmitting information across generations. This study marks a significant advance in understanding epigenetic inheritance, highlighting RNAi-exclusive epimutations as a widespread phenomenon in the pathogenic M. circinelloides species complex. Understanding how epimutations occur, propagate, and confer resistance may enable their detection in other eukaryotic pathogens, provide solutions for challenges posed by rising antimicrobial drug resistance (AMR), and also advance research on phenotypic adaptability and its evolutionary implications.

Results of a large-scale humeri pair matching study.

During the analysis of a commingled human remains assemblage, pair-matching is often conducted as part of the inventory to inform the DNA sampling strategy and to calculate the minimum or most likely number of individuals. As commingled assemblages become larger, pair-matching becomes more difficult, and it is unknown whether accuracy declines. Therefore, a study to determine the accuracy rates of visual pair-matching for multiple observers was conducted on a large, commingled human assemblage. The sample consisted of 580 left and right humeri (n = 287 and n = 293, respectively) from the commingled remains of the USS Oklahoma, which were undergoing analysis at the Defense POW/MIA Accounting Agency. Five anthropologists completed this study, determining humeri pairs with varying degrees of confidence and non-pairs. The overall precision was 81.0% and ranged from 74.1% to 95.9% for participants, including all confidence levels. When considering only confident matches, the overall precision for all participants increased to 91.4%.

HiCDiffusion - diffusion-enhanced, transformer-based prediction of chromatin interactions from DNA sequences

Prediction of chromatin interactions from DNA sequence has been a significant research challenge in the last couple of years. Several solutions have been proposed, most of which are based on encoder-decoder architecture, where 1D sequence is convoluted, encoded into the latent representation, and then decoded using 2D convolutions into the Hi-C pairwise chromatin spatial proximity matrix. Those methods, while obtaining high correlation scores and improved metrics, produce Hi-C matrices that are artificial - they are blurred due to the deep learning model architecture. In our study, we propose the HiCDiffusion, sequence-only model that addresses this problem. We first train the encoder-decoder neural network and then use it as a component of the diffusion model - where we guide the diffusion using a latent representation of the sequence, as well as the final output from the encoder-decoder. That way, we obtain the high-resolution Hi-C matrices that not only better resemble the experimental results - improving the Fréchet inception distance by an average of 11 times, with the highest improvement of 56 times - but also obtain similar classic metrics to current state-of-the-art encoder-decoder architectures used for the task.

Clinical Utility of Metagenomic Next-Generation Sequencing of Plasma Microbial Cell-Free DNA: a Single-Center Retrospective Study

Abstract Infectious diseases account for significant morbidity and mortality in healthcare, and metagenomic next-generation sequencing of plasma microbial cell-free DNA has emerged as a promising non-invasive diagnostic tool in this scenario. However, the available data on the real impact of this test in clinical practice is limited and conflicting. In this context, we performed a retrospective evaluation of the utilization of this test at our institution since February 17, 2019. A total of 76 tests were performed in this period in 68 different patients. The mean age of the patients was 9 years old, with 40 being male (58.82%) and 28 female (41.18%). The ordering specialties included infectious diseases (82.90%), hematology/oncology (9.21%), neurology (3.95%), pulmonology (2.63%), and dermatology (1.31%). The vast majority of the tests were ordered as inpatient (73 tests, 96.05%). 45 tests (59.21%) resulted as positive, of which 19 (42.22%) were polymicrobial. The tests were concordant with conventional microbiological studies 47 times (61.84%). Most tests (67 tests, 88.16%) had no significant clinical impact, 8 tests (10.53%) had a positive impact, and one test (1.31%) had an indeterminate impact. No tests were determined to have a negative clinical impact. Overall, our data is consistent with most studies which assessed the clinical utility of metagenomic next-generation sequencing of plasma microbial cell-free DNA in the literature, showing that testing did not have a significant impact on patient care for the majority of cases. There were some cases, however, in which testing showed a positive impact in escalating or de-escalating antibiotic therapy. In conclusion, despite being mostly a bystander, in selected cases and in the right clinical context, the test can be useful to guide antimicrobial treatment without causing significant harm. More studies are needed to further investigate the real utility of the test.

Mathematics behind the identifying CpG islands

The major objective of the paper is to review the theory for an hidden Markov model, a very general type of probabilistic model for sequences of symbols. In order for the hidden Markov model to be applicable to real-world applications, three key problems about the model must be addressed, and to do this, first we go over how to choose the best state sequence to explain an observation sequence, then we go over how to calculate the probability of an observation sequence, and finally we go over how to compute the maximization of the probability of the observation sequence. From these three angles, we review the mathematical concept behind the identification of CpG islands. The entire process and study of the outcomes have been tackled by examining both hypothetical and real DNA sequences side by side. We use well-known biological sequence analysis servers to carry out the experiment. Analytical and algorithmic approaches are compared while taking the hypothetical DNA sequence example into consideration.

Cytosine Methylation and Demethylation Model: Equilibrium Point and the Influence of Enzymes on Cytosine Forms Levels

Epigenetic changes, apart from changes in the DNA sequence, are one of the most important factors determining the fate of cells in living organisms. The main epigenetic change is cytosine methylation in relevant regions of DNA. Recently, we published an article in which we proposed a mathematical model of cytosine methylation and demethylation. In present work, we present an equilibrium analysis of this model. We present the conditions for its stability and aperiodic nature of the system’s free response. In particular, we focus on the influence of the levels of enzymes involved in cytosine methylation and demethylation on the equilibrium coordinates. The conclusions from this analysis are important in understanding the biological basis of the behavior of cells in living organisms.

Recombinant Plasminogen Activator of the Sandworm (Perinereis aibuhitensis) Expression in Escherichia coli.

As an essential thrombolytic agent, the tissue plasminogen activator receives increasing attention due to its longer half-life, lower immunogenicity, and easier administration, which are superior to other thrombolytic agents. In this study, the isolated and purified plasminogen activator from the sandworm (Perinereis aibuhitensis) was expressed in E. coli (Escherichia coli) to investigate its potential for simplifying the development process. The sandworm plasminogen activator was previously successfully cloned and expressed in E. coli with low yield and activity in the culture supernatant. This low yield and activity prompted us to optimize its DNA sequence. Furthermore, to raise the efficiency in the separation of the target protein, the protein's solubility was enhanced by fusing it with maltose-binding protein (MBP) tags. Eventually, the fibrinolytic activity was successfully restored after digestion with tobacco etch virus (TEV) protease. This study provides an innovative method of efficiently expressing and purifying plasminogen activators from sandworm in E. coli and broadens its applications in therapeutic treatment of cardiovascular diseases, including thrombosis, stroke, and coronary atherosclerotic heart disease.

Revisiting ELISA with in situ amplification of biomarkers to boost its sensitivity

The enzyme-linked immunosorbent assay (ELISA) stands as a pivotal instrument in diagnostics and biomedical research, widely used for the detection and quantification of specific proteins associated with diseases. Despite its critical role, ELISA's ability to identify low-concentration proteins remains a significant challenge, hindering its effectiveness in early diagnostics, an area of growing importance. This study introduces a novel approach termed nucleic acid-templated amplification (NATA) to enhance ELISA's sensitivity without significantly modifying the common practices of the present ELISA. Drawing inspiration from viral proliferation mechanisms, our approach integrates a cell-free protein synthesis step using DNA sequences that encode the target biomarkers. This approach enables an in situ 'amplification' of target proteins during ELISA, boosting their concentrations to detectable levels. Our results demonstrate a substantial enhancement in sensitivity, with ELISA now being able to detect protein biomarkers at femtomolar concentrations—approaching the sensitivity range typically reserved for PCR in nucleic acid analysis. This breakthrough not only broadens ELISA’s applicability in early disease diagnosis and monitoring therapeutic interventions but also marks a significant advancement in protein biomarker detection technology. With its modular design, our method also offers high versatility and sensitivity, offering an advanced approach for protein detection in clinical diagnostics and research.

Competition between homologous chromosomal DNA and exogenous donor DNA to repair CRISPR/Cas9-induced double-strand breaks in Aspergillus niger

BackgroundAspergillus niger is well-known for its high protein secretion capacity and therefore an important cell factory for homologous and heterologous protein production. The use of a strong promoter and multiple gene copies are commonly used strategies to increase the gene expression and protein production of the gene of interest (GOI). We recently presented a two-step CRISPR/Cas9-mediated approach in which glucoamylase (glaA) landing sites (GLSs) are introduced at predetermined sites in the genome (step 1), which are subsequently filled with copies of the GOI (step 2) to achieve high expression of the GOI.ResultsHere we show that in a ku70 defective A. niger strain (Δku70), thereby excluding non-homologous end joining (NHEJ) as a mechanism to repair double-stranded DNA breaks (DSBs), the chromosomal glaA locus or homologous GLSs can be used to repair Cas9-induced DSBs, thereby competing with the integration of the donor DNA containing the GOI. In the absence of exogenously added donor DNA, the DSBs are repaired with homologous chromosomal DNA located on other chromosomes (inter-chromosomal repair) or, with higher efficiency, by a homologous DNA fragment located on the same chromosome (intra-chromosomal repair). Single copy inter-chromosomal homology-based DNA repair was found to occur in 13–20% of the transformants while 80–87% of the transformants were repaired by exogenously added donor DNA. The efficiency of chromosomal repair was dependent on the copy number of the potential donor DNA sequences in the genome. The presence of five homologous DNA sequences, resulted in an increased number (35–61%) of the transformants repaired by chromosomal DNA. The efficiency of intra-chromosomal homology based DSB repair in the absence of donor DNA was found to be highly preferred (85–90%) over inter-chromosomal repair. Intra-chromosomal repair was also found to be the preferred way of DNA repair in the presence of donor DNA and was found to be locus-dependent.ConclusionThe awareness that homologous chromosomal DNA repair can compete with donor DNA to repair DSB and thereby affecting the efficiency of multicopy strain construction using CRISPR/Cas9-mediated genome editing is an important consideration to take into account in industrial strain design.

Phage Display Selection of Antibody Libraries: Screening of Selected Binders.

Phage display selection of antibody libraries is a powerful method for generating and evolving monoclonal antibodies. The pComb3 phagemid family of phage display vectors facilitates the mining of antibody libraries in Fab format from human and nonhuman antibody repertoires. Here, we describe the screening for monoclonal Fab binders after selection of a polyclonal pool of Fab binders to an antigen of interest, with the goal of identifying and sequencing monoclonal antibodies that bind the antigen with high affinity and specificity. The screening cascade involves a phage ELISA, followed by a crude Fab ELISA and DNA fingerprinting and sequencing. The protocol outlines phage and crude Fab ELISAs using purified antigen immobilized on microplates, native antigen expressed on eukaryotic cells, or both.

Identification of mutations in canine oral mucosal melanomas by exome sequencing and comparison with human melanomas

Oral mucosal melanomas (OMMs) are aggressive neoplasms commonly found in dogs but rare in humans. Utilizing whole exome sequencing (WES), which focuses on protein-coding regions to reveal mutation profiles, we conducted a comparative analysis of canine OMM and human melanomas. This study involved DNA extraction, exome enrichment, and sequencing from three canine OMM cell lines (CMGD-2, CMGD-5, TLM-1), five canine OMM frozen samples, a human OMM cell line (MEMO), and a human commercial skin melanoma cell line (SK-MEL-28). The sequencing and subsequent analysis of FASTQ files yielded final variant files, leading to the identification of mutations. Our findings revealed a total of 500 mutated genes in canine OMM, including significant ones such as EP300, FAT4, JAK3, LRP1B, NCOR1, and NOTCH1. Notably, 82 shared mutations were identified between human melanomas and canine OMM genomes. These mutations were categorized based on the gene functions. The identification of these mutations provides critical insights that can pave the way for the development of novel therapeutic strategies for both canine and human OMM, offering hope for more effective treatments in the future.

"Amplification and DNA Sequencing of the Nuclear Ribosomal Internal Transcribed Spacer Region from Dried Black Pepper Samples for Traceability"

"Amplification and DNA Sequencing of the Nuclear Ribosomal Internal Transcribed Spacer Region from Dried Black Pepper Samples for Traceability"

TE-Seq: A Transposable Element Annotation and RNA-Seq Pipeline.

The recognition that transposable elements (TEs) play important roles in many biological processes has elicited growing interest in analyzing sequencing data derived from this 'dark genome'. This is however complicated by the highly repetitive nature of these sequences in genomes, requiring the deployment of several problem-specific tools as well as the curation of appropriate genome annotations. This pipeline aims to make the analysis of TE sequences and their expression more generally accessible. The TE-Seq pipeline conducts an end-to-end analysis of RNA sequencing data, examining both genes and TEs. It implements the most current computational methods tailor- made for TEs, and produces a comprehensive analysis of TE expression at both the level of the individual element and at the TE clade level. Furthermore, if supplied with long-read DNA sequencing data, it is able to assess TE expression from non-reference (polymorphic) loci. As a demonstration, we analyzed proliferating, early senescent, and late senescent lung fibroblast RNA-Seq data, and created a custom reference genome and annotations for this cell strain using Nanopore sequencing data. We found that several retrotransposable element (RTE) clades were upregulated in senescence, which included non-reference, intact, and potentially active elements. TE-Seq is made available as a Snakemake pipeline which can be obtained at https://github.com/maxfieldk/TE-Seq . All software dependencies besides Snakemake and Docker/Singularity are packaged into a container which is automatically built and deployed by the pipeline at runtime.

The Revolution of Genetic Diagnosis: An Example from Rare Disorders

Since the advent of DNA sequencing, genetic analyses have been increasingly incorporated into clinical practice to support the diagnosis of rare disorders [...]

A Comparison of DNA-Methylation during Protoplast Culture of Ponkan Mandarin (Citrus reticulata Blanco) and Tobacco (Nicotiana tabacum L.)

The epigenetic variation in protoplast regeneration is a topic that has attracted interest recently. To elucidate the role of DNA methylation in the regeneration of protoplasts from the ponkan (Citrus reticulata), this study employs the methylation-sensitive amplification polymorphism (MSAP) molecular marker technique to analyze changes in DNA methylation levels and patterns during the isolation and culture of protoplasts from ponkan and tobacco. Additionally, differential DNA methylation fragments are cloned, sequenced, and subjected to bioinformatics analysis. The results reveal that, for non-regenerable ponkan mesophyll protoplasts, DNA methylation levels increase by 3.98% after isolation and then show a trend of initial decrease followed by an increase during culture. In contrast, for regenerable ponkan callus protoplasts and tobacco mesophyll protoplasts, DNA methylation levels decrease by 1.75% and 2.33%, respectively, after isolation. During culture, the DNA methylation levels of ponkan callus protoplasts first increase and then decrease, while those of tobacco mesophyll protoplasts show an opposite trend of initial decrease followed by an increase. Regarding DNA methylation patterns, ponkan mesophyll protoplasts exhibit primarily hypermethylation changes accompanied by a small amount of gene demethylation, whereas ponkan callus protoplasts are dominated by demethylation changes with some genes undergoing hypermethylation. The methylation exhibits dynamic changes in protoplast isolation regeneration. By recovering, cloning, sequencing, and performing BLASTn alignment analysis on specific methylation modification sites in the ponkan, 18 DNA sequences with high homology are identified which are found to be involved in various biological functions, thereby establishing a foundational basis for genetic editing in protoplasts.

THE MOLECULAR HISTORY OF IDH-MUTANT ASTROCYTOMAS WITHOUT ADJUVANT TREATMENT

Abstract AIMS Hypermutation and malignant transformation are potential complications arising from temozolomide treatment of IDH-mutant gliomas. However, the natural history of IDH-mutant low-grade gliomas without temozolomide treatment is relatively un-studied. We set out to study the molecular history of paired IDH-mutant astrocytomas without adjuvant treatment in the interim. METHOD We retrieved retrospectively from our hospitals paired tumors from 19 patients with IDH-mutant, 1p19q noncodeleted Grade 2 astrocytomas where no interim adjuvant treatment with either temozolomide or radiotherapy was given between primary and first recurrences. Two patients had multiple resected recurrences and radiotherapy but not temozolomide was given before the last specimens were resected. We studied the natural molecular history of these low-grade IDH-mutant astrocytomas without pressure of temozolomide with methylation profiling and copy number variation (CNV) analyses, target DNA sequencing, TERTp sequencing, FISH for ALT and selected biomarkers. RESULTS Recurrences were mostly higher grades (15/19 patients) and characterized by new CNVs not present in the primary tumors (17/19 cases). Few novel mutations were identified in recurrences. Tumors from 17/19 (89.5%) patients showed either CDKN2A homozygous deletion, MYC or PDGFRA amplification at recurrences. There was no case of hypermutation. Phylogenetic trees constructed for tumors for the two patients with multiple recurrences showed a conserved evolution pattern. CONCLUSION In summary, our studies demonstrated in contrast to the phenomenon of temozolomide-induced hypermutation, IDH-mutant, 1p19q non-codeleted Grade 2 astrocytomas which were not treated by temozolomide instead acquired new CNVs at tumor recurrences. These findings improve our understanding of the molecular life history of IDH-mutant astrocytomas.

Xoconochcothelphusa, a new genus for Ehecatusa chiapensis (Rodríguez & Smalley, 1972), with notes on Spirothelphusa Pretzmann, 1965 (Crustacea: Decapoda: Pseudothelphusidae)

The species of the genus Ehecatusa Ng & Low, 2010, E. chiapensis (Rodríguez & Smalley, 1972) and E. mixtepensis (Rodríguez & Smalley, in Smalley, 1970), were referred to as incertae sedis in the classification system of the tribe Pseudothelphusini Ortmann, 1897, proposed by Villalobos-Hiriart (2005) and Villalobos & Alvarez(2010) and as members of the subfamily Pseudothelphusinae in the recent phylogenetic proposal by Álvarez et al. (2020), supported with morphology and molecular information. The recent discovery of a new specimen of E. chiapensis, from Chiapas, Mexico, comes to expose again the unresolved taxonomic situation of this species in the genus Ehecatusa. New morphologic evidence from the male first gonopod and a phylogenetic analysis based on partial DNA sequences of mitochondrial and nuclear genes (COI, 16S and H3), support the placement of the two species in different genera. Consequently, Xoconochcothelphusa n. gen. is erected to receive X. chiapensis n. comb. The phylogenetic relationships of X. chiapensis n. gen., n. comb. and E. mixtepensis with other genera of the subfamily Pseudothelphusinae Ortmann, 1893 (Ehecatusa, Smalleyus Alvarez, 1989, and Spirothelphusa Pretzmann, 1965) distributed in Mexico, are examined and a key to the identification of the genera of this subfamily is provided.

Human Schistosomiasis due to Schistosoma bovis in Nigeria.

Schistosomiasis is a global public health challenge, especially in sub-Saharan Africa, where Nigeria has the highest burden of disease. Schistosoma hybrids have been discovered in various countries, including Nigeria, where livestock and human beings share common water resources. This study, carried out in three communities, two of which are endemic for urinary schistosomiasis, aimed to identify the urinary schistosomiasis causative agent by using a species-specific molecular technique and their evolutionary relationship. Polymerase chain reaction using primers specific for a partial DNA sequence of the mitochondrial cox1 gene of Schistosoma haematobium was carried out on pooled urine sediments preserved in 70% ethanol. DNA from the high-intensity pooled samples from Onye-Uku camp amplified, and a BLAST search identified the pooled samples as Schistosoma bovis, whereas S. haematobium DNA did not amplify. The phylogenetic relationship of the sequence showed that it clustered with an S. bovis hybrid obtained from a human host in Côte d'Ivoire and had close ancestry with isolates from cattle in Cameroon. This finding revealed the prevalence of S. bovis among some inhabitants in a zone in Nigeria where schistosomiasis is endemic.